MetaMorph Drop-Ins

MetaMorph Drop-Ins
Meta Imaging Series®
MetaMorph
Drop-in Commands
Version 7.0 for
®
Microsoft Windows XP
User’s Guide
1020 2102-03
ii
Copyrights, Notices, and Trademarks
© 2004 – 2006 Molecular Devices Corporation. All rights reserved. Printed in the
U.S.A.
Information in this document is subject to change without notice and does not
represent a commitment on the part of Molecular Devices Corporation. The
software described in this document, including information contained in any
databases, is furnished under a license agreement and may be used or copied
only in accordance with the terms of the agreement. It is illegal to copy the
software, except as specifically allowed in the license agreement. No part of this
manual may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopying and recording, for any purpose,
without the express written permission of Molecular Devices Corporation.
MetaMorph and ImageXpress are registered trademarks and Discovery-1,
MetaXpress, MDCStore and ImageXpress Micro are trademarks of Molecular
Devices Corporation.
All other trademarks are the property of their respective owners.
Disclaimer
Molecular Devices Corporation reserves the right to change its products and
services at any time to incorporate technological developments. This user guide
is subject to change without notice.
Although this user guide has been prepared with every precaution to ensure
accuracy, Molecular Devices Corporation assumes no liability for any errors or
omissions, nor for any damages resulting from the application or use of this
information.
iii
iv
User’s Guide
Table of Contents
List of Available Drop-ins
15
Open 4D Series into Stack (File Menu)
26
4D Selection Grid Command Shortcuts ........................................................ 32
Run User Program (File Menu)
32
Send Image as Email Attachment (File Menu)
34
Configure Email Settings (File Menu)
36
Image Properties (Edit Menu)
37
Convert Regions to Lines (Regions Menu)
40
Resequence Region Labels (Regions Menu)
41
Create Segment Regions (Regions Menu)
42
Select Region (Regions Menu)
44
Acquire Timelapse (Acquire Menu)
46
Acquire Spectral Scan (Acquire Menu)
49
Multi-Journal Timelapse (Journal Menu)
52
Equalize Light (Stack Menu)
54
Keep Planes (Stack Menu)
55
Stitch Stack (Stack Menu)
57
Make Movie (Stack Menu)
59
Kymograph (Stack Menu)
63
Montage Stacks (Stack Menu)
65
Convert Stacks to TIFFs (File Menu)
66
Interleave Stacks (Stack Menu)
67
Stereographic Views (Stack Menu)
69
Acquire (Acquire Menu)
74
Digital Camera Adjustments
92
Hamamatsu C2400-60 (Acquire Menu)......................................................... 93
MetaMorph
1
Version 7.0
Drop-in Commands
Configure Digital Camera (Acquire Menu)
95
Acquire from Digital Camera (Acquire Menu)
97
Understanding Binning ................................................................................ 100
Stop Focusing (Acquire Menu)
108
Basic Digital Acquire (Acquire Menu)
109
Error Conditions That Generate Error Messages in Basic Digital Acquire.. 111
MetaMorph
2
Version 7.0
User’s Guide
Set BNC Output Trigger (Device Menu)
113
Acquire with Frame Transfer Camera (Acquire Menu)
114
Twain Configure (Acquire Menu)
118
Acquire from Spot Camera (Acquire Menu)
120
Acquire from Flashbus (Acquire Menu)
132
Sum 16-Bit Image (Acquire Menu)
140
Select Camera/Board (Acquire Menu)
141
Acquire Color Camera (Acquire Menu)
141
Stream Acquisition (Acquire Menu)
153
Digital Camera Video Control (Acquire Menu)
159
Acquire Multiple Wavelengths (Acquire Menu)
160
Configure Intensifier Gain Control (Acquire Menu)
166
Set Intensifier Gain (Acquire Menu)
168
Set Camera Level and Gain (Acquire Menu)
168
PI Video ICCD Settings (Acquire Menu)
169
Nikon Microscope
171
Olympus Microscope
175
Zeiss MTB Microscope
180
Leica DMR Microscope
184
Linkam MDS600/TMS93 Stage
189
Heating Sequence
191
Kodak MotionCorder (Devices Menu)
194
Kodak MotionCorder Procedures ................................................................ 194
Kodak - Dialog Box Options ........................................................................ 198
MetaMorph
3
Version 7.0
Drop-in Commands
Auto-Focus via Hardware (Devices Menu)
200
Auto-Focusing the Microscope
201
Configure Auto-Focus via Hardware (Devices Menu)
201
Auto-Focus via Software (Devices Menu)
202
Adjust Focus (Auto-Focus via Software)
203
Find Focus (Auto-Focus via Software) (Devices Menu)
206
Resync Focus Dialog with Olympus Z-Motor (Devices Menu)208
Custom I/O Control (Devices Menu)
208
Send Serial Data (Devices Menu)
209
Wait for Serial Data (Devices Menu)
210
Set Digital I/O (Devices Menu)
212
Wait for Digital I/O (Devices Menu)
213
Syntax Rules................................................................................................ 215
ASCII Control Code Chart ........................................................................... 216
MetaMorph
4
Version 7.0
User’s Guide
MMKeyPad
216
Color Align (Display Menu)
218
Color Mosaic (Display Menu)
223
Split View (Display Menu)
226
Interlace Images (Display Menu)
232
Duplicate as Displayed (Edit Menu)
233
Arrow (Display Menu)
234
Grid (Display Menu)
238
Stretch and Mirror (Display Menu)
240
Boxes on Binary Image (Display Menu)
241
Text (Display Menu)
243
Gray Wedge (Display Menu)
245
Show/Hide Image at Full Screen (Display Menu)
248
Graph Settings
250
Set Color Threshold (Measure Menu)
257
Combine into B&W + Color (Display Menu)
261
Log Color Threshold (Log Menu)
264
Save Original and Result Loop (Journal Menu)
265
Set Image Zoom (Display Menu)
268
FFT (Process Menu)
269
The FFT Pattern Removal Filter .................................................................. 271
The Blur FFT Filter ...................................................................................... 272
The High Pass FFT Filter ............................................................................ 273
The Homomorphic FFT Filter ...................................................................... 274
Convolve with Image Kernel (Process Menu)
275
2D Deconvolution (Process Menu)
278
The Process of Nearest Neighbors Deconvolution ......................... 278
Measured PSF Decon (Process Menu)
282
Batch Deconvolution (Process Menu)
285
Summary Procedure ........................................................................... 285
MetaMorph
5
Version 7.0
Drop-in Commands
3D Deconvolution (Process Menu)
289
Sub-Pixel Shift (Process Menu)
302
Optical Density (Scaled) (Process Menu)
303
Ratio Images (Process Menu)
305
IMD Display ................................................................................................. 306
MetaMorph
6
Version 7.0
User’s Guide
Use Region for Background (Legacy)
307
Overlay Images (Display Menu)
309
Produce Background Correction Image (Legacy)
314
Log Image Annotation (Log Menu)
317
Log Image Histogram (Log Menu)
318
Open Object Log (Log Menu)
320
Close Object Log (Log Menu)
322
Pause Object Logging (Log Menu)
322
Resume Object Logging (Log Menu)
323
View Current Object Log (Log Menu)
323
Log All Object Data (Log Menu)
324
Open Summary Log (Log Menu)
324
Close Summary Log (Log Menu)
326
Pause Summary Logging (Log Menu)
326
Resume Summary Logging (Log Menu)
326
View Current Summary Log (Log Menu)
327
Open EdgeList Log (Log Menu)
327
Close EdgeList Log (Log Menu)
329
Pause EdgeList Logging (Log Menu)
330
Resume EdgeList Logging (Log Menu)
330
View Current EdgeList Log (Log Menu)
330
Display EdgeList Log as Image (Log Menu)
331
Configure Object Classifiers (Measure Menu)
333
Configure Object Measurements (Measure Menu)
337
Annotate Measured Objects (Measure Menu)
341
Measure Objects (Measure Menu)
343
Recalculate Object Parameters (Measure Menu)
344
MetaMorph
7
Version 7.0
Drop-in Commands
Measure Objects with Mask (Measure Menu)
345
Measure Single Object (Measure Menu)
347
Morphometry Histogram (Measure Menu)
349
Cut Objects (Measure Menu)
350
Join Objects (Measure Menu)
352
Integrated Morphometry Analysis (Measure Menu)
353
Internally Threshold Objects (Measure Menu)
363
Create Regions Around Objects (Regions Menu)
364
Show Classifier Statistics (Measure Menu)
365
Show Individual Object Data (Measure Menu)
367
Reset Object Measurements (Measure Menu)
368
Create Classifier Stack (Measure Menu)
369
Measure Distance with Annotation (Measure Menu)
370
Graph Intensities (Apps Menu)
371
Calipers (Measure Menu)
377
Count Cells (Apps Menu)
380
Count 2 Types of Cells (Apps Menu)
387
Measure Colocalization (Apps Menu)
398
Morphology Filters
401
Segment Image (Process Menu)
409
Segment Image Modifications
412
Correlation Plot (Apps Menu)
417
Log Pixels in Region (Log Menu)
420
Measure Grid (Apps Menu)
423
Measure XYZ Distance (Apps Menu)
429
Measure Volume (Apps Menu)
435
Measure Object Distance (Measure Menu)
438
MetaMorph
8
Version 7.0
User’s Guide
Track Objects (Apps Menu)
440
Track Objects: Stat Table - Dialog Box Options
457
Track Points (Apps Menu)
460
Track Points Data Type Display .................................................................. 464
Show Zeiss Image Info (Edit Menu)
468
Start Recording (Journal Menu)
470
Stop Recording (Journal Menu)
471
Pause Recording (Journal Menu)
472
Resume Recording (Journal Menu)
472
Run Journal (Journal Menu)
472
Edit Journal (Journal Menu)
473
Application Note — How to use image file variables to save images in a journal
without user interaction................................................................... 474
MetaMorph
9
Version 7.0
Drop-in Commands
Loop a Journal (Journal Menu)
479
Loop for All Planes (Journal Menu)
481
Loop for All Regions (Journal Menu)
482
Loop for All Images in Directory (Journal Menu)
483
Toggle Interactive (Journal Menu)
485
Show Message and Wait (Journal Menu)
486
Record Image State (Journal Menu)
487
Select Image (Journal Menu)
488
Change Plane (Journal Menu)
490
Beep (Journal Menu)
490
Delay (Journal Menu)
491
Pick Point (Journal Menu)
492
Pick Point - Dialog Box Options
492
Banch on User Input (Journal Menu)
493
Branch on Object Measurement (Journal Menu)
494
Introduction to the Use of Variables
498
The Variables Commands:................................................................. 500
Using Variables - Procedures...................................................................... 500
MetaMorph
10
Version 7.0
User’s Guide
Assign Variable (Journal Menu)
500
Enter Variable (Journal Menu)
506
511
Delete Variable
Log Variable
512
Branch on Variable
513
Loop Variable
515
StartUp Journal (Journal Menu)
519
Import Journal Suite
520
Export Journal Suite (Journal Menu)
521
Create Taskbar (Journal Menu)
523
Edit Taskbar (Journal Menu)
526
Load Taskbar (Journal Menu)
529
Taskbar Shortcuts (Journal Menu)
530
Show Taskbar (Journal Menu)
530
Hide Taskbar (Journal Menu)
531
Graph Variable Value (Journal Menu)
531
Find Spots
534
Tissue MicroArray Acquisition
536
TMA: Set Alignment
541
Multi Dimensional Acquisition (Apps Menu)
549
Binning
563
Acquiring Multiple Dimensions .................................................................... 564
Multi Dimensional Acquisitions - Options .................................................... 564
MetaMorph
11
Version 7.0
Drop-in Commands
Review Multi Dimensional Data (Apps Menu)
564
Review Multi Dimensional Data - Options
565
Create Rotational Sequence (Apps Menu)
573
Multi Dimensional Data Set Utilities (Apps Menu)
574
Build .nd Set (Apps Menu)
578
Run Journal for Multi Dimensional Data
581
Scan Slide
584
AQI 3D Visualizer
597
AQI 3D Visualizer - Dialog Box Options
597
Using the AQI 3D Visualizer
597
Menu Commands
599
Menu Commands - View
599
Menu Commands - Color Map
603
The X-, Y-, and Z- axes of the stack are represented by the red, green, and
blue lines, respectively.
616
This is the orthogonal slices view of a stack. To change views, click the View
menu and select from the list.
616
The floor is used as a 3-D point of reference. To remove the floor, click
Options and uncheck Display Floor.
616
616
FRET Analysis (Apps Menu)
628
628
Plate Acquisition Setup
628
Plate Acquisition and Control
665
Configure Fluidic Stations
669
Define Tips
672
Reset Tips
674
Fluidics System Properties
675
MetaMorph
12
Version 7.0
User’s Guide
Fluidic Event
676
Fluidic Event Steps...................................................................................... 677
Fluidic Control
681
Plate Acquisition Toolbar
683
Plate Acquisition
685
Shading Correction...................................................................................... 687
Load Plate Acquisition Settings
687
Save Acquisition Setting
688
Configure Laser Sensor
690
Plate Data Utilities
695
Add Analysis to Database
698
Import Images
700
Import Cellomics Data
702
Monopole Detection
703
Multi Wavelength Cell Scoring (Apps Menu)
708
General Procedures ............................................................................ 708
Neurite Outgrowth (Apps Menu)
716
Angiogenesis Tube Formation (Apps Menu)
726
Cell Proliferation HT
729
Cell Scoring (Apps Menu)
732
General Procedures ............................................................................ 733
Cell Cycle
740
Cell Health (Apps Menu)
749
Mitotic Index (Apps Menu)
757
General Procedures ............................................................................ 758
Live Dead (Apps Menu)
764
Granularity (Apps Menu)
770
Nuclear Translocation HT
775
Using Nuclear Translocation HT ....................................................... 775
MetaMorph
13
Version 7.0
Drop-in Commands
Transfluor®
779
Cellular Results
785
Adaptive Background Correction™ System
786
Making the Best Use of the Adaptive Background Correction™ System
788
Count Nuclei (Apps Menu)
789
Transfluor® HT
793
Translocation (Apps Menu)
797
Using Translocation............................................................................ 798
Translocation-Enhanced (Apps Menu)
801
Review Plate Data (DB)
809
Plate Dialog Box
819
Sharing and Security
821
Review Screen Data
823
Screen Data Utilities
832
Auto Run Mode
834
Auto Run Status
835
Run Analysis on Plates
837
Run Assay on Plates (Apps Menu)
838
Screen Acquisition (Legacy)
839
Load Screen Acquisition State
859
Configure Focus Sensor (Version 1)
861
Configure Focus Sensor (Version 2)
862
Configure Plate Loader
868
Screen: Set Alignment (Apps Menu)
872
MetaMorph
14
Version 7.0
User’s Guide
List of Available Drop-ins
The following table lists all MetaMorph Drop-ins, the associated commands, the menus from
which they are accessed, the assigned drop-in category, and a brief description.
Drop-in
Menu
Command Name
Category
Description
ACQUIRE
Acquire
Acquire
Common
Acquires images
from digital
cameras.
ACQUIRECOLOR
Acquire
Acquire Color
Common
Configures
acquisition
parameters for
single-chip color
cameras such as
the CoolSNAP
camera, and
acquires color or
grayscale images.
ACQUIREULTRA
Acquire
Acquire from
ImageXpress Ultra
Special
Acuires images from
an ImageXpress
Ultra.
DCAMVID
Acquire
Digital Camera
Video Control
Common
Dialog which
controls standard
video output of
digital cameras.
DIGITAL4
Acquire
Acquire from
Digital Camera
Common
Acquires from digital
camera. Can be
used with any digital
camera.
FLASHBUS
Acquire
Acquire from
Flashbus
Common
Acquires images
from the video
camera using the
Flashbus board.
SPOTCAM
Acquire
Acquire from Spot
Camera
Common
Acquires 24-bit color
images or stacks of
12-bit singlechannel
monochrome
images from a
SPOT camera.
STREAM
Acquire
Stream Acquisition
Common
Configures and
controls high-speed
stream acquisition
data to RAM or
realtime hard disk.
TLAPSE
Acquire
Acquire Timelapse
Common
Acquires a series of
frames at a
specified interval
and duration.
VIDEVICE
Acquire
Set Acquisition
Board-Camera
Common
Selects the video
device and switches
MetaMorph
15
Version 7.0
Drop-in Commands
between cameras.
CELLVIEW
Acquire
Cellview
Acquisition
Legacy
Controls devices
and acquisition from
Scanalytics EPR
system. Use Multi
Dimensional
Acquisition
(ndacquir) instead.
this is a legacy
drop-in.
CFGCCD
Acquire
Configure Digital
Camera
Legacy
Configures a digital
CCD camera. This
is a legacy drop-in.
These settings are
now made in the
Meta Imaging
Series
Administrator.
ZOOMPAN
Acquire
Zoom, Pan and
Scroll Video
Legacy
Zooms, pans, and
scrolls the video
monitor display
(requires board).
MULTIWAC
Acquire
Acquire Multiple
Wavelengths
Occasional
Configures and
performs acquisition
of images using up
to six different sets
of settings for
wavelength,
intensity, and
exposure.
C240060
Acquire
Hamamatsu
C2400-60
Special
Allows control of
Hamamatsu C240060 camera
controller.
DIGADJ
Acquire
Digital Camera
Adjustments
Special
Adjusts black level
for Hamamatsu
ORCA-Series
cameras.
DWS
Acquire
Acquire DWS
Images
Special
Dual wavelength
strobe custom
application.
FRAME
Acquire
Acquire Image with
Frame Transfer
Camera
Special
Acquires images
with a Frame
Transfer Camera.
SPECTRAL
Acquire
Acquire Spectral
Scan
Special
Acquires a series of
images in a range of
wavelengths from
monochromators.
SUM16
Acquire
Sum 16-bit Image
Special
Sums video into a
16-bit image.
(requires Flashbus)
TWAINCFG
Acquire
Configure TWAIN
Special
Provides a TWAIN
driver configuration
dialog to select a
Twain-compliant
device for image
MetaMorph
16
Version 7.0
User’s Guide
acquisition and
specify whether to
use the device's
user interface.
SCANSLIDE
Apps
Scan Slide
Special
Automatically scans
a user-defined
section of a slide
and displays the
result as a single
image.
BTIME
Apps
Graph Intensities
Common
Graphs multiple
regions from stacks
or live video.
NDACQUIR
Apps
Multi Dimensional
Acquisition
Common
Acquires images or
image stacks in
multiple dimensions
with mulltiple
parameters and
saves the images in
multi dimensional
data sets.
NDPLAYER
Apps
Review Multi
Dimensional Data,
Multi Dimensional
Set Utilities
Common
Reviews, performs
file operations, and
runs journals on
multi dimensional
data sets.
FINDSPOTS
Apps
Find Spots
Occasional
Finds Spots On An
Image And Highlight
Them With Regions
TRACKOBJ
Apps
Track Objects
Common
Performs motion
analysis by
automatically
tracking one or
more objects
through an image
stack, or a
sequential series of
single images.
TRACKPTS
Apps
Track Points
Common
Performs motion
analysis by
manually tracking
points through
planes in a stack.
ASTM
Apps
ASTM Grain Size
Legacy
Performs limited
metallurgical ASTM
grain size
measurements.
OPEN4D
Apps
Open 4D Series
into Stack
Legacy
Creates a single
stack of images
selected from a grid
that references a
series of images on
disk. This is a
legacy drop-in.
Suggest using
Review MultiDimensional Data
MetaMorph
17
Version 7.0
Drop-in Commands
(ndplayer dropin)
instead.
CELLCNT
Apps
Count 2 Type of
Cells, Count Cells
Occasional
Counts cells, or two
groups of cells
distinquished by
staining.
COLOCAL
Apps
Meaure
Colocalization
Occasional
Measure
colocalization two
fluorescent probes
in B&W images.
CORRPLOT
Apps
Correlation Plot
Occasional
Creates a
correlation plot
between the
intensities of
corresponding
pixels in two
images.
MEASGRID
Apps
Measure Grid
Occasional
Performs
measurements or
runs journals over a
user definable grid
pattern.
MEASXYZD
Apps
Measure XYZ
Distance
Occasional
Measures manually
the distance
between points in
different Z-axis
planes.
MVOLUME
Apps
Measure Volume
Occasional
Measures the
volume of a
thresholded object
through a stack.
ODSCALE
Apps
Optical Density
(Scaled)
Occasional
Creates scaled
optical density
images and
performs scaled
optical density
measurements.
AQI3DVIEW
Apps
AQI 3D Visualizer
Occasional
AQI 3D Visualizer
enables you to more
fully visualize 3D
stacks.
TMACQUIRE
Apps
Tissue MicroArray
Acquisition
Special
Locates , identifies,
and acquires
images of tissue
microarray spots, on
a semi automated
basis.
FRET
Apps
FRET Analysis
Occasional
Performs
background and
bleed through
correction to
Fluorescence
Resonance Energy
Transfer (FRET)
image sets.
MetaMorph
18
Version 7.0
User’s Guide
AUTOFO_S
Devices
Autofocus via
Software > Adjust
Focus, Find Focus
Common
Finds an optimum
focal position for the
microscope using
an intensity
measurement
algorithm. Used for
both Adjust Focus
and Find Focus.
SHUTTER
Devices
Shutter
Common
Opens/closes/toggle
s active shutter.
CENTROID
Devices
Move Motors,
Configure Motors,
Refocus Motors,
Acquire Centroid Z
Series
Legacy
Controls the
Centroid motion
controller. This is a
legacy drop-in for a
device that is no
longer supported by
Molecular Devices.
CUSTOMIO
Devices
Custom I/O Control
Occasional
Controls parallel
and serial
Input/Output
devices. This is a
custom drop-in.
AUTOFCUS
Devices
Autofocus via
Hardware
Special
Triggers Ludl or
Prior autofocus
controller cards to
run.
KODAK
Devices
Kodak
MotionCorder
Special
Acquires and plays
back images with
the Kodak
MotionCorder digital
video device.
LINKAM
Devices
Linkam
MDS600/TMS93
Stage
Special
Linkam
MDS600/TMS93
Stage temperature,
flow rate, and XY
control.
MCU
Devices
MCU Driver
Parameters
Special
Sets the Zeiss MCU
26/27 XYZ device
settings.
PIBNC
Devices
Set BNC Output
Trigger
Special
Set the BNC trigger
on Princeton
Instruments
cameras.
SETICCD
Devices
Intensifier Gain
Special
Configures and
controls an
intensified CCD and
provides ICCD gain
control.
LEICADM
Devices
Leica DMR
Microscope
Special
Leica DMR
automated
microscope control.
NIKON
Devices
Nikon Microscope
Special
Nikon automated
microscope control.
OLYMPUS
Devices
Olympus
Microscope
Special
Olympus automated
microscope control.
MetaMorph
19
Version 7.0
Drop-in Commands
ZEISSMTB
Devices
Zeiss MTB
Microscope
Special
Zeiss automated
microscope control.
You must have the
Zeiss MicroToolbox
(MTB) software
installed. Version
2.13g or later of the
MTB is required.
LINKAM
Devices
Linkam Heated
Stage
Special
Linkam
MDS600/TMS93
Stage temperature,
flow rate, and XY
control.
MMKEYPAD
Devices
MMKeypad
Special
Provides an
interface for a
Remote Control
Keypad
ARROW
Display
Graphics > Arrow
Common
Stamps arrows onto
image.
CALIGN
Display
Color Align
Common
Shifts the red,
green, and blue
planes of an image
independently to
bring them into
alignment Can be
used to align
fluorescent images
acquired with
separate cubes.
OVERFLUO
Display
Overlay Images
Common
Combines a
grayscale image
and up to six
fluorescence
images into a single
image and assigns
a different color to
each image.
SETZOOM
Display
Set Image Zoom
Common
Specifies image
zoom, or zooms a
region to the size of
the image and
applies a selected
magnification level
(1 - 800%).
TEXT
Display
Graphics
Common
Draws text on an
image.
WEDGE
Display
Graphics > Gray
Wedge
Common
Draws a gray wedge
on the image.
COMB_BWC
Display
Combine Into B&W
+ Color
Legacy
Creates a simple
overlay of black and
white and color
images This is a
legacy dropin.
Recommend using
Overlay Images
(overfluo) instead.
MetaMorph
20
Version 7.0
User’s Guide
FULLSCR
Display
Show/Hide Image
at Full Screen
Occasional
Displays the
selected image
centered against a
black background.
Useful for
presentations.
GRID
Display
Graphics > Grid
Occasional
Draws a grid on an
image or live video.
GRIDBIN
Display
Graphics> Boxes
on Binary Image
Occasional
Draws grid/boxes on
binary images.
ILACE
Display
Interlace Images
Occasional
Interlaces two
images into a single
image (opposite of
de-interlace).
STRETCH
Display
Stretch and Mirror
Occasional
Stretches and/or
mirrors an image.
SUBRGN
Display
Use Region For
Background
Occasional
Uses the Region
For Background by
subtracting the
grayscale value in a
selected region from
each pixel in an
image.
SUBSHIFT
Display
Sub-Pixel Shift
Occasional
Shifts an image in
sub-pixel
increments in
horizontal and/or
vertical directions.
MOSAIC
Display
Color Mosaic
Special
Creates color
images from black
and white images
acquired using the
Sony color mosaic
CCD.
SPLITVIEW
Display
Split View
Occasional
Splits apart multiple
images from a beam
splitter or twin
cameras.
24BITCPY
Edit
Duplicate > As
Displayed
Common
Duplicates image as
a 24 bit color image
with overlays.
PROPMGR
Edit
Image Properties
Occasional
Defines and or
removes image
annotation
properties to an
image or a stack of
images.
ZEISSINF
Edit
Show Zeiss Image
Info
Special
Displays Zeiss
confocal image
information.
EMAIL
File
Send Image as
Email Attachment
Occasional
Send image as
email attachment.
STK2TIFF
File
Convert Stack to
TIFF's
Occasional
Saves all stacks in a
directory as
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Version 7.0
Drop-in Commands
sequential TIFF
files.
RUNUSER
File
Run User Program
Special
Runs a user-written
Visual Basic
program from within
MetaMorph.
MMVAR
Journal
Variables
Common
Creates and applies
user-defined
variables for
advanced
journaling.
JOURNAL
Journal
Common
Journal
menu
commands.
Contains most of
the Journal
commands
SAVELOOP
Journal
Loop > Save
Original and Result
Loop
Legacy
Applies one or two
journals to each
image in a selected
directory, saving
both original and
result images in a
separate directory.
JNLYESNO
Journal
Journal Control >
Branch On User
Input
Occasional
Chooses one of two
journals to run
based on user input
.
LPALLDIR
Journal
Loop > Loop for all
Images in
Directory
Occasional
Loop for all images
in a directory.
MBRANCH
Journal
Journal Control >
Branch on Object
Measurement
Occasional
Runs one journal if
an object
measurement meets
a set of criteria, and
can run a different
journal if it does not.
PICKPT
Journal
Recording Tools >
Pick Point
Occasional
Special journal
function to click on
an image and
record the
coordinates to
variables.
SELRGN
Journal
Recording Tools >
Select Region
Occasional
Specialized function
used by journals to
select a region by its
label.
SOUND
Journal
Journal Tools >
Play Sound File
Occasional
Plays a sound file.
STARTUP
Journal
Journal Control >
StartUp Journal
Occasional
Configures a journal
to run automatically
whenever you start
MetaMorph.
TLAPSE2
Journal
Journal Control >
Multi-Journal
Timelapse
Occasional
Plays multiple
journals at varying
intervals.
EDGELIST
Log
Display EdgeList
Occasional
Creates centroid
image and binary
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User’s Guide
Log as Image
stack by reading an
edgelist log file.
EXTHRESH
Log
Log Color
Threshold
Occasional
Logs the color
threshold setting as
text.
LOGAN
Log
Log Image
Annotation
Occasional
Logs image
annotation
information.
LOGHISTO
Log
Log Image
Histogram
Occasional
Logs image
histogram data.
LOGPIX
Log
Log Pixels in
Region
Occasional
Logs pixel numeric
data.
CLRTHRES
Measure
Set Color
Threshold
Common
Sets the color
threshold range for
24-bit color images
using RGB, HSI, or
HSL color models.
IMA
Measure
Integrated
Mophometry and
Analysis (IMA)
Common
Counts, measures,
and logs
measurement data
of thresholded
objects.
ANDIST
Measure
Measure Distance
with Annotation
Legacy
Measures distance
with annotation to
data log.
AUTOMEAS
Measure
Morphometry
Legacy
Performs
morphometric
measurement,
analysis, and
logging of
measurement data
from image objects
This is a legacy
dropin for
performing
automated
measurements.
Recommend using
the IMA drop-in
instead.
MSTACK
Measure
Morphometry >
Create Classifier
Stack
Legacy
Creates a measured
stack from the result
of Measure Objects
Note: This drop-in
requires the
AUTOMEAS drop-in
to be installed. The
Create Classifier
Stack command
cannot be used in a
journal.
OBJDIST
MetaMorph
Measure
Measure Object
Distance
Legacy
23
Measures the
distance of a line
region across a
thresholded object.
Version 7.0
Drop-in Commands
ANMEAS
Measure
Annotate
Measured Objects
Occasional
Stamps IMA
measurements on
image.
CALIPERS
Measure
Calipers
Occasional
Measures the
straight line distance
between a pair of
movable "caliper"
lines.
CUTJOIN
Measure
Cut Objects, Join
Objects
Occasional
Cuts or join objects.
Use this command
before using the
Integrated
Morphometry
Analysis command.
THRESHOB
Measure
Internally
Threshold Objects
Occasional
Separates objects
measured in IMA
according to each
object's intensity
information.
TOCKMAN
Measure
Optical Density
Application
Special
Customized optical
density application.
BACKCORR
Process
Produce
Background
Correction Image
Occasional
Produces a
Background
Correction Image
using a variablemedian filter.
FFT
Process
FFT
Occasional
Performs Fast
Fourier Transform
(FFT) filtering of
images.
KERNEL
Process
Convolve with
Image Kernel
Occasional
Creates and
convolves large
image kernels.
2DDECON
Process
3D DeconvolutionNo Neighbors, 2D
DeconvolutionNearest Neighbors
Common
Functions for
performing 2d
subtractive
deconvolution
MEASPSF
Process
Measured Point
Spread Function
Occasional
Performs ThreeDimensional
iterative
deconvolution.
3DDECON
Process
AutoQuant 3D
Deconvolution
Occasional
Performs ThreeDimensional
iterative
deconvolution.
MORPHOLOGY
Process
Morphology Filters
Common
Filters and smooths
binary and
grayscale images.
RATIO
Process
Ratio Images
Occasional
Ratio images from
two sources (like
MetaFluor).
TRACEOBJ
Regions
Create Region
Around Objects
Common
Creates regions
around objects in
the currently active
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image.
LINERGNS
Regions
Convert Regions to
Lines
Occasional
Converts polygon
regions to line
regions.
RGNSEQ
Regions
Resequence
Region Labels
Occasional
Renumbers regions
in an image,
eliminating "gaps" in
the sequence left
after removing one
or more regions..
SEGMENTS
Regions
Create Segement
Regions
Occasional
Creates polygon
segments along a
line region.
PLATEACQUIRE
Screening
Plate Acquisition,
Special
Acquires images
from multi well
plates. For
MetaXpress
systems only.
Plate Acquisition
and Control,
Plate Acquisition
Setup
HTPLAYER
Screening
Review Screen
Data, Screen
Utilities
Special
Reviews, performs
file operations, and
runs journals on
multi well plate data.
HTDB_PLAYER
Screening
Review Plate Data,
Plate Utilities for
Database
Special
Accesses and
retrieves images
from the database.
Reviews, performs
file operations, and
runs journals on
multi well plate data.
Runs assays on
stored images.
3D
Stack
3D Reconstruction,
View Orthoganol
Planes,
Topographic
Surface. Process >
Remove Haze.
Common
Acquires, performs
3D reconstruction
of, and analyzes Zseries stacks.
KEEPPLN
Stack
Keep Planes
Common
Selects the subset
of planes from a
stack; chooses
which planes in a
stack to
keep/discard
PAIR
Stack
Montage Stacks
Common
Combines 2-4
stacks into a
montage stack of 24 panels.
EQUALIZE
Stack
Equalize Light
Occasional
Equalizes the light
levels of a stack of
images.
INTLEAVE
Stack
Interleave Stacks
Occasional
Interleaves planes
from two stacks into
a single stack.
KYMO2
Stack
Kymograph
Occasional
Creates a cross-
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Version 7.0
Drop-in Commands
sectional view of the
intensity values of a
line region through
the planes in a
stack.
MAKEAVI
Stack
Make AVI
Occasional
Creates an AVI
video file from an
image stack.
STITCH
Stack
Stitch Stack
Occasional
Takes a stack of
calibrated images
and stitches them
into one image.
STEREO
Stack
Stereographic
Views
Special
Creates
stereographic image
pairs from a Zseries stack.
Open 4D Series into Stack (File Menu)
Creates a single stack of images from a collection of images on disk. Allows you to
preview images while making your selection.
Drop-in: OPEN4D
Use this command when you want to create a new stack of images that you have selected from a series
of image files. This command allows you to create a selection grid that references individual image
planes from an image stack, images from files with sequentially numbered names or extensions, and a
variety of individual image files. Images can then be selected from this 4D Selection Grid, and selected
images will be placed in a new stack. All image file formats supported by MetaMorph can be accessed
by the 4D Selection Grid.
Open 4D Series into Stack is best used in situations where a four-dimensional series of images has
been acquired. For example, you may have acquired a Z-series (depth) image stack at each of several
time points, or may have images that have been acquired at a number of different wavelengths at each
of a number of Z-positions.
Note: Be sure to choose Grid Use to view a display of the keyboard shortcuts and mouse
actions for selecting images from the grid.
Open 4D Series into Stack - Overview
Configuring the 4D Grid Layout - Single Series Mode
Configuring the 4D Grid Layout - User Defined Mode
Selecting Images from the 4D Grid
Open 4D Series into Stack - Overview
To create a single stack of images from a collection of images on disk, use the following
procedure:
Step
1
Action
From the File menu, choose Open 4D Series
into Stack. The Open 4D Series Into Single
Stack dialog box and 4D Selection Grid will
appear.
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2
Choose Base File and use the Select Base
File dialog box to select the first file to be
displayed in the 4D Selection Grid. The base
file should be the file for the image that you
wish to be the first one being referenced in
the grid. If necessary use the Look In list or
Up One Level icon button to select a different
folder. The selected base file will be
displayed in the status line next to the Base
Line button.
3
If (1) the base file is a stack or is the first in a
continuous series of files with sequentially
numbered names or extensions, and (2) this
file will be the exclusive source of images for
the 4D Selection Grid, select 4D Images
Stored as Single Sequential Series, so that
an "X" appears in the check box. The dialog
box will change to reflect the Single Series
mode.
OR
If you plan to add image files that have a
different base name from the selected Base
File, leave the 4D Images Stored as Single
Sequential Series check box cleared. The
format of the dialog box will stay in User
Defined mode.
4
Decide on a layout for storage of images in
the 4D Selection Grid. This will be
determined by the number of images in each
series or image stack. The configuration
process and steps involved in adding images
to the 4D Selection Grid will be different
depending on whether you are in Single
Series mode or User Defined mode (see
Step 3).
5
When the 4D Selection Grid has been
configured and images have been added to
it, you may begin selecting images from
the grid to be used in creating a new image
stack.
6
When you have finished selecting images,
choose OK. Your new stack of selected
images will appear. Images will appear in the
stack in the order in which they were
selected.
7
Choose Close to close the Open 4D Series
into Single Stack dialog box.
Configuring the 4D Grid Layout - Single Series Mode
To configure and add images to the 4D Selection Grid in "Single Series" mode, use the following
procedure:
Step
1
Action
In the X Max spin box, enter the number of
images available in each row. This will affect
the maximum number of columns being
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Drop-in Commands
displayed in the 4D Selection Grid.
AND
In the Y Max spin box, enter the maximum
number of rows to be displayed in the 4D
Selection Grid.
2
If you wish, you may change the X-axis title
from the default "Time" by typing a new
name in the X Axis group's Title text box.
Similarly, you may change the Y-axis title
from the default "Z" by typing a new name in
the Y Axis group's Title text box.
3
Select the method for indexing the files
(Name, Directory, Extension, or Plane) from
the X Axis group’s Source drop-down menu.
The files to be added will start at the base file
and will be indexed incrementally by the
selected file attribute.
4
If image planes increment from row to row
and then by column, select Incr X by Y Max
from the X-Y Interaction group. This selection
will make the Incr. By and Y Max options
interactive: changing one will change the
other.
OR
If image planes increment from column to
column and then by row, select Incr Y by X
Max. This selection will enforce a value of 1
in the X Axis group's Incr. By spin box.
Configuring the 4D Grid Layout - User Defined Mode
To configure and add images to the 4D Selection Grid in User Defined mode, use the following
procedure:
Step
Action
1
If you wish, you may change the X-axis title
from the default "Time" by typing a new
name in the X Axis group's Title text box.
Similarly, you may change the Y-axis title
from the default "Z" by typing a new name in
the Y Axis group's Title text box.
2
Select the method for indexing the files
(Name, Directory, Extension, or Plane)
across rows and down columns from the X
Axis and Y Axis groups' Source pull-down
menus, respectively. For example, you may
wish to index the Y-axis by incrementing the
Extension numbers and index the X-axis by
file Name.
3
If you are only adding images from
sequentially indexed files, skip to Step 7.
OR
If you are adding images from a user-defined
list (i.e. non-sequential), select the List check
box for the appropriate axis. The Set List
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Version 7.0
User’s Guide
button will now be enabled. You will be able
to add files from a user-defined list to only
one axis. The List check box and Set List
button for the other axis will be unavailable
and will appear dimmed.
4
Choose Set List. The Select File List by
Hand dialog box will appear.
5
Choose Add File and select a file to be
added to the Files list.
AND
Choose OK, and the file will be added to the
list.
6
Repeat Step 5 for all image files you wish to
add, and choose OK from the Select File List
by Hand dialog box.
7
Use the X Axis and Y Axis groups' Incr. By
spin boxes to specify how to increment the
indexing of images (as specified in Step 2)
across rows and down columns, respectively.
These values typically will both be 1.
8
Choose Auto Range to set the range to be
displayed in the 4D Selection Grid, based on
the available files.
Selecting Images from the 4D Grid
To select images from the 4D Selection Grid for placement in a new image stack, use the
following procedure:
Step
1
Action
If you have a relatively small number of
images being referenced in the 4D Selection
Grid, you will probably want to verify that the
Grid Range in the Open 4D Series into
Single Stack dialog box is showing all rows
and columns. If you see a box-in-box image
in the Grid Range region, drag the borders of
the inner box until they match the borders of
the outer box.
OR
If you have a large number of images being
referenced, you will want to verify that the
box-in-box image in the Grid Range region is
highlighting the area which references
images you wish to select next. If necessary,
move the box-in-box outline within the Grid
Range boundaries so that the smaller box
encloses the desired images. This smaller
region of the entire grid will be represented in
the 4D Selection Grid window.
2
If you wish, you can use the Grid Size slider
in the Open 4D Series into Single Stack
dialog box to change the size of the 4D
Selection Grid window.
3
If you wish to turn off the grid display in the
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Drop-in Commands
4D Selection Grid window, clear the Grid
check box in the Open 4D Series into Single
Stack dialog box. Selecting it again will
reenable the grid display.
4
If you want to see a preview of selected
images in a separate window as each image
is selected, select the Show Image check
box. Clearing the check box will disable the
preview.
5
Use your mouse or keyboard to select
images from the 4D Selection Grid. If you are
using keyboard shortcuts, you will need to
make the 4D Selection Grid the active
window ( [CTRL] + [TAB] ). Be sure to
choose Grid Use for a display of all
Selection Grid commands.
6
Images will be added to the new image stack
in the order in which they were selected. If
you wish to automatically reorder the
numbering of selected images, choose
Renumber. Images will be renumbered, with
"top-to-bottom" renumbering having priority
over "left-to-right" renumbering.
7
When satisfied with your image selection,
choose OK. A new image stack will appear,
containing your selected images. The image
stack window will have a title that is based on
the specified Base File name, with "4D" and
the 4D Selection Grid's starting and ending
X-Y coordinates appended to the name.
8
Choose Close.
Open 4D Series into Stack - Dialog Box Options
Base File
Brings up a file selector for choosing the first file. This file will form the basis from which subsequent files are
selected.
4D Images Stored as Single Sequential Series
Select this check box if all images of the 4D series are stored in a single stack or are stored in one
continuous set of files with sequentially incremented file names or extensions. When you enable this option,
the Open 4D Series dialog will be reconfigured for Single Series mode. The List, Set List, Source, and Incr.
By options in the Y Axis group will be disabled, and a new control, X-Y Interaction, will appear.
X Max
Sets the maximum number of available columns.
Title (X Axis)
This editable text field sets the title for the X-axis of the 4D Selection Grid.
List (X Axis)
This check box determines if the images to be added to each column will be from a user-defined list. Only
one axis may be defined from such a list. When this check box enabled in the X Axis group, the
corresponding List check box and Set List button will be disabled in the Y Axis group.
Set List (X Axis)
This button brings up a dialog for adding image files to a user-defined list. The image files will be added to
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Version 7.0
User’s Guide
the 4D Selection Grid, one to a column. The Source option for the X Axis group will automatically be
configured to Name. Accordingly, the Source option for the X Axis group will be unavailable.
Source (X Axis)
This pull-down menu selects the method for sorting image files across the X-axis of the 4D Selection Grid.
Sorting can be performed according to the file’s Name, the Directory, the file’s Extension, and the Plane of
an image stack. Sorting will be incremented by the number of steps specified in the Incr. By option.
Incr. By (X Axis)
Specifies the step size for incrementing the file indexing (as selected from the Source option) across the
rows of the 4D Selection Grid. If the dialog is configured for Single Series mode, changing the number in this
Incr. By field will set the X-Y Interaction option to Incr X by Y Max and bring about a corresponding numeric
change in Y Max.
Y Max
Sets the maximum number of available rows.
Title (Y Axis)
This editable text field sets the title for the Y-axis of the 4D Selection Grid.
List (Y Axis)
This check box determines if the images to be added to each row will be from a user-defined list. Only one
axis may be defined from such a list. When this check box enabled in the Y Axis group, the corresponding
List check box and Set List button will be unavailable in the X Axis group.
Set List (Y Axis)
This button brings up a dialog for adding image files to a user-defined list. The image files will be added to
the 4D Selection Grid, one to a row. The Source option for the Y Axis group will automatically be configured
to Name. Accordingly, the Source option for the Y Axis group will be unavailable.
Source (Y Axis)
This pull-down menu selects the method for sorting files down the Y-axis of the 4D Selection Grid. Sorting
can be performed according to the file’s Name, the Directory, the file’s Extension, and the Plane of an image
stack. Sorting will be incremented by the number of steps specified in the Incr. By option.
Incr. By (Y Axis)
Specifies the step size for incrementing the file indexing (as selected from Source) down the columns of the
4D Selection Grid.
Grid Range
This box-in-box control is used to select which portions of the range of images will be displayed in the 4D
Selection Grid.
X-Y Interaction
This option, which only appears when the dialog is in Single Series mode, will determine how images are
added to the 4D Selection Grid. When Incr X by Y Max is selected, each column will consist of as many
rows as is specified in Y Max, and each image series (planes of a stack, or sequence of files with the same
base file name) will be added to the grid down a column. When Incr Y by X Max is selected, each row will
consist of as many columns as is specified in X Max, and each image series will be added to the grid across
a row.
Status line
Displays the last action taken.
Auto Range
Automatically sets the displayed Grid Range based on the available files. The files are determined by Set
List and by the settings in the X Axis and Y Axis groups. If the appropriate files do not exist, an error
message will appear. If neither List check box has been selected, the range will be determined by the values
specified in X Max and Y Max.
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Drop-in Commands
Renumber
Reorders the numbering of images that have been selected from the 4D Selection Grids. "Top-to-bottom"
renumbering has priority over "left-to-right" renumbering.
Grid Use
This button displays the keyboard shortcuts and mouse actions for selecting images from the 4D Selection
Grid.
Clear All
Deselects all images from the 4D Selection Grid.
Grid
Determines if indicator lines separating each element will be displayed in the 4D Selection Grid. If this check
box is cleared, only the external outline of the grid will be displayed.
Show Image
When this check box is selected, a preview of each image will be displayed as it is selected from the 4D
Selection Grid.
Grid Size
This slider selects the size of the grid display. This will not affect the range that is displayed.
Undo Click
Undoes the effects of the previous mouse click in the grid.
OK
Creates a stack of the images that have been selected from the 4D Selection Grid. Images will be placed in
the stack in the order in which they were selected (unless rearranged by choosing the Renumber
command). If any selected file can not be found, an error message will be displayed. You may then cancel
the operation, fill the stack with images acquired up to the error point, or continue, skipping the missing
range altogether.
Close
Closes the dialog.
4D Selection Grid
This separate window displays the grid from which images are selected. The appearance of the grid will be
determined by the options that have been selected (X Max and Y Max settings, Title entries for the X Axis
and Y Axis groups, etc.).
4D Selection Grid Command Shortcuts
If you want to…
Then use this shortcut:
Or use this mouse action:
Select/deselect an image
[SPACEBAR]
Left-click on the image.
Move the cursor
Corresponding cursor key
Click the desired image.
Deselect all images
[BACKSPACE]
Right-click anywhere.
Undo the previous action
[U]
Choose Undo Click.
Select a row of images
[R]
Click just to the left of the row.
Select a column of images
[C]
Click just above the column.
Run User Program (File Menu)
Runs a user-defined Visual Basic routine from within MetaMorph.
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User’s Guide
Drop-in: RUNUSER
Use this command to run a previously created set of Visual Basic functions while in MetaMorph. This
command is useful for processing and analyzing your images with MetaMorph's extended set of Visual
Basic functions, and provides a powerful and flexible addition to the MetaMorph armament of imaging
tools.
The full complement of Microsoft Visual Basic functions are available to you with the Run User Program
command, including the ability to run "If…Then…Else" routines, create nested subroutines and loops,
and to pass and return values with the imaging system. For a complete description of the use of this
command and of creating Visual Basic routines with the MetaMorph programming functions, please refer
to the Visual Basic Reference Guide, a written manual which is available to you from Molecular Devices
upon request.
Note: You can also use MetaMorph built-in variables in the Run User Program command
line. You must enclosing the variables in percent signs when entering them into the
command line — for example, %Acquire.FPS% is the correct way to enter the
Acquire.FPS variable. For more information about MetaMorph’s built-in variables, refer to
the Introduction to the Use of Variables help page.
Running a User-Defined Visual Basic Program
To run a Visual Basic program from MetaMorph, use the following procedure:
Step
Action
1
From the File menu, choose Run User
Program. The Run User Program dialog box
will appear.
2
From the Program Name drop-down list,
select the user program you want to run.
OR
If the name of the program you want to run
does not appear in the Program Name list,
choose the Browse button. The Select Start
File dialog box will appear. Select the icon for
the program you want to run. If necessary,
use the Look In drop-down list or the Up One
Level icon button to find the appropriate drive
and folder. Then choose Open to return to
the Run User Program dialog box.
Note: If you need to remove the currently
highlighted program from the Program Name
list and unregister it with the system, choose
Remove.
3
In the Command Line text box, type any
parameter you want to pass to the user
program. For example, you may wish to
specify the name of an image. (See the
Visual Basic Reference Guide for details.)
4
If you want the user program to stay in
memory after running, select the Keep
Program in Memory After Execution check
box.
OR
If you want the user program to be unloaded
after running, clear the Keep Program in
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Drop-in Commands
Memory After Execution check box.
5
When you are ready to run the user program,
choose OK. The program will run, and the
Run User Program dialog box will close
automatically.
Run User Program - Dialog Box Options
Program Name
Contains a list of all of your user programs that are currently registered with the system. The entries in the
list will be descriptions that were entered in Visual Basic when you created your program. If there is no
description, the project name entered in Visual Basic will appear instead.
Command Line
This is a text field which will be passed to your user program as the parameter for the Startup and
DoCommand functions. You might use this field, for example, to specify the name of an image that your
program will then load, convolve with an image filter, threshold, measure, log measurement data from, and
close.
Keep Program in Memory After Execution
This check box determines whether your user program will stay in memory after running, or if it will be
unloaded when the routine is completed. If you select the check box, the program will stay in memory, and
will therefore run more quickly on subsequent runs. If you clear this check box, the program will be unloaded
after running. This may be useful when you are debugging your program, as you can leave both MetaMorph
and Visual Basic running at the same time, alternating between running your program and editing it. Visual
Basic would not be able to recompile your program if the check box were still selected.
When the Keep Program in Memory check box is selected, the Startup function will be called the first time
your program is run after being loaded. Subsequent runs will call the DoCommand function. When this
check box is cleared, the Shutdown function will be called after the program finishes. If this check box is
cleared before you run the program for the first time, the Startup and Shutdown functions will be called
each time you run the program.
Browse
If the program you want to run does not appear in the Program Name list, this command button will allow
you to search your system for it. This button opens the Select Start File dialog box, which is a standard fileselection dialog box that has a Look In drop-down list, Up One Level icon button, File Name text box, and a
table that displays the files in the current folder.
When you create a user program, Visual Basic registers it with the system when it is compiled, and it should
then be available in the Program Name list. However, if you obtain a program that was compiled elsewhere,
it will not have been registered on your system. The Browse command button will register the program and
insert it in the Program Name list.
Remove
Choosing this button will remove the currently highlighted user program from the Program Name list and
unregisters it with the system.
OK
Loads the program selected in the Program Name list, passes the parameter you specify in the Command
Line text box, runs the user program, and closes the Run User Program dialog box.
Cancel
Cancels any changes made in the Run User Program dialog box and closes it.
Send Image as Email Attachment (File Menu)
Generates an email message with the active image attached, then sends the message to
the address you specify.
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Drop-in: EMAIL
Use this command when you want to attach the active image to an email message. This opens a new
message and attaches the image automatically without requiring you to open a separate email
application.
Note:
You must configure your email settings using before sending mail.
Sending Images as Email Attachments
To send an image as an email attachment, use the following procedure:
Step
Action
1
From the File menu, choose Send Image as
Email Attachment. The Send Image as Email
Attachment dialog box will appear.
2
Type the email address you would like to
send the image to in the TO: text box or
select a previously used address from the
History list. An address chosen from this list
will automatically appear in the TO: text box.
3
In the Subject box, type a brief description of
your message, such as the name of the
image file you are sending. (optional)
4
The Image list box displays the names of all
open images; click on the name of the image
you want to send. If the active image is a
stack an additional list box will appear. In
this box you can choose whether to send the
entire stack or just the current plane.
5
Under Send As, select a file type for the
image you are sending.
6
If you want to include a description or other
message, enter it in the Message box.
(optional)
7
Click the Send button to send the email
message with the image attached.
8
Click Cancel if you do not wish to send the
message.
Send Image as Email Attachment - Dialog Box Options
To
Specifies the address of the message recipient.
History
Lists recently used addresses that the user can select to be entered in the To: field. Select "Use email
address entered in To: field" if you want to send the message to the address listed in the To: field.
Subject
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This space allows the user to type in a brief description of the message and its attachment.
Image
This lists all open images and allows the user to select the name of the image to be sent.
Send As
Click one of these buttons to determine the file format for the attached image.
Message
This text box provides a space for the user to enter a message to be sent with the image.
Send
Click this button to send the email message and attached image.
Cancel
Click this button to cancel the operation.
Configure Email Settings (File Menu)
Configures settings to enable the user to send images as email attachments.
Drop-in: EMAIL
Use the Configure Email Settings command before using the Send Image as Email Attachment
command. This function allows you to determine how the messages will be sent.
Configuring Email Settings
To configure your email settings and enable sending images as email attachments, use the
following procedure:
Step
Action
1
From the File menu, choose Configure Email
Settings. The Configure Email Settings
dialog box will appear.
2
Type the name of your local mail server into
the SMTP Server text box. If you are not sure
of the name of your mail server please see
your system administrator.
3
In the Port box, type the number of BLANK
4
Enter the sender's email address in the
Sender's E-mail Address box. For example
[email protected]
5
From the Dial-Up Entry to Use list select the
dial-up networking profile you want to use.
Note: If you are not using a modem you can
leave this field blank.
6
If you are using a dial-up entry and want to
provide a password, enter it into the Optional
Password for Dial-Up Entry box.
7
If you want to log information about
messages (such as error messages) type the
path name of the file you want to log the
information to.
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8
To save your settings and close the
Configure Email Settings dialog box, click the
Okay button. To cancel your settings, click
the Cancel button.
Configure Email Settings - Dialog Box Options
SMTP Server
The name of your outgoing mail server, for example mail.yourcompany.com.
Port
The number of the TCP/IP port used to direct mail to and from your site. If you do not know the number of
the port you are using, contact your system administrator.
Sender's Email Address
Enter your email address (or the address of the user). This will appear as the reply to address for your
message.
Dial-Up Entry to Use
If you have a modem and are using dial-up networking connections select the name of the connection to use
when sending mail.
Optional Password for Dial-Up Entry
You can choose to store a password for the dial-up entry in this field.
Path to Log File
If you want to keep a record of all information (such as error messages) you receive about your mail, type
the path (location) of the file you want to use to store this information. For example C:\log\filename.
Okay
Click okay to save your settings and close the dialog box.
Cancel
Click cancel to exit the dialog box without saving your settings.
Image Properties (Edit Menu)
Creates and attaches user-defined properties to an image or a stack of images.
Drop-in: PROPMGR
Use this command to create custom-defined "labels" and their values, and to attach them to a
single-plane image or an entire stack of images. Examples of such labels might be the name
of the person performing the experiment or the grant associated with the experiment. In these
examples, the Name for the properties could be "Experimenter" and "Grant," and the Value
might be "Dr. N. E. Boddy" and "NSF 29758-03," respectively. Defined properties are
particularly useful when used by the Find Image Files command (File menu).
Image properties can be text-based or numeric. The Define Image Property secondary dialog
box which appears when you choose the Define button will retain the last ten definitions that
you have created, allowing you quickly to update the Value associated with the property
(Name) for the current image. After you attach a user-defined property to an image or stack,
you can remove it easily by highlighting its entry in the Image Properties dialog box and
choosing Remove. The image properties can be viewed either by choosing the Image
Properties command, which allows you to edit the properties, or by choosing the Get Info
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command (which does not) from the File menu.
QUICK TIP: The pop-up context menu that appears when you right-click in the Define
Image Property secondary dialog box contains commands that allow you to cut, copy,
paste, or delete text, as well as to undo any changes you make.
For More Information on Managing Images:
Find Image Files
Get Info
Annotate Image
Creating and Attaching Image Properties
To create, edit, remove, or simply view user-defined image properties for an image or stack, use
the following procedure:
Step
Action
1
From the Edit menu, choose Image
Properties. The Image Properties dialog box
will appear.
2
If you will be editing or reusing a previously
created image property, choose More >> to
expand the dialog box.
3
If necessary, use the Image selector to select
the image whose image properties you want
to view, edit, or create.
4
If the image you select already has userdefined properties attached to it, these will
appear in the Image Properties table. If you
want to remove an existing property,
highlight its entry and choose Remove.
5
If you want to create a new property or edit
an existing one, choose Define. The Define
Image Property dialog box will appear.
OR
If you have finished viewing or removing the
image properties, skip to Step 11.
6
In the Name text box, type the name for the
property label (e.g., "Experimenter"), or
select an existing entry from the Most
Recently Used Properties table.
7
Depending on whether the user-defined
property is text-based or numeric, select
either String or Number, respectively, from
the Value Type option button group.
8
In the Value text box, type the value for the
user-defined label.
9
Choose Define to attach the property to the
current image.
OR
Double-click the property's entry in the Most
Recently Used Properties table to attach the
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property to the image.
10
Repeat Steps 5 - 9, as needed, for any
additional properties you want to attach to
the current image.
11
When you have finished, choose Close to
close the Define Image Property dialog box.
Then choose Close to close the Image
Properties dialog box.
Image Properties - Dialog Box Options
Image
Selects the image whose image properties you want to view, edit, create, or remove.
Image Properties
Lists the user-defined properties currently attached to the selected image.
Define
Opens the Define Image Property dialog box, from which you can create a new property or edit an existing
one.
Remove
Detaches the property currently highlighted in the Image Properties table from the selected image.
Close
Closes the dialog box.
Define Image Property - Dialog Box Options
Name
The label for the custom image property. For example, this might be "Experimenter." If you select a property
from the Most Recently Used Properties, the Name text box will update to display the associated label for
the property.
Value
The "value" for the currently selected property. To use the preceding "Experimenter" example, this might be
"Dr. John Doe." If you select a property from the Most Recently Used Properties, the Value text box will
update to display the associated value for the property. If you select Number from the Value Type radio
button group, your Value entry must be numeric.
Value Type
Selects a format for the Value text box: String (text-based) or Number.
Most Recently Used Properties
Displays previously created image properties, up to a maximum of ten entries. When you select a previously
defined property by clicking it in this table, the Name and Value text boxes will update to display the label
and value for the property, thereby allowing you to edit them.
Define
Attaches the current property (Name and Value) to the selected image.
More >>
Expands the dialog box.
Less <<
Condenses the dialog box.
Close
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Closes the dialog box.
Convert Regions to Lines (Regions Menu)
Automatically develops line regions from binary skeletonized images in order to use the
line regions for line scans or kymographs.
Drop-in: LINERGNS
This function allows you to take a region that isn't a line and convert it to a line region.
Convert Regions to Lines is useful if you have odd regions such as pointing branches in
your image. As regions, the lines may be analyzed with a linescan or kymograph.
Creating line regions from a skeletonized image is a two-part process. First you need to
create regions in your image. There are three basic ways to do this:
Create Regions around Objects
Skeletonize your Image
Trace Regions
Once you have created regions in your image, you can convert them into lines using
Convert Regions to Lines.
Converting Regions to Lines
To convert regions to lines, use the following procedure:
Step
Action
1
Once you have created regions in your image
that you want to convert into lines, select
Convert Regions to Lines from the Regions
menu.
2
Next to Image, select the name of the image
you would like to use.
3
In the Line Regions to Keep group, select
which lines in the image you want to keep
when converting them to line regions.
4
If you have selected Lines Longer than
Minimum, type a value to use for the
minimum length a line must be to be included
in the conversion.
5
In the Convert group, select whether you
want to apply the conversion to an active
region or to all regions.
6
Click the Convert button to convert the
regions to lines.
7
Click the Close button to close the dialog box
and cancel the conversion process.
Convert Regions to Lines - Dialog Box Options
Image
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Select the image you want to apply the conversion to.
Line Regions to Keep
From this group, select whether you would like to include all lines, the longest line only, or lines longer than
minimum.
Minimum Length
Select the minimum length in pixels of the lines to include in the conversion process.
Convert
Select whether to apply the conversion to all of the regions in the image or only to the active region.
Convert
Click this button to convert the regions to lines.
Close
Click this button to close the dialog box and cancel the conversion process.
Resequence Region Labels (Regions Menu)
Renumbers the regions of interest in an image, starting from "1".
Drop-in: RGNSEQ
Use this command when you have removed one or more of the regions that have been defined on an
image, and want to eliminate the "gaps" in the numbering sequence. Regions will be reassigned
numbers in the order in which they were created.
For More Information about Regions:
Open Regions
Save Regions
Clear Regions
Transfer Regions
Region Tools
Resequencing Region Labels
To renumber your regions of interest, use the following procedure:
Step
Action
1
From the Regions menu, choose Sequence
Region Labels. The Sequence Region
Labels dialog box will appear.
2
From the Image selector, select the image
containing the regions that need to be
renumbered.
3
Choose OK. The region numbers will be
resequenced.
4
When you have finished, choose Close.
Resequence Region Labels - Dialog Box Options
Image
Selects the image with the regions that you want renumbered.
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OK
Renumbers the regions in the order in which they were created, starting with "1."
Close
Closes the dialog box.
Create Segment Regions (Regions Menu)
Creates rectangular regions of a selected length and width along a line region.
Drop-in: SEGMENTS
Use this command to create rectangular regions of a specified length and width along the entire length of
a line region. The line region can be drawn with any of the line region tools: Single Line Tool, Multi-Line
Tool, or Traced Line Tool. You can configure the command to draw the regions above, below, or along
the midline of the line region. If your line region is perfectly vertical, selecting Top will draw the regions
on the right side of the line region, and selecting Bottom draws them on the left side.
For More Information about Region Tools:
Single Line Tool
Multi-Line Tool
Traced Line Tool
Moving a Line Region
Resizing or Reshaping a Line Region
Creating Segment Regions from a Line
To create segmental regions of interest along a line region, use the following procedure.
Note: If you want to label the segment regions, be sure to select the Draw Labels Next to
Regions check box in the Region Labels tab page of the Preferences dialog box (Edit
menu).
Step
Action
1
From the Regions menu, choose Create
Segment Regions. The Create Segment
Regions dialog box opens.
2
If necessary, select the target image with the
Image selector.
3
Use one of the line region tools (Single Line
Tool, Multi-Line Tool, or Traced Line Tool) to
draw your line region in the target image.
4
If you are using region labels, type a label
prefix in the Segment Label Prefix text box.
The default prefix is "Segment." The prefix
will be given an incrementing region number.
5
Use the Segment Length and Segment
Width spin boxes to select a length and
width, respectively, for each of the segment
regions that will be created.
6
Select the geometric configuration of the
segment regions from the Set Regions Along
radio button group:
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Top will draw the segments above the
line region. If the line region is perfectly
vertical, the segments will be drawn to
the right of the line region.
Bottom will draw the segments below
the line region. If the line region is
perfectly vertical, the segments will be
drawn to the left.
Top and Bottom draws pairs of
segments both above and below the line
region.
Center draws the segments with their
midlines centered along the line region.
7
If you are creating segment regions
successively from more than one line region
and want to continue the numbering of the
segments from one line region group to the
next, select the Incr. Labels check box.
OR
If you want to start each line region's set of
segments with number 1, clear the Incr.
Labels check box.
8
If you want to remove the original line region
during the creation of the segment regions,
select the Delete Line check box.
9
When you are ready to create the segments,
choose Create Regions. The segments will
be drawn for the currently active line region.
Note: If you need to clear the segments for
the currently active line region, choose Clear
Polygons.
10
When you have finished, choose Close.
Create Segment Regions - Dialog Box Options
Image
Selects the image in which the segment regions are to be created.
Segment Label Prefix
Specifies the prefix for the segment region labels. The prefix will be given an incrementing region number.
The default prefix is "Segment."
Segment Length
Specifies the length (parallel to the line region) of each segment region.
Segment Width
Specifies the width (perpendicular to the line region) of each segment region.
Set Regions Along
Selects the geometric configuration of the segment regions:
Top will draw the segments above the line region. If the line region is perfectly vertical, the
segments will be drawn to the right of the line region.
Bottom will draw the segments below the line region. If the line region is perfectly vertical, the
segments will be drawn to the left.
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Top and Bottom draws pairs of segments both above and below the line region.
Center draws the segments with their midlines centered along the line region.
Incr. Labels
Continues the numbering of the segments from one line region group to the next when you are creating
segment regions successively from more than one line region. If you want to restart each line region's set of
segments with number 1, leave this check box cleared.
Delete Line
Removes the original line region during the creation of the segment regions.
Clear Polygons
Clears the segments for the currently active line region.
Create Regions
Draws the segment regions along the line region.
Close
Closes the dialog box.
Select Region (Regions Menu)
Programmatically selects a region from defined regions in open images during the running
of a journal or lets a user specify a region selection during a programmed pause while
running the journal.
Drop-in: SELRGN
Use this command to select a region programmatically in a journal. Place this command in your journal
to select a predefined region or use it to enable the person running the journal to select a region within
an image during a programmed pause. You can specify the name of the region in the Regions Label
field or you can pass the name of the region you want to select to the Regions Label field as a variable.
Selecting Regions
The following procedure assumes that you are recording a journal or editing a journal.
To select a region in a journal, use the following procedure:
Step
Action
1
If you are recording a journal, ensure one or
more images are open in MetaMorph and
that you have defined one or more regions.
If you are editing a journal using the Journal
Editor, go to step 2.
2
From the Regions menu, choose Select
Region. The Select Region dialog box
opens.
OR
From the Journal Editor, drag or add the
Select Region function to your journal dialog.
When the Add Function dialog box opens,
choose Yes or No for Interactive Mode. The
Select Region dialog box opens.
3
Click the Image box to identify the image
that you want the journal to select. The
image drop-down list opens.
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4
Click the image you want to have the journal
select, or choose the appropriate method of
image selection for the journal to use.
• Choose Last Result to use the last
resulting image created by this journal.
• Choose Current at Start to use the image
open and selected at the start of running
the journal. (This option is active only in
journal edit mode.)
• Choose Specified to enable you to
change the image selection in the journal
editor.
• Choose Select on Playback if you want
to enable the user running the journal to
be able to select or change the image
selection while playing the journal.
Note: See Image Selectors and Image
Selector Structure for Journals for more
information about image selectors.
5
In the Region Label box, type the name of
the region that you want to select or enter
the variable name for the region. Be sure to
enclosed the variable name in percent
symbols (%variable_name%).
6
Click Select to complete adding this function
to your journal.
7
Click Cancel to discontinue adding this
function to your journal.
Select Regions - Dialog Box Options
Image
Identifies the image containing the region that you want to select and to be used by the journal you are
running. You can apply a region selection while recording your journal, apply or change an region selection
using the Image Editor, or enable the user to apply an region selection when running the journal. Click the
image you want to have the journal select, or choose the appropriate method of image selection for the
journal to use.
Last Result uses the last resulting image created by this journal.
Current at Start uses the image open and selected at the start of running the journal. (This option
is active only in journal edit mode.)
Specified enables you to change the image selection in the journal editor.
Select on Playback enables the user running the journal to be able to select or change the image
selection while playing the journal.
Note: See Image Selectors and Image Selector Structure for Journals for more information about image
selectors.
Region Label
Specifies the name of the region that you want to select. As an alternative, you can enter a variable in this
field and programmatically replace the variable with the name of the region you want to select.
Select
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Applies the Select Region function and the associated settings to your journal.
Cancel
Discontinues applying the Select Region function to your journal.
Acquire Timelapse (Acquire Menu)
Acquires a series of frames at a specified interval and duration. The acquired frames can
be placed in a stack or stored on disk. If you are acquiring image(s) using a journal, you
are not required to save the acquired images.
Drop-in: TLAPSE
Use Acquire Timelapse to acquire timelapsed images of an experiment. If you are using a RS-170 video
camera, this command uses the acquisition settings from the Acquire Image command. If you are using
a digital camera, Acquire Timelapse uses the acquisition settings from the Acquire from Digital Camera
command.
Note: Acquire Z Series, Acquire Timelapse and Acquire Spectral Scan can be running at the same time.
However, you will not be able to carry out a command within itself (such as running a timelapse within a
timelapse).
Acquiring Timelapsed Images
Configuring a Timelapse Acquisition
Acquiring Timelapsed Images
Configuring a Timelapse Acquisition
To configure a timelapse acquisition, use the following procedure:
Step
Action
1
From the Acquire menu, choose Acquire
Timelapse. The Acquire Timelapse dialog
box opens.
2
Select the desired amount of time between
frames using Time Interval. Select the
desired unit of time from the drop-down list.
Note: Use Time Interval = 0 to acquire
images as rapidly as possible. In this case,
Acquire Timelapse cannot be guaranteed to
acquire at precisely regular intervals.
3
Type the number of frames you want to
acquire in the Number of Planes to Acquire
text box. When you make a change to this
setting, the Duration settings will be updated
automatically.
OR
Select the desired duration for the entire
timelapse acquisition using Duration. Select
the desired unit of time from the drop-down
list.
Note: Use Duration = 0 to acquire images for
an unlimited duration (subject to running out
of memory or disk space).
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4
Select the desired type of storage from the
Image Storage group. Use None to perform a
timelapse operation without storing images.
5
If you selected Stack, select the destination
stack using the Destination image selector.
OR
If you selected Disk, choose the Save File
Name command button to select a file name.
Type the file name in the File Name text box
and select the desired file type using Files of
Type. Choose Save when you have finished.
Note: If Acquire Z Series on Each Interval is
set, the file type must be a multiple image
format, such as .stk or .pic.
6
If you want to run a journal between
acquisitions, choose the command button
next to Journal to Run. Select the desired
journal name from the File Name list. Choose
OK.
Note: To clear a journal from the selection,
choose this command button again and
choose Cancel. The command button will
display Press to Select rather than the
journal name.
7
Select any of the desired check box options if
necessary. (See Acquire Timelapse Dialog
Box Options for more information.)
8
If you are using a shutter, select the
illumination setting associated with your
illumination hardware from the Illumination
list.
OR
Otherwise, select "[None]."
9
Choose OK. The Timelapse Acquisition
dialog box will appear.
Note: If you are recording this command in a
journal, choosing OK will display a dialog box
that asks whether or not you want to record
the journal without actually acquiring the
images. Choose Yes if you want to merely
save the journal without acquiring. Choose
No if you want to both save the journal and
acquire the images.
Acquiring Timelapsed Images
To collect images using the Timelapse Acquisition dialog box, use the following procedure:
Step
Action
1
Configure the acquisition using the
procedure presented in Configuring a
Timelapse Acquisition.
2
The Timelapse Acquisition dialog box will
appear, and the acquisition will start
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immediately.
3
If you want to change the Duration and Time
Interval settings during acquisition, choose
Interval. When you have adjusted the
duration and time Interval as necessary in
the Change Timelapse Interval dialog box,
choose OK to begin acquisition again.
4
To control the acquisition process, you can
use the Pause/Resume, Acquire, and Stop
command buttons. Use Acquire when you
want to acquire some of the specified frames
manually, rather than at the specified
interval.
5
MetaMorph will close the Timelapse
Acquisition dialog box automatically when
the acquisition is completed.
Acquire Timelapse - Dialog Box Options
Time Interval
Specifies the amount of time between acquired frames. The drop-down list next to the text box specifies the
unit of time. Use a Time Interval setting of 0 if you want to acquire images as rapidly as possible. However, if
you select 0, Acquire Timelapse cannot be guaranteed to acquire at precisely regular intervals. For best
results in acquiring images as rapidly as possible, disable frame-averaging, Update Image Window, and
Show Live Between Acquisitions.
Duration
Specifies the duration for the entire timelapse acquisition. The drop-down list next to the text box specifies
the unit of time. Use a Duration setting of 0 if you want to acquire images for an unlimited duration of time
(subject to running out of memory or disk space).
Number of Planes to Acquire
Specifies the number of frames to acquire. The maximum number of frames that can be acquired is 32767.
When you make a change to this setting, the Duration settings will be updated automatically. If you selected
Acquire Z-Series on Each Interval, this option title will change to "Number of Stacks to Acquire."
Image Storage
Specifies the type of image storage for the timelapsed images. Select
None if you want to perform a timelapse acquisition without storing images.
Stack to create a stack.
Disk to store images in a file on disk.
Save File Name
Specifies the file name for image storage when Disk is selected as the Image Storage type. If a file name
has not been selected, the words "Press to Select" will appear on the command button. Otherwise the file
name will appear on the command button.
Destination
Specifies the destination stack if Stack is selected as the Image Storage type.
Journal to Run for Each Time Interval
Specifies the journal to run between timelapse intervals If a journal file name has not been selected, the
words "Press to Select" will appear on the command button. Otherwise, the journal name will appear on the
command button. Note: If you are recording this command in a journal, choosing OK will display a dialog
box that asks whether or not you want to record the journal without actually acquiring the images. Choose
Yes if you want to merely save the journal without acquiring. Choose No if you want to both save the journal
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and acquire the images.
Update Image Window
Updates the image window after each acquisition when images are stored to a stack. If this option is
disabled, the image window will not update until after the timelapse is completed.
Warn If Interval Time Exceeded
Displays a warning if the selected time interval is exceeded and prompts you to change the interval and
duration.
Acquire Z Series on Each Interval
Expands the dialog box to include the appropriate options from the Acquire Z Series dialog box. Acquires a
Z-series of images at specified time intervals. This option will not be available if the "3D" drop-in has not
been installed. The options that will appear include
Number of Planes - Specifies the number of planes that will be acquired using the spacing
selected with the Step(s) option. The default is 1.
Step(s) - Specifies the spacing between planes. This option is based on the Z-axis device driver
presently configured. The units of measurements are calibrated using the Focus command.
Start At - specifies the starting location of the stage for the Z-series acquisition. Current Position is
the stage's present location: Origin, Top, Bottom, and Home are positions you set with the Focus
command.
Move To - Specifies the ending location of the stage for the Z-series acquisition. Planes * Spacing
specifies the ending location as the Number of Planes x Step(s). The Origin, Top, Bottom, and
Home options are positions you set with the Focus command.
After - Specifies the location to which the stage will be moved after an acquisition is successfully
completed. The Origin, Top, Bottom, and Home options are positions you set with the Focus
command.
Journal to Run for Each Plane in Z-Series Acquisition - Specifies the journal to run between
acquisitions. If a journal file name is not selected, the words "Press to Select" will appear on the
command button. Otherwise, the journal name will appear on the command button.
Illumination
Selects the illumination setting as defined in the Configure Illumination dialog box.
OK
Opens the Timelapse Acquisition dialog box and starts the timelapse acquisition. This dialog box displays
the progress of the timelapse acquisition. You can change the interval during acquisition from the Timelapse
Acquisition dialog box. An Acquire button is provided so that you can manually acquire some of the images
at any interval, rather than at the specified interval. You can also Pause, Resume, and Stop the acquisition.
Close
Closes the dialog box.
Acquire Spectral Scan (Acquire Menu)
Acquires a series of images in a range of excitation wavelengths. The acquired images can
be placed in a stack or stored on disk. If you are acquiring image(s) using a journal, you
are not required to save the acquired images.
Drop-in: SPECTRAL
Use this command to acquire images while changing excitation wavelengths at a specified increment.
For example, you could use this command to acquire images from the wavelengths of 310 to 420 nm
using increments of 10 nm to evaluate a system's ability to illuminate a fura-2 sample. (This task could
be performed manually by changing the wavelength in the Illumination dialog box and then acquiring the
image.)
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If you are using a RS-170 video camera, this command will use the acquisition settings from the Acquire
Image command. If you are using a digital camera, Acquire Spectral Scan will use the acquisition
settings from the Acquire from Digital Camera command.
This command requires a hardware device that uses continuous wavelength control, such as a
monochromator. You must first install and configure the hardware using the Meta Series System
Administrator.
Note: Acquire Z Series, Acquire Timelapse, and Acquire Spectral Scan can be running at
the same time. However, you will not be able to perform a command within itself (such as
running a timelapse within a timelapse).
Acquiring a Spectral Scan
To configure a spectral scan acquisition, use the following procedure:
Step
Action
1
From the Acquire menu, choose Acquire
Spectral Scan. The Acquire Spectral Scan
dialog box opens.
2
Select the starting wavelength for the
acquisition using Starting Wavelength.
3
Select the ending wavelength for the
acquisition using Ending Wavelength.
4
Select the wavelength increment between
acquisition using Wavelength Increment.
5
If you are using a shutter, select the
illumination setting from the Illumination list.
OR
Otherwise, select "[None]."
6
Select the desired type of storage from the
Image Storage group. Use None to perform
an acquisition without storing images.
7
If you selected Disk, choose Save File Name
to select a file name. Type the file name in
the File Name text box and select the desired
file type using Files of Type. Choose Save
when you have finished.
8
If you want to run a journal between
acquisitions, choose the command button
next to Journal to Execute. Select the
desired journal name from the File Name list.
Then choose OK.
Note: To deselect a journal, choose this
command button again and choose Cancel.
The command button will display Press to
Select, rather than the journal name.
9
Select Update Image Window if you want the
image window updated after each acquisition
(for a stack).
10
Select Show Live Between Acquisitions if
you want to display the live video on the
video monitor between acquisitions.
11
Choose OK. The Spectral Scan Acquisition
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dialog box will appear.
Note: If you are recording this command in a
journal, choosing OK will display a dialog box
that asks whether or not you want to record
the journal without actually acquiring the
images. Choose Yes if you want to merely
save the journal without acquiring. Choose
No if you want to both save the journal and
acquire the images.
To collect images using the Spectral Scan Acquisition dialog box, use the following procedure:
Step
Action
1
Configure the acquisition using the
procedure presented in the preceding table.
2
The Spectral Scan Acquisition dialog box will
appear.
3
Choose Begin to start the acquisition.
3
If you want to change the Ending
Wavelength or the Wavelength Increment
during acquisition, choose Change. When
you have adjusted the options as needed in
the Change Scan dialog box, choose OK to
begin acquisition again.
4
To control the acquisition process, you can
use the Pause/Resume and Stop command
buttons.
5
MetaMorph will close the Spectral Scan
dialog box when the acquisition is completed.
Acquire Spectral Scan - Dialog Box Options
Starting Wavelength
Specifies the starting wavelength (nanometers) for the spectral scan acquisition.
Ending Wavelength
Specifies the ending wavelength (nanometers) for the spectral scan acquisition.
Wavelength Increment
Specifies the number of wavelengths to move the wavelength changer between each acquisition.
Status
Displays the number of images that will be acquired during the spectral scan acquisition.
Image Storage
Specifies the type of image storage for the spectral scan images. Select
None if you want to perform a spectral scan acquisition without storing images,
Stack to create a stack,
Disk to store images in a file on disk.
Save File Name
Specifies the file name for image storage when Disk is selected as the Image Storage type. If a file name is
not selected, the words "Press to Select" will appear on the command button. Otherwise the file name will
appear on the command button.
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Illumination
Selects an illumination setting as defined in the Configure Illumination command.
Journal to Run
Specifies the journal to run between acquisitions If a journal file name has not been selected, the words
"Press to Select" will appear on the command button. Otherwise the journal name will appear on the
command button.
Note: If you are recording this command in a journal, choosing OK will display a dialog
box that asks whether or not you want to record the journal without actually acquiring the
images. Choose Yes if you want to merely save the journal without acquiring. Choose No
if you want to both save the journal and acquire the images.
Update Image Window
Updates the image window after each acquisition when images are stored to a stack. If this option is
disabled, the image window will not update until after the spectral scan is completed.
Show Live Between Acquisitions
Displays live video between image acquisitions. If this option has not been selected, the last acquired image
will remain frozen on the video monitor until the next acquisition.
OK
Starts the spectral scan acquisition and opens the Spectral Scan Acquisition dialog box. This dialog box
displays the progress of the acquisition. You can change the ending wavelength and the wavelength
increment during acquisition from the Spectral Scan Acquisition dialog box. You can also Pause, Resume,
and Stop the acquisition.
Close
Closes the dialog box.
Multi-Journal Timelapse (Journal Menu)
Runs up to five journals simultaneously. Similar to Define Stopwatch Sequences (Journal
menu), but uses a much simpler interface with fewer features.
Drop-in: TLAPSE2
Use this command when you want to run a number of journals at the same time. This command is used
both to create a timelapse sequence and to run it.
Define Stopwatch Sequences
Creating a Multi-Journal Timelapse
To create and use a multi-journal timelapse, use the following procedure:
Step
Action
1
From the Journal menu, choose Journal
Control>Multi-Journal Timelapse. The MultiJournal Timelapse dialog box will appear. If
the command has been run previously, the
last group of settings will still be available for
immediate execution.
2
Choose Add Jnl. The Select a Journal to Run
During Timelapse dialog box will open.
3
Select the desired journal name from the File
Name list.
AND
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Choose OK. The name of the journal will be
displayed in the Journal list and will appear
highlighted.
4
Use the Interval spin box and drop-down list
box to define the time between executions of
the journal highlighted in the Journal list. The
time interval will appear in the Interval (sec.)
list, across from its associated journal name
in the Journal list.
5
If necessary, repeat Steps 2 - 4 for all
journals you want to run simultaneously. If
you want to remove a journal from the list,
click on its name in the Journal list and
choose Remove.
6
Use the Expt. Duration spin box and dropdown list box to define the overall length of
time that you want the journals to run.
7
To run the journals, choose Start. The MultiJournal Timelapse dialog box will close, the
Timelapse Control dialog box will open, and
the journals will run. The Timelapse Control
dialog box will allow you to Pause (and
Resume) or Stop journal timelapse
execution.
When the timelapse is completed or stopped,
the Timelapse Control dialog box will close
automatically.
Multi-Journal Timelapse - Dialog Box Options
Journal
Lists the names of journals that have been added to the Multi-Journal Timelapse sequence.
Interval (sec.)
Lists the time between executions of its corresponding journal listed in the Journal list.
Interval
Defines the time unit (Seconds, Minutes, or Hours) for the interval (time between executions) of the journal
highlighted in the Journal list. Use the spin box to set the number of time units (maximum of 999).
Expt. Duration
Defines the time unit (Seconds, Minutes, or Hours) for the duration (total time of execution) of the entire
Multi-Journal Timelapse sequence. Use the spin box to set the number of time units (maximum of 999).
Start
Opens the Timelapse Control dialog box and starts execution of the Multi-Journal Timelapse sequence.
Cancel
Cancels all current changes made to the Multi-Journal Timelapse dialog box and closes the dialog box.
Add Jnl
Opens the Select a Journal to Run During Timelapse dialog box. Allows you to add a previously created
journal to the timelapse sequence.
Remove
Removes the journal highlighted in the Journal list from the timelapse sequence.
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Equalize Light (Stack Menu)
Changes the average, minimum, or maximum light intensity in each plane in an image
stack to equal the average, minimum, or maximum intensity, respectively, of the current
plane or of a user-defined active region of interest in the current plane.
Drop-in: EQUALIZE
Use this command when the background levels vary greatly between the various planes in an image
stack. Equalize Light will change the intensities in an image stack so that the average, minimum, or
maximum intensity in each plane in the stack will be equal to the average, minimum, or maximum
intensity, respectively, in the current stack or in a user-defined region of the current stack. You can
specify how this command is carried out by choosing between an algorithm that adds a constant value to
the entire image and one that multiplies each plane by a constant.
WARNING:
This command should not be used if you are performing a quantitative analysis of intensities or optical
densities, because it will alter the grayscale data in your image.
Equalizing Light Levels in a Stack
To equalize the light levels throughout all planes in a stack, use the following procedure:
Step
Action
1
From the Stack menu, choose Equalize
Light. The Equalize Light dialog box will
appear.
2
Select a source image stack using the
Source image selector.
3
Select the destination image using the Dest
image selector. You can overwrite or add to
the existing image or you can place the
results in a new image window.
4
If you want to use a region of interest within
the current image plane to determine the
intensity characteristics of the entire stack,
draw the region using a Region Tool.
OR
If you want to use the entire current plane to
determine the light levels of the stack, move
on to Step 5.
5
Select an equalization function from the
Equalize group: Average, Minimum, or
Maximum.
6
If you wish, you can select the algorithm to
be used to perform the light level
equalization, either by Addition of a constant
to each plane or by Multiplication of each
plane by a constant.
7
Choose Apply to carry out the equalization
operation.
8
Choose Close to close the dialog box.
Equalize Light - Dialog Box Options
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Source
Selects the source image stack.
Dest
Selects the destination for the equalized stack. You can overwrite the existing stack or place the results in a
new image window.
Equalize
Specifies the intensity characteristic that you wish to make equal between all of the planes in the selected
image stack.
Average: Sets the average intensity of the entire image stack to equal the average intensity in the
current plane or region of interest. Typically used for transmitted light images.
Minimum: Sets the minimum intensity in the stack to equal the minimum intensity in the current
plane or region of interest. Typically used for fluorescent images.
Maximum: Sets the maximum intensity in the stack to equal the maximum intensity in the current
plane or region of interest.
Equalize By
Selects the algorithm by which the equalization is performed. You can equalize either through Addition
(arithmetic addition or subtraction of a constant to or from the grayscale value of each pixel) or through
Multiplication (arithmetic multiplication of the grayscale value of each value by a constant).
Apply
Carries out the equalization operation. The dialog box will stay open.
Close
Closes the dialog box.
Keep Planes (Stack Menu)
Selects planes in an image stack that are to be kept and discards the rest.
Drop-in: KEEPPLN
Use the Keep Planes command when you want to remove unwanted planes from a stack of images.
Using this command will allow you to use a journal to remove planes within a specified range within the
stack. The advantage to using this command rather than the Remove Plane command is that you do not
need to know the total number of planes in a stack before removing them in a journal.
Using Keep Planes
To use the Keep Planes command, use the following procedure:
Step
Action
1
From the Stack menu, choose Keep Planes.
The Keep Planes dialog box will appear.
2
Select a source image stack using the Image
Stack image selector.
3
Select the image planes to be retained by
clicking on the desired images' plane
numbers in the Plane Selection Table. A
check mark will appear next to each selected
plane number.
OR
Select a range of image planes to be
retained by specifying the number of the
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lowest plane to be kept in the Low Range
spin box and number of the highest plane in
the range in the High Range spin box and
choose Select Planes in Range. You can
choose every nth plane by selecting the
number of planes to jump, from the Step Size
spin box. This method of selecting planes
can be used in journals.
4
Choose Apply. The dialog box will close and
the non-selected planes will be removed
from your image stack.
Note: If you have configured the Keep
Planes dialog box to remove all planes (that
is, no planes have been selected), a
message box will appear, stating that nothing
will be done to the image stack. Similarly, if
you configure the Keep Planes dialog box to
retain all planes, a message will appear,
stating that the image stack will not be
altered and asking if you wish to continue.
Keep Planes - Dialog Box Options
Image Stack
Selects the source image stack for processing by the Keep Planes command.
Low Range
Specifies the lowest numbered plane in a range of planes to be kept.
Step Size
Selects every nth plane in the range that is specified by the values in Low Range and High Range.
High Range
Specifies the highest numbered plane in a range of planes to be kept.
Select Planes in Range
Selects the planes to be discarded. The range will start with the plane specified by Low Range and end with
the plane specified by High Range. Every nth plane will be marked, as specified by the Step Size setting. A
check mark will also appear in the Plane Selection Table next to the numbers of the planes in the selected
range.
Clear All
Deselects all planes and unchecks all plane numbers in the Plane Selection Table.
Plane Selection Table
Displays the numbers of the planes in the image stack. Individual planes can be selected and deselected
directly from this list with a single mouse click. The numbers of the planes to be kept will have a check mark
next to them.
Apply
Removes and discards all but the selected planes in the image stack, and closes the dialog box.
Close
Closes the dialog box.
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Stitch Stack (Stack Menu)
Attaches and combines individual images of a calibrated stack into one image.
Drop-in: STITCH
Use this dialog box to combine related images in a calibrated stack into one complete image. This
dialog takes into account the orientation of the camera. By selecting the setting that indicates the
relative angle of the camera to the stage, you can correct for different camera orientations. You can also
compensate for reversed left-to-right orientation that has resulted from a mirror image effect.
In addition, you can apply a Flatten background algorithm to compensate for an uneven background in
individual images. As a result, the background of the combined images will be continuously smooth and
even.
Note: The calibrated stack image must use the same calibration units as your stage. The
stack image can be calbrated using the Calibrate Distances command. The stage is
calibrated using the Meta Imaging Series Administrator program.
Stitching Stacks
To combine separate related images of a stack in to a single image, complete the following steps:
Step
Action
1
Ensure that at least one appropriate,
calibrated image stack is open.
2
From the Stack menu, choose Stitch Stack.
The Stitch Stack dialog box opens.
3
If more than one image stack is open, click
Source stack to select the appropriate
calibrated image stack.
4
Click the image selector for Dest image to
start a new stack or add images to an
existing stack.
5
In the Camera Orientation area, click Images
are mirror to specify that you want to change
the left-to-right image orientation.
6
In the Camera Orientation area, click the
appropriate relative camera angle.
7
Click Flatten background if the background
intensity is uneven and you want to have an
even background intensity in the destination
image.
8
In the Flatten Background area, click
Fluorescence if the image stack was
acquired with a fluorescence light source;
click Transmitted if the image stack was
acquired with a transmitted light source.
9
In the Object size box, type or select the
smallest size object in microns that you want
to consider as image data.
10
Click Quick Paint to create the completed
stitched image without applying smoothing.
11
In the Background pixel fill value area, click
the appropriate setting to fill any non-image
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areas with a value that closely matches
either the darkest (for fluorescence),
brightest (for brightfield), or average intensity
found among all the images to be stitched.
12
After all settings have been made, click
Stitch to generate the new stitched image as
the destination image.
13
Click Close to close the Stitch Stack dialog
box.
Stitch Stack - Dialog Box Options
Source stack
Selects the image stack that you want to stitch from the available open stacks.
Dest image
Selects whether you want to start a new stack or add new images to an existing stack.
[Warning Text]
Alerts you that the loaded stack is not a calibrated stack.
Camera Orientation
Provides options to adjust for the camera’s relative orientation to the stage.
Images are mirror images – Reverses images that are mirrored or flipped left-to right from the
desired orientation.
Relative angle of camera to stage – Selects the appropriate camera angle setting of either 0, 90,
180, or 270 degrees, depending on the camera’s orientation to the stage. By selecting the correct
angle, the stitching algorithm can correctly determine which edges of the images are to be
compared.
Flatten background
When checked, applies the Flatten Background algorithm.
Illumination
Specifies the type of illumination used to acquire the image, either Fluorescence or Transmitted.
This selection determines the appropriate Flatten Background algorithm to be applied to the image
stack.
Fluorescence – Applies the Fluorescence Flatten Background algorithm.
Transmitted – Applies the Transmitted Flatten Background algorithm.
Object size
Specifies the object size of the smallest object in the image for the Flatten Background algorithm.
Quick Paint
Creates the completed stitched image without applying any smoothing. This option enables you to create
the stitched image faster than with smoothing applied.
Background pixel fill value
Fills non-image areas with an appropriately selected maximum dark or light value. When images are
stitched, areas can occur where there is no original image information. These areas are usually close the
edges of the new stitched image or in small spaces created where three or four overlapping images
intersect. Choose one of the following settings appropriate for your images:
Min – Fills non-image areas of Fluorescence stitched images with the lowest intensity value of all
the images.
Max – Fills non-image areas of Brightfield stitched images with the highest intensity value of all the
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images.
Average – Fills non-image areas of stitched images with the average intensity value of all the
images.
Stitch
Applies the stitching algorithm and creates a stitched Dest image.
Close
Closes the Stitch Stack dialog box.
Make Movie (Stack Menu)
Creates multimedia .avi video or QuickTime movie files from a stack of image planes.
Drop-in: MAKEAVI
Use this command to create full motion Video for Windows (*.avi) files or QuickTime movie (*.MOV) files
of the images in a stack. These video files can be played back similarly to a MetaMorph Movie, but do
not require the use of MetaMorph for playback. AVI (audio video interleaved) files can be replayed
automatically with the Media Player (Mplayer.exe) that comes bundled with Microsoft Windows. These
multimedia files can also be embedded in a word processor document or spreadsheet, or placed on a
Web page.
Digital video information can consume large amounts of memory, particularly if the video frames are in
24-bit color. Because of this, you may want to use one of the compression formats, or codecs
(compression-decompression engines), if you save your movie as an .avi file. The .avi format has
several standard compression codecs to choose from after creating an .avi movie. The number and type
of codecs available will vary depending on your operating system version, video card, and other software
installed on your system. The following codecs are installed with all versions of Windows:
Radius Inc. Cinepak: A 32-bit video codec that works best for compressing 24-bit color video
images. This format provides greater compression, higher resolution, and faster playback than the
Microsoft Video codec. You can specify your desired tradeoff between image quality and
compression.
Intel Indeo Video: Another 32-bit video codec that works best for compressing 24-bit color video
images. As with the Cinepak codec, you can specify your desired tradeoff between image quality
and compression.
Microsoft Video 1: A 32-bit lossy compressor that works best with 8-bit and 16-bit images. You
can specify separate settings for your desired tradeoffs between (1) compression and image
quality, and (2) compression and temporal resolution.
Microsoft RLE: A 16-bit compressor that uses run-length encoding. Best for binary or 8-bit highcontrast images.
There is also an option to save an uncompressed, full frames version of the .avi file.
Note: If you are saving the movie as an .avi file or a QuickTime file and want to play it
back on a Macintosh or use the Apple QuickTime (TM) player, you must use the included
Radius, Inc. Cinepak codec so that the appropriate look-up table values are saved along
with the .avi or QuickTime information.
Making a Movie Video File
To create an .avi or QuickTime movie file from a stack of images, use the following procedure:
Step
1
Action
From the Stack menu, choose Make Movie.
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The Make Movie dialog box opens.
2
Use the Source Stack image selector to
select the stack containing the images you
want to include in the .avi or QuickTime file.
3
In the Play Each Frame for… 1/30th of a
Second text box, type the frame time,
measured in 1/30ths of a second.
Note: The default value is 1 ("video rate").
This option allows you to play back the
frames at a slower rate.
4
In the Save radio button group, select
Selected to include the planes you select in
Step 5 in the .avi file.
OR
Select Unselected to exclude the planes you
select from the .avi file.
Note: To use the Make Movie command in a
Journal, you must ensure that no planes are
selected in the Plane Selection Table and
that Unselected is selected in the Save field.
This ensures the journal will create complete
movies from stacks containing different
numbers of planes.
5
To select the planes that you want to include
in the .avi video file, you can click the
number of the plane in the Plane Selection
Table.
OR
Alternatively, you can use the Low Range
and High Range edit boxes to specify the
lowest and highest plane number,
respectively, in the range of planes to be
included. You can choose every nth plane by
selecting the number of planes to jump from
the Step Size edit box. Then choose Select
Planes in Range.
Note: Choose the Clear All button to
deselect all planes.
6
In the Movie Formats box, click AVI to make
an .avi format movie,
OR
Click QuickTime to make a QuickTime
movie.
7
Choose Save. The Make Movie dialog box
will close, and the Save AVI or Save
QuickTime movie dialog box will open. Type
a name for the new .avi or .mov file in the
File Name text box. If necessary, use the
Save In drop-down list or Up One Level icon
button to select an appropriate drive and
folder for storing the file. Then choose Save.
8
If you are creating a QuickTime movie, the
make movie command will build the .mov file
using the Radius, Inc. Cinepak codec and
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save it to the selected folder.
OR
If you are creating an .avi movie, the Save
AVI dialog box will close, and the Video
Compression dialog box will appear.
Select the desired compression format from
the Compressor drop-down list. Where
available, use the Compression Quality slider
to select a setting for the tradeoff between
compression and image quality. A setting of
100 will provide the highest image quality at
the expense of compression. You can also
use the Configure option on some codecs to
open a dialog box specific to that codec.
9
Choose OK. The selected compression
codec will automatically build the .avi file and
save it in the location you specified in Step 7.
Make Movie - Dialog Boxes
Make Movie
Video Compression
Make Movie - Dialog Box Options
Source Stack
Selects the stack of images to be used for creating the .avi full motion video file or QuickTime video file.
Play Each Frame for… 1/30th of a Second
Specifies a display time, in one-thirtieths of a second, for each frame in the .avi or QuickTime file. The
default value is 1 ("video rate").
Low Range
Selects the lowest plane number for a range of planes to be selected.
Step Size
Selects every nth plane in the range specified by Low Range and High Range.
High Range
Selects the highest plane number for the range of planes to be selected.
Select Planes in Range
Selects the planes to be marked. The range will start with the plane specified by Low Range and end with
the plane specified by High Range. Every nth plane will be marked, as specified by the Step Size setting. A
check mark will also appear in the Planes List Box next to the numbers of the planes in the selected range. If
the Save radio button group has been set to Selected, the marked planes will be included in the .avi file. On
the other hand, if the Save radio button group has been set to Unselected, the marked planes will be
excluded from the .avi file.
Clear All
Deselects all planes and unchecks all plane numbers in the Plane Selection Table.
Plane Selection Table
Displays the numbers of the planes in the image stack. Individual planes can be marked and unmarked
directly from this list with a single mouse click. The numbers of the selected planes will have a check mark
next to them.
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Note: To use the Make Movie command in a Journal, you must ensure that no planes are
selected in the Plane Selection Table and that Unselected is selected in the Save field.
This ensures the journal will create complete movies from stacks containing different
numbers of planes.
Movie Formats
Specifies the video format that you want to create when you export an image stack using this command.
Click AVI to create an AVI format movie, click QuickTime to create a QuickTime movie.
Save (radio button group)
Specifies whether marked planes are to be excluded (Unselected) or included (Selected)
in the .avi or QuickTime file.
Note: To use the Make Movie command in a Journal, you must ensure that no planes are
selected in the Plane Selection Table and that Unselected is selected in the Save field.
This ensures the journal will create complete movies from stacks containing different
numbers of planes.
Save (command button)
Opens the Save AVI of Save QuickTime dialog box, from which you can select a name and storage location
for the file. After you choose Save from the Save AVI dialog box, the Save AVI dialog box will close, and the
Video Compression dialog box will appear.
Note: If you are creating a QuickTime movie, the Save command will build the .mov file
using the Radius, Inc. Cinepak codec and save it to the selected folder without opening
the Video Compression dialog box.
Close
Closes the Make Movie dialog box without creating the movie.
Video Compression - Dialog Box Options
Compressor
Selects a codec (compression/decompression engine) for creation and playback of the .avi file. The number
and type of codecs available will vary depending on your operating system version, video card, and other
software installed on your system. There will always be the option to choose an uncompressed, full frames
version of the .avi file.
Compression Quality
Selects a setting that determines the tradeoff between compression and image quality. Moving the slider
"thumb" to the right selects a higher quality image at the expense of storage space.
Configure
Opens a proprietary configuration dialog box for codecs that support them.
About
Displays a proprietary message box for codecs that support them.
OK
Closes the Video Compression dialog box and creates the configured .avi file, saving it in the location
specified in the Save AVI dialog box.
Cancel
Cancels the command.
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Kymograph (Stack Menu)
Creates a cross-sectional view of the intensity values of a user-defined line region through
the planes in a stack. This can be used to create either a Z-axis view of objects in a
through-focus sequence of images or a view of object movement in a time-based series of
images.
Drop-in: KYMO2
Use this command to create a cross-sectional view through the planes in an image stack. Such images
typically represent either a Z-axis through-focus series of images or a sequence of images taken over
time. The grayscale intensity values along a line region, or "transept," which has been drawn in the
source stack image window will be displayed in the result image. The values from the first plane will be
displayed in the first row of the result image, the values from the second plane in the second row, and so
on. The effect of this command is somewhat similar to that of the View Orthogonal Planes command,
but additionally allows you to configure the line region to have any length, angle, or location that you
wish.
The Kymograph dialog box contains an option to increase the width of the line region from its default
setting of one pixel. Accordingly, you can select between the average or the maximum grayscale value
within that line width.
Note: If you are simultaneously using the Linescan command (Measure menu) and the
line region you have drawn on the image is being shared by both the Kymograph and
Linescan commands, the Line Width setting in the Kymograph dialog box will follow the
width set in the Linescan dialog box. Conversely, if you make a change to the width of the
line in the Kymograph dialog box, the Scan Width setting in the Linescan dialog box will
be updated.
Using Kymograph
To create a cross-sectional display of the intensity values of a line region as it passes through a
stack, use the following procedure:
Step
Action
1
From the Stack menu, choose Kymograph.
The Kymograph dialog box will appear.
2
Use the Kymograph Image selector to
specify the destination image. You can
overwrite or add to the existing source stack,
or you can place the results in a new image
window.
3
Select the source image stack with the
Source Stack image selector.
4
With the Single Line Tool (or any other openended line region tool), draw a line region
across the area of interest in the image
window.
Note: Because the Kymograph command
draws the result image starting with the line
region values in the topmost plane in the
stack, you should verify that you are looking
at the top plane in your stack; otherwise,
your result image may appear to be inverted.
5
If you want to display the grayscale
intensities from all planes in the source
stack, select the All Planes check box.
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OR
If you want to use a restricted range of
planes in the source stack, clear the All
Planes check box. Then use the Plane…
to… spin boxes to select the starting and
ending plane.
6
By default, line regions drawn with a line
region tool will have a width of one pixel. If
you want to use a wider line region, select
the width with the Line Width spin box.
AND
From the Value Across Width option button
group, select which grayscale value across
the width of the line that you want to display:
Average or Maximum.
7
Choose Create Kymograph. The crosssectional image will be displayed.
8
When you have finished, click the Close
button in the upper right corner of the
Kymograph dialog box.
Kymograph - Dialog Box Options
Kymograph Image
Selects the destination for the cross-sectional image. You can overwrite the existing image or place the
results in a new image window. Or you can add the result image as a plane to the existing stack.
Source Stack
Selects the source image.
All Planes
Specifies whether all planes in the source stack are to be included in the cross-sectional image. When
selected, all planes will be used, and the Plane… to… spin boxes will be unavailable. When this check box
is cleared, the Plane… to… spin boxes will become available.
Plane… to…
Selects a starting and ending plane for inclusion in the result image. When the All Planes check box is
selected, these spin boxes will be unavailable.
Line Width
Selects a width for the line region to be used as the cross-sectional transept. When a width greater than 1 is
specified, the Value Across Width option button group will become available.
Value Across Width
Selects which grayscale value across the width of the line region is to be displayed: Average or Maximum. If
a Line Width of 1 has been specified, this option button group will be unavailable.
A line has not been selected in the source image stack.
Displayed if no line region is present and selected on the stack.
Create Kymograph
Creates the cross-sectional image, using a width corresponding to the length of the line region and a height
corresponding to the number of selected planes in the source stack.
Cancel
Cancels the command.
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Montage Stacks (Stack Menu)
Uses two to four stacks to create a two- or four-pane montaged stack that consists of one
image from each original stack in each plane of the montaged stack.
Drop-in: PAIR
Use this command when you want to compare two stacks, plane-by-plane, in a montage format. This
command is useful for presentations because you can create a movie of a montaged stack. You can use
the Select Plane command to see each plane in the montaged stack.
Montage
Montaging Stacks
To create a montage from two stacks, use the following procedure:
Step
Action
1
From the Stack menu, choose Montage
Stacks. The Montage Stacks dialog box will
appear.
2
If the desired Resultant Montage is not
displayed, select it using the image selector.
You can overwrite or add to an existing
image or you can place the results in a new
image window.
3
Select two to four source stacks for the
montage using the Source Stacks image
selectors. If you want arrange a side-by-side
montage, use the Top Left and Top Right
selectors (or the Bottom Left and Bottom
Right), and select None from the other image
selectors. If you want to create a vertically
arranged montage (one above the other) use
the Top Left and Bottom Left selectors (or
the Top Right and Bottom Right).
4
Choose Apply.
5
When you have finished, choose Close.
Montage Stacks - Dialog Box Options
Resultant Montage
Specifies the destination montage. You can add to or overwrite an existing image or stack. You can also
specify a new stack.
Source Stacks
Use these image selectors to specify the source stacks for the montage: Top Left, Top Right, Bottom Left,
and/or Bottom Right. You can select from two to four stacks.
Apply
Creates the montage from the specified source stacks.
Close
Closes the dialog box.
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Convert Stacks to TIFFs (File Menu)
Converts each stack image file in a source directory to TIFF files with sequentially
numbered file names or extensions.
Drop-in: STK2TIFF
Use this command to save each plane in your stack image (*.stk) files as separate .tif files. You can save
the single-plane image files using either a sequential file name format (e.g., file001.tif, file002.tif, etc.) or
a sequential file extension format (e.g., filename.001, filename.002, etc.). This command will be
particularly useful when you need to convert your stack files for processing by a third-party software
program, such as a deconvolution program, that does not recognize the Molecular Devices Corporation
stack file format.
Converting Stacks to Sequential TIFF Files
To save the individual planes in all of the stacks in a directory as separate sequential .tif files, use
the following procedure:
Step
Action
1
From the File menu, choose Convert Stacks
to TIFFs. The first page of the Convert
Stacks to TIFFs wizard opens.
2
Choose Source Directory. The Browse for
Folder dialog box opens.
3
In the Select Source Directory tree window,
find the folder containing the stack files you
want to convert and click it so that the closed
folder icon is displayed as an open folder
icon.
AND
Choose OK. The Browse for Folder dialog
box will close and the Convert Stacks to
TIFFs wizard reopens.
4
If you want to save the stack planes as
sequentially named .tif files (e.g., file001.tif,
file002.tif, etc.), select Save with Sequential
Names from the Destination TIFF Images
option button group.
OR
If you want to save the stack planes as
sequentially numbered .tif files (e.g.,
filename.001, filename.002, etc.), select
Save with Sequential Extensions.
5
Choose Destination Directory. The Browse
for Folder dialog box opens.
6
In the Select Destination Directory tree
window, find the folder in which you want to
save the .tif files and click it so that the
closed folder icon is displayed as an open
folder icon. If necessary, you can create a
new subfolder under an "open" folder by
choosing New.
AND
Choose OK. The Browse for Folder dialog
box will close and the Convert Stacks to
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TIFFs wizard reopens.
7
Choose Next >>. The second page of the
Convert Stacks to TIFFs wizard opens. This
page contains information about the number
of stack files in the source directory, the
amount of disk space to be used by the new
.tif files, and the amount of free space that
will remain on your hard disk.
If necessary, choose << Back to return to the
preceding page of the wizard.
8
When you are ready to start the conversion
process, choose OK. The conversion will
proceed automatically. A third page of the
wizard will appear, providing you with a realtime progress report as the files are
processed.
9
When you have finished, choose OK to close
the Convert Stacks to TIFFs dialog box.
Convert Stacks to TIFFs - Dialog Box Options
Source Directory
Opens the Browse for Folder dialog box, from which you select the folder containing the stack files to be
converted to single-plane .tif files.
Destination TIFF Images
Selects a name format for the .tif files. Save with Sequential Names saves the files using sequential file
names (e.g., file001.tif, file002.tif, etc.). Save with Sequential Extensions saves the files using sequential file
extensions (e.g., filename.001, filename.002, etc.).
Destination Directory
Opens the Browse for Folder dialog box, from which you select the folder in which you want to save the
sequential .tif files. You can create a new subfolder by selecting an existing folder and choosing New.
Information
This status text box, which appears in the second page of the Re-Save Stacks as TIFFs wizard, provides
information regarding the number of stack files in the source directory, the amount of disk space to be used
by the new .tif files, and the amount of free space that will remain on your hard disk.
Status
Provides real-time feedback regarding the progress of the file conversion process. A progress meter will
indicate the status of the conversion of each stack file, and a status line will display the stack file and plane
currently being converted.
Next >>
Proceeds to the next page in the wizard.
<< Back
Returns to the preceding page in the wizard.
Cancel
Cancels the Re-Save Stacks as TIFFs command and closes the dialog box.
Interleave Stacks (Stack Menu)
Creates a new stack of interleaved images from two source stacks, with all odd-numbered
planes coming from one source stack and all even-numbered planes coming from the
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other source stack.
Drop-in: INTLEAVE
Use this command to combine two stacks of images into a single stack. The interleaved planes can be
placed in a new destination stack, or can be added to an image stack that is currently open on the
desktop.
The number of interleavings is limited by the number of planes in the smaller source stack. Thus, for
example, if you combine a stack of four images with a stack of 50 images, the resulting stack will consist
of eight planes, four from each source stack.
If you combine two stacks with different sizes, the new stack planes will have dimensions corresponding
to the larger width and the larger height. For example, if you combine a stack of 200 x 300 images with a
stack of 300 x 200 images, the resulting stack will consist of 300 x 300 planes. The additional space
around the image data will be filled with black pixels.
If you combine two stacks with different bit-depths, the new stack will consist entirely of images of the
greater bit-depth. For example, if you combine a binary stack with a 24-bit color stack, the planes in the
resulting stack that came from the binary source stack will consist of pixels with "color values" of either
0,0,0 or 255,255,255.
Interleaving Stacks
To interleave two stacks of images into a single stack, use the following procedure:
Step
Action
1
From the Stack menu, choose Interleave
Stacks. The Interleave Stacks dialog box will
appear.
2
With the Source Stack 1 (Odd Planes) image
selector, select the source stack whose
images will give rise to Planes 1, 3, 5, etc., in
the new set of stack planes.
3
With the Source Stack 2 (Even Planes)
image selector, select the source stack
whose images will give rise to Planes 2, 4, 6,
etc., in the new set of stack planes.
4
With the Destination Stack image selector,
specify a target destination for the
interleaved planes. You can specify a New
stack or Add To an existing one.
5
Choose OK to start the interleaving.
6
When you have finished, choose Close.
Interleave Stacks - Dialog Box Options
Source Stack 1 (Odd Planes)
Selects the source stack whose images will give rise to Planes 1, 3, 5, etc., in the new set of stack planes.
Source Stack 2 (Even Planes)
Selects the source stack whose images will give rise to Planes 2, 4, 6, etc., in the new set of stack planes.
Destination Stack
Specifies the destination for the new, interleaved stack of images. You can specify a New stack or Add To
one that is currently open in the MetaMorph application workspace.
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OK
Carries out the interleaving process.
Close
Closes the dialog box.
Stereographic Views (Stack Menu)
Creates paired stereographic images from an image stack.
Drop-in: STEREO
Use this command to create stereo pair images for viewing (1) with stereographic goggles, (2) with
red/green or red/blue glasses, or (3) as side-by-side stereo pairs. The Stereographic Views command
works best when the source stack that it uses is one that was created with the 3D Reconstruction
command. You can configure the arrangement of the stereo pair images for optimal viewing with either
Stereographics Viewer synchronizing goggles (contact your Molecular Devices Corporation
representative for information), red/green viewing glasses, or red/blue glasses. The output of this
command is particularly suited for viewing as side-by-side stereo pairs or for use in creating a movie .
The # of Planes Between Image Pairs option determines the way in which images are paired up. For
example, if you select 1, the starting image pair will consist of planes 1 and 2 of the original source stack.
If you select 2, the starting image pair will consist of planes 1 and 3. If you select 3, the starting pair will
comprise planes 1 and 4, and so on.
The Create Pair for Every nth Image option determines which source stack planes will be used as the
first image of each resulting pair. Thus, if you select 1, the first of the paired images in the result stack
will be planes 1, 2, 3, etc., from the original source stack. If you select 2, the first member of the pairs will
be planes 1, 3, 5, etc., from the source stack, and so on.
Result images can be saved as an individual stack or as separate TIFF files.
Creating Stereographic Views
To create a set of stereographic image pairs from a stack of images, use the following procedure:
Step
Action
1
From the Stack menu, choose Stereographic
Views. The Stereographic Views dialog box
will appear.
2
Select the source image stack with the
Image Stack from Which to Create Stereo
Pairs image selector.
3
If you are using an external monitor, select
the Transfer Image to Video While
Previewing check box so that a check mark
appears in it. You should use this option if
you wish to export the images to an optical
memory disk recorder.
4
Use the # of Planes Between Image Pairs
spin box to specify the way in which images
are paired up.
EXAMPLES:
If you select 1, the first image pair that is
created will come from planes 1 and 2 of the
original source stack. If you select 2, the first
image pair will be made up of planes 1 and
3. If you select 3, the first pair will be planes
1 and 4, and so on.
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AND
Use the Create Pair for Every nth Image spin
box to specify which source stack planes will
be used as the first image of each resulting
pair.
EXAMPLES:
If you select 1, the first image in the resulting
pairs will be come from planes 1, 2, 3, etc., of
the original source stack. If you select 2, the
first image in the resulting pairs will come
from planes 1, 3, 5, etc., from the source
stack, and so on.
5
Select the number of resulting image pairs
you want to create using the # of Stereo
Pairs to Create spin box.
6
If there are not enough planes in the source
stack to create a complete set of image pairs
from a single pass using the configuration
you specified in the preceding two steps,
select the Wrap Around Until All Planes Are
Exhausted check box so that a check mark
appears in it.
This will prompt MetaMorph to begin using
source planes at the beginning of the stack
when there would otherwise not be enough
planes in the stack to complete the stereo
pairs as configured.
7
By default, stereo pairs will be arranged
vertically in a stack, and the destination
image will be 512 x 480. If you want to
reconfigure the size, placement, color
rendering or spacing of the stereo pairs, or
specify a different format for saving the
images, choose More >>. The dialog box will
expand, revealing more options.
OR
If you wish to use these default configuration
settings, skip to Step 13.
8
Depending on your method of viewing,
specify an arrangement and color rendering
of the stereo pairs by selecting from the Type
of Stereo Pairs to Create list. The selection
you make will determine the spacing options
that appear just below this box. Select
Stereographics Stacked to create pairs that
are arranged vertically (one above the other)-select this if you are using a pair of
Stereographics Viewer goggles;
Side by Side to create pairs that are
arranged horizontally--select this if you will
be using another method to view the stereo
pairs such as "cross-eyed" viewing;
Red/Green Anaglyph to create a pair of red
and green "superimposed" stereo images-select this if you are using a pair of red/green
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viewer glasses; or
Red/Blue Anaglyph to create a pair of red
and blue "superimposed" stereo images-select this if you are using a pair of red/blue
viewer glasses.
9
If you selected Stereographics Stacked in
Step 8, use the Vertical Spacing Between
Images spin box to specify the space, in
pixels, between the upper and lower image.
Then use the Horizontal Offset of Bottom
Image spin box to specify the shift of the
lower image relative to the upper image, in
pixels.
AND
Use the X and Y spin boxes in the Dest.
Image Size option group to specify the
horizontal and vertical dimensions, in pixels,
of the destination image. You can choose
Preview to see the arrangement of the
images and make adjustments as necessary
while wearing your viewer goggles. Then
skip to Step 12.
10
If you selected Side by Side in Step 8, a
resizable region of interest will be defined on
the source image. If you wish, choose
Center, Set Region to Half Image Width. This
will automatically center the region and
resize it horizontally so that it is half the width
of the original image (the height will stay the
same). When the stereo pairs are created,
only this region of the source image planes
will be used in the result images.
AND
Use the Spacing Between Images spin box
to specify the space, in pixels, between the
left and right image. If you wish, you can
switch the left and right images by selecting
the Flip Views check box. You can choose
Preview to see the arrangement of the
images while wearing your viewer goggles
and make adjustments as necessary. Now
skip to Step 12.
11
If you selected Red/Green Anaglyph or
Red/Blue Anaglyph in Step 8, select the
offset between the red image and the green
or blue image with the Horizontal Offset of
Bottom Image spin box. You can choose
Preview to see the arrangement of the
images while wearing your viewer goggles
and make adjustments as necessary.
12
From the Save radio button group, select the
format for saving the stereographic image(s).
Select
Stack if you want the pairs saved in a single
stack (*.stk) file (you will perform the actual
Save operation when you close the image).
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Disk if you want to save the images as
separate TIFF files. Then choose the Select
command button and type a base name for
the series of images in the File Name text
box of the Select File for Timelapse Storage
dialog box that appears and choose Save.
13
When you are satisfied with your
configuration, choose OK. The stereographic
pair images will be generated and the
Stereographic Views dialog box will close
automatically.
Note: If you want to hide the Image Window
Tools, right-click in the stereographic image
window and choose Hide Image Window
Toolbar from the pop-up context menu that
appears.
Stereographic Views - Dialog Box Options
Image Stack from Which to Create Stereo Pairs
Selects the image stack from which planes will be used to generate the stereographic pair image(s).
Wrap Around Until All Planes Are Exhausted
If there are not enough planes in the source stack to create a complete set of image pairs from a single pass
using the current configuration, this option prompts MetaMorph to begin using planes at the beginning of the
stack to complete the stereo pairs as configured. This option is only valid if the original 3D stack was created
for the full 360 degrees, such that the last image differs from the first image by the same number of degrees
as the first image differs from the second.
# of Planes Between Image Pairs
Specifies the number of planes by which the images in a stereo pair differ. For example, if you start with
plane 1 in the source stack, setting this option to 1 will result in the first pair being taken from planes 1 and 2
of the source stack. If you set this option to 2, the first pair will consist of planes 1 and 3 from the source
stack.
Create Pair for Every nth Image
Specifies which planes from the source stack will be used as the first image of each resulting pair. For
example, if you set this option to 1, the first image in the resulting pairs will come from planes 1, 2, 3, etc., in
the source stack. If you set this option to 3, the first image in the resulting pairs will come from planes 1, 4, 7,
etc.
# of Stereo Pairs to Create
Specifies the number of stereographic image pairs that will be created.
Transfer Image to Video While Previewing
If you are using an external video monitor, selecting this check box will send the Preview image to the
monitor, rather than to an image window on the computer monitor.
Preview
Places a sample view of the first stereo pair in an image window on the computer monitor (or on an external
monitor if you selected the Transfer Image to Video While Previewing check box). This will allow you to
obtain quick visual feedback on the arrangement that will result from your current set of configuration
settings.
More >>
Expands the dialog box, revealing additional options for image pair configuration, arrangement, and saving.
Less <<
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Condenses the dialog box.
Type of Stereo Pairs to Create
Selects the format for the stereographic pair images.
Stereographics Stacked arranges the images in a column, one above the other. This format is
best when you are using a pair of Stereographics Viewer goggles. (Note: Because of their
synchronization rate, these goggles work best when you use an external video monitor.)
Side by Side arranges the images in a horizontal row. This format is used if you will be using
another method to view the stereo pairs such as "cross-eyed" viewing.
Red/Green Anaglyph arranges the image pair in a "superimposed" configuration, with one image
rendered in red and the other in green, with a user-specified offset between the two. This format is
ideal when you are using a pair of red/green viewing glasses.
Red/Blue Anaglyph is similar to the Red/Green Anaglyph, but renders the second image in blue,
rather than green. This format is used when you have a pair of red/blue viewing glasses.
Vertical Spacing Between Images
Selects the space between the upper and lower image, in pixels. This spin box appears only when
Stereographics Stacked is selected as the Type of Stereo Pairs to Create. This option is used to set the
alignment of the images when they are superimposed by the stereographics hardware.
Horizontal Offset of Bottom Image
Selects the shift of the lower image, in pixels, relative to the upper image. This spin box appears only when
Stereographics Stacked is selected as the Type of Stereo Pairs to Create. This option is used to set the
alignment of the images when they are superimposed by the stereographics hardware.
Dest. Image Size
Selects the horizontal (X) and vertical (Y) size of the result image, in pixels. This set of spin boxes appears
only when Stereographics Stacked is selected as the Type of Stereo Pairs to Create.
Save
Selects a format for saving the resulting sets of image pairs.
Stack saves the pairs as planes in a single stack (*.stk) file. The actual saving operation will be
carried out when you close the stack.
Disk saves the image pairs as separate TIFF files on disk.
Spacing Between Images
Selects the space between the left and right image, in pixels. This spin box appears only when Side by Side
is selected as the Type of Stereo Pairs to Create. This option is useful for creating a border between the
images.
Region
Indicates the starting (upper left) X and Y coordinate (in the first set of parentheses) and the ending (lower
right) X and Y coordinate (in the second set of parentheses) for the currently defined region of interest on
the source image. This region will appear automatically, and this status line will only appear, when Side by
Side is selected as the Type of Stereo Pairs to Create.
Flip Views
Switches the placement of the left and right images. When this option is disabled, images will be "read in" to
the image pair from left to right. When this option is selected , images will be "read in" from right to left. This
check box appears only when Side by Side is selected as the Type of Stereo Pairs to Create. This allows
you to determine whether the pairs are to be viewed "cross-eyed" or "wall-eyed."
Center, Set Region to Half Image Width
Centers the region of interest within the source image and resizes it horizontally so that it is half the width of
the original image (the height will stay the same). When the stereo pairs are created, only this region of the
source image planes will be used in the result images. This command button appears only when Side by
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Side is selected as the Type of Stereo Pairs to Create.
File Name (Select command button)
Displays the Select File for Timelapse Storage dialog box, from which you can specify the base name for
images being saved as TIFF files on disk. This command button appears only when Disk is selected as the
Save format.
OK
Creates the stereo image pairs as configured. The Stereographic Views dialog box will close automatically.
Close
Closes the dialog box.
Acquire (Acquire Menu)
Configures image acquisition for and acquires images from a variety of digital cameras.
Drop-in: ACQUIRE
Use this command for acquiring images from several different types of digital cameras. This dialog box
combines multiple functions and options into a single, multi-tabbed dialog box. It enables you to specify
settings that control image acquisition, image correction, image annotation, image display, and special
settings including digitizing speed, gain, and camera shutter selection. Also use this command to
acquire 24-bit color images. Acquisition of 24-bit color images is enabled when the Bit Depth on the
Special tab is set to 24-Bit, and the correct video channel is selected in the Set Video Channel dialog
box.
The Acquire command now features a Live Replay tab for capturing real time events such as in FRAP
studies and other laser-based events, time lapse experiments, live cell imaging and digital video
microscopy. When Live Replay is enabled, MetaMorph buffers the live stream to memory. This enables
you to start recording a stream when something of interest occurs. You can configure Live Replay to
include frames in the stack that occurred before the capture point.
Live Replay also enables you to select an optional journal to run at the capture point. One example of
using Live Replay with a journal is to record recovery data during a FRAP experiment. You can configure
a journal to photobleach at the capture point. The resulting stack can be used to measure recovery
because it contains frames from before and after the photobleaching.
Note: The Live Replay feature is only available for supported cameras. Refer to the
MetaMorph Support site for a list of supported cameras.
The Acquire dialog box has the following two formats:
•
A minimized format that contains only the controls for acquisition region selection, exposure,
binning, live activation (Show Live), plus Acquire, and Save Image.
•
A standard format that contains all the controls in the minimized dialog box, plus tabbed areas
containing controls for Acquisition (Acquire), Display, Image Correction (Correct), Image
annotation (Annotate), and special settings for Digitizer speed (Digitizer), Gain, Bit Depth, and
Camera Shutter.
Using the default image selector settings, the acquire command acquires images to an image window
called "Acquired." If additional images are acquired, and the last image acquired is not saved and
closed, the name of newly acquired image becomes Acquired-2. Subsequently acquired images are
incremented with Acquired-3, Acquired-4, and so on. The image selector enables you to specify the
name of the acquired image. The assigned name is also automatically incremented when additional
images are acquired. If you set the Image Selector to Overwrite, acquired images are called "Acquired"
or a name that you designate in the image selector, and will overwrite the currently displayed image.
Optionally, using the image selector, you can specify that subsequently acquired images are
accumulated into an image stack (.stk) file.
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Note: You can cancel an ongoing acquisition at any time by pressing the [Esc] key.
Procedures
Dialog Box Options
Acquiring Images
Acquiring Images - Main Dialog Box
Acquiring Images - Display Tab
Acquiring Images - Acquire tab
Acquiring Images - Correct Tab
Acquiring Images - Annotate Tab
Acquiring Images - Special Tab
Acquiring Images - Live Replay Tab
Acquiring Images - Color Tab (Brightfield)
Acquiring Images - Color Tab (Fluorescence)
Acquiring Images – Main Dialog Box
The configuration steps for many types of simple image acquisitions can be completed on the
Acquire main, minimized (Less<<) dialog box. Additional acquisition requirements can be
specified on one or more of the five Acquire dialog box tabs. This Acquire dialog box enables you
to configure and acquire images rapidly because some of the buttons simultaneously set a
specific acquisition requirement and acquire an image.
To Configure acquisition and acquire one or more images, complete the following procedure:
Step
Action
1
From the Acquire menu, click Acquire, the
Acquire dialog box opens.
2
If the dialog box is not maximized, click the
More>> button.
3
If you are loading a previously save group of
settings in a setting file, click Load… The
Acquire: Load Setting dialog box opens. If
you are making a new group of Acquire
settings, Skip to step 5.
4
From the Acquire: Load Setting dialog box,
check the settings that you want to load from
your saved settings file. Click once to check
a setting, click again to uncheck. Then click
Load.
5
Determine if you will be acquiring a single
image, an image stack, or a sequence of
images. If you are acquiring single images,
an image stack, or are overwriting your
acquired images, configure the image
selector accordingly. If you are acquiring a
sequence of images, click Save w/
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Sequence. If you are acquiring only a single
image, Skip to step 8.
6
If you clicked Save w/ Sequence, Click Set
Save…to specify the sequence base name
and specify Set Saving options.
7
In the Acquire: Set Saving dialog box, type
the base file name in the Base Name box.
In the If image already exists box, choose an
option. Click Directory… to specify the
directory where you want to save the
images. Click Show Saved Image to have
the saved image displayed in an Image
window. Click OK to close the Acquire: Set
Saving dialog box.
8
Click the Acquire tab to make settings for
auto exposure and to select an external
shutter.
9
On the Acquire Main dialog, type or select
an exposure time and unit of measure.
10
If you are auto exposing, click AutoExpose.
The acquire command calculates the
exposure time and acquires an image.
11
If you need to shorten the exposure time,
type or enter a value in the Binning box.
12
In Camera Area, specify the acquisition
region. Remember, each of these buttons
simultaneously sets the acquisition region
and acquires an image.
Click Full Chip to set the acquisition region
to the entire area of the chip, and acquire an
image.
Click Center Quad to set the acquisition
region to the center quadrant of the chip,
and acquire an image.
Click Use Active Region to assign the
acquisition region to the current active
region, and acquire an image.
13
Click Show Live to continuously acquire
images to enable you to set the exposure
time and to focus the microscope.
14
If Show Live is active, type or select a value
in the Live Bin box. Increasing the Live Bin
increases the frame rate within the live
window.
15
To acquire the image after configuring the
settings as needed (see the help file for each
Acquire tab) click Acquire to begin acquiring
the image.
Note: You can cancel an
ongoing acquisition at any time
by pressing the [Esc] key.
16
To save settings made in the Acquire dialog
box, click Save As… when saving to a new
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setting file. Click Save to save settings to the
setting file shown in the Setting (Modified):
box. To choose a different setting file, click
the arrow button in the Setting box, and click
the mane of the setting file.
Acquiring Images - Display Tab
Use the settings on this tab to configure the images shown on your display.
To configure the Acquire Display dialog box, complete the following procedure:
Step
Action
1
Click the Display tab. The display options
and settings move to the front.
2
If autoscale is on, type or select values for
the Low percent and the High percent.
OR
If Autoscale is off, in the Image Scaling area,
type or select values for the Low and High
limits.
3
If you want scaling to be performed in the
active region on acquire, click Scale within
the active region.
4
To apply gamma correction to your viewed
image, move the Image Gamma slider left or
right to decrease or increase the image
gamma below or above 1.00. To reset the
gamma to 1.00, click =1. When you
decrease or increase the gamma, the line
chart indicates the change.
5
To select a different chart or turn off the
chart, click the appropriate button on the
right side of the chart.
6
To change the chart configuration, click the
down arrow button under the lower left
corner of the chart. A list box opens
containing a list of options that you can use
to customize the chart, print the chart, copy
the chart, or save it as a bitmap file.
7
From the list box, click Configure Plot. The
configure plot dialog box opens. Use this
dialog box to change the visual and physical
attributes of the chart including the
background and border colors.
8
From the list box, you can also select Y axis,
X axis, Title, X Title, and Y Title. Use these
options to modify and customize settings
and colors for each of these individual parts
of the graph.
9
Click Reset Display to reset all values in the
display to their previous default values.
Acquiring Images - Color Tab (Brightfield)
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To set the color balance for acquiring 24-bit brightfield color images, complete the following
procedure:
Step
Action
1
Click the Color tab, the Color options move
to the front.
2
Select Brightfield if you are acquiring
brightfield images illuminated by a
transmitted light source (dark objects against
a white background).
3
Click Show Live to begin acquiring images
from which to preview your settings.
4
Without a specimen in place or with an
active region created that defines an area to
be interpreted as white, Click Measure White
Balance to establish an initial white balance
level.
5
With a specimen in place, adjust the
Brightness control to achieve the best overall
level of brightness.
6
Adjust the controls for each individual color
(Red, Green, Blue) to obtain the best color
balance.
7
Click Stop Live, and acquire a sample image
to verify the accuracy of your settings.
Acquiring Images - Color Tab (Fluorescence)
To set the color balance for acquiring 24-bit fluorescence color images, complete the following
procedure:
Step
Action
1
Click the Color tab, the Color options move
to the front.
2
Select Fluorescence if you are acquiring
fluorescent images illuminated by a reflected
light source (light objects against a dark
background).
3
Click Show Live to begin acquiring images
from which to preview your settings.
4
With a specimen in place, create an active
region that defines an area that should be
interpreted as white, Click Measure White
Balance to establish an initial white balance
level.
5
For each color (Red, Green, Blue), adjust
the Min and Max values to obtain the best
color balance and image quality.
6
Click Stop Live, and acquire a sample image
to verify the accuracy of your settings.
Acquiring Images - Acquire tab
To configure for auto exposure settings and select an external shutter, complete the following
procedure:
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Step
Action
1
Click the Acquire tab. The acquire options
and settings move to the front.
2
In the Target Intensity box, type or select a
target intensity that is the desired
percentage of the maximum intensity
capability of the camera. (Typically, 75
percent of maximum is chosen.)
OR,
Optionally, set a value in the % of Max box
to specify what percentage of the maximum
intensity the target intensity should be.
3
In the Maximum Exposure box, type or
select an exposure time that you do not want
to exceed.
4
In the Increment Exposure by box, type or
select a value by which you want the
exposure time setting on the main dialog box
to increment.
5
In the Shutter box, type of select the name of
the external shutter you are using (If
applicable).
6
Click Zoom live image if binning is different
to keep the image windows for Show Live
and Acquire the same size when Live and
Acquire have different binning values.
7
Click Use setting name as image name to
assign the name of the Save Setting file as
the name of the image file.
Acquiring Images - Correct Tab
To apply corrections to your image(s) during acquisition, complete the following procedure:
Step
Action
1
Click the Correct tab. The Correction options
and settings move to the front.
2
If you want to apply background subtraction,
click the background subtraction option that
you want to apply.
3
In the Offset Value box, type or select the
amount of offset you want to apply to the
image to set the level considered to be
"black" above the background noise level.
4
Click Keep Shutter Closed if you need to
have complete darkness for acquiring a
background image.
5
Click Acquire Background to acquire the
background image that you want subtracted
from your image.
6
Click Display Background Image to see the
background image that was acquired.
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7
Click Do correction when live is running to
apply Background Subtraction and Shading
Correction to the live image while it is
actively acquiring images.
Acquiring Images - Annotate Tab
To apply annotations to your image(s) during acquisition, complete the following procedure:
Step
Action
1
Click the Annotate tab. The Annotate options
and settings move to the front.
2
Under Automatic Image Annotation click to
add or delete any annotations that you want
included with your image(s).
3
Under User Annotation type any additional
annotations that you want included with your
image(s). Be sure to enclose any variables
within percent symbols.
Acquiring Images - Special Tab
To set special controls on your camera, complete the following procedure:
Step
Action
1
Click the Special tab. The Special options
and settings move to the front.
2
Set the appropriate controls for your specific
camera. (The following steps guide you
through the example.)
3
In the Digitizer box, select the appropriate
digitizer speed (Slow or Fast).
4
In the Gain box, select the appropriate gain
level (Low, High, or Super High).
5
In the Bit Depth box, select the appropriate
bit depth (10, 12, 14, or 16-bit).
6
In the Camera Shutter box, select the
appropriate Camera Shutter option (Open for
Expose, Always Closed, or Always Open).
7
In the Digital4 Visibility box, click Hide
Digital4 menu to exclude the Digital4
commands from the Acquire menu.
OR
Click Show Digital4 and set as acquisition
handler to include the Digital4 commands on
the Acquire menu.
Acquiring Images - Live Replay tab
To configure and start capturing live images, complete the following procedure:
Step
1
Action
Click the Live Replay tab to bring it to the
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front.
2
Select Enable Live Replay.
Note: This option should be unchecked
when you finish using Live Replay to free up
system memory.
3
To specify a name for the resulting stack,
click Untitled in the Image Stack field, then
select Specified and enter or select a name
for the stack.
4
To select a journal to run when Capture Live
Images is activated, click Browse and
navigate to the journal.
5
In the Before the capture point field, enter
the number of frames to add to the stack that
were acquired before activating Capture Live
Images mode.
Note: The number of frames possible in the
stack is dependent on the amount of
memory on your computer. See the Memory
Acquisition Information fields for the amount
of memory available.
6
In the After the capture point field, enter the
number of frames to add to the stack that
were acquired after activating Capture Live
Images mode.
7
Click Show Live to open a live image
window. The Timing Acquisition Information
will update.
8
Click Capture Live Images (or press F11) to
start recording based on the current settings.
The stack image window opens when the
capture is complete.
9
Click F2: Stop Live to close the live image
window.
10
After you are done using the Live Replay
tab, uncheck Enable Live Replay to free up
system memory.
Acquire - Dialog Box Options
Acquire - Main Dialog Box Options
Acquire Dialog Box Options - Display Tab
Acquire Dialog Box Options - Acquire Tab
Acquire Options Dialog Box -- Annotate Tab
Acquire Dialog Box- Correct Tab
Acquire Dialog Box Options - Special Tab
Acquire Dialog Box Options - Live Replay Tab
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Acquire Dialog Box Options - Color Tab
Acquire – Main Dialog Box Options
Acquire
Acquires an image using the current settings. The image is always acquired into an image window called
"Acquired." Newly acquired images automatically overwrite previously acquired images in this window. The
"Acquired" window stores information about its last position, size, zoom, gamma, LUT, and other image
display attributes. Journals that include the Acquire button do not record any information about the settings.
On playback, the acquisition occurs using the dialogs latest configuration.
Note: You can cancel an ongoing acquisition at any time by pressing the [Esc] key.
Save Image
Saves the image currently in the "Acquired" window. If there is no "Acquired" window, this button is inactive.
If Save w/Sequence is not checked, this opens a "Save as" dialog box to save the image. The image
defaults to TIFF format using the Configure Default Paths last saved path for Save Images. If Save
w/Sequence is checked, the image is saved to the file location indicated on the "Save to" line in the
expanded dialog box. Click More to fully expand the Acquire dialog box. Click Set Save to open the
Acquire: Set Saving dialog box and specify an image sequence name.
Note: If the Show Saved Image option is checked, when an image is saved, it is
displayed in a new window, and the Acquire window remains open.
Save w/Sequence
Enables the Save with Sequence option. This option allows you to save sequentially acquired images as a
sequentially numbered group of images in a single directory. Click Save w/ Sequence, then acquire an
image. In the upper right corner of the maximized (More>>) Acquire dialog box, click Set Save…
Set Save
Opens the Acquire: Set Saving dialog box. Use this dialog box to name a sequential sequence of images.
Type the Base Name (for example, Image001) in the Base Name field. Set the option that specifies what to
do if the image name already exists. Click Show Image when Overwrite Saved if you want overwritten
saved images to open in a new window.
Image
Specifies the name and destination for the acquired image and selects whether the image should be saved
as a new image, overwrite an existing image with the same name, or add the image to an existing image in
order to make a stack of two or more images. This is a standard Meta Imaging Series Image Selector.
Though this image selector is not visible in Less<< mode, settings made to the image selector are still active
in Less<< mode.
Exposure Time
Selects and indicates the exposure time for the current acquisition. Possible units are, ms, sec, and min.
This will be available depending on what range the camera driver supports. The exposure time will be the
basis for the exposure of the live window. If live binning is different than the acquire binning, the time used
for live exposure will be a value calculated from the displayed exposure.
Note: If you set an exposure time greater than five seconds, a status bar will display on
the bottom of the MetaMorph desktop when you click Acquire to indicate the progress of
the exposure.
AutoExpose
Calculates and sets the autoexposure time for individually acquired images and for the Set Live image
window. The AutoExpose button works differently depending on whether the Live window is open. If the
Live window is open, AutoExpose seeks an exposure time that achieves the auto-expose parameters
specified on the Acquire tab and uses the resulting exposure time. If the Live window is not open, the same
AutoExpose calculations occur and an image is acquired just as if the Acquire button was pressed. The
initial exposure attempted by AutoExpose is the value specified in the Exposure field. If AutoExpose can not
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achieve the target intensity, the status line shows an error message accompanied by a red error "light". The
following are the possible warnings that can be displayed.
Low Signal – If no exposure reached or exceeded the target intensity this message appears.
Several circumstances can produce this message, including: The camera or controller is not turned
on; the light source is not turned on; the shutter is not turned on; or the wrong shutter is specified
on the Acquire tab, and the maximum exposure duration is too small for the actual sample present.
Target Not Achieved – If an exposure exceeded the target intensity but no exposure was within
range of the target this message appears. Possible causes including a changing image due to
changes in illumination or sample while the exposure was being calculated.
No Hardware Image – If AutoExpose can not obtain an image from the camera this message will
appear. The most likely cause of this message is that the device is not properly configured in the
Meta Imaging Series Administrator.
Binning
Sets the binning used by the Acquire command. Horizontal and vertical binning are always set the same.
The settings are limited to binning choices supported by the camera driver.
Camera Area
Defines the area that will be the acquisition region and acquires an image from the defined region. Use one
of the following buttons to define the acquisition region and acquire an image:
Full Chip – Defines the entire chip area as the acquisition region and acquires an image.
Center Quad. – Defines the center quadrant of the chip as the acquisition region and acquires an
image.
Use Active Region – Defines the designated active region as the acquisition region and acquires
an image.
When one of the buttons is pressed, the defined camera area is used and a new image is acquired, resizing
the image to fit the appropriate acquire image.
Note: For the Active Region button to perform any action, there must be an active region
on the active window on the desktop.
Show Live (and F2: Stop Live)
Rapidly acquires new image data in to a new window; in effect, showing a live image. Pressing F2 or this
button again stops updating the image.
Note: If you change the position of the stage (X, Y, or Z axes) using any of the
commands within MetaMorph the live window will pause until the stage reaches its
destination.
Live Bin
Sets the binning value for live acquisition. If live binning is different than acquire binning, the time used for
live exposure will be a value calculated from the acquire exposure. For example, if the live binning is 2 and
the acquire binning is 1, then the exposure used when acquiring the live image will be one-fourth the
displayed exposure. This compensates for the intensity increase that results from combined pixels and
enables the live image to update at a faster rate. This feature is not available if the Do correction when live is
running checkbox is checked in the Correct tab.
Status Line
Displays the current camera status, such as, "Exposing…" or "Transferring…". If the camera is not in a
process, it displays the camera CCD chip temperature. It also displays an error message if AutoExpose
could not reach its target.
Status Lights
Indicates whether background subtraction or shading correction are in use. If a setting is not active, no light
or text will be displayed. A green light is displayed if the setting is active and valid. A yellow light indicates
the setting was modified (and serves as a reminder that to keep the modifications, the setting should be
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resaved). A red light indicates the active setting cannot be used due to a configuration error. Explanations
that accompany warnings and errors can be seen on the "Correct" tab.
Setting
Displays the names of the most recently saved or loaded settings. Up to eight settings can be displayed in
the order of most recent usage. Selecting a setting load the values for the setting into the dialog for the next
acquisition, immediately updating the controls and the display of the current "Acquired" image. If any value
in the dialog is changed after a setting has been loaded, the title of the setting control will change from
"Setting:" to "Setting [Modified]:" to indicate that the dialog differs from the currently listed settings.
When loading a setting the load dialog may appear so that the portion of the setting to be loaded can be
configured. If the load dialog does not appear, the portion used will be the same as the portion used the last
time a setting was loaded. To get the Load dialog to appear when selecting a setting from the popup, hit the
"Load…" button on the more section of the dialog and set the "When using the pop list to load" radio
buttons.
Load
Opens the Acquire Load Setting dialog box.
Save
Saves the current settings state into the existing state file for subsequent re-use. The previously saved
settings are overwritten.
Save as
Opens the Acquire State save dialog box. Use this option and dialog box to assign a name to a new setting
file or to create a duplicate setting file that you can modify.
Close
Closes the Acquire dialog box.
More>>/Less<<
Expands (maximizes) or reduces (minimizes) the Acquire dialog box.
Acquire: Load Setting - Dialog Box Options
Exposure
Check this box to use the Exposure Time value from the setting you are loading.
Binning
Check this box to use the Binning value from the setting you are loading.
Camera Area
Check this box to use the Camera Area selection from the setting you are loading.
Illum
Check this box to use the Illumination setting defined in the Acquire tab from the setting you are loading.
Use Setting for name
Check this box to set the image name of acquired images to the name of the state file when the state file is
loaded. The state file name appears in the Setting drop-down box.
Display
Check this box to use the values defined in the Display tab from the setting you are loading.
Image Saving
Check this box to use the Save values from the setting you are loading. The Save values affected are set in
the following commands: Save w/Sequence, Set Save, and Image.
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Correction Settings
Check this box to use the values defined in the Correct tab from the setting you are loading.
Correction Images (if applicable)
Check this box to use the correction images acquired in the Correct tab from the setting you are loading.
Annotation
Check this box to use the values defined in the Annotation tab from the setting you are loading.
Special Parameters
Check this box to use the values defined in the Special tab from the setting you are loading.
Scaling for Color Camera
Check this box to use previously saved color camera scaling settings.
When using the popup list to load
Show this dialog
Check this box to view this dialog when selecting a setting from the Setting list.
Use latest selections. Skip this dialog.
Check this box to immediately switch to the selected settings without opening this dialog box. The
default (all) settings will be loaded.
Load
Opens the Acquire: Load Setting dialog box used to select a saved Acquire state (.AST) file.
Cancel
Cancels the command.
Acquire Dialog Box Options – Display Tab
Image Scaling
Sets 16-bit image scaling and specifies the range within which to either automatically or manually scale the
image bit density.
Note: As you make settings changes, the Acquired window (if open) reflects the
changes. Any subsequent images acquired will use these settings.
Low
Specifies the scaling value for the lower end of the range. If Autoscale is selected, the value is specified as
a percentage of the range to be excluded from the lower end of the scale. If Autoscale is not selected, the
value is specified as the exact low cutoff point on the image scale.
High
Specifies the scaling value for the upper end of the range. If Autoscale is selected, the value is specified as
a percentage of the range to be excluded from the upper end of the scale. If Autoscale is not selected, the
value is specified as the exact high cutoff point on the image scale.
Autoscale
Activates or deactivates image autoscaling. If Autoscale is on, the low and high percentage values specify
the percentages of the lower and upper areas of the image histogram to ignore; if Autoscale is off, the low
and high values specify the direct gray range within which to scale.
Scale within the active region
Scales the acquired image based on its active region. The scaling is applied immediately upon checking the
box or adding an active region to a live or acquired image.
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Image Gamma
Displays and sets the Gamma for the "Acquired" image. Use the slider control to set the gamma value.
=1
Resets the gamma value to 1.
Graph
Shows the histogram of the current "Acquired" image or the Gamma curve used in displaying the image.
The histogram will be scaled to the available bit depth of the camera in use. The histogram enables you to
identify which portion of the dynamic range of the camera is in use. This is useful for setting the exposure
while in live mode.
Reset Display
Resets the display of the "Acquired" image to defaults. This includes the scaling and gamma values set on
this tab as well as the other display values such as LUT, zoom and contrast settings.
Acquire Dialog Box Options - Color Tab
Image Type
Selects either Brightfield or Fluorescence as the image type based on the illumination source the you
intend to use. The default for this setting is Brightfield, which enables you to make settings for Brightness
and/or the color intensity of individual primary colors. If you select Fluorescence as the image type, the
settings options on the Color tab change to a different group of settings.
Settings for Fluorescence
Red
Provides controls to set the minimum and maximum scaling values to control the red sensitivity.
Move the pointed sliders along the red scale or type or select minimum and/or maximum scaling
values in the associated boxes.
Green
Provides controls to set the minimum and maximum scaling values to control the green sensitivity.
Move the pointed sliders along the green scale or type or select minimum and/or maximum scaling
values in the associated boxes.
Blue
Provides controls to set the minimum and maximum scaling values to control the blue sensitivity.
Move the pointed sliders along the blue scale or type or select minimum and/or maximum scaling
values in the associated boxes.
Min
Sets the lower limit of the scaling range for the associated color. This specifies the minimum gray
scale sensitivity for this color. Type or select a value in this box, or move the associated pointer
along the associated color scale slider.
Max
Sets the upper limit of the scaling range for the associated color. This specifies the maximum gray
scale sensitivity for this color. Type or select a value in this box, or move the associated pointer
along the associated color scale slider.
Settings for Brightfield
Brightness
Adjusts the overall intensity levels of all three color channels simultaneously. Moving this slider is
equivalent to moving the Red, Green, and Blue sliders by equal amounts.
Red
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Adjusts the intensity levels of the red values in the image.
Green
Adjusts the intensity levels of the green values in the image.
Blue
Adjusts the intensity levels of the blue values in the image.
Reset
Resets all sliders to the default midpoint position.
Measure Black Reference
Measures a black reference region in the image and records the measurement in the Black Reference
window.
Measure White Balance
Measures a white reference image, which is used to correct for the differences between red, green, and blue
values of the image. For the region that you specify as white, MetaMorph scales the intensity value
indicated for each channel to equal the maximum intensity for that color.
Acquire Dialog Box Options – Acquire tab
Auto-Expose Settings
Sets the target and limits for auto-expose calculations.
Target Intensity
Sets the maximum intensity value for the acquired image. The target intensity value default is 75 percent of
the maximum gray level that the camera driver reports as possible to obtain (For example, 75 percent of
4096 is 3072).
% of Max
Specifies the percentage of the maximum gray level needed to achieve the target intensity. The default
value is 75 percent.
Maximum Exposure
Sets the maximum exposure time that you want to allow. Time can be in milliseconds (ms), seconds (sec),
or minutes (min).
External Shutter Linked to Camera
Specifies the name of the external shutter that is in use.
Illumination
Lists the available shutters for you to use to acquire images. When acquiring an image, the shutter is
opened, the image is acquired, and then the shutter is closed. When in Live mode, the shutter is locked
open.
Preferences
Provides settings for available preferences.
Amount to adjust exposure when using the arrows in the edit box
Sets a value by which the exposure time can be incremented for each up or down click of the Exposure
Time settings buttons. The default value for this is 25.
Zoom live image if binning is different
Enables you to maintain the Live image window at the same size as the Acquire image window. If the Live
binning value is greater or less than the Acquire binning value the image window size will be increased or
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decreased by a proportionate percentage.
Use setting name as image name
Sets the image name in the image selector to the name of the state file when the state file is loaded. The
state file name appears in the Setting drop-down box.
Acquire Dialog Box Options – Correct tab
Background Subtraction
This can be off or use a constant, region, or image as a background.
None – Applies no background subtraction.
Constant -- Applies a constant value for background subtraction. Select or type a value in the
Constant Value box.
Region -- Applies a background subtraction value that is the average value of the defined region.
You must first define a region on the Acquire image. The average value of the region is subtracted
from the image. If you specify a value in the Offset Value box, this value can be added to the
image. This is useful for transmitted light images in which the background level should be high and
not 0.
If a region has never been established or is not valid for the image, the Define Region button
places a region on the "Acquired" image, acquiring the image if necessary. Once the background
region is on the image it can be moved or resized (but not deleted) as necessary. If the binning
changes and a new image is acquired the region will resize to position itself so that it covers the
same portion of the image. The region will maintain its proper position if the camera area changes.
If the camera region area changes so that the background is no longer on the image the region will
become invalid. Only the region created through the Define Region button can be used as the
subtraction region, other user defined regions will not be used.
Image – Acquires an image for the background using the current exposure and binning. The
background image acquired will always be the full chip area of the camera. When an acquisition is
done using some portion of the image, the correct portion of the background image is subtracted. In
this way, you can select new camera regions without having to acquire new background images.
Acquire Background – Acquires a background image.
Keep Shutter Closed – Prevents the shutter from opening during acquisition. Use this option to
acquire a background image to be used for background subtraction.
Display Background Image – Shows the portion of the background image that corresponds to the
current acquisition area. Altering, deleting, or saving the displayed image will have no effect on the
actual background image. If a new background image is acquired the window displaying the
previous background will not close, update or change. To see the newly-acquired background
image, click Display Background Image.
Load Background Image – Loads the acquired background image to be used for background
subtraction. If you acquire a new background image, you must click Load Background Image again
to use the most recently acquired background image.
(Error and Warning Text) – Provides error and warning information about the image background.
Under some circumstance a background setting may not be functional or appropriate. In these
cases a light matching that on the main tab will appear with some explanatory text. The following
messages may appear for Background subtraction:
[Red] Background image not acquired or loaded
[Red] Background binning differs from acquire
[Red] Background image size differs from camera
[Red] Background region not valid for acquisition
[Yellow] Background exposure differs from acquire
Errors, [Red], indicate that no subtraction will occur when the image is acquired. Warnings [Yellow]
indicate that subtraction will be performed but may not be appropriate under all circumstance.
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Note: Background and shading images are saved when exiting MetaMorph and
reloaded when MetaMorph restarts. In addition these images are saved with Setting
files and can be loaded with the Setting files.
Shading Correction
None – Applies no shading correction.
Image – Enables the shading correction option and applies shading correction to your image.
Acquire Shading Reference -- Acquires an image for shading correction using the current
exposure and binning. The shading image acquired will always be the full chip area of the camera.
When an acquisition is done using some portion of the image, the correct portion of the shading
image is used for correction. In this way you can select new camera regions without having to
acquire new shading images.
Display Shading Image -- Shows the portion of the shading image that corresponds to the current
acquisition area. Altering, deleting, or saving the displayed image will have no effect on the actual
shading image. If a new shading image is acquired the window displaying the previous shading
image will not close, update or change. To see the newly-acquired shading image, click Display
Shading Image.
Load Shading Image – Loads the most recently acquired shading image for use by the Shading
Correction option. If you acquire a new shading image, you must click Load Shading Image again
to use the most recently acquired shading image.
Error and Warning Text -- Provides error and warning information about the image shading.
Under some circumstance a shading setting may not be functional or appropriate. In these cases a
light matching that on the main tab will appear will some explanatory text. The following messages
may appear for shading correction:
[Red] Shading image not acquired or loaded
[Red] Shading binning differs from acquire
[Red] Shading image size differs from camera
Errors, [Red], indicate that no shading correction will occur when the image is acquired.
Do correction when live is running
Applies Background Subtraction and Shading Correction to the live image when "live" is actively acquiring
images. Activating this disables the Live Bin field.
Acquire Dialog Box Options – Annotate tab
Automatic Image Annotation
Enables you to select and include information that can automatically be placed into the acquired image’s
annotation. Click each checkbox for the information that you want to include.
User Annotation
Enables you to append text and/or variables you want to the acquired image’s annotation. All variables
must be enclosed between percent symbols (%variable%). Type the text and/or variable into the User
Annotation box.
Acquire Dialog Box Options – Special tab
Various Controls
Any control unique to the camera driver can appear on this tab. The options available will vary depending
on the camera installed.
Sensor Mode
Defines the operational mode of the camera.
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Digitizer
Sets the range of speed with in which the digitizer will acquire images.
Gain
Sets the range of sensitivity and signal strength for the camera. The gain setting works both for image
acquisition and Show Live. In Show Live mode, the value displayed is the live gain. In non-live mode, the
value displayed is the gain value to be used for image acquisition.
Intensifier Gain
Controls the output gain on specific cameras or controls the gain of image intensifiers on certain cameras.
Bit Depth
Sets the image bit depth. Enables you to expand the camera image bit depth range to fit within the bit depth
range of the acquired image. Set this value to 24-Bit to enable color image acquisition from qualified color
video cameras.
Sharpness
Controls camera hardware to increase or decrease camera sharpness.
Camera Shutter
Sets the state of the camera shutter to one of the following states: Open for Exposure, Always Closed, and
Always Open.
Clear Mode
Defines when to clear the camera chip.
Clear Count
Specifies the number of frames to clear when clearing the camera chip.
Frames to Average Field
Specifies the number of frames to combine for frame averaging cameras.
Offset
Adjusts the black level reference above the zero level to reduce or eliminate background noise.
Sensitivity
Turns on/off camera sensitivity capability and specifies a sensitivity value to control the camera’s internal
image intensifier.
Light Mode
Specifies the relative brightness of the light source and raises or lowers the camera’s sensitivity accordingly.
Cooler On
Activates internal camera cooling.
Use Contrast Knobs
Enables the camera-mounted contrast controls.
External Trigger/Trigger Mode
Enables the camera’s external trigger capability.
Flat Field Correction
Activates the camera’s internal shading correction capability.
Get Flatfield
Acquires an image for flatfield (shading) correction.
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Noise Filter
Activates the camera’s internal noise correction capability.
Compute exposure and gain on live startup
Computes exposure and gain whenever the Live button is pressed.
Quality/Speed
Enables you to achieve an ideal balance between Quality and Speed when continuously updating "live"
images. The better the quality, the slower the speed; conversely, the faster the speed, the lesser the quality.
Image Type
Selects the image's type of illumination: Brightfield or Darkfield.
Show Focus Indicator
Displays a value on the live image that reflects focus accuracy, where the highest value equals the most
accurate focus.
Reset
Resets certain camera settings to default values.
Digital4 Visibility
Enables a special option for those users who have Digital4 loaded in addition to the Acquire drop-in. Users
may want to switch to the new Acquire dialog, but have journals they want run that were recorded with
Acquire from Digital Camera. In this case the "Hide Digital4 menu" may be appropriate.
Hide Digital4 menu
Hides digital4 from the menu, but journals using Digital4 will still run. The acquire icons on the toolbar and
other functions such as Acquire Timelapse will run through the settings of the Acquire dialog.
If the user selects the option to show the digital4 menu then the acquire icons on the toolbar and other
functions such as Acquire Timelapse will run through the settings in the Acquire from Digital Camera dialog.
Show Digital4 and set as acquisition handler
Makes the Digital4 commands Acquire From Digital Camera and Basic Digital Acquire visible on the Acquire
menu, and sets them to function as the valid acquisition handler.
Acquire Dialog Box Options - Live Replay Tab
Enable Live Replay
Enables the Live Replay command when in Live mode. This checkbox must be selected to use the Live
Replay feature.
Image Stack
Specifies the name and destination for the Live Replay image stack and selects whether the stack should be
saved as a new image, overwrite an existing image with the same name, or add the image to an existing
image in order to make a stack of two or more images. This is a standard Meta Imaging Series Image
Selector.
Capture point journal
Displays the name of the journal to be run at the capture point. This is an optional step.
Browse
Enables you to select a journal to run at the capture point.
Number of frames to capture
Before the capture point
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Sets the number of images to add to the stack that were acquired before activating Capture Live
Images mode.
After the capture point
Sets the number of images to add to the stack that were acquired after activating Capture Live
Images mode.
Timing Acquisition Information
Note: The Timing Acquisition Information is determined by the values set in the Number
of frames to capture fields. The time is also effected by acquisition factors such as
binning, camera area, and digitizer speed.
Amount of time before capture point
Displays the amount of time (in seconds) from before the capture point that will be in the resulting
stack. This information is only displayed while in Live mode.
Amount of time after capture point
Displays the amount of time (in seconds) from after the capture point that will be in the resulting
stack. This information is only displayed while in Live mode.
Memory Acquisition Information
Displays memory usage statistics based on the current settings.
Capture Live Images
Begins capturing the live images into a stack based on the current settings. The Number of frames to
capture fields must be set before starting this command. You can also use the F11 key to activate this
command.
Digital Camera Adjustments
Eliminates background noise and redefines the black level value by repositioning the zero
reference level above the background noise.
Drop-in: DIGADJ
Use this command to eliminate background noise in images acquired with MV-1500 ORCA cameras.
Moving the slider or typing a new value in the settings box enables you to reposition the image's zero
pixel level reference point. When repositioned at a value above zero, all chip pixel information below the
new level setting is ignored, and the new level value is considered to be zero.
Adjusting Digital Camera Offset
To use the Digital Camera Adjustments, perform the following procedure:
Step
Action
1
From the Acquire menu, choose Digital
Camera Adjustments. The Digital Camera
Adjustments dialog box opens.
2
Acquire continuous images using Show Live
in the Acquire from Digital Camera dialog
box, the Basic Digital Acquire dialog box, or
the Acquire dialog box.
3
Close the shutter, cover the lens, or place a
lens cap on the lens to eliminate any outside
light source.
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4
While observing the image view, adjust the
offset control (slider) to obtain the best
possible black image.
5
Click Close when you are finished setting the
offset value. The offset dialog box closes.
Digital Camera Adjustments - Dialog Box Options
Offset
Adjusts the black level reference above the zero level to reduce or eliminate background noise.
Close
Closes the dialog box.
Hamamatsu C2400-60 (Acquire Menu)
Allows you to control the Hamamatsu C2400-60 camera controller from either the
controller or the computer.
Drop-in: C240060
You can use this command to specify Remote (computer) control or Local (controller box) control of
some of the camera controller functions, such as contrast enhancement. (There are several functions of
the controller that cannot be controlled from the computer. These include the SHADING control knobs
and the DETAIL control knob.)
Note: You must complete the following steps before using this command:
• Close MetaMorph and open the Meta Imaging Series Administrator program.
• Load the video camera driver using the Configure Acquisition command.
• Close the Meta Imaging Series Administrator program and open MetaMorph.
• Select the camera with the Select Camera/Board command (Acquire menu).
Local mode disables all of the dialog box options except the Local/Remote toggle command button. All of
these functions can then be controlled using the controller. Remote mode enables the dialog box
options. Except for Gain and Offset, all functions that can be controlled by the computer will be disabled
on the controller. The Contrast list allows you select whether the Gain and Offset functions are adjusted
using the dialog box, the controller knobs, or by Auto Enhance.
Functions Controlled by the Hamamatsu C2400-60 Command:
Controller Name
Dialog Box Option
REMOTE (LED)
GAIN
OFFSET
AUTO ENHANCE
BOOST - HIGH
BOOST - LOW
BOOST - OFF
NEGA
AGC
GRAY SCALE
SHADING MODE DIAG
SHADING MODE -
Remote/Local toggle command button
Gain
Offset
Auto Enhance selection in Contrast list
High (0.45) selection in Gamma list
Low (0.75) selection in Gamma list
Off (1.00) selection in Gamma list
Negate
Auto-Gain
Gray Scale
Diagonal selection in Shading list
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NORM
SHADING MODE OFF
Off selection in Shading list
Using the Hamamatsu C2400-60 Camera Controller
To control the Hamamatsu C2400-60 camera controller from MetaMorph, use the following
procedure:
Step
Action
1
From the Acquire menu, choose Hamamatsu
C2400-60.
2
If this is the first time that this command has
been used, the Serial Communications
dialog box will appear.
Select the communication port being used by
the camera controller from the Comm. Port
list. Select 9600 baud from the Baud Rate
list. Choose OK when you have finished.
This allows MetaMorph to set up the
appropriate Data Stream that it will create
exclusively for this command. This step will
be skipped during subsequent uses of the
Hamamatsu C2400-60 command.
3
Using the Remote/Local toggle command
button, select whether the controller (Local)
or computer (Remote) controls the
Hamamatsu C2400-60 functions. The dialog
box options will be available when the button
is labeled as Remote. The dialog box options
will be unavailable when the button is labeled
as Local.
4
If you selected Local in Step 3, you can
operate the controls using the controller box.
Otherwise, continue to Step 5.
5
To set the Gain and Offset manually, choose
either Set via Dialog Controls or Set via
Controller Knobs from the Contrast list.
OR
Select Auto Enhance from the Contrast list if
you want the controller to determine the best
Gain and Offset settings. Then skip to Step
7.
6
Use the Gain and Offset options to set the
gain and offset levels. If you selected Set via
Controller Knobs in Step 5, use the knobs on
the controller box instead.
7
Select the desired Shading Mode (Normal,
Diagonal, or Off) from the Shading list.
8
Select the desired Boost setting (High, Low,
or Off) from the Gamma list.
9
You can select the Auto-Gain, Negate,
and/or Gray Scale options to turn on their
respective functions (AGC, NEGA, and
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GRAY SCALE) on the controller box.
To turn off one of these functions, deselect
the appropriate option so that its check box is
cleared.
10
Choose Close when you have finished.
Hamamatsu C2400-60 - Dialog Box Options
Remote/Local
Specifies whether the applicable camera controller functions are set from the controller box (Local) or from
this dialog box (Remote). When Remote is displayed as the label on the button, the functions will be
controlled by the computer. When Local is displayed as the label, the dialog box options (except this one)
will be disabled.
Contrast
Specifies how the Gain and Offset will be set: with the control knobs on the controller box, with the dialog
box options, or to be determined by the controller using Auto Enhance to select the best settings. If you
select Auto Enhance, Gain and Offset will be unavailable.
Gain
Specifies the gain setting. You can select a value from 0 to 10.
Offset
Specifies the offset setting. You can select a value from 0 to 10.
Shading
Specifies the shading mode. You can select Normal, Diagonal, or Off.
Gamma
Specifies the boost mode. You can select High, Low, or Off.
Auto Enhance
Enables and disables the AUTO ENHANCE function.
Negate
Enables and disables the NEGA function.
Gray Scale
Enables and disables the GRAY SCALE function.
Close
Closes the dialog box.
Configure Digital Camera (Acquire Menu)
Configures basic settings for use of a digital camera.
Drop-in: CFGCCD
Use this command to set a digital camera's temperature, shutter speed, and sensor mode. This
command is used after installation of a charge-coupled device (CCD), or "digital camera." After the
camera's use has been configured, you probably will not need to use this command again for that
camera.
Note: These configuration settings are currently only supported by the Photometrics PVCam driver and
by Princeton Instruments cameras (EXAMPLES: MicroMAX, PentaMAX).
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Note: You must complete the following steps before using this command:
• Close MetaMorph and open the Meta Imaging Series Administrator program.
• Load the video camera driver using the Configure Acquisition command.
• Close the Meta Imaging Series Administrator program and open MetaMorph.
• Select the camera with the Select Camera/Board command (Acquire menu).
Configuring a Digital Camera
To configure the use of your digital camera, use the following procedure:
Step
Action
1
From the Acquire menu, choose Configure
Digital Camera. The Configure Digital
Camera dialog box will appear.
2
The temperature setting of your camera is
shown in the Current Camera Temperature
Is text line. To change the temperature
setting for your camera, enter the new
temperature with the Set Camera
Temperature To spin box. This will be
determined by the particular camera driver
being used. Typical settings for the
Photometrics PVCam camera are in the
range of -5 to -25 degrees Centigrade.
3
Select a sensor mode from the Sensor Mode
drop-down list box. Again, the particular
camera driver you are using will determine
your choice. Possible choices are: Normal,
FT (frame transfer), MPP (a camera option
available on certain PVCam cameras), and
FT MPP (a frame transfer version of an MPP
camera).
4
If you are using a camera that has a shutter,
set the shutter's open and close delays in the
Shutter Open Delay and Shutter Close Delay
text boxes. Typical delays for the PVCam are
15 ms to open and 30 ms to close.
5
Choose OK.
Configure Digital Camera - Dialog Box Options
Current Camera Temperature Is
Indicates the current temperature setting for your digital camera.
Set Camera Temperature To
Changes the temperature setting of your digital camera to your specified new temperature.
Sensor Mode
Sets the sensor mode of your camera. The particular camera driver you are using will determine your
choice. The choices are: Normal, FT (frame transfer), MPP (a camera option available on certain PVCam
cameras), and FT MPP (a frame transfer version of an MPP camera).
Shutter Open Delay
Sets the delay time for the opening of your camera's shutter. The typical delay for a Photometrics PVCam
camera is 15 ms.
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Shutter Close Delay
Sets the delay time for the closing of your camera's shutter. The typical delay for a Photometrics PVCam
camera is 30 ms.
OK
Closes the dialog box and implements the option changes that were selected.
Cancel
Closes the dialog box and cancels the option changes that were selected.
Acquire from Digital Camera (Acquire Menu)
Acquires and transfers images from a digital camera. Allows focusing with a digital
camera.
Drop-in: DIGITAL4
Use this command when you want to acquire images from a digital camera.
Expose acquires the current image, placing it in memory (replacing the image previously stored in
memory).
Transfer transfers the most recently acquired image stored in the video board's memory to an image
window in MetaMorph.
Expose & Transfer performs both of these tasks.
WARNING:
You must run all mechanical shutters at a cycle time greater than 25 ms. Uniblitz, Lambda 10, Metaltek,
Ludl, and cooled CCD shutters are driven by a high voltage which takes time to dissipate. Running these
shutters at a cycle length shorter than 25 ms will cause a build-up of heat, leading to eventual jamming.
Neither Molecular Devices nor any manufacturers of these shutters will honor warranties on equipment
that has been damaged by improper use. Operation of these shutters at a cycle length shorter than 25
ms is considered improper use.
Note: You must complete the following steps before using this command:
• Close MetaMorph and open the Meta Imaging Series Administrator program.
• Load the video camera driver using the Configure Acquisition command.
• Close the Meta Imaging Series Administrator program and open MetaMorph.
• Select the camera with the Select Camera/Board command (Acquire menu).
Acquire from Digital Camera allows you to create and define multiple sets of acquisition settings that you
can select quickly as needed during an acquisition work session. Each set of acquisition settings can
include specific settings for exposure time, binning, gain, speed, bit-depth, region size, shutter,
background subtraction, and shading correction. After you define them using the Define Acquisition
Settings dialog box, three separate sets of acquisition settings can be selected using the Acquisition
Settings lists. You can then switch between these three sets as needed by selecting the check box next
to the desired set. You can save and load settings.
You can also use this command to focus the image while acquiring from the digital camera. The Focus
command quickly updates and displays the image in an image window. This allows you to see an image
while focusing on your specimen. The focusing image will be displayed in the Focus window at 100%
zoom, regardless of the zoom you select for the acquisition image window. The Focus command derives
its acquisition settings from those that are selected in the Focus Acquisition Settings list. Acquisition
configurations such as binning settings will be disabled while you are focusing.
If you include the Focus command as part of a journal, you will have the option of configuring a brief
message that will appear during playback. This message will then be displayed in a dialog box that
provides you with two buttons: Cancel Journal and Stop Focusing.
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The Acquire from Digital Camera dialog box expands to include three image selectors. The first one,
Destination Image, is used to select the destination image for Expose & Transfer and Transfer. The
Background Reference image selector is used to specify the background reference image to be used if
you selected Subtract Background from the Define Acquisition Settings dialog box. Likewise, the
Shading Reference image selector is used to specify the shading reference image if you selected
Correct Shading. (These image selectors will be unavailable if you did not select the pertinent option in
the Define Acquisitions Settings dialog box. Background and shading reference images are acquired as
for any other image, using the Expose & Transfer command. You may wish to save them for future work
sessions using the Save command in the File menu.)
Acquiring Digital Images
Opening the Acquire from Digital Camera Dialog Box
Defining Acquisition Settings
Configuring Region Settings
Focusing a Digital Camera
Acquiring Images
Acquiring Reference Images
Opening the Acquire from Digital Camera Dialog Box
To transfer an image or a stack to video, use the following procedure:
Step
Action
1
From the Acquire menu, choose Acquire
from Digital Camera. The Acquire from
Digital Camera dialog box opens.
2
If you want to set the preferences for the
digital camera, select Prefs. The Digital
Camera Preferences dialog box will appear.
AND
Select Warn If Exposure Time Is Long if you
want to be warned when the camera's
exposure is longer than the Exposure Time
Warning Threshold value. Then select the
desired number of milliseconds for the
Exposure Time Warning Threshold.
3
Choose OK.
Defining Acquisition Settings
To define the acquisition settings for Acquire from Digital Camera, use the following procedure:
Step
Action
1
Open the Acquire from Digital Camera
dialog box.
2
Choose Define Acquisition Settings. The
Define Acquisition Settings dialog box will
appear.
3
To create a new set of acquisition settings,
choose New Setting. "Untitled 1" will appear
in the Setting Name text box. You can
replace this name with a name of your
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choice.
OR
Select an existing set of settings from the
Stored Setting list. Its name will appear in the
Setting Name text box.
4
Select the desired exposure time and the unit
of time using Exposure Time.
5
Use the Configure Region command to
configure the desired region size.
AND
Select the desired region from the Region
list.
6
If your camera supports binning, you can
select the desired type of binning from the
Binning list. Select Sep H and V for separate
horizontal and vertical binning, Same H and
V for the same horizontal and vertical
binning, or None for no binning. (The options
available in this drop-down list box will vary
depending on the type of binning supported
by your camera.)
AND
Select the desired Horz. and Vert. values.
7
You can select settings for Gain, Speed, and
Bit Depth if your camera supports any of
these options. The settings will vary
depending on the type of camera. In some
cases, the settings available in one of these
options will depend on the setting(s) in the
other option(s).
Bit-depth specifies the gray level scale
resolution of the camera. Although setting
the bit-depth to a lower value may result in
faster acquisition, the image quality will suffer
if you do not use the highest bit value.
8
If you are using an external shutter, select a
shutter state from the Shutter list: Open for
Expose, Always Closed, or Always Open.
AND
Select the shutter associated from the
External Shutter list.
9
If much of your image data resides in a
narrow band at the lower or upper end of the
grayscale range, you may need to rescale
the image to be able to discriminate intensity
differences. You can select Auto Scale 16-Bit
Image if you want MetaMorph to choose the
range of gray levels used for scaling.
OR
Deselect Auto Scale 16-Bit Image to choose
the range manually. Then select the darkest
gray level for the image using the Low text
box and select the brightest gray level using
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the High text box.
10
Select Subtract Background if you want to
enable background subtraction. The
background image will be subtracted
whenever an image is acquired. This option
will use the reference image selected from
the Background Reference image selector in
the Acquire from Digital Camera dialog box.
AND
If the image resulting from background
subtraction is too dark (less than gray level
0), select a Subtraction Offset value to be
added back to the image's values.
(See also: Acquiring Reference Images.)
11
If your hardware supports camera chip
clearing, use the Clear Chip Count spin box
to specify the number of times the chip is to
be cleared before exposure.
12
Select Correct Shading if you want to enable
shading correction. This option will use the
reference image selected from the Shading
Reference image selector in the Acquire from
Digital Camera dialog box.
13
Repeat Steps 3 - 12 for as many sets of
acquisition settings as you need for
acquisition and focusing. You can use the
Delete Setting command to delete any
settings if necessary.
14
Choose Close when you have finished.
Understanding Binning
Binning is the process of combining data from groups of image pixels into a single pixel
during acquisition. Binning results in a higher signal to camera noise ratio in the resulting
image, with a corresponding increase in intensity, or brightness, for the resulting image.
For example, 2 x 2 binning causes the signal to camera noise ratio to increase 4x in the
resulting image, with a 4x decrease in the image size. 3 x 3 binning results in a 9x
increase in signal to camera noise ratio and a 9x decrease in image size. Because the
image is smaller, the time required to transfer the image and the image file size are
significantly reduced.
In the example below, two horizontal pixels and two vertical pixels are combined in 2 x 2
binning. Usually the number of horizontal and vertical pixels that are used are the same.
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When you select the Horz. and Vert. binning values, it is important that you consider the
final value of the resulting pixel. If your camera is a 12-bit camera, you would calculate
the maximum brightness as: 2 * 2 12 – 1 = 4095. Thus, the sum of the binned pixels
cannot exceed that value for a 12-bit camera. Binned pixels will lose some spatial
resolution but no intensity data loss unless the resulting pixel values exceed the
maximum brightness value.
Configuring Region Settings
To configure the region settings for Acquire from Digital Camera, use the following procedure:
Step
Action
1
Open the Acquire from Digital Camera
dialog box.
2
Choose Define Acquisition Settings. The
Define Acquisition Settings dialog box will
appear.
3
Choose Configure Regions. The Configure
Regions dialog box will appear.
4
To create a new region, choose New Region.
"Default" will appear in the Region Name text
box. You can replace this name with a name
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of your choice.
OR
Select an existing region from the Stored
Region list. Its name will appear in the
Region Name text box.
If you want to use the active rectangular
region from an image on the desktop, rather
than defining the region with the dialog box
options, select the desired image using the
Image selector.
5
AND
Choose Use Action Region Defined on
Image.
You can use Left, Top, Width, and Height to
specify the size and location of the region on
the chip. Choose Entire Chip to create a
region that is the size of the chip, or choose
Center Quadrant to create a region centered
on the chip that is the size of one quadrant.
Choose Ctr to center the region. You can use
the << or >> options to shrink or enlarge a
region by a factor of 2.
6
OR
Specify the size and location of region using
the box-in-box option on the left side of the
dialog box. The smaller box can be sized and
moved just as for a region of interest.
7
Repeat Steps 4 - 6 for each region you want
to create.
8
Choose Close when you have finished.
Focusing the Microscope with a Digital Camera
The Focus command instructs MetaMorph to acquire an image continuously into an image
window while you are focusing the microscope, so that you can verify that your specimen is
visible and in focus. It is important to use the Focus command because what can be seen through
the microscope's eyepiece and what the camera acquires are not always the same. A few digital
cameras have gain and offset controls. These too can be adjusted while using the Focus
command.
MetaMorph will use the acquisition settings defined in the selected Focus Acquisition Setting to
acquire images continuously until you choose Stop Focusing (or press the [F2] key). Acquisition
configurations such as binning settings will be disabled while you are focusing.
Step
Action
1
Open the Acquire from Digital Camera
dialog box.
2
Choose Define Acquisition Settings. The
Define Acquisition Settings dialog box will
appear.
3
Define a set of acquisition settings suitable
for focusing using the procedure outlined in
Defining Acquisition Settings. When you
select a region size for focusing, the smaller
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the region, the faster the Focus image
window can update.
After you have finished defining the settings
for focusing, choose Close to close the
Define Acquisition Settings dialog box.
4
Select the desired acquisition settings from
the Focus Acquisitions Settings list.
5
Choose Focus. The Focus image window will
appear.
6
Focus your microscope.
7
Choose Stop Focusing or press the [F2] key
to stop the focusing acquisition. MetaMorph
will stop acquiring images and will close the
Focus image window.
Acquiring Images from a Digital Camera
To acquire images from Acquire from Digital Camera, use the following procedure:
Step
Action
1
Open the Acquire from Digital Camera
dialog box.
2
Define the desired set(s) of acquisition
settings using the procedure outlined in
Defining Acquisition Settings.
3
Choose More >> to select the desired
images for your acquisition.
AND
Select the desired destination image using
the Destination image selector. If you
selected Subtract Background as one of your
acquisition settings, you can select a
reference image using the Background
Reference image selector. If you selected
Shading Correction as one of your
acquisition settings, you can select a
reference image using the Shading
Reference image selector.
4
Select the desired sets of acquisition settings
that you want to display in the three
Acquisition Settings lists during acquisition.
AND
Select the check box next to the set that you
want to use first during the acquisition. You
can quickly change to a different set at any
time by selecting the appropriate check box.
5
Choose Expose & Transfer to acquire an
image and transfer the image to an image
window.
OR
Choose Expose if your camera supports the
video monitor display of the image in the
video board's memory. Then choose
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Transfer when you want to place the
displayed image in an image window.
If you plan to switch between Acquisition
Settings sets that have different region sizes,
you should use Clear command before each
Expose command to clear the contents of
video board's memory. (Otherwise, your
monitor will display part of the previous
image.)
6
Choose Close when you have finished.
Acquire from Digital Camera - Dialog Box Options
Acquisition Settings
The Acquire from Digital Camera command allows you to create and define multiple sets of acquisition
settings that you can select quickly as needed during an acquisition work session. Each set of acquisition
settings can include specific settings for exposure time, binning, gain, speed, bit-depth, region size, shutter,
background subtraction, and shading correction. After you have configured them using the Define
Acquisition Settings dialog box, you can select from three separate sets of acquisition settings using the
Acquisition Settings lists. You can switch between these three sets as needed by selecting the check box
next to the desired set.
Focus Acquisition Settings
Specifies the set of acquisition settings MetaMorph will use for focusing.
Define Acquisition Settings
Opens the Define Acquisition Settings dialog box.
Expose & Transfer
Acquires the current image from the digital camera, placing it in the video board's memory (replacing the
previous image stored in memory) and then transfers it to an image window in MetaMorph.
Expose
Acquires the current image from the digital camera, placing it in the video board's memory (replacing the
image previously stored in memory). If the digital camera supports display of the video board's memory on a
video monitor, the image will be displayed on the monitor.
Transfer
Transfers the last acquired image stored in the video board's memory to an image window in MetaMorph.
Clear
Clears the contents of the video board's memory. If you plan to switch between Acquisition Settings sets that
have different region sizes, you should use Clear command before each Expose command to clear the
monitor. Otherwise, your monitor will display part of the previous image.
More >>
Expands the dialog box.
Less <<
Condenses the dialog box.
Focus
The Focus command instructs MetaMorph to acquire an image continuously into an image window while you
are focusing the microscope so that you can verify that your specimen is visible and in focus. It is important
to use the Focus command because what can be seen through the microscope's eyepiece and what the
camera acquires are not always the same. A few digital cameras have gain and offset controls. These too
can be adjusted while using the Focus command. MetaMorph will use the acquisition settings defined in the
selected Focus Acquisition Setting to acquire images continuously until you choose Stop Focusing (or press
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the [F2] key). The journal function for this option, "ADC: Focus," acts as a toggle--the first time it is activated,
images will be acquired into a Focus window. A subsequent call to the function or a press of the [F2] key will
terminate acquisition.
Stop Focusing
Stops the digital camera acquisitions used for focusing. Pressing the [F2] key also stops the acquisitions.
Choosing Expose & Transfer, Expose, Transfer, or selecting an Acquisition Setting check box will also stop
the focus acquisitions.
Prefs
Opens the Digital Camera Preferences dialog box.
Destination
Specifies the destination image for the acquisition. You can add to or overwrite an existing image or stack.
You can also specify a new image.
Background Reference
Specifies the background reference image for the acquisition. You can add to or overwrite an existing image
or stack. You can also select None. This image selector can be used only if you selected Subtract
Background from the Define Acquisition Settings dialog box. You will not be able to select the same image
for both background reference and shading reference.
Shading Reference
Specifies the shading reference image for the acquisition. You can add to or overwrite an existing image or
stack. You can also select None. This image selector can be used only if you selected Correct Shading from
the Define Acquisition Settings dialog box. You will not be able to select the same image for both
background reference and shading reference.
Load
Loads a set of acquisition settings previously saved with the Save command. This command opens the Load
Acquisition Settings dialog box.
Save
Saves the current acquisition settings on disk. You can open the settings at a later date using the Load
command. This command opens the Save Acquisition Settings dialog box.
Close
Closes the dialog box.
Digital Camera Preferences - Dialog Box Options
Warn If Exposure Time Is Long
Instructs MetaMorph to warn you before an acquisition starts if the camera's exposure setting is longer than
the value specified in the Exposure Time Warning Threshold.
Exposure Time Warning Threshold (ms)
Specifies the minimum exposure time that is to be considered a long exposure. You will be warned if the
exposure time equals or exceeds this limit.
OK
Sets the digital camera preferences.
Cancel
Cancels the command.
Define Acquisition Settings - Dialog Box Options
Stored Setting
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Specifies the stored setting currently displayed in the Define Acquisition Settings dialog box.
Setting Name
Lists the name of the new or existing setting that you are editing. You can edit the name of the current
setting using this option's text box. The name for each setting must be unique.
Delete Setting
Deletes an acquisition setting set from the Stored Setting list. Will not allow you to delete the last remaining
setting.
New Setting
Creates a new setting based on the last setting displayed in the dialog box.
Exposure Time
Specifies the length of the exposure and unit of time for each acquisition.
Region
Specifies the region for the acquisition.
Configure Regions
Opens the Configure Regions dialog box which configures regions for use with various sets of acquisition
settings.
Binning
Specifies the type of binning used if your camera supports binning.
Horz
Specifies the horizontal value for binning if your camera supports binning.
Vert
Specifies the vertical value for binning if your camera supports binning.
Gain
Specifies the gain used if your camera supports this option. Select a higher gain value if you want a brighter
image. The settings will vary depending on the type of camera. In some cases, the settings available will
depend on other setting(s) selected.
Speed
Specifies the speed used if your camera supports this option. The settings will vary depending on the type of
camera. In some cases, the settings available will depend on other setting(s) selected.
Bit Depth
Specifies the grayscale resolution of the camera if your camera supports this option. Although setting the bitdepth to a lower value results in faster acquisition, the image quality will be better if you use the highest bit
value. The settings will vary depending on the type of camera. In some cases, the settings available will
depend on other setting(s) selected.
Shutter
Selects a shutter state for an external shutter, if one is available. This option is available only if your camera
has its own shutter and it can be controlled from MetaMorph.
Open for Expose will open the shutter only during active acquisition of an image.
Always Closed leaves the shutter closed. This can be used for acquiring a dark reference image.
Always Open leaves the shutter open continuously.
Auto Scale 16-Bit Image
Instructs MetaMorph to choose the range of gray levels used for scaling a 16-bit image.
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Low and High
Sets the range for scaling a 16-bit image manually. Select the darkest gray level for the image using the Low
text box and select the brightest gray level using the High text box option.
Subtraction Offset
If the image resulting from background subtraction is too dark ("less" than gray level 0), this option specifies
the value that will be added back to the image's values.
Clear Chip Count
This option erases the camera chip to gray level 0 the specified number of times before each acquisition. If
your camera supports this option, consult your camera's documentation to determine the correct value for
this option.
Subtract Background
Enables background subtraction.
Shading Correction
Enables shading correction.
Use Contrast Knobs
This option is only available if your camera's control box has contrast knobs. When this option is selected,
the camera will use the settings from the contrast knobs. Otherwise, these settings will be ignored.
External Shutter
Selects the shutter. If you are not using a shutter, select "[None]."
Transfer as 8-Bit Image
Scales the image to 8-bit depth and transfers it to the destination image window. This option uses the Auto
Scale 16-Bit Image settings.
Close
Closes the dialog box.
Acquire from Digital Camera: Configure Regions - Dialog Box Options
Stored Region
Specifies the stored region currently displayed in the Define Region dialog box.
Region Name
Lists the name of the new or existing region that you are editing. You can edit the name of the current region
using this option's text box. The name for each region must be unique.
Delete Region
Deletes a region from the Stored Region list.
New Region
Creates a new region based on the last region displayed in the dialog box.
Box-in-Box Interactive Display of Region
Allows you to click on the smaller box with the left mouse button and then drag the pointer to resize and
move the chip region box, as you would for a region of interest.
Image
Specifies the image to use for the Use Active Region Defined on Image command.
Use Active Region Defined on Image
Defines a region for the chip based on the active region of interest in the image selected with the Image
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selector. The box-in-box display is updated as well as the region's Left, Top, Width, and Height values.
Left
Specifies the region's leftmost point.
Top
Specifies the region's topmost point.
Width
Specifies the region's width.
Height
Specifies the region's height.
Entire Chip
Creates a region that is the size of the entire chip.
Center Quadrant
Creates and centers a region that is the size of one quadrant of the chip.
<< and >>
Shrinks or enlarges the region by a factor of two.
Ctr
Centers the region on the chip.
Close
Closes the dialog box.
Stop Focusing (Acquire Menu)
Stops acquiring images used during focusing with a digital camera.
Drop-in: AUTOFCUS, ACQSCCC
Use this command after you have finished using the Focus command in the Acquire from Digital Camera
or Acquire Color dialog boxes. This command is the same as the Stop Focusing command button in that
dialog box.
Note: This command has no relationship with the Device menu's Auto-Focus or Focus commands or
with the Stack menu's Acquire Z Series command.
Note: You must load the appropriate digital camera driver in the Meta Imaging Series Administrator and
select it with the Select Camera/Board command (Acquire menu) for this command to be available.
Shortcut: [F2]
Stop Focusing a Digital Camera
To stop focusing a digital camera, use the following procedure:
Step
Action
1
From the Acquire menu, choose Stop
Focusing.
2
MetaMorph will stop image acquisition and
close the Focus image window.
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Basic Digital Acquire (Acquire Menu)
Acquires an image from a digital camera. Can be configured to calibrate the exposure time
automatically.
Drop-in: DIGITAL4
This command allows you to acquire an image from a digital camera. You can also use the command to
calculate the exposure time for you automatically.
WARNING:
You must run all mechanical shutters at a cycle time greater than 25 ms. Uniblitz, Lambda 10, Metaltek,
Ludl, and cooled CCD shutters are driven by a high voltage which takes time to dissipate. Running these
shutters at a cycle length shorter than 25 ms will cause a build-up of heat, leading to eventual jamming.
Neither Molecular Devices nor any manufacturers of these shutters will honor warranties on equipment
that has been damaged by improper use. Operation of these shutters at a cycle length shorter than 25
ms is considered improper use.
Note: You must complete the following steps before using this command:
• Close MetaMorph and open the Meta Imaging Series Administrator program.
• Load the video camera driver using the Configure Acquisition command.
• Close the Meta Imaging Series Administrator program and open MetaMorph.
• Select the camera with the Select Camera/Board command (Acquire menu).
This command has a simpler interface than the powerful Acquire from Digital Camera and, as such, is
particularly useful for newer users. It is also useful for streamlining the acquisition process because it
allows you to calculate the exposure for the session automatically. Once you have performed this at the
beginning of a session, you can condense the dialog box and acquire images by using the options in the
upper half of the dialog box. This command is ideal for use in journals.
This command uses the settings that were set in the Acquire from Digital Camera command. Basic
Digital Acquire provides access to Acquire from Digital Camera's Define Acquisition Settings dialog box
so that you can define settings as needed. The Configure Regions dialog box is also accessible from the
Basic Digital Acquire dialog box so that you can specify the size of the region used for acquisition.
When the exposure is calculated, multiple acquisitions are performed until MetaMorph produces an
exposure value that satisfies the image intensity criteria that you have defined in the dialog box.
Typically, the targeted intensity value will be obtained by the third or fourth acquisition in the series.
The first exposure uses the Initial Exposure Duration value set in the dialog box. Based on the brightest
pixel in this image and the desired Target Intensity value, MetaMorph will adjust the exposure time,
extrapolating from the values of the first exposure, to arrive at a calculated exposure value that meets
your criteria. It then acquires the second exposure. An image will be deemed acceptable if the maximum
pixel intensity in it is within 10% (+/-) of the target intensity value.
A safety feature has been included. If the calculated exposure is greater than a previous one which
exceeded the target intensity or is shorter than one which fell short of the target, the command will resort
to a binary search. The newly calculated exposure will be halfway between the longest exposure that
didn't reach the target intensity and the short exposure that went beyond the target.
Using Basic Digital Acquire
Setting Autoexposure Options
Acquiring Images
Setting Autoexposure Options
To set up the autoexposure options for Basic Digital Acquire, use the following procedure:
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Step
Action
1
From the Acquire menu, choose Basic Digital
Acquire. The Basic Digital Acquire dialog box
opens. Then choose More >> to expand the
dialog box.
2
Select your camera's maximum intensity
value from the first Target Intensity list.
Then use the second text box to select the
percentage of the maximum value that the
brightest pixel in the image should equal.
This will update the third text box to display
the brightest pixel's target intensity value. (If
desired, you can select actual value first, and
let MetaMorph update the percentage for
you.)
3
Select the length of the first exposure
attempt from Initial Exposure Duration.
4
Select the maximum and minimum duration
lengths from Duration of Exposure.
5
Select the size of the region used to test the
brightest pixel value against its neighbors
from Region Size for Testing Noisy Pixel.
6
If you want to change the acquisition settings
choose Acquisition Settings.
Note: Background Subtraction and Shading
Correction are not available in Basic Digital
Acquire.
7
When you are satisfied with the settings, you
are ready to acquire images using Basic
Digital Acquire.
Acquiring Images Using Basic Digital Acquire
To acquire images using Basic Digital Acquire, use the following procedure.
Note: If you are using the autoexposure options and want to log the exposure
calculations, open a data log file prior to using this procedure.
Step
Action
1
From the Acquire menu, choose Basic Digital
Acquire. The Basic Digital Acquire dialog box
opens.
2
Select the desired image from the
Destination Image selector.
3
Select the desired set of stored acquisition
settings that you want to use for the
acquisition from the Acquire Digital Camera
Settings list.
To change the acquisition settings for a
particular set, you can expand the dialog box
using More >> and choose Acquisition
Settings.
Note: Background subtraction and shading
correction are not available in Basic Digital
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Acquire.
4
Choose Configure Regions if you want to
define the region size used for the
acquisition.
5
If you want to acquire images using the Basic
Digital Acquire auto exposure feature, set up
the Auto Exposure options, if you have not
already done so.
Select Calculate Exposure and Update
Settings to enable the autoexposure options.
Then skip to Step 7.
OR
If you do not want to use the autoexposure
options, skip this step.
6
If you are not using the autoexposure
options, use the Next Exposure text box to
set the length of your next exposure.
7
Choose Acquire.
If you have enabled Calculate Exposure and
Update Settings, the command will perform
multiple acquisition until your criteria are met.
If it cannot meet your criteria, an error
message will appear, explaining the cause
of the error. In most cases, you will be
allowed to continue trying or you can accept
the last acquisition which will be placed in the
destination image window.
If you are not using the autoexposure
options, the acquisition will be placed the
destination image window.
8
Choose Close when you have finished.
Error Conditions That Generate Error Messages in
Basic Digital Acquire
The following conditions will generate error messages when using autoexposure options in Basic
Digital Acquire.
• Null image: A camera has not yet been selected using the Meta Imaging Series Administrator.
• Several saturation exposures have occurred.
• Initial value is very low compared to camera's maximum value on attempts of increasing
duration.
• Repeat values of the maximum pixel intensity occurred more than once.
• The exposure to be performed has a duration that is outside the duration range set by the
user.
• Too many attempts have been performed.
These error messages appear because the command's algorithm tests for the following items:
• No camera selected in the Video Driver Manager: Indicated by presence of null image.
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• Saturation: If 1% or more of the image has a value equal to the maximum pixel intensity.
• Initial low values: If the initial exposure result is low then it is not a good choice for choosing a
maximum pixel.
• Repeat values of maximum pixel: Indicates that camera is turned off or some other type
camera error has occurred.
• Duration outside of range: Next exposure is longer than selected maximum value.
• Duration outside of range: Next exposure is shorter than selected minimum value.
• Number of attempts without success: A limit on the number of times that the command will
attempt to reach the target without success has been defined. This prevents the program from
oscillating between a several exposures.
Print this page
Basic Digital Acquire - Dialog Box Options
Destination Image
Specifies the destination for the acquired image. You can add to or overwrite an existing image or stack.
You can also specify a new image.
Acquire Digital Camera Settings
Displays the names of the stored settings in the Acquire from Digital Camera dialog box so that you can
select the desired set to be used by the Basic Digital Acquire command. If you want to use whichever set is
currently active in the Acquire from Digital Camera dialog box, rather than selecting a specific set, you can
choose Current. The name of the current set will then be displayed below the Acquire Digital Camera
Settings list and will be updated if you change it with Acquire from Digital Camera. (The Current option is
useful for creating a journal that performs an autoexposure using the current settings.)
Last Exposure/Next Exposure
If you select Calculate Exposure and Update Settings, "Last Exposure" will be displayed, along with the
maximum pixel value of the last exposure. The duration of the last exposure will be displayed in its text box.
If you leave the Calculate Exposure and Update Settings check box cleared, or if you change the duration
value for the text box (which disables Calculated Exposure and Update Settings), the Last Exposure label
will change to "Next Exposure." The duration value will then be used for the next exposure.
Configure Region
Opens the Configure Regions dialog box which configures regions for use with various sets of acquisition
settings.
Calculate Exposure and Update Settings
This option causes Acquire to calculate the length of time necessary for a proper exposure and acquire an
image for this exposure. If Acquire is successful in reaching the target intensities, the exposure time for the
setting is set to the exposure time used in the automatic exposure.
Acquire
Performs an exposure. If you select Calculate Exposure and Update Settings, multiple exposures will be
performed until the program produces an exposure that satisfies the criteria set using the Auto Exposure
options.
More >>
Expands the dialog box to include the auto exposure options in addition to the Configure Log and Acquisition
Settings buttons.
Less <<
Condenses the dialog box.
Target Intensity % Of (autoexposure option)
Specifies a maximum gray value for the image and two text boxes. The first text box allows you to specify
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the percentage of the maximum gray value that you want the brightest pixel in the image to equal. Changing
this value will update the second text box, which selects the actual value that you want to use as the
brightest pixel. You can change either the first or second text box. The command will adjust the other value
for you.
Initial Exposure Duration (autoexposure option)
Specifies the length of the first exposure attempt when you select Calculate Exposure and Update Settings.
Duration of Exposure: Maximum and Min (autoexposure option)
Specifies the longest and shortest durations to use for calculated automatic exposures before issuing a
warning.
Region Size for Testing Noisy Pixel (autoexposure option)
Because bad pixels in a camera can give falsely high values, MetaMorph tests each pixel against its
neighbors to ensure that the values are consistent when determining the brightest pixel in the image. This
option specifies the region size around the brightest pixel used to test it against its neighbor's values. If the
region around the brightest pixel is not at least 80% of the brightest pixel, it will be determined that the
brightest pixel is giving a false value due to some type of noise. In such cases, the program then tests the
next brightest pixels in order until it finds one that passes this test. That pixel is used for the calculations.
Acquisition Settings
Opens the Define Acquisition Settings dialog box.
Configure Log (for logging exposure calculations)
Opens the Configure Log dialog box so that you can select the parameters to be logged to the log file.
Close
Closes the dialog box.
Set BNC Output Trigger (Device Menu)
Configures the signal state for the BNC output from a Princeton Instruments PentaMAX
camera.
Drop-in: PIBNC
Use this command to set the BNC output trigger state for the PentaMAX camera. An example of such an
output might be that to a Sutter DG4 filter wheel controller. This configuration step will only need to be
performed once at the beginning of an acquisition session.
Note: You must complete the following steps before using this command:
• Close MetaMorph and open the Meta Imaging Series Administrator program.
• Load the video camera driver using the Configure Acquisition command.
• Close the Meta Imaging Series Administrator program and open MetaMorph.
• Select the camera with the Select Camera/Board command (Acquire menu).
Setting the BNC Output Trigger State
To set the signal state for the BNC output from a PentaMAX camera, use the following procedure:
Step
Action
1
From the Acquire menu, choose Set BNC
Output Trigger. The Set BNC Output Trigger
dialog box will appear.
2
Select the trigger state from the BNC Output
Trigger list. Your selection will depend on
your hardware configuration. (See the user's
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manual for your camera for more details.)
3
Choose Close.
Set BNC Output Trigger - Dialog Box Options
BNC Output Trigger
Specifies the trigger state used in BNC output of the camera: Not Scan, Cleaning, Shutter, Not Ready to
Accept Sync, Not Frame Transfer Shift, Logic 0, Logic 1, or Reserved. Your selection will depend on your
hardware configuration. (For details, consult the user's manual for your camera.)
Close
Closes the dialog box.
Acquire with Frame Transfer Camera (Acquire
Menu)
Controls the sequencing and acquisition of images from a frame transfer camera.
Drop-in: FRAME
This command controls the sequencing and acquisition from a Frame Transfer camera through the use
of scripts. A script is a sequence of acquisition and device control commands. You can define up to four
scripts of commands. Once defined, you can then acquire images from the frame transfer camera by
running the desired script. You can select the script commands to be added and the order of the script
commands for each script. You can add the same script command more than once to a script. Each time
you add a command to a script, its parameters will appear at the bottom of the dialog box, where they
can be configured.
You can run a script by choosing the appropriate Acquisition Scripts button or you can record the script
to a journal and then run it from the journal. The journal will only record the button to be used, not the
actual script commands.
Sample Script 1
Sample Script 2
Because this command provides a great deal of flexibility in the order of the script commands, you
should check your scripts carefully to make sure that the commands are in a sensible order.
Note: You can also use the Acquire from Digital Camera or Basic Digital Acquire commands. These
commands automatically perform the exposure, shift, transfer, and copy to image window. However, when
acquiring multiple images as rapidly as possible, the Acquire with Frame Transfer Camera command will
perform faster.
Acquiring Images with a Frame Transfer Camera
To acquire images with a frame transfer camera, use the following procedure:
Step
Action
1
From the Acquire menu, choose Acquire with
Frame Transfer Camera. The Acquire with
Frame Transfer Camera dialog box will
appear. Choose More >> to expand the
dialog box so that you can access the script
definition options.
2
Select the script that you want to define from
the Script # list.
3
Select the first script command you want to
add to the script from the Acquisition
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Commands list. Then choose Add.
OR
Double-click the script command name in the
Acquisition Commands list.
The script command will appear in the script
list box and its parameter setting options will
appear in the bottom of the dialog box.
(Refer to Acquire with Frame Transfer
Camera - Dialog Box Options for more
information.)
4
Select the desired parameter settings for the
script command.
5
Repeat Steps 3 and 4 for each script
command you want to add. The command
will be added above the currently highlighted
item in the script list box. Therefore, if you
want to add an command to the end of the
script, highlight ***End of Script*** before you
choose Add.
6
Type the name of the script in the Script
Name text box and press the [TAB] key. The
name will appear in the Script # list and on
the Acquisition Script button.
7
Repeat Steps 2 - 6 for each script that you
want to define. You can then choose
<< Less to condense the dialog box, if
desired.
8
The image selectors are used to define the
image windows used by the Copy to Image
Window commands in your script. Select the
desired image for each. You can overwrite or
add to the existing image or you can place
the results in a new image window.
9
To run a particular script, choose its
Acquisition Scripts button.
Acquire with Frame Transfer Camera - Dialog Box Options
Acquisition Scripts
Runs the script assigned to its button. You can define up to four scripts.
Image #1, Image #2, Image #3, and Image #4
These image selectors are used to hold images from the four frames that can be acquired and copied to
image windows. The image windows are assigned when you define the Copy to Image Window parameters.
You can overwrite the existing image or place the results in a new image window. Or you can add the image
as a plane to an existing image or stack.
More >>
Expands the dialog box.
Less <<
Condenses the dialog box.
Close
Closes the dialog box.
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Acquisition Commands
Lists the acquisition commands available for scripts.
Script #
Specifies the script that is currently being defined in the script list box.
Script List Box
Lists the script commands that have been defined and added to the script selected from the Script # list. The
parameters for the currently selected command will appear at the bottom of the dialog box.
Add
Adds the currently selected command in the Acquisition Command list to the script list box.
Remove
Removes the currently selected command from the Script List Box.
Clear
Clears all commands from the currently selected script and returns it to a state of "<Undefined>."
Script Name
Specifies the name of the current script and displays that name in the Script # list and the corresponding
Acquisition Scripts button.
Parameters
This option group is used to configure the individual Acquisition Commands. The options that appear here
will be determined by the script command that is currently selected and by your hardware set-up.
Acquisition Commands for Acquire with Frame Transfer Camera
Set Exposure
Sets the Exposure Time of the camera in milliseconds, seconds, or minutes.
Set Illumination
Selects the illumination setting as defined in the Configure Illumination dialog box.
Run Journal
Allows you to run any journal.
Expose
Exposes the unmasked portion of the camera's chip. The video driver determines the number of frames that
are available. For example, the Princeton Instruments Video Driver (PI.vin) has storage for four frames. Use
the Frame slider to select the number of frames you want to acquire.
The camera will be exposed for the exposure time set by the Set Exposure command (if in the script) or, if
did not select Set Exposure, the exposure last set in the Acquire from Digital Camera command will be used.
When exposed, the image will be shifted under the camera's mask. If Expose is used again or Transfer is
used, the image that is currently under the mask will be read out of the chip and into the appropriate frame
memory in the camera driver. Therefore, you must perform a Transfer command after the last Expose
command. See Example
Transfer
Transfers the area of the chip under the camera's mask to the host computer. You must use this command
after the last Expose command. See Example
Copy to Image Window
Copies image data from the host computer's video driver to one of the image windows. You can specify
which frame is to be copied and the window its image is to be placed in from one of the four image selectors.
There may be fewer image selectors than there are frames on the video driver.
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A frame transfer camera has a factory-installed mask over half of its imaging
sensor. When an image is acquired, it is quickly transferred to the side of the sensor
behind the mask. A new image can then be acquired. This setup makes it possible
to perform high speed acquisition of multiple images.
To capture two images, you would write a script that used Expose 1,
followed by Expose 2, followed by Transfer. The first Expose will capture
an image and shift it underneath the camera's mask. That image will be
"tagged" to go into frame 1 of the video driver's memory. The second Expose
will cause the image already under the mask to go into the desired frame buffer
(in this case, frame 1) and will capture a second image and shift it underneath
the mask. The second image is now "tagged" to go into frame 2. The Transfer
command will force the second image, which is still under the mask, to be read
into its frame buffer.
Sample Script 1
One typical operation is to acquire two wavelengths. Available
hardware for this might consist of the frame transfer camera,
a shutter on the light source, and a filter wheel. An acquisition
script would look like this:
Set Exposure
(100
milliseconds)
Set Wavelength
(wavelength
#1)
Set Shutter
(open)
Expose
(frame 1)
Set Shutter
(closed)
Set Exposure
(500
milliseconds)
Set Wavelength
(wavelength
#2)
Set Shutter
(open)
Expose
(frame 2)
Set Shutter
(closed)
Transfer
Copy to Image
Window
(frame 1,
image 1)
Copy to Image
Window
(frame 2,
image 2)
Sample Script 2
If you wanted to acquire three wavelengths, you could use the
following acquisition script:
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Set Exposure
(100
milliseconds)
Set Wavelength
(wavelength
#1)
Set Shutter
(open)
Expose
(frame 1)
Set Shutter
(closed)
Set Wavelength
(wavelength
#2)
Set Shutter
(open)
Expose
(frame 2)
Set Shutter
(closed)
Set Exposure
(500
milliseconds)
Set Wavelength
(wavelength
#3)
Set Shutter
(open)
Expose
(frame 3)
Set Shutter
(closed)
Transfer
Copy to Image
Window
(frame 1,
image 1)
Copy to Image
Window
(frame 2,
image 2)
Copy to Image
Window
(frame 3,
image 3)
Twain Configure (Acquire Menu)
Selects a TWAIN-compliant device for image acquisition and specifies whether to use the
device's user interface.
Drop-in: TWAINCFG
Use this command to select a different TWAIN device and user interface mode (interactive or noninteractive) for image acquisition without the need for exiting MetaMorph to use the external Meta
Imaging Series Administrator program. The Configure Twain Driver dialog box that this command
displays is the same as that used in the Configure Acquisition program.
Note: You must complete the following steps before using this command:
• Close MetaMorph and open the Meta Imaging Series Administrator program.
• Load the TWAIN driver using the Configure Acquisition command.
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• Close the Meta Imaging Series Administrator program and open MetaMorph.
• Select the TWAIN driver with the Select Camera/Board command (Acquire menu).
The dialog box that this command displays provides a list of the TWAIN-compliant devices that you have
installed on your system. This list will reflect the TWAIN device files that reside on your system,
regardless of whether the equipment is still actually connected. Examples of TWAIN-compliant devices
include certain CCDs, video cameras, and flat-bed scanners.
A typical use of this command is to select the Show Device User Interface check box so that the
acquisition options for the TWAIN device will appear before each acquisition. Most TWAIN devices can
save the acquisition settings from their respective device user interfaces. You can use this feature to
fine-tune your acquisition settings, save the settings, and then clear the Show Device User Interface
check box in this dialog box and proceed to acquire your images in non-interactive mode, typically with
the use of a journal.
Configuring Use of a TWAIN-Compliant Device
To configure use of a TWAIN device for image acquisition, use the following procedure:
Step
Action
1
From the Acquire menu, choose Twain
Configure. The Configure Twain Driver dialog
box will appear.
2
Select the TWAIN device you want to use
from the Installed Devices table.
3
Select the Show Device User Interface check
box so that a check mark appears in it. Then
choose OK. The Configure Twain Driver
dialog box will close.
4
From the Acquire menu, choose Acquire
Image. The Acquire Image dialog box will
appear. The image selector and the Acquire
Image command button will be the only
options that are valid for use with the TWAIN
device.
5
Choose Acquire Image. The user interface
dialog box for your selected TWAINcompliant device will appear.
6
Make any adjustments to the acquisition
settings that are necessary, and acquire and
transfer some images through the use of the
device's user interface.
7
If you want to acquire images using the
device's user interface, you are done. Simply
proceed to acquire your experimental images
using the user interface.
OR
If you want to acquire images automatically
in non-interactive mode, choose Twain
Configure again from the Devices menu. The
Configure Twain Driver dialog box will
reappear.
8
Clear the Show Device User Interface check
box. Then choose OK.
9
If desired, create a journal for image
acquisition that uses the Acquire Image
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journal function.
10
When you are ready to acquire your images
automatically, run the image acquisition
journal you created in Step 9, or choose
Acquire Image from the Acquire Image dialog
box.
Twain Configure - Dialog Box Options
Installed Devices
Lists the TWAIN-compliant devices (EXAMPLES: FlashPoint video acquisition board, scanners, etc.) that
you have installed on your system. Select the device that you want to use from this list before acquiring
images with the TWAIN device.
Show Device User Interface
Selecting this check box will configure your system to display the TWAIN device's interface when you
choose the acquisition command. You should acquire an image in this fashion at least once to adjust your
image acquisition settings before you subsequently acquire images in non-interactive mode with this check
box cleared.
OK
Accepts your selection of a TWAIN-compliant device from the Installed Devices table and closes the
Configure Twain Driver dialog box.
Cancel
Cancels any changes you have made to the settings in the Configure Twain Driver dialog box and closes the
dialog box.
Acquire from Spot Camera (Acquire Menu)
Acquires 24-bit true color images or stacks of 12-bit single-channel monochrome images
with the Spot camera. Configures the exposure balance between the red, green, and blue
acquisition channels.
Drop-in: SPOTCAM
Use this command to configure acquisition from the Spot camera and to acquire images. You can
acquire images (1) as a single-plane, 24-bit color image consisting of image data from one, two, or all
three color channels, (2) as a single-plane, single-color 12-bit image, or (3) as a two- or three-plane
stack of single-channel, single-color (red, green, and/or blue) 12-bit images.
Note: To use the Spot camera to acquire images with MetaMorph, you must first load the
Spot Camera driver Using the Meta Imaging Series Administrator’s Configure Acquisition
command and select it with the Select Camera/Board command (Acquire menu). You
must also install the SPOTCAM drop-in for this command to be available. The DIGITAL4
drop-in commands (Acquire from Digital Camera, Basic Digital Acquire) and other
commands that rely on them (for example, Acquire Multiple Wavelengths) will be
unavailable unless you select at least one of the three 12-bit color channels from the
Acquire from Spot Camera dialog box.
Note: The Spot camera driver automatically bins at 2x2 to increase the frame rate. This
means that the live image window is half width and half height. However, the Region of
Interest (ROI) used by the live image is the same as the full chip when you have Full Chip
selected as the Camera Area.
Depending on your camera model, you can acquire single-plane images using the Acquire 24-Bit Image
command or the Acquire 12-Bit Image command. You can use the Acquire 24-Bit Image command for
three-color or single-color images. Single-plane images will always be acquired as 24-bit images,
regardless of the number of color channels you activate. For example, if you select just the red channel
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for acquisition, the brightest possible pixel will have a color value of 255,0,0.
As an alternative, you can acquire 12-bit images, using the Acquire 12-Bit Image command, either as a
single plane or as a stack of planes. This mode of acquisition relies on the Acquire 12-Bit Image/Acquire
12-Bit Stack command button. When you select a single color channel for acquisition, the command
button will read "Acquire 12-Bit Image." If you select two or three color channels for acquisition, the
button title will change to "Acquire 12-Bit Stack." If you are conducting fluorescence imaging, it may not
be necessary to acquire a 24-bit color image--such images are limited to 255 intensity levels per color
channel. You can select a single channel through which to acquire a 12-bit image, and in this way use
the full dynamic range of the camera (4096 intensity levels).
QUICK TIP: If your image moves between acquisitions from the various color channels, you can use the
Color Align drop-in command (Display menu) to align the component colors in a 24-bit color image. If
you are acquiring images in a stack, you can use the Align Stack command (Stack menu).
Configuration of acquisition time is a two-stage process. The first step is to compute the relative ratio
between the exposures of the red, green, and blue channels. This is performed by choosing the
Compute White Balance command button while your Exposure Settings are in Auto-Exposure mode.
Your image should contain at least some white regions for this to be effective. Once the white balance
has been computed, you will need to compute the actual exposure times for the three color channels.
Exposure time is determined automatically by choosing the Compute /Gain command button while your
Exposure Settings are in User-Defined mode. Exposure time selection is an iterative process that uses
the ratios which you just obtained from the white balance computation.
You can also use this command to focus the image while acquiring live video from the Spot camera. The
Focus command is used to display and update the image quickly from the camera and display it on the
video monitor. This allows you to see an image while focusing on your specimen. It is important to use
the Focus command because what can be seen through the microscope's eyepiece and what the
camera acquires are not always the same. Acquisition configurations such as binning settings will be
disabled while you are focusing.
Acquiring Images with a Spot Camera
Acquiring Images with a Spot Camera - Main Dialog
Acquiring Images with a Spot Camera - Acq Setup Tab
Acquiring Images with a Spot Camera - Live Setup Tab
Acquiring Images with a Spot Camera - Display Tab
Acquiring Images with a Spot Camera - Process Tab
Computing Exposure Times (Spot Camera)
Acquiring Images with a Spot Camera - Main Dialog
To acquire images using the Spot Camera, complete the following procedure:
Step
Action
1
From the Acquire Menu, choose Acquire
from SPOT. The Acquire from SPOT dialog
box opens.
2
If you have a settings file with a group of
previously saved settings that you want to
load, click Load Settings. The Load Spot
Camera State dialog box opens. Select the
settings file that you want to use, and click
Open.
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3
Set the Acquisition region that you want to
use to acquire images by clicking on the
appropriate area.
• To use the entire chip, Click Full Chip.
• To use the center quadrant of the chip,
click Center Quad.
4
• To use a custom area, first acquire an
image using Full Chip or Center Quad,
then use an appropriate region tool to
select a custom area. After you define an
active region, click Use Active Region.
If the Acquire from SPOT Camera Dialog
box is not fully expanded, click More>> to
expand the dialog box.
5
Click the Acq Setup tab to make acquisition
settings.
6
Click the Live Setup tab to make live
acquisition settings.
7
Click the Display tab to make display
settings.
8
Click the Process tab to make process
settings.
9
Click Show Live to continuously acquire
images that will enable you to set or check
the microscope focus.
10
Click Acquire to acquire the type of image for
which the Acquire from SPOT dialog box is
configured.
11
To save your Spot Camera settings, click
Save Settings. The Save Spot Camera State
dialog box opens. Assign a name to the
state file, and click Save.
Acquiring Images with a Spot Camera - Acq Setup Tab
To configure the Acq Setup tab, complete the following procedure:
Step
Action
1
Click the Acq Setup tab. The Acq Setup
options move to the front.
2
In the Image Bit Depth box, select the image
bit depth that you need. Color 24BPP yields
24-bit color images that consist of three
separate exposures that are combined by
the SPOT Camera program. Monochrome
12BPP are 12-bit images that can be
acquired separately and used as individual
monochrome images or can be acquired into
a stack through color filters and combined
into a single, high quality color image.
3
In the Exp Mode box, select the exposure
mode that you want to use.
4
In the Gain box, type or select the camera
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gain that you want to apply to your image.
5
In the Binning box, type or select the level of
binning that you want to apply.
6
If you selected Auto for the exposure mode,
in the Exp Adjust box, type or select the
amount of exposure adjustment that you
want to apply.
7
If you selected Auto for the exposure mode,
in the Image Type box select the image type
for the image you are acquiring. For
brightfield images, select Brightfield, for
darkfield, select Darkfield, For fluorescence
images, select Fluorescence.
8
If you selected Auto for the exposure mode
and you are acquiring a color image, in the
White Balance area you need to set the
color order and compute the white balance.
Click the arrow in the Color Order box, and
choose the appropriate color order. One
reason for selecting a different color order
might be that in your experiment, a particular
color fades faster than the others.
9
To compute the white balance for a color
image, ensure that the Red, Green, and Blue
check boxes are checked, then click
Compute White Balance. The Compute
White balance Values information box opens
and reports the results. If the white balance
values are acceptable, click Yes, otherwise
click No.
10
If you selected Manual for the Exp. Mode
and you are acquiring a color image, a
Compute Exp/Gain button will be in place of
the Compute White Balance button, and the
area will be called Exposure.
11
To compute the exposure, ensure that the
Red, Green, and Blue checkboxes are
checked, set the color order that you want to
use, then click Compute Exp/Gain. A
Compute Exposure Time and Gain
information box is displayed. If the exposure
times are acceptable, click Yes, otherwise
click No.
12
In the Illumination box, click the down arrow,
and select the illumination setting that you
want to use.
Acquiring Images with a Spot Camera - Live Setup Tab
Note: Settings you make on the Live Setup tab affect only the appearance of the Live
(continuously acquired) image. This image is for aligning the position of your specimen,
selecting an appropriate objective, or setting the microscope focus. Settings made on this
tab do not affect your acquired image result.
To configure the Live Setup tab, complete the following procedure:
Step
Action
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1
Click the Live Setup tab. The Live Setup
options move to the front.
2
Click Compute exposure and gain on live
startup if you want these values computed
each time you click Show Live.
3
If Compute exposure and gain on live startup
is checked, move the Quality/Speed slider to
an appropriate setting.
4
Click Show Live. If Compute exposure and
gain on live startup is not checked, type or
select an appropriate exposure value in the
Exp. Time box.
5
Click Show Live. In the Gamma box, type or
select an appropriate gamma setting to
improve the appearance of the image.
6
Click Show Live. In the Brightness box, type
or select an appropriate brightness setting to
improve the appearance of the image.
7
In the Binning box set an appropriate binning
value if you need to reduce the live mode
exposure time.
8
In Camera area for live mode, select the
appropriate acquisition region.
• To use the entire chip, Click Full Chip.
• To use the center quadrant of the chip,
click Center Quad.
9
• To use a custom area, first acquire an
image using Full Chip or Center Quad,
then use an appropriate region tool to
select a custom area and click Use
Active Region. Then, select Same as
Acquire Area on the Live Setup tab.
In the LC Filter Position box, click the Liquid
Crystal filter that you want to use for live
mode. Choose Red, Green, or Blue. If your
camera is the SPOT RT, you can also
choose Clear or RGB.
Note: RGB continuously cycles through the
Red, Green, and Blue filters.
Acquiring Images with a Spot Camera - Display Tab
Note: The settings on this tab apply to 12-bit Monochrome images only.
To configure the Display tab, complete the following procedure:
Step
Action
1
Click the Display tab. The Display options
move to the front.
2
In the Monochrome (12 BPP) Image scaling
box, click Autoscale to select or deselect
autoscaling, as appropriate.
Note: If autoscaling is not selected, the
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values you enter into the low and high
settings boxes are high and low limits in bit
density. If autoscaling is selected, the low
and high settings are a percentage of the
overall scale range.
3
In the Lo box, enter a lower scaling limit for
the scaling range.
4
In the Hi box, enter an upper scaling limit for
the scaling range.
Acquiring Images with a Spot Camera - Process Tab
To configure the Process tab, complete the following procedure:
Step
Action
1
Click the Process tab. The Process options
move to the front.
2
Click Chip Defect Correction to automatically
apply the Chip Defect Correction Table to all
images.
3
Click Fluorescence Color Handling to apply
fluorescence color handling to darkfield
images that are fluorescence images.
4
Click Flatfield Correction, then click Get
Flatfield to apply Flatfield correction to the
acquired images.
Flatfield correction has two purposes,
depending on the type of camera used. Use
Flatfield correction with a RT or SPOT
camera to adjust images with uneven
intensity or coloration in illumination, or to
correct for artifacts in the optical system.
OR
Use Flatfield correction with an Insight
camera to correct uneven color density
("halo" effect) in low contrast images.
Note: Flatfield correction using
the SPOT camera refers only to
the correction of uneven
lighting. It should not be
confused with the correction of
optical field flatness
5
In the Gamma Adjustment area, click
Gamma Adjust and type or select a gamma
adjustment value in the Gamma Adjust box.
6
If you checked Fluorescence Color Handling,
click RGB to apply the gamma correction to
the Red, Green, and Blue channels, or click
Luminance to apply the gamma correction to
the Luminance channel.
Computing Exposure Times (Spot Camera)
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To compute the exposure times for image acquisition with the Spot camera, use the following
procedure:
Step
Action
1
Choose the Compute Exp/Gain command
button. The Computing Exposure Time and
Gain dialog box opens.
2
The White Balance option group will display
the current selection of active color channels,
as well as the current white balance ratio
values. If desired, you can alter these
settings before computing the exposure
times.
3
Verify that the Image Type drop-down list
indicates the appropriate selection of image
illumination: Brightfield or Darkfield.
4
If desired, you can adjust the overall
brightness of the acquired images with the
Exp Adjust spin box.
The default value is 1.00. Increasing the
value will yield a brighter image, while
decreasing the value will result in a darker
image.
5
When you are ready, choose Compute.
The Spot Camera Status message box will
appear, displaying messages that indicate
the progress of the procedure. MetaMorph
will perform an iterative process to determine
the optimum exposure times for each color
channel, using the current set of white
balance ratios and exposure adjustment
values, and taking the image type into
account.
7
When the process is complete, the Spot
Camera Status message box will close, and
a second message box will appear, asking
whether you want to save the computed
values. Choose Yes to store the exposure
times.
The exposure times will appear in the
Exposure option group spin boxes of the
Acquire from Spot Camera dialog box.
Acquire with Spot Camera - Dialog Box Options
Acquire from Spot Camera - Main Dialog Box Options
Acquire from SPOT Camera Dialog Box Options - Acq Setup tab
Acquire from SPOT Camera Dialog Box Options - Live Setup tab
Acquire from SPOT Camera Dialog Box Options - Display tab
Acquire from SPOT Camera Dialog Box Options - Process tab
Computing Exposure Times (Spot Camera) - Dialog Box Options
Acquire from Spot Camera - Main Dialog Box Options
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Acquire 12-Bit Image / Acquire 24-Bit Image / Acquire 12-Bit Stack
Acquires a single-plane, single-channel 12-bit image, a single-plane 24-bit image, or a two-, three-, or fourplane 12-bit image stack (depending on which color channels are selected), where each plane corresponds
to a color channel. Images are acquired using the current settings. By default, the image is acquired into an
image window with the name "acquired." Click the Image Name button to rename the file.
Image Name (Destination)
Specifies the image name and destination to apply to the image. You can add to or overwrite an existing
image or stack, or you can specify a new image.
Status
Indicates the Acquisition status.
Show Live/F2: Stop Live
Acquires a continuously updating image into an image window. Use this command to interactively set the
microscope's focus or color balance. Click F2: Stop Focus to discontinue live image capture.
Camera Area
Specifies the active region.
Full Chip – Specifies that the entire chip be used to acquire the image.
Center chip – Specifies that only the center portion of the chip be used to acquire the image.
MetaMorph creates and centers a region that is the size of one quadrant of the chip.
Use Active Region – Specifies that the active region is used to acquire the image.
Load Settings
Opens the Load SPOT Camera State dialog box to enable you to load a saved settings file.
Save Settings
Opens the Save SPOT Camera State dialog box to enable you to save your current settings to a file.
Close
Closes the Acquire from SPOT RT or SPOT Camera dialog box.
More>>/Less<<
Expands the Acquire from SPOT RT Camera dialog box to full size; if expanded, Less<< sets the dialog box
to its minimized size.
Acquire from SPOT Camera Dialog Box Options - Acq Setup tab
Image Bit Depth
Selects either Color (24BPP) or Monochrome (12BPP)
Exp Mode
Selects an acquisition mode: Manual or Auto:
Manual mode, acquisition will proceed based on the specified exposure times for each color
channel. The Exp Adjust option is unavailable in this mode.
Auto mode, acquisition will be an iterative process that determines the optimum exposure for each
image. This mode is used for computing white balance and for acquisition of images in which the
speed of acquisition is not a concern.
Gain
Specifies a conversion factor for transforming acquired 12-bit source images to an 8-bit range. Increasing
the Gain value will yield a brighter image. You may need to adjust the Gain value if the brightness of the
specimen changes or if the exposure times that are computed are too long to be practical. This option is
available only in Manual exposure mode.
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Binning
Selects a number of pixels in the horizontal and vertical direction to be binned during acquisition. This will
increase the speed of acquisition and conserve memory.
For example, if you select 2, a 2 x 2 binning region will be used. This will nearly quadruple acquisition speed
while consuming just 25% of the memory space that an unbinned image would use.
Note: As binning is increased, image resolution decreases.
Exp Adjust
Adjusts the calculated exposure times. For example, if you select 1.25 and MetaMorph has calculated an
exposure time of 100 ms, the returned value will be 125 ms. This option will be available in this dialog box
only in Auto exposure mode.
Image Type
Selects the image's type of illumination: Brightfield, Darkfield, or Fluorescence. This option is available only
in Auto exposure mode.
Exposure (Monochrome mode only)
Use Filter
Enables use of color filters for capturing separate monochrome (gray scale) images that can be
recombined later into a color image.
Red – Controls the image exposure time through red filtration. If Manual exposure mode
is selected, sets the exposure time in the red filter box.
Green – Controls the image exposure time through green filtration. If Manual exposure
mode is selected, sets the exposure time in the green filter box.
Blue – Controls the image exposure time through blue filtration. If Manual exposure mode
is selected, sets the exposure time in the blue filter box.
Clear – Controls the image exposure time with no filter in place. If Manual exposure mode
is selected, sets the exposure time in the clear box (RT only).
B/W
Specifies image capture in monochrome (gray scale) only. No color filters are used for this option.
White Balance (Color mode only)
Provides the controls for setting the white balance for Color (24-bit) images.
Note: To change these values while in Live mode, ensure that Exp. Mode is set to Auto
and Compute exposure and gain on live startup is enabled in the Live Setup tab.
Red
Enables the red color channel. In Manual mode, the spin box specifies an exposure time or adjusts
the computed exposure time. In Auto mode, the spin box specifies a white balance value or adjusts
the computed one.
Green
Enables the green color channel. In Manual mode, the spin box specifies an exposure time or
adjusts the computed exposure time. In Auto-Exposure mode, the spin box specifies a white
balance value or adjusts the computed one.
Blue
Enables the blue color channel. In Manual mode, the spin box specifies an exposure time or
adjusts the computed exposure time. In Auto mode, the spin box specifies a white balance value or
adjusts the computed one.
Color Order
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Selects the order in which the camera's color channels will be sampled during image acquisition:
RGB, RBG, etc.
Compute White Balance
Calculates the relative exposure ratios between the color channels that are necessary to achieve
the same intensity at each channel. This command button appears when the system is in AutoExposure mode, and becomes the Compute Exposure Times button when you switch to UserDefined mode. Be sure to move the specimen to a blank area of the slide before you compute the
white balance.
Compute Exp/gain
Opens the Computing Exposure Time and Gain dialog box under the following conditions:
ƒ
The Exp. Mode is set to Manual
ƒ
The camera is acquiring 24-bit color images or 12-bit Red, Green, Blue, and/or Clear
stacks.
Use the Computing Exposure Times and Gain dialog box to turn on/off colors and adjust the White
Balance, Image Type, and Exp Adjust values to calculate the exposure times and gain values for
the active color channels.
If camera is in monochrome mode, The Compute Exp/Gain command will directly compute
exposure time and gain values for image acquisition without opening the Compute Exposure Time
and Gain dialog box.
Use ‘Acquire’ as default when channel is 12bit
Enables the Acquire drop-in to be used as well as the Acquire from SPOT drop-in when acquiring images
with a SPOT camera. When this is selected the settings from the Acquire dialog are used by default when
acquiring images using other commands in MetaMorph.
Illumination
Selects the desired illumination setting from among those that are available. To use the active setting, select
[Current Shutter]. If you are not using an illumination device, select "[None]."
Acquire from SPOT Camera Dialog Box Options - Live Setup tab
Compute exposure and gain on live startup
Automatically calculates the correct exposure and gain values whenever you click Show Live. Uncheck this
box to manually set exposure and gain settings for Live mode.
Recalc Exposure Time
Recalculates exposure time and gain in Live mode.
Quality/Speed
Enables you to achieve an ideal balance between Quality and Speed when continuously updating "live"
images. The better the quality, the slower the speed; conversely, the faster the speed, the lesser the quality.
This feature is disabled if Compute exposure and gain on live startup is selected.
Gamma
Sets the image gamma value for live-mode image viewing.
Binning
Sets the camera pixel binning value for live-mode image viewing.
Note: The binning value can be changed while in Live mode.
Live Gain
Sets the gain for the live-mode viewing. This feature is disabled if Compute exposure and gain on live
startup is selected.
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Exp Time
Sets the exposure time (in milliseconds) for live images. This feature is disabled if Compute exposure and
gain on live startup is selected.
Camera area for live mode
Provides controls to designate the live-mode image viewing area.
Full Chip
Assigns the entire chip as the live-mode image viewing area.
Center Quad
Assigns the chip's center quadrant as the live-mode image viewing area.
Same as Acquire Area
Assigns the selected active region as the live-mode image viewing area.
LC Filter Position
Selects the filter to have in place for live-mode image viewing.
Note: If the Image Bit Depth is set to Color (24BPP) in the Acq Setup tab, you can
change the LC Filter Position while in Live mode and it will update.
Red
Selects the red filter for live-mode image viewing.
Green
Selects the green filter for live-mode image viewing.
Blue
Selects the blue filter for live-mode image viewing.
Clear
Selects no filters to be in place for live-mode image viewing.
RGB
Selects all filters to be in place for live-mode image viewing.
Acquire from SPOT Camera Dialog Box Options - Display tab
Monochrome (12BPP) Image Scaling
Provides controls to set image scaling for monochrome images.
Low
Selects the gray value to be used as the lower limit of the range being autoscaled.
High
Selects the gray value to be used as the upper limit of the range being autoscaled.
Autoscale
Enables the autoscale function. When active, autoscaling calculates the correct exposure limited by high
and low limits specified as a percentage of the total range. When autoscale is off, Low and High are fixed
scaling values on a linear scale.
Acquire from SPOT Camera Dialog Box Options - Process tab
Black Level Subtraction (12 BBP)
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Turns off the automatic Black Level Subtraction function. This function can be deactivated only when
capturing 12 BBP (12-bit mono) images. For other capture formats, this option is always on. When active,
this function ensures that any part of the image that is attributed to the image background (such as noise or
minor pixel level differences) will be black (level 0). However, image accuracy might be sacrificed when
Black Level Subtraction is on. To ensure greater image accuracy, turn off Black Level Subtraction.
Chip Defect Correction
When this check box is selected, MetaMorph will correct for chip defects automatically, based on the
settings in the ChipInfo.dat file. However, if you have not copied the ChipInfo.dat file correctly to the Spot
camera's VInput folder and this check box has been selected, your image acquisition may be considerably
slowed.
Flatfield Correction
Applies Flatfield correction to the acquired image. Click Get Flatfield, then acquire an image to apply the
Flatfield correction.
Flatfield correction has two purposes, depending on the type of camera used. Use Flatfield correction with a
RT or SPOT camera to adjust images with uneven intensity or coloration in illumination, or to correct for
artifacts in the optical system.
OR
Use Flatfield correction with an Insight camera to correct uneven color density ("halo" effect) in low contrast
images.
Get Flatfield
Click this button when the Flatfield Correction box is checked, then acquire an image to apply the Flatfield
correction.
Color Enhancement
Applies automatic color enhancement of acquired image.
Note: Color enhancement will not be applied to live images.
Fluorescence Color Handling
Applies compensations to the settings to optimize image capture for fluorescence color images. The Color
Enhancement checkbox must be checked for this option to be enabled.
Gamma Adjustment
Provides controls for enabling and setting a Gamma correction value for monochrome images or RGB or
Luminance to color images. Typical gamma values range between 1.3 and 1.7.
Gamma Adjust
Enables and sets the value for the gamma correction curve to be applied to images.
RGB
Check this button to apply the gamma correction curve separately to the red, green, and blue pixel
values. Use this option for monochrome images
Luminance
Check this button to apply the gamma correction curve to HSL (hue, saturation, and luminance)
values. Use this option for all color images except fluorescence images.
Computing Exposure Times (Spot Camera) - Dialog Box Options
Red
The check box enables use of the red channel during calculation of exposure time. The spin box indicates
the calculated white balance value, which you can adjust as desired.
Green
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The check box enables use of the green channel during calculation of exposure time. The spin box indicates
the calculated white balance value, which you can adjust as desired.
Blue
The check box enables use of the blue channel during calculation of exposure time. The spin box indicates
the calculated white balance value, which you can adjust as desired.
Image Type
Selects the image's type of illumination: Brightfield or Darkfield.
Exp Adjust
Adjusts the calculated exposure times. For example, if you select 1.25 and MetaMorph calculates an
exposure time of 100 ms, the returned value will be 125 ms.
Compute
Begins calculating the exposure time for each active channel, based on the current set of white balance
ratios and exposure adjustment values, and taking the image type into account. The Spot Camera Status
dialog box will appear, and will close automatically when acquisition is complete.
Abort
Terminates the exposure time calculation process.
Acquire from Flashbus (Acquire Menu)
Acquires and displays images from a video camera connected to the Flashbus MV video
acquisition board. Configures the acquisition region, frame integration and averaging, and
image display.
Drop-in: FLASHBUS
Use this command to configure acquisition with the Flashbus MV video acquisition board, and to display
"live video" or acquire single-frame images (NTSC = 33 ms/frame exposure duration, PAL = 40
ms/frame) with a video camera. This easy-to-use interface enables integration and averaging of both the
"live" display and of acquired frames.
The Flashbus frame grabber board supports the use of RS-170, CCIR, RGB, Composite, and S-Video
input signals. Acquisition will be performed using the channel that you select with the Set Video Channel
command (Acquire menu). If you select the NTSC (US) format when you configure the Flashbus driver in
the Meta Imaging Series Administrator, you can select an RS-170, RGB, Composite, or S-Video input
channel from the Set Live Video Channel dialog box. If you select the PAL (EC) format when you
configure the driver, you can select CCIR, RGB, Composite, or S-Video.
The Flashbus frame grabber and video driver also support the use of a VCR as the video input device. If
you are using a VCR for transmission of images to the Flashbus board, it is recommended that you
configure the Flashbus driver for optimum synchronization, using the Configure Acquisition command
(see below).
Note: You must complete the following steps before using this command:
• Close MetaMorph and open the Meta Imaging Series Administrator program.
• Load the video camera driver using the Configure Acquisition command.
• Close the Meta Imaging Series Administrator program and open MetaMorph.
• Select the camera with the Select Camera/Board command (Acquire menu).
The Acquire from Flashbus command uses the concept of State files to restore previous settings. This
enables you to configure acquisition settings and save them to a State file that can be loaded the next
time you use the command. There is no limit to the number of State files you can save. This help file
contains procedures for both manually configuring the dialog box or loading a saved State file:
Performing Acquisition with the Flashbus Video Board using Manual Settings
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Performing Acquisition with the Flashbus Video Board Using a State File
The Acquire from Flashbus dialog box settings enable you to reduce or eliminate background image
information that is constant and not part of your experiment image information. This feature incorporates
three settings designed to work together. The Acquire Background button acquires and stores an image
of the background data. When you check the Subtract Background function, this information is
subtracted from the acquired image information to remove any constant background information
associated with the microscope, its objectives, or the illumination sources. If the image intensity is
excessively reduced from background subtraction, type or select a value in the Offset box to restore
image brightness and add a base level of intensity to the image.
Performing Acquisition with the Flashbus Video Board Using a State File
To configure acquisition and acquire images with a camera connected to the Flashbus video
acquisition board using a previously saved State file, use the following procedure:
Step
Action
1
From the Acquire menu, choose Acquire
from Flashbus. The Acquire from Flashbus
dialog box opens.
2
Click Load State to open the Load
Acquisition State dialog box.
3
Click the selections for the settings you want
to load from the state file. The settings that
are not checked will not be loaded. Click
Select All if you want to load all saved
settings.
4
Click Load. The Open dialog box opens.
5
Navigate to the state file (.fbs) you want to
open and click Open. The state file you
selected will load and the Load Acquisition
State dialog box will close.
6
Click the Dest image selector to select a
destination image. To acquire images into a
stack, configure the selector to acquire
images into an image stack.
7
If you need to make adjustments to the
image display, click Start Live to display a
"live" image. (Adjust the microscope focus, if
necessary). Then click Adjustments. The
Video Adjustments dialog box opens.
Depending on the type of signal your video
input device generates, different sets of
adjustment options will be available in the
dialog box.
OR
If you do not need to adjust the image, skip
to Step 11.
8
For all signal types, adjust the Contrast and
Brightness sliders for an optimal image.
If you are using the 8-bit RS-170 signal, you
can select Use Saturation LUT to display the
image with undersaturated pixels (grayscale
value 0 or "below") displayed in blue and
saturated pixels (grayscale value 255 or
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"above") displayed in red.
9
For Composite or S-Video signals, use the
Hue, Saturation, and Gain sliders to adjust
the image display. If you are using a
Composite signal, you can also use the
Sharpness slider.
10
When you have finished adjusting the
display, choose Close to return to the
Acquire from Flashbus dialog box.
Note: To return all video adjustment settings
to their defaults, choose Reset before closing
the Video Adjustments dialog box.
11
To acquire the Active Region, define the
active region for the image area that you
want on an image window using the
Rectangular Region tool, then Click Set
using Active Region.
OR
To acquire the entire image area after you
have already specified the active region as
the acquisition, click Reset to Full Frame.
12
To display a "live" video image at any time,
such as when you want to adjust the focus or
change the visual field, choose Start Live.
OR
To acquire an image, choose Acquire. An
image will be captured and saved using the
name you specified with the Dest image
selector.
13
When you have finished your acquisition
session, choose Close to close the Acquire
from Flashbus dialog box.
Performing Acquisition with the Flashbus Video Board using Manual
Settings
To manually configure acquisition and acquire images with a camera connected to the Flashbus
video acquisition board, use the following procedure:
Step
Action
1
From the Acquire menu, choose Acquire
from Flashbus. The Acquire from Flashbus
dialog box opens.
2
Click the Dest image selector to select a
destination image. To acquire images into a
stack, configure the selector to acquire
images into an image stack.
3
To perform frame averaging, in the Average
box, type or select the number of frames to
average for each image.
Note: If you are performing both integration
and averaging, MetaMorph will first perform
the frame integration and then perform
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averaging on the integrated images.
4
If you used the Configure Acquisition
command to configure the Flashbus driver
for integration and want to integrate your
acquired images, click Enable Integration.
Then, in the Integrate box, type or select the
number of frames to be integrated for each
image.
Note: Integration is supported only by
RS-170, CCIR, and RGB inputs, but not by
Composite or S-Video.
5
Click Acquire Background to store a
background image to be subtracted from
your acquired images.
Note: Ensure that no specimen is in place
and that all illumination and objective settings
are already made before acquiring a
background image.
6
If you need to make adjustments to the
image display, click Start Live to display a
"live" image. (Adjust the microscope focus, if
necessary). Then click Adjustments. The
Video Adjustments dialog box opens.
Depending on the type of signal your video
input device generates, different sets of
adjustment options will be available in the
dialog box.
OR
If you do not need to adjust the image, skip
to Step 10.
7
For all signal types, adjust the Contrast and
Brightness sliders for an optimal image.
If you are using the 8-bit RS-170 signal, you
can select Use Saturation LUT to display the
image with undersaturated pixels (grayscale
value 0 or "below") displayed in blue and
saturated pixels (grayscale value 255 or
"above") displayed in red.
8
For Composite or S-Video signals, use the
Hue, Saturation, and Gain sliders to adjust
the image display. If you are using a
Composite signal, you can also use the
Sharpness slider.
9
When you have finished adjusting the
display, choose Close to return to the
Acquire from Flashbus dialog box.
Note: To return all video adjustment settings
to their defaults, choose Reset before closing
the Video Adjustments dialog box.
10
Click Subtract Background to enable
subtraction of the stored background image.
11
In the Offset box, type or select a base
intensity value to be added to the image
intensity value. Use this setting to
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compensate for image intensity reduced by
the Subtract Background function.
12
Select Continue Live after Acquisition to
leave the Live window open after acquiring
an image.
13
Select a live average method from the Live
Average drop-down list, or select No
Average.
14
If you are using an illumination device, select
its setting from associated from the Illum
drop-down list.
OR
If you are not using an illumination device,
select "[None]."
15
To acquire the Active Region, click Start Live
to open a live image window. Define the
active region for the image area that you
want using the Rectangular Region tool, then
Click Set using Active Region.
Note: You can also press the F2 key to
start and stop live acquisition.
OR
To acquire the entire image area after you
have already specified the active region as
the acquisition, click Reset to Full Frame.
16
To display a "live" video image at any time,
such as when you want to adjust the focus or
change the visual field, choose Start Live.
OR
To acquire an image, choose Acquire. An
image will be captured and saved using the
name you specified with the Dest image
selector.
17
To save the current settings, click Save State
to open the Save Acquisition State dialog
box and save the State file.
18
When you have finished your acquisition
session, choose Close to close the Acquire
from Flashbus dialog box.
Acquire from Flashbus - Dialog Box Options
Acquire from Flashbus
Video Adjustments
Load Acquisition State
Acquire from Flashbus - Dialog Box Options
Dest
Specifies or selects the name of the acquired image and the file location for the new image file. You can
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create a new image file or stack, overwrite the existing image file or stack, or add images to an existing
image stack.
Average
Selects the number of frames (integrated or non-integrated) to be averaged together for each image. This
can be used to reduce image pixel "noise."
Acquire
Acquires a single image, using any integration and averaging settings and the acquisition region you have
selected, and saves it using the name you specified with the Dest image selector.
Enable Integration
Enables integration of images using the number of frames specified in the Integrate box. When you select
the Enable Integration check box, the Integrate spin box will become available. You must configure the
Flashbus driver for integration using the Configure Acquisition command in the Meta Imaging Series
Administrator for the Enable Integration check box to be available.
Note: Integration is supported only by RS-170, CCIR, and RGB inputs, but not by
Composite or S-Video.
Note: If you are configuring the Acquire from Flashbus command from within the Journal
Editor, there is a function in the Builtin Functions Tab called Acquire from FlashBus Adjust FTI that accepts fractional numbers and, when run in a journal, sets a value such
that each subsequent acquisition from the Flashbus has the integration value
incremented by that amount. For example, if you enter 0.50 in the Adjustment factor field
of the Adjust FTI function dialog box and run the journal, each subsequent acquisition
from the FlashBus has the integration value incremented by 0.50 (whether acquiring from
within a journal or in normal mode). Because frame integration requires an integer value,
the value used in the acquisition is truncated to an integer. So, if you start with integration
= 2 and acquire 5 images, the following parameters are used:
Im
ag
e
Integration
Value
Actual
Number of
Frames
Integrated
1
2.0
2
2
2.5
2
3
3.0
3
4
3.5
3
5
4.0
4
Start Live
Acquires continuous "live" images of the preparation at video rate (NTSC = 30 frames/s, PAL = 25 frames/s)
and displays them in the Live Image window.
Note: You can also press the F2 key to start and stop live acquisition.
Integrate
Selects the number of frames to be "added" together for each image. This may be particularly useful if you
are acquiring images under low light conditions.
Adjustments
Opens the Video Adjustments dialog box, from which you can adjust the contrast, brightness, hue,
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saturation, sharpness, and gain of your image display. (Depending on the type of signal your video input
device generates, different sets of adjustment options will be available in the dialog box.)
Subtract Background
Enables background subtraction. Use this function to subtract the image background data captured with the
Acquire Background function from the entire acquired image area. The result is an image absent of all
constant background image information. This option is only enabled if a background has been acquired
using the Acquire Bkgd command.
Offset
If the image resulting from background subtraction is too dark ("less" than gray level 0), this option specifies
a base image intensity value to be added back to the image's values.
Acquire Bkgd
Acquires and stores a background image. With no specimen present, click Acquire Background to acquire a
background image to be stored in memory. When Subtract Background is selected, this image data is
subtracted from the captured specimen image data to remove all information that is not part of the specimen
image.
Continue Live after Acquisition
Leaves the Live image window open after acquiring an image.
Live Average
Determines the method of averaging frames while in Live mode. Valid choices include:
ƒ
ƒ
ƒ
No Average — no averaging is performed.
Jumping Average — Applies average to groups of frames in sequence, such as 0-2, 3-5, and 6-8.
Running Average — Applies average to groups of frames that overlap, such as 0-2, 1-3, and 2-4.
Acquire Region
Contains settings to define the limits of the region to be acquired.
Reset to Full Frame
Sets the acquisition region to include the entire image frame.
Set Using Active Region
Configures image acquisition within the image area defined by an active region of interest. Use the
Rectangular Region Tool, in the Region Tools window, to define the acquisition region in the image
window.
Illum
Selects an Illumination Setting to use when acquiring. If you are not using an illumination device, select
"[None]."
Load State
Opens the Load Acquisition State dialog box. This option enables you to reuse Flashbus dialog box options
that you previously saved to a state file.
Save State
Saves the current Flashbus dialog box settings to a file. Load these settings from the file to reuse them
using the Load State option.
Close
Closes the Acquire from Flashbus dialog box (and the Video Adjustments dialog box, too, if it is open).
Acquire from Flashbus: Load Acquisition State - Dialog Box Options
These checkboxes enable or disable the loading of specific settings from a state file. The Settings listed in
the Load Acquisition State dialog box are configured on the Acquire from Flashbus dialog box. The following
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table lists each setting and where they are set in the Acquire from Flashbus dialog box:
Setting
Fields/Comments
Enable Integration
Enable Integration
You must configure the Flashbus driver
for integration using the Configure
Acquisition command in the Meta Imaging
Series Administrator for the Enable
Integration check box to be available.
Frames to Integrate
Integrate
Enable Integration must be checked for
this option to be valid.
Enable Bkgd
Subtraction
Bkgd Offset
Illumination Setting
Acquisition Region
Subtract Background
Offset
Enable Bkgd Subtraction must be
checked for this option to be valid.
Illum
This loads the region used when the
state file was created.
Continue Live after
Acquisition
Continue Live after Acquisition
Live Average Mode
Live Average
Load
Opens the Load Flashbus state dialog box used to select a saved Flashbus state (.fbs) file.
Select All
Selects all checkboxes.
Clear All
Deselects all checkboxes.
Cancel
Cancels the command and closes the dialog box.
Video Adjustments (Flashbus) - Dialog Box Options
Contrast
Adjusts the contrast (degree of change of the grayscale or color intensity levels) of the image.
Brightness
Adjusts the overall brightness or darkness of the image.
Hue
Adjusts the color in the image. This option is available only for Composite and S-Video inputs.
Saturation
Adjusts the level of color saturation (degree of "dilution" or vividness of the color) in the image. This option is
available only for Composite and S-Video inputs.
Sharpness
Adjusts the sharpness of the display of grayscale transitions, or object edges, in the image. This option is
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available only for Composite inputs.
Gain
Adjusts the level of signal amplification. To some extent, this is equivalent to increasing the contrast and
brightness of the image. This option is available only for Composite and S-Video inputs.
Use Saturation LUT
Displays the image with a look-up table in which undersaturated pixels (grayscale value 0 or "below") are
displayed in blue and saturated pixels (grayscale value 255 or "above") are displayed in red. This option is
applicable only for RS-170 inputs.
Reset
Reverts all settings to their default values. The actual values will depend on the specific input type (RS-170,
CCIR, RGB, Composite, or S-Video).
Close
Closes the Video Adjustments dialog box.
Sum 16-Bit Image (Acquire Menu)
Sums the specified number of images together to create a 16-bit image.
Drop-in: SUM16
Use this command to enhance the contrast and reduce the noise of faint or dim images.
Note: This command can be used only if you are using a Matrox Image Series frame-grabber board and
a compatible RS-170 or CCIR camera.
The images summed together should have a narrow range of gray levels for this command to work well.
For very dim images, you may need to sum together a large number of frames. For this reason, Sum 16Bit Image may not be ideal for fast-moving specimens. To determine how much time is needed to
acquire an image using an RS-170 camera, divide the number of frames by 30 (divide by 25 for a CCIR
camera).
When the image is acquired, you can adjust its contrast using the Scale 16-Bit Image command.
Summing a 16-Bit Image
To sum acquired 8-bit frames into 16-bit images, use the following procedure:
Step
Action
1
From the Acquire menu, choose Sum 16-Bit
Image. The Sum 16-Bit Image dialog box will
appear.
2
Select the desired destination image using
the Destination image selector.
3
Select the desired number of frames to sum
together using Number of Frames.
4
Choose Acquire.
5
Choose Close when you have finished.
Sum 16-Bit Image - Dialog Box Options
Destination
Specifies the destination image for the 16-bit image. You can add to or overwrite an existing 16-bit image or
stack. You can also specify a new 16-bit image.
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Number of Frames
Specifies the number of frames to sum together to create the 16-bit image.
Acquire
Acquires the specified number of frames and sums them together to create the 16-bit image.
Close
Closes the dialog box.
Select Camera/Board (Acquire Menu)
Selects a current video device from the list of devices that have been installed using the
Meta Imaging Series Administrator.
Drop-in: VIDEVICE
Use this command to change the current video device from the default selection without using the Configure
Acquisition command in the Meta Imaging Series Administrator. This command's dialog box will display only
those video devices that are currently installed. If you want to use a video device that is not currently
installed, you must quit MetaMorph and use the Meta Imaging Series Administrator program to install it. This
command temporarily changes the current video device for the current work session.
Selecting a Camera/Board
To select a video device as the current device, use the following procedure:
Step
Action
1
Before starting MetaMorph, install the
desired video device drivers using the
Configure Acquisition command in the Meta
Imaging Series Administrator.
2
From the Acquire menu, choose Select
Camera/Board. The Select Camera/Board
dialog box opens.
3
Select the desired device from the Video
Device list.
4
Choose OK.
Select Camera/Board - Dialog Box Options
Video Device
Selects an active video device from the list of currently installed video device drivers. Alternatively, the
dummy driver, "Demonstration Video," can be selected.
OK
Changes the current video device based on the Video Device selection.
Cancel
Cancels the command.
Acquire Color Camera (Acquire Menu)
Configures acquisition parameters for single-chip color cameras and acquires color or
grayscale images. Configures the exposure balance between the red, green, and blue
acquisition channels, and interactively sets the focus.
Drop-in: ACQUIRECOLOR
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Use this command to configure single-chip color camera acquisition parameters and acquire color or
grayscale images.
Note: To use this command, before you start MetaMorph, load the Photometrics Camera device driver
using the Configure Acquisition dialog box in the Meta Imaging Series Administrator, and click This
camera uses a color mosaic chip. After you start MetaMorph, from the Acquire menu, click Select
Camera/Board, then choose the name of the Video Device that you are using.
Single-chip Color Cameras typically acquire images at 12 bits per channel or 10 bits per channel. When
you acquire color images, input from the three color channels will be converted into 24-bit true color
images (8 bits per channel). The Color Balancing tab page of the Acquire Color dialog box contains an
option for measuring a "white reference" image. This measurement determines the contributions from
the red, green, and blue channels. This will in turn be used to balance the input from each channel,
thereby achieving true color balance. Other options allow you to "tweak" the settings by adjusting the
brightness of each color channel separately, or by adjusting the brightness of the entire image.
Grayscale images retain their native 12-bit image depth, which MetaMorph handles as 16-bit images. If
you configure acquisition of grayscale images, an additional set of options will appear in the Acquire tab
page which allow you to configure image intensity scaling either manually or automatically and apply the
scaling to all images during focusing or acquisition. In addition, you can scale the image display after
acquisition with the Scale 16-Bit Image command.
Acquiring Color Images
Overview of Configuration and Acquisition
Setting Acquisition Parameters
Acquire Color - Color Balancing for Brightfield Images
Acquire Color - Color Balancing for Fluorescence Images
Focusing
Overview of Configuration and Acquisition from Single-chip Color
Cameras
These procedures guide you in setting up your Single-chip Color Camera configuration, saving and/or
loading a configuration, making adjustments to color and focus, and acquiring images. This procedure
contains links to subordinate procedures for setting acquisition parameters, setting color balance, and
setting focus. Be sure to follow the links to complete these procedures, and then return to the main
procedure after you have finished the subordinate procedure.
Note: Many of the options and controls in the Acquire Color dialog box will function
interactively during the focusing procedure.
To acquire images using Single-chip Color Camera, complete the following procedures:
Step
Action
1
From the Acquire menu, choose Acquire
Color. The Acquire Color dialog box opens.
2
On the Acquire tab, click the Image selector
to select the destination image window.
3
If you have previously saved a set of Singlechip Color Camera acquisition settings and
you want to use these settings, choose Load
to open the Load Settings dialog box and
configure which features of a setting to load,
or select a setting from the Settings dropdown box. Then, skip to Step 5.
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OR
If you are configuring a new set of acquisition
settings, continue with Step 4.
4
Complete the procedure for setting
acquisition parameters. The settings you
use depend on whether you are acquiring
color or grayscale images and your type of
illumination.
5
If you are acquiring brightfield color images,
you need to check the brightfield color
balance for your images.
6
If you are acquiring fluorescence color
images, you need to check fluorescence
color balance for your images.
7
Before you begin acquiring images, check
the focus of the microscope. Follow the
procedure for focusing the image sample.
8
Now you are ready to acquire your
experimental images. Click Acquire. A single
image will be acquired.
9
If you want to save your acquisition settings,
click Save As. The Save Single-chip Color
Camera Settings As dialog box opens.
AND
Type a name for the settings (.csn) file in the
File Name text box and click Save.
10
Click Close.
Setting Acquire Color Acquisition Parameters
To configure acquisition parameters for the Single-chip Color Camera, use the following
procedure:
Step
1
Action
On the Acquire Color dialog box Acquire tab,
select the Image Type and Exposure that
you want to use.
Click Color Image if you are acquiring a color
image; click Intensity Image if you are
acquiring a grayscale image.
2
Type or select an exposure time interval in
milliseconds (ms) in the active exposure time
box.
OR
If you want MetaMorph to calculate your
exposure time, click Auto Expose. This
exposure time is based on the exposure time
in milliseconds that you entered limited by
the Target maximum pixel value on the
Preferences tab.
3
If you selected Intensity Image, the Intensity
Image settings became active. If you want
MetaMorph to automatically set the
grayscale image intensity range, click
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Autoscale Image.
4
If you selected Autoscale image, you can
specify the low and high limits of the intensity
range as a percentage. Image data below
the low value will be interpreted as no signal
and above the high value will be interpreted
as maximum signal; thus, both will be
excluded. Type or select percentages for
either or both the low and high ends of the
image intensity scale.
OR
If you did not select Autoscale Image, you
can specify the low and high limits of the
image intensity range. Type or select a
value for either or both the low and high ends
of the image intensity scale.
Note: The low value must be less than the
high value.
5
If you are using an illumination device, select
the device from the Illum drop-down list.
OR
If you are not using an illumination device,
select "[None]."
6
Click the Preferences tab.
7
If you are acquiring color images, select a
target maximum pixel intensity value from
one of the Color Image boxes. You can
select the target value either as a percent of
the 4095-level maximum for the 12-bit range
(left box) or as an actual pixel color intensity
value (right box).
OR
If you are acquiring grayscale images, select
a target maximum pixel intensity value from
one of the Intensity Image boxes. You can
select the target value either as a percent of
the 4095-level maximum for the 12-bit range
(left box) or as an actual pixel grayscale
value (right box).
8
If you want MetaMorph to display an
overexposure warning, select the Warn If
Exposure Time Is Long check box. Then
select the desired Exposure Time Warning
Threshold (ms).
AND
If you want to display a warning graphic on
overexposed focusing images, select the
Draw Warning Text Onto Focus Image If
Image Is Overexposed check box.
9
Click the Region tab.
10
If you want to use the active rectangular
region from an image on the desktop, rather
than defining the region with the dialog box
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options, select the desired image using the
Image selector.
AND
Use the Rectangular Region Tool from the
Region Tools window to draw an acquisition
region in the image window. Then choose
Use Active Region Defined on Image.
12
You can use Left, Top, Width, and Height to
specify the size and location of the region on
the chip. Choose Entire Chip to create a
region that is the size of the chip, or you can
choose Center Quadrant to create a region
centered on the chip that is the size of one
quadrant. Choose Ctr to center the region.
You can use the << or >> options to shrink or
enlarge a region by a factor of two.
OR
Specify the size and location of region using
the Box-in-Box option on the left side of the
dialog box. The smaller box can be sized and
moved similarly to a region of interest.
13
Now you are ready to proceed with
configuring the image display scaling,
focusing, and acquisition.
Acquire Color - Color Balancing for Brightfield Images
Color balancing adjusts the relationship between the red, green, and blue channels. This is
necessary only if you are acquiring color images. To adjust color balance effectively, you need to
expose the camera to an evenly illuminated, all-white visual field, or have a white region within
the visual field. If your field does not contain a clear (white) region, you might only need to move
the microscope slide to a clear region.
To configure the color balance for image acquisition with the Single-chip Color Camera, use the
following procedure:
Step
Action
1
Click Start Focusing. A focus image window
opens.
2
Click the Place Active Region for Color
Balancing button (next to the Close button).
The Color Balancing tab moves to the front,
and an active square region is placed in the
focus image window.
3
In the focus image window, move and/or size
the region so it is surrounds an all-white area
or an area that you want to define as white.
OR
Provide an evenly illuminated, all-white visual
field, or move the specimen slide to a clear
region.
4
Set the Image Type to Brightfield.
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5
Click Measure White Ref. MetaMorph
assigns the intensity value of the active
region as the white value for the image. All
other color intensity values should fall below
this. All values higher will also be considered
as white.
6
Adjust the Brightness slider to obtain the
level of brightness you want in your image.
To cancel the adjustment you made, click the
associated Reset button.
Note: Moving the Brightness slider is
equivalent to moving all three individual color
sliders an identical distance.
7
Adjust the three individual sliders for Red,
Green, and Blue to fine tune the color
balance that you want in your image. To
cancel the adjustment you made, click the
associated Reset button.
8
If it was necessary to move your specimen
slide to a clear area to achieve correct white
balance, return the specimen to its original
position.
9
Click the Preferences tab.
10
If an active color balance region is still
present, move the region and size it to a
black area in your image or an area that you
want to define as black.
11
Click Measure Black Reference.
OR
Type or select a value in the Black
Reference box.
Note: You can also use this setting to finetune the black reference value after you have
clicked the Measure Black Reference button.
12
Click the Color Balancing tab.
13
Make any necessary final adjustments to the
Brightness or to the individual color settings.
14
Check Scale Every Image if you want
MetaMorph to recalculate scaling for each
acquired image. MetaMorph will
compensate for image intensity variations
from one image to the next, while retaining
the color balance setting that you made.
15
When you have finished making your
adjustments, click F2: Stop Focusing, or
press the [F2] function key on your keyboard.
The Focus window will close.
Acquire Color - Color Balancing for Fluorescence Images
If you are acquiring fluorescence images, you need to set the color balance (color channel scaling). The
MetaMorph Acquire Color Color Balancing tab enables you to select an image type of either Brightfield or
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Fluorescence. When you choose Fluorescence, the settings on the Color Balancing tab change to controls
that enable you to either automatically or manually set fluorescence color channel scaling. Because
fluorescence images are darkfield images, a white balance setting is not applicable.
To set the color balance for fluorescence images, complete the following procedures:
Step
Action
1
From the Acquire Color dialog box, click the
Color Balancing tab.
2
In the Image Type list, click Fluorescence.
The fluorescence color balancing controls
appear.
3
On the Scale Range list, select the camera
scale range to use to scale your images.
MetaMorph converts the camera scale of 12bits to 8-bit (0-255) format. By using 12-bits
per channel instead of 10-bits per channel,
you can achieve greater control over the
range you select to convert to 8-bit format.
4
In the Image Scaling box, click on Auto to
enable automatic image scaling for all color
channels. If you need to manually control
image scaling for one or more channels,
click Custom.
5
If you selected Auto Image Scaling, skip to
step 7.
OR
If you selected Custom Image Scaling, click
the Autoscale check box for the channels
that you want to control manually.
6
Click Start Focusing, then using the focus
image as a guide, set the minimum (Low)
and maximum (High) levels for each channel
for which you turned off Autoscale. You can
change the low and high values by typing
the value into the box, clicking the scroll
buttons on the right side of the box, or
moving the sliders under the channel's color
strip.
7
For channels that you allowed MetaMorph to
autoscale, you can type or select percentage
values that specify the low and high limits
into the Low and High boxes.
8
Click F2:Stop Focusing when you have
finished setting your color balance.
Focusing with Single-chip Color Cameras
The Start Focusing command instructs MetaMorph to acquire an image continuously into an image
window while you are focusing the microscope to verify that your specimen is visible and in focus.
Some digital cameras have gain and offset controls which can be adjusted while using the Start
Focusing command.
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Note: Because of a potential difference between the microscope eyepiece view and the
camera view, it is recommended that you complete the following procedure to ensure
accurate focus and image content.
To focus the microscope, use the following procedure:
Step
Action
1
From the Acquire Color dialog box, click the
Acquire tab.
2
Focusing is more effectively performed with
intensity images, rather than color images. If
Color Image is currently selected, change the
selection to Intensity Image.
3
Click Start Focusing.
MetaMorph begins to acquire a continuous
stream of images that are displayed and
updated in a Focus Image window.. The
Start Focusing button becomes the F2: Stop
Focusing button, and the Stop Focusing
command entry in the Devices menu is
active.
4
Focus your microscope while monitoring the
appearance of the images in the Focus
window.
Note: Exposure, scaling, and color
balancing options will remain available.
5
When you have finished adjusting your
microscope's focus, click F2: Stop Focusing,
or press the [F2] function key on your
keyboard. The Focus window closes.
6
Now you are ready to acquire your
experiment images. If you are acquiring color
images, select Color Images as the Image
Type.
Acquire Color Dialog Box Options
Acquire (tab)
Moves the Acquire tab to the front and enables the functions and settings on the Acquire tab.
Region (tab)
Moves the Region tab to the front and enables the functions and settings on the Region tab.
Color Balancing (tab)
Moves the Color Balancing tab to the front and enables the functions and settings on the Color Balancing
tab.
Preferences (tab)
Moves the Preferences tab to the front and enables the functions and settings on the Preferences tab.
Start Focusing/ F2: Stop Focusing
Acquires an image continuously into an image window while you are focusing the microscope to verify that
your specimen is visible and in focus. Images will be acquired continuously until you choose F2: Stop
Focusing, which discontinues acquisition of focusing images. As an alternative to using this button in the
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dialog box, you can press the [F2] function key on your keyboard or choose the Acquire Color Stop Focusing
command from the Acquire menu.
(Adjust Digital Contrast)
Opens the Adjust Digital Contrast dialog box.
(Place Region for Color Balance)
Places a preset size rectangular region in the center of the continuously updating focusing image to be used
as the color balancing region. You can resize or move this region anywhere within the acquisition region.
Click this button after you click Start Focusing. This function is active only when the focusing function is
active and when acquiring a color image. When you click this button, the Color Balancing tab is
automatically displayed.
Close
Closes the dialog box.
Acquire Color: Acquire - Dialog Box Options
Image
Selects the destination image window.
Image Type and Exposure
Selects the type of images that you plan to acquire and an exposure time for the selected image type. If you
select Color Image, the Color Balancing options are available. If you select Intensity Image, additional
intensity scaling options (Autoscale Image, Low, High) appear. However, the options on the Color Balancing
tab page will not be operative.
Intensity Image
Contains settings for monochrome images, including Autoscale Image and Low (or Low %) and High (or
High%).
Autoscale Image
Configures Acquire Color to scale the acquired images automatically to a 256-level intensity display. The
lowest grayscale value in the image will be assigned grayscale value 0 (black), the highest value will be
assigned value 255 (white) and all intensity values between the lowest and highest value will be scaled
accordingly. The result will be an increase in image contrast. This may be necessary if much of your image
data resides within a relatively narrow range of the camera's 12-bit (4096 intensity levels) overall intensity
range. If you select the Autoscale Image check box, the Low and High spin boxes will become Low% and
High% spin boxes, respectively. The Autoscale Image check box will be available only when Intensity Image
has been selected as the image type.
Note: If image intensity settings are set too high, the image will be over-saturated and a
warning message will appear in each corner of the image window to indicate that the
image will be over exposed if the current settings are used to acquire an image.
Low / High
If the Autoscale Image check box is unchecked, the Low and High boxes are used for selecting an intensity
range manually, to be scaled to a 256-level image intensity display. Low selects the image's lowest
grayscale value, which will be reassigned to grayscale value 0 (black), and the High spin box selects the
highest grayscale value, which will be reassigned to grayscale value 255 (white). The result will be an
increase in image contrast. This may be necessary if much of your image data resides within a relatively
narrow range of the camera's 12-bit (4096 intensity levels) overall intensity range. If you select the Autoscale
Image check box, the Low and High spin boxes will become Low% and High% spin boxes, respectively. The
Low / High spin boxes will be available only when Intensity Image has been selected as the image type and
the Autoscale Image check box has been left cleared.
Low% / High%
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If you select the Autoscale Image check box, the Low and High boxes become Low% and High% boxes,
respectively. These exclude a selected percentage of pixels in the image from the lower and upper end,
respectively, of the range of values being autoscaled. For example, you could exclude the bright nucleus of
a cell in a fluorescence image by setting the High% spin box to 10. The Low% / High% spin boxes will be
available only when Intensity Image has been selected as the image type and the Autoscale Image check
box has been selected.
Acquire
Acquires an image with the Color camera, using the current acquisition region and exposure settings. The
image will be placed in the video board's memory (replacing the previous image stored in memory) and then
transferred to an image window, where it will be displayed using the scaling settings you configured.
Note: In certain instances, if you press the Esc key, stream acquisition or other processes in progress may
be halted.
Auto-Expose
Initiates an autoexposure and adjusts the exposure time as determined by the target intensity preference.
Load
Opens the Load Setting dialog box. Use this dialog box to load all or part of a set of Color Camera
acquisition settings, previously saved as a .csn file with the Save or Save As command. You can also
configure this dialog to determine if it is opened each time a setting is changed using the Setting drop-down
box.
Save
Save changes to a previously loaded .csn file. If you want to save the changes to a new or different .csn file,
click Save As.
Save As
Saves all current acquisition settings on disk, including color balance settings. You can open the settings at
a later date using the Load command. This command opens the Save Acquire Color Settings As dialog box.
Setting
Displays the names of the most recently saved or loaded settings. Up to eight settings can be displayed in
the order of most recent usage. Selecting a setting load the values for the setting into the dialog for the next
acquisition, immediately updating the controls and the display of the current "Acquired" image.
When loading a setting, the Load Setting dialog may appear so that the portion of the setting to be loaded
can be configured. If the Load Setting dialog does not appear, the portion used will be the same as the
portion used the last time a setting was loaded. To get the Load Settings dialog to appear when selecting a
setting from the popup, open the Load setting dialog box and select Show this dialog from the When using
the pop list to load fields.
Illumination
Selects the illumination setting. If you are not using a shutter, select "[None]." If a shutter is selected, it will
open the shutter before acquiring an image and close the shutter after exposure (before transfer.)
Acquire Color: Region - Dialog Box Options
Acquisition
Selects the region settings that are to be applied to the acquisition region.
Focus
Selects the region settings that are to be applied to the focus region.
Use Single Region
Indicates that a single region will be used for all acquired images, including images acquired in focusing
mode.
Note: When this box is checked (default state), the Focus radio button is inactive.
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Chip Size
Indicates the size of the camera chip, in pixels.
Box-in-Box Interactive Display of Region
Allows you to click on the smaller box with the left mouse button and then drag the pointer to resize and
move the chip region box, as you would for a region of interest.
Image
Specifies the image to use for the Use Active Region Defined on Image command.
Use Active Region Defined on Image
Defines a region for the chip based on the active region of interest in the image selected with the Image
selector. The box-in-box display is updated as well as the region's Left, Top, Width, and Height values.
Left
Specifies the region's leftmost point.
Top
Specifies the region's topmost point.
Width
Specifies the region's width.
Height
Specifies the region's height.
Entire Chip
Creates a region that is the size of the entire chip.
Center Quadrant
Creates and centers a region that is the size of one quadrant of the chip.
<< and >>
Shrinks or enlarges the region by a factor of two.
Ctr
Centers the region on the chip.
Acquire Color: Color Balancing - Dialog Box Options
Image Type
Selects either Brightfield or Fluorescence as the image type based on the illumination source the you intend
to use. The default for this setting is Brightfield, which enables you to make settings for Brightness and/or
the color intensity of individual primary colors. If you select Fluorescence as the image type, the settings
options on the Color Balancing tab change to a different group of settings.
Brightness
Adjusts the overall intensity levels of all three color channels simultaneously. Moving this slider is equivalent
to moving the Red, Green, and Blue sliders by equal amounts.
Adjustment
Adjusts color intensity levels of each individual color:
Red – Adjusts the intensity levels of the red values in the image.
Green – Adjusts the intensity levels of the green values in the image.
Blue – Adjusts the intensity levels of the blue values in the image.
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Reset
Resets the associated slider(s) to the original midpoint position setting. If you choose the Reset button for
the Brightness slider, this will also simultaneously reset all three of the color sliders.
Scale Every Image
Forces MetaMorph to recalculate the minimum and maximum scaling values for all color channels every
time you acquire an image. This effectively recalculates the brightness for the view or region to compensate
for variations in image brightness, while maintaining the original, overall color balance of the image.
Measure White Ref
Measures a "white reference" image, which is used to correct for the differences between red, green, and
blue values of the image. For the region that you specify as white, MetaMorph scales the intensity value
indicated for each channel to equal the maximum intensity for that color.
Image Scaling
Selects either automatic scaling for the entire image or selected image region or custom scaling that you can
set for each color.
Scale Range
Selects the appropriate custom scaling range to apply to your color image. Select either 10 bits (1024
colors) or 12 bits (4096 colors).
Autoscale
Enables automatic scaling of a color. When Autoscale is selected for a specific color, type or select a low
and high percentage value. When Autoscale is not selected, type or select a both a low and high scaling
range or move the pointers on the color scale to select the scaling range that you want to use.
Scaling Range
Accepts both low and high scaling range values. If Auto scale is selected, type or select a low and high
scaling range as a percentage value. If Autoscale is not selected, type or enter appropriate low and high bit
range values, or move the sliders to select appropriate low and high values.
Acquire Color: Preferences - Dialog Box Options
Warn If Exposure Time Is Long
Instructs MetaMorph to warn you when an acquisition starts if the camera's exposure setting is longer than
the value specified in the Exposure Time Warning Threshold.
Exposure Time Warning Threshold (ms)
Specifies the minimum exposure time that is to be considered a long exposure. You will be warned if the
exposure time equals or exceeds this limit.
Draw Warning Text Onto Focus Image If Image Is Overexposed
Draws an overexposure warning in the corners of the Focus image if any portion of the acquired image is
saturated.
Color Image
Selects a maximum pixel intensity for image acquisition. The target intensity will determine the exposure
time. You can select the target value either as a percent of the 4095-level maximum for the 12-bit range (left
spin box) or as an actual pixel color intensity value (right spin box).
Intensity Image
Selects a maximum pixel intensity for image acquisition. The target intensity will determine the exposure
time. You can select the target value either as a percent of the 4095-level maximum for the 12-bit range (left
spin box) or as an actual pixel grayscale value (right spin box).
Black Reference
Establishes the maximum image intensity value for black in the image.
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Measure Black Reference
Initiates a measurement of a black reference region in the image and records the measurement in the Black
Reference window.
Stream Acquisition (Acquire Menu)
Enables you to configure MetaMorph for rapid acquisition of images as a continuous data
stream, activate image streaming, and transfer the images into a stack.
Drop-in: STREAM
Use this command to configure Stream Acquisition settings and to acquire an image stream based on
the settings. When configuring Stream Acquisition, you can specify the number of frames to acquire, the
camera state, shutter mode, and the clear mode settings. Stream Acquisition captures images as a
continuous data stream at the fastest possible rate and stores the image data in RAM, or directly to the
hard drive. Once all of the frames have been acquired, MetaMorph transfers the image data into an
image stack, performing the image saving and data logging tasks as specified by the settings in the
Configure Digital Camera dialog box.
Note:
Be sure to configure your digital camera correctly in the Configure Digital Camera dialog box before
beginning stream acquisition.
After Configure Stream Acquisition and Configure Digital Camera settings are defined, use the Acquire
button on the Acquire tab to begin streaming your image data. When all of the frames have been
acquired, MetaMorph will transfer the image data into planes in a stack.
Stream Acquisition also enables you to acquire through-focus Z-series images as a stream. To do so,
both your video driver and focus motor driver must be capable of supporting streaming sequences.
Currently, only the Princeton Instruments digital camera driver and the Physik focus motor device driver
support streaming. When these conditions have been met, an enhanced version of the Configure Stream
Acquisition dialog box is displayed. This provides additional options for setting the starting and ending
position of the focus motor, based on the position settings that have been configured with the Focus
command (Devices menu).
Note: To use this command, your camera must support image streaming.
WARNING:
If you attempt to use a streaming exposure time that is shorter than the fastest readout time your camera
can handle, your actual exposure time will be limited by the readout time. If this occurs, MetaMorph will
display a warning message.
WARNING:
You must run all mechanical shutters at a cycle time greater than 25 ms. Uniblitz, Lambda 10, Metaltek,
Ludl, and cooled CCD shutters are driven by a high voltage which takes time to dissipate. Running these
shutters at a cycle length shorter than 25 ms will cause a build-up of heat, leading to eventual jamming.
Neither Molecular Devices nor any manufacturers of these shutters will honor warranties on equipment that
has been damaged by improper use. Operation of these shutters at a cycle length shorter than 25 ms is
considered improper use.
Stream Acquisition - Procedures
To acquire images as a stream, complete the following procedure:
Step
Action
1
From the Acquire menu, choose Stream
Acquisition. The Stream Acquisition dialog
box opens.
2
In the Acquisition Mode box on the Acquire
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dialog tab, choose the image stream storage
method. If sufficient RAM memory is
available, choose Stream to RAM, otherwise
choose Stream to Hard Disk.
Note: The default acquisition method,
Stream to RAM, sends the entire image
stream to RAM and transfers the images to
disk afterwards. You can also stream the
acquired frames to hard disk in real-time by
selecting Stream to Hard Disk.
3
In the Number of Z sets box, select the
number of z sets (frames) you want to
acquire.
Note: MetaMorph displays the amount of
memory the stream will use (based on the
size and number of images that you want to
acquire). It will also display the amount of
memory the resulting stack will use and the
amount of memory available. This
information will help you to determine how
many frames you can acquire in one stream.
4
Select an illumination setting from the Initial
Illum list.
OR
If you are not using a shutter, leave this
selection as "[None]."
5
Select Use with High-Speed Focus Motor to
enable the Focus Motor options. The settings
in this option group will be based on the
position settings that have been configured
with the Focus command (Devices menu).
6
Select Use with high-speed Wavelength
changer, if you have a high-speed
wavelength changer installed and configured.
7
Click the Focus tab, The focus tab dialog is
displayed.
8
From the Start At drop-down list, select the
starting position for the Z-series image
stream: Current Position, Origin, Top,
Bottom, or Home.
9
From the Move to list select the Z-position for
the last image in the stream series from one
of the following: Origin, Top, Bottom, or
Home.
10
From the After drop-down list, select the
position to which the focus motor should
return after the Z-series stream has been
acquired: Current Position, Origin, Top,
Bottom, or Home.
11
Click the Wavelength tab. The Wavelength
tab dialog is displayed.
12
From the Wavelength tab dialog, type or
choose the number of wavelengths to include
in your image stream in the Number of
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Wavelengths box.
13
In the Wavelengths Selection area, set the
wavelength value for each wavelength that
you want to acquire in your image stream.
14
Click the Camera Parameters tab. The
Camera Parameters tab dialog is displayed.
15
Select the acquisition mode from the
Acquisition Mode section. The options
available will vary depending on the camera
used.
16
Select the desired state for the camera from
the Camera State list. The options available
will vary depending on the camera used. The
suggested setting for the PVCam camera is
HALT, CLEAR.
17
Select the desired shutter mode for the
camera from the Shutter Mode list. The
options available will vary depending on the
camera used. The suggested setting for a
frame transfer camera operating in the FT
mode is OPEN PRE SEQUENCE. If you do
not have a frame transfer camera, you
should set this option to OPEN PRE
EXPOSURE.
18
Select the desired clear mode for the camera
from the Clear Mode list. The options
available will vary depending on the camera
used. The suggested setting for a frame
transfer camera operating in the FT mode is
CLEAR PRE EXPOSURE.
19
After you complete all settings on all tabs,
click the Acquire tab, and then click Acquire
to capture your image stream based on your
settings.
Note: To cancel a Stream
Acquisition before it finishes,
press the Esc key. If your
camera driver supports it, you
are given the option to save the
images already collected to a
stack on the MetaMorph
desktop.
20
When you have finished, click Close to close
the dialog box.
Stream Acquisition – Main Dialog Box Options
Acquire Tab
Select the dialog box tab that contains the principle acquire configuration settings and displays the
Acquisition Information.
Focus Tab
Selects the dialog box tab that contains the focus motor configuration settings.
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Wavelength Tab
Selects the dialog box tab that contains the wavelength configuration settings.
Camera Parameters Tab
Selects the dialog box tab that contains the camera parameter settings.
Status
Displays a message regarding the status of the stream acquisition and alerts you to any problems that may
occur.
Note: The types of messages that display depend on your camera and driver. For
example, the Orca ERG supports the display of Readout time per frame information.
Record Configuration State
Applies any new configuration settings.
Acquire
Begins image streaming acquisition.
Note: To cancel a Stream Acquisition before it finishes, press the Esc key. If your
camera driver supports it, you are given the option to save the images already collected
to a stack on the MetaMorph desktop.
Close
Closes the Stream Acquisition dialog box and disregards the new configuration settings.
Stream Acquisition - Acquire Dialog Box Options
Stream Acquisition - Focus Dialog Box
Stream Acquisition - Wavelength Dialog Box
Stream Acquisition - Camera Dialog Box
Stream Acquisition - Acquire Dialog Box Options
Acquisition Mode
This option enables you to select between (1) the default method of acquiring frames rapidly to RAM and
then processing the images afterwards on disk (Stream to RAM), and (2) acquiring the image frames directly
to disk by high-speed streaming (Stream to Hard Disk).
Number of Z Sets
Specifies the number of image frames to acquire.
Browse
Opens the Select Stream File dialog box to enable you to select or assign a file name for the acquired
stream. By default, the file will be saved as a stack (.stk) file. This option is only available when Stream to
Hard Disk is selected in the Acquisition Mode field.
Filename
Displays the path and name of the stack file for the streamed acquisition. You can edit this path directly, or
use the Browse command to choose the path. This option is only available when Stream to Hard Disk is
selected in the Acquisition Mode field. This field must be filled before you can acquire a stream using the
Stream to Hard Disk option.
Acquisition Information:
Your Current Acquisition Region Is
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Displays the size of the acquisition region. (The acquisition region is defined in the Configure Digital
Camera dialog box.)
Each Pixel Will Use
Displays the amount of memory that each pixel will use. This value depends on the capabilities of
the channel used to acquire the stream. Channels that acquire 8-bit images will use 1 byte per
pixel.
Each Frame Will Use
Displays the amount of memory that each frame will use. This value is a result of the size of the
acquisition region and the number of bytes used per pixel.
Total Number of Frames
Indicates the number of frames planned to acquire. Change the number of frames in the Number
of z sets window. As you change the Number of z sets value, the value displayed by the Amount of
Memory Stream Will Use is automatically updated.
Amount of Memory Stream Will Use
Displays the amount of memory needed to complete the stream acquisition. If the amount of
memory used by the stream exceeds the total amount of free memory, acquisition will not be
possible. When this situation occurs, a message will be displayed on the Status line.
Amount of Memory Stack Will Use
Displays the amount of memory that will be used by the resulting stack.
Amount of Memory Available
Displays the amount of memory available for use by the stream during acquisition.
Readout Time Per Frame
Displays the amount of time it takes to read the image out of the camera and into system memory.
Note: This field is only displayed if your camera can provide frame readout times.
Acquisition Time Per Stream
Displays the total amount of time that the stream acquisition will require.
Note: This field is only displayed if your camera can provide frame readout times.
Initial Illum
Selects the illumination setting to use when acquiring the stream. Illumination settings are defined using the
Configure Illumination command. If you are not using a shutter, select "[None]."
Use with High-Speed Focus Motor
Enables the Focus Motor options (Start At, Move To, After), which are used for selecting the starting,
ending, and return position settings of a Z-series of through-focus images. The Focus Motor options and
Use with High-Speed Focus Motor check box will be displayed only if your video and focus motor device
drivers support Z-streaming. Currently, only the Princeton Instruments video driver and the Physik focus
motor device driver support streaming.
Use with High-Speed Wavelength Changer
Indicates that a high speed wavelength changer is connected and enabled.
Stream Acquisition - Focus Dialog Box
Focus Motor:
Specifies the parameters that determine the focus motor Z plane movements.
Start At
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Selects the starting position for the Z-series image stream: Current Position, Origin, Top, Bottom, or Home.
These settings are based on the position settings that have been configured with the Focus command
(Devices menu).
Move To
Selects the Z-position for the last image in the stream series: Origin, Top, Bottom, or Home. These settings
are based on the position settings that have been configured with the Focus command.
After
Selects the position to which the focus motor should return after the Z-series stream has been acquired:
Current Position, Origin, Top, Bottom, or Home. These settings are based on the position settings that have
been configured with the Focus command.
Plane Distance
Indicates the between-plane distance of the Z-series images, based on the starting and ending Z-positions
and the Number of Frames to Acquire.
Total Distance
Indicates the total distance between the starting and ending position in the Z-series.
Stream Acquisition - Wavelength Dialog Box
Number of Wavelengths
Indicates the number of wavelengths that you want to acquire.
Wavelength Selection:
Displays a control for each wavelength that you want to acquire.
Wavelength #1…4
Indicates the wavelength value in nanometers for the first wavelength, and provides a slider for setting the
wavelength value.
Stream Acquisition - Camera Dialog Box
Acquisition Mode
Acquire Images at frame Rate
Specifies image acquisition to occur at the frame rate determined by the current exposure time.
Acquire Images from Each External Trigger
Specifies image acquisition to occur in synchronization with a separate external trigger source for each
frame of the stream.
Acquire Images from First External Trigger
Specifies image acquisition to occur in synchronization with a single external trigger source.
Number of Frames to Skip
Specifies the number of frames to skip before beginning to acquire a continuous stream of frames.
Camera State
Specifies the camera state used during acquisition. The options available will vary depending on the camera
used. The suggested setting for the PVCam camera is HALT, CLEAR.
Shutter Mode
Specifies the shutter mode used during acquisition. The options available will vary depending on the camera
used. The suggested setting for a frame transfer camera operating in the FT mode is OPEN PRE
SEQUENCE. In this mode, the shutter will open before the acquisition and stay open until the acquisition is
done. If you do not have a frame transfer camera, you should set this option to OPEN PRE EXPOSURE.
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This will open the shutter before each frame is acquired and then close it after the acquisition.
Clear Mode
Specifies the mode used to clear the camera chip. The options available will vary depending on the camera
used. The suggested setting for a frame transfer camera operating in the FT mode is CLEAR PRE
SEQUENCE. This setting clears the chip prior to starting the first exposure. If you do not have a frame
transfer camera, you should set this option to CLEAR PRE EXPOSURE. This setting clears the chip before
each frame is exposed.
Digital Camera Video Control (Acquire Menu)
Controls the RS-170 video output of a digital camera.
Drop-in: DCAMVID
Use this command to focus digital cameras that use an external video monitor. The command allows you
to set the zoom level, panning, exposure time, and intensity scaling for the video output. The Digital
Camera Video Control command also allows you to focus the camera so that you can set up the video
output while focusing.
Note: This command is supported only by the Princeton Instruments MicroMAX and PentaMAX
cameras.
WARNING:
You must run all mechanical shutters at a cycle time greater than 25 ms. Uniblitz, Lambda 10, Metaltek,
Ludl, and cooled CCD shutters are driven by a high voltage which takes time to dissipate. Running these
shutters at a cycle length shorter than 25 ms will cause a build-up of heat, leading to eventual jamming.
Neither Molecular Devices nor any manufacturers of these shutters will honor warranties on equipment that
has been damaged by improper use. Operation of these shutters at a cycle length shorter than 25 ms is
considered improper use.
Configuring Digital Camera Video Control
To configure digital camera video control, use the following procedure:
Step
Action
1
From the Acquire menu, choose Digital
Camera Video Control. The Digital Camera
Video Control dialog box will appear.
2
If you are using an external shutter, select
the Illumination setting associated with your
shutter from the Illumination setting list. The
shutter will open automatically when focusing
begins, and will restore itself to its previous
state when focusing ends.
OR
If you are not using a shutter, leave the
Illumination setting list selection as "[None]."
3
From the Zoom group, select the desired
zoom level at which you want to display the
video output.
The 2x and 4x zoom levels come with the
additional option of using Binning, in which
the camera chip pixels are binned in X-Y
fashion to produce the video image, or
Decimation, in which every Nth pixel is used
for the final image.
4
If you selected one of the 2x or 4x options
from the Zoom group, the Pan group will
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become available, as you will not be able
display the entire zoomed image. You can
pan to one of nine separate positions in the
image.
5
In the Exposure Time (ms) text box, type the
amount of time that you want the chip to be
exposed.
6
From the Intensity Scaling list, select the
scaling factor that you want to use to scale
the camera's 12-bit image to an 8-bit display
(if necessary).
7
Choose Focus to focus the image.
When you have finished, choose Stop Focus.
8
Choose Close.
Digital Camera Video Control - Dialog Box Options
Zoom
Zooms the image displayed on the video monitor. You can select 1x, 2x, or 4x. The 2x and 4x zoom levels
come with the additional option of using Binning, in which the camera chip pixels are binned in X-Y fashion
to produce the video image, or Decimation, in which every Nth pixel is used for the final image. If you select
one of the 2x or 4x options, the Pan radio buttons will become available, allowing you to select which part of
the image to display.
Pan
Allows you to pan to one of nine separate positions in the image when the image is zoomed up to 2x or 4x.
This option is available only when the Zoom factor is either 2x or 4x.
Exposure Time (ms)
Specifies the amount of time the chip is exposed, in milliseconds. The Princeton Instruments camera
optimizes the focus algorithm internally, so that it may not seem as though the value selected here
corresponds to the exposure time of the chip.
Intensity Scaling
Specifies the scaling factor to use to scale the camera's 12-bit image to an 8-bit display. This may be
necessary when your images' intensity values are skewed toward one end of the grayscale. If you are using
a MicroMAX camera, you will be able to choose the range of gray levels to display. However, the PentaMAX
camera uses look-up tables instead to determine the display.
Illumination
Selects the Illumination setting associated with the external shutter you are using.
Focus
Starts camera focusing. This button's text will change to "Stop Focus."
Stop Focus
Stops camera focusing.
Close
Closes the dialog box.
Acquire Multiple Wavelengths (Acquire Menu)
Configures and performs acquisition of images using up to six different sets of settings
for wavelength, intensity, and exposure duration.
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Drop-in: MULTIWAC
Use this command to acquire images automatically, using up to six sets of illumination, exposure, and
offset settings. The images can be saved as planes in a stack, or acquired as images of individual
wavelengths.
For digital cameras, you can set different exposure times for the acquisitions, or you can specify that
autoexposures be performed for each.
Note: If you use the Acquire command to acquire images, this command uses the Acquire acquisition
settings. If you use the Acquire from Spot or Acquire from Digital Camera commands to acquire images,
this command uses acquisition settings from those commands. If you are using a Spot Camera, you
must also select at least one of the three color channels from the Acquire from Spot Camera dialog box
to be able to use Acquire Multiple Wavelengths. Background subtraction and shading correction will not
be performed when images are acquired with this command, regardless of the setting used.
The planes in the acquired stack can be shifted relative to one another to align the objects in the images.
This is similar to the process used in the Align Stack command. When you perform an alignment, only
that the area of the image that is common to all wavelengths will be saved. Thus, the result image may
be smaller than the original acquisition area.
Aligning a Stack
Aligning a Stack
Reference
Plane
Shifting Plane
Reference
Plane
Shifting Plane
1. A horizontal shift of ten pixels to
the left is selected and applied to
the middle plane, using the top
plane as a reference.
2. A horizontal shift of ten pixels to
the left is selected and applied to
the bottom plane, using the middle
plane as a reference.
Acquiring Multiple Wavelengths - Procedures
Acquiring Multiple Wavelengths
Aligning a Multiple-Wavelength Stack
Acquiring Multiple Wavelengths
To configure and perform multiple wavelength acquisitions, use the following procedure. If you
are using a digital camera, the acquisition settings not configured here will be taken from the
settings in the Acquire from Digital Camera dialog box.
Step
Action
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1
From the Acquire menu, choose Acquire
Multiple Wavelengths. The Acquire Multiple
Wavelengths dialog box opens.
2
If you have previously saved a state file
containing the dialog box settings that you
want to use, choose Load State and select
the desired .amw file from the Load Acquire
Multiple Wavelength File dialog box that
appears. Then choose OK to return to the
Acquire Multiple Wavelengths dialog box and
skip to Step 9.
OR
If you will be using a new set of dialog box
settings, continue to Step 3.
3
With the # of Waves spin box, specify the
number of different sets of illumination
settings that will be used.
4
Select an illumination setting for each of the
wavelengths in the Illumination drop-down
boxes.
5
If you are using a digital camera, you can
specify an exposure duration in the Exposure
(ms) spin box for each wavelength.
OR
If you want MetaMorph to determine an
appropriate exposure duration, select the
Auto check box for the first wavelength.
Then specify a maximum grayscale value for
the acquired image in the Target Intensity
spin box.
6
If you want to specify an exposure duration
for each wavelength, continue to Step 7.
OR
Select an exposure duration mode. Choose
Auto Balance if you want all wavelength
acquisitions to use the autoexposure feature.
A check mark will appear in the Auto check
boxes for all wavelengths.
Balance = 1 if you want all wavelength
acquisitions to use the exposure duration set
for Wavelength 1. A check mark will appear
in the Balance check boxes for all
wavelengths, and the Balance spin boxes will
all update to display a "1".
Use Balance if you want Wavelengths 2 and
higher to use an exposure duration that is a
specific ratio or multiple of the exposure for
Wavelength 1 (to be specified in the next
step).
7
Repeat Steps 4 - 8, as appropriate, for each
successive wavelength acquisition set.
If you want to configure an exposure duration
for each wavelength acquisition that is a
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specific ratio or multiple of the exposure for
Wavelength 1, verify that a check mark
appears in its Balance check box and enter a
ratio value in its Balance spin box.
OR
If you want to specify an exposure duration
for each wavelength acquisition, verify that
there are no check marks in either its
Balance or Auto check boxes, and type the
duration, in milliseconds, in the Exposure
(ms) spin box.
8
If you want to save the current set of dialog
box settings, choose Save State. Type a file
name for the .amw state file in the File Name
text box of the AMW State dialog box that
appears. Then choose Save.
9
To acquire all configured wavelength images
as planes in a single stack, select the
destination stack with the Stack image
selector and click the Acquire icon in the All
Wavelengths section (upper-left corner of
dialog box).
OR
If you want to acquire an image using a
single wavelength’s settings, click the
Acquire icon next to the settings for that
wavelength.
If you have enabled autoexposure, the
Exposure and/or Balance settings will be
updated following the acquisition(s).
10
If you want to align the acquired set of
images, follow the procedure for aligning
the stack planes.
11
Choose Close when you have finished.
Aligning a Multiple-Wavelength Stack
To align the planes in a multiple-wavelength image stack, use the following procedure.
Step
Action
1
From the Acquire Multiple Wavelengths
dialog box, choose Set Alignment. The
AMW: Set Alignment dialog box will appear.
2
Select the desired image from the Stack
image selector.
3
If you are starting with a stack that was not
aligned during acquisition, select Set Initial
Alignment from the Change to Multiple
Wavelengths option button group.
OR
If you are editing a stack that was aligned
during acquisition with the main dialog box's
Image Alignment and Cropping spin boxes
(X and Y), and you want to add a set of offset
values to the values already in the main
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dialog box, select Adjust Current Values.
4
Select an alignment display from the Display
group:
Subtract uses subtraction to show the
difference between the reference plane and
the shifting plane.
Average uses averaging to display this
difference.
5
Use the Horizontal Shift and Vertical Shift
text boxes or sliders to adjust the alignment
of the plane displayed in the alignment image
window. The plane will be moved in one-pixel
increments.
If you are using the Subtract display, the
plane will be aligned when there is a nearly
uniform grayscale level throughout the entire
image.
If you are using Average, the aligned plane
should look like the original plane with as
little blurring as possible.
If you need to start over, choose Zero Shift to
reset the sliders to zero and repeat Step 5 as
needed.
6
Choose the Next or the Previous command
button to advance to the next or the
preceding plane (depending on whether you
are starting with the first or last plane).
7
Repeat Steps 5 and 6 until you have aligned
each plane in the stack.
8
Choose Close when you have finished.
Acquire Multiple Wavelengths - Dialog Box Options
Acquire Multiple Wavelengths (main dialog box)
AMW: Set Alignment
Acquire Multiple Wavelengths - Dialog Box Options
All Wavelengths
# of Waves
Sets the number of separate wavelengths which can be configured.
Auto Balance
Enables Auto for all wavelengths. Not recorded in journals.
Use Balance
Sets all wavelengths to use their respective Balance values. For each wavelength, excluding Wavelength 1,
the Balance check box will be enabled. This is used to best effect after an autoexposure is performed for all
wavelengths.
Balance = 1
Sets the exposure time to be the same for all wavelengths. For each wavelength, excluding Wavelength 1,
the Balance check box will be selected, Auto will be deselected, the Balance value will be set to 1, and the
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exposure of the wavelength being configured will be set to the exposure duration of Wavelength 1.
Stack
Sets the destination for the stack of images generated by Acquire in the All Wavelengths section.
Specific Wavelengths
Illumination
Selects the illumination setting for the wavelength being configured.
Auto
Determines whether a new exposure duration is to be calculated for the current wavelength. If you select the
check box for this option, MetaMorph will perform multiple exposures to determine an appropriate exposure
setting. The algorithm used is the same as that used for Basic Digital Acquire. If you deselect this option and
an appropriate exposure is achieved, the new exposure duration will be represented in the ms spin box, and
the Balance value for the wavelength will be set.
Exposure (ms)
Specifies the exposure duration for the corresponding wavelength. If you select Auto, this option will merely
display the duration to be used once an acquisition time has been determined.
Balance (check box)
Determines whether changes to the settings for Wavelength 1 are to affect the current wavelength. If this
check box is selected while Wavelength 1 is calculated or simply changed, the exposure for the current
wavelength will be calculated as a ratio of the exposure duration of Wavelength 1. If this check box is
cleared while Auto is simultaneously disabled, the exposure value for the current wavelength will be the
time, indicated in milliseconds, and the Balance value will simply reflect the calculated ratio.
Balance (spin box)
Displays and sets the ratio of the exposure time for the current wavelength, relative to Wavelength 1.
Changing this value will change the exposure time. If All to Use Balance has been chosen, changes to the
settings for Wavelength 1 will automatically affect the exposure duration of the current wavelength. If Auto is
enabled, the Balance spin box will merely display the ratio to be used once an acquisition time has been
determined.
Offset
X
Specifies a horizontal alignment shift for the currently active image plane. Positive numbers will shift the
image to the right, negative numbers to the left. When you perform an alignment, only that the area of the
image that is common to all wavelengths will be saved. Thus, the result image may be smaller than the
original acquisition area.
Y
Specifies a vertical alignment shift for the currently active image plane. Positive numbers will shift the image
downwards, negative numbers will shift upwards.
Destination
Determines the destination for acquisition of the current wavelength only.
Acquire
Acquires an image for the current wavelength, based on the active settings.
Target Intensity
Sets the maximum value of the intensity during autoexposures. This field affects all wavelengths.
Set Alignment
Opens the AMW: Set Alignment dialog box, from which you can configure alignment of the planes in the
acquired multiple-wavelength stack.
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Load State
Opens the Load Acquire Multiple Wavelength File dialog box, from which you can select and load a saved
state file (*.amw) which stores the Acquire Multiple Wavelengths settings. These settings include the
exposure times, the balances, the wavelengths to autoexpose, illumination information for each wavelength,
and image alignment information.
Save State
Saves the current set of dialog box settings in an .amw state file.
Close
Closes the dialog box.
AMW: Set Alignment (Acquire Multiple Wavelengths) - Dialog Box Options
Stack
Selects the stack that will be aligned.
Change to Multiple Wavelengths
Determines whether the stack being edited was aligned with the X and Y spin box values in the main dialog
box's Image Alignment and Cropping option group. If you are starting with a stack that was not created with
a set of alignment values, select Set Initial Alignment. If the stack was acquired with the X and Y offset
values and you want to add a value set to the existing values, select Adjust Current Values.
Display
Selects the method to be used to display differences between the reference plane and the shifting plane:
Subtract uses subtraction to show the difference between the reference plane and the shifting plane. The
planes will be aligned when there is a nearly uniform grayscale level throughout the entire image.
The Average display uses averaging to display the offset between the two planes. The aligned plane
should look like the original plane with as little blurring as possible.
Horizontal Shift (text box and top slider)
Adjusts the horizontal alignment of the plane in one-pixel increments.
Vertical Shift (text box and left slider)
Adjusts the vertical alignment of the plane in one-pixel increments.
Previous
Places the previous plane in the alignment image window.
Next
Places the next plane in the alignment image window.
Zero Shift
Resets the Horizontal Shift and Vertical Shift to zero.
Cancel
Cancels the command and returns to the main Acquire Multiple Wavelengths dialog box.
OK
Accepts the new settings and closes the dialog box. Any values you enter in the Horizontal Shift and Vertical
Shift boxes will be added to the Y and X spin boxes, respectively, in the main dialog box's Image Alignment
and Cropping option group.
Configure Intensifier Gain Control (Acquire Menu)
Configures the control of the intensifier CCD camera settings when using computer-
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controlled gain.
Drop-in: SETICCD
Use this command before using the Set Intensifier Gain command and the Set Camera Level and Gain
command with an intensified CCD camera (and the PI Video ICCD Settings command, if you are using a
Princeton Instruments video camera). Configure Intensifier Gain Control allows you to specify the
camera model, serial port, and baud rate. It also allows you to select whether the camera is controlled by
the computer or by the front-panel knobs on the intensifier. The camera must be controlled by the
computer to use the intensifier gain commands in MetaMorph.
You must use this command before using the Set Intensifier Gain, the Set Camera Level and Gain, and
PI Video ICCD Settings commands. These three commands will be unavailable until you do so.
Prior to using this command, you must install its drop-in, SETICCD, using the Configure Drop-ins/Toolbars
Command in the Meta Imaging Series Administrator.
Configuring the Intensifier Gain Control
To configure the intensifier gain control for use with an intensified CCD camera, use the following
procedure:
Step
Action
1
From the Acquire menu, choose Configure
Intensifier Gain Control. The Configure
Intensifier Gain Control dialog box will appear.
2
Select your camera's model name from the
Intensifier Model drop-down list.
3
Select the serial port used to connect the
camera from the Serial Port drop-down list.
4
Select the appropriate baud rate for the
connection from the Baud drop-down list.
5
To control the intensified CCD camera using
the other intensified gain control commands in
MetaMorph, select Computer from the Camera
Control group.
Note: If your camera requires manual control
for some features on the camera, select
Manual before performing those operations.
6
Choose OK.
Configure Intensifier Gain Control - Dialog Box Options
Intensifier Model
Specifies the intensified CCD camera model name.
Serial Port
Specifies the serial port used to connect the camera to the computer.
Baud
Specifies the baud rate used for the connection between the camera and the computer.
Camera Control
Switches between manual control and computer control of the camera. Use Manual when you want to
change settings on the camera that can only be accessed on the camera when it is not controlled by the
computer. Use Computer when you want to control the camera from MetaMorph.
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OK
Configures the intensifier gain control options.
Cancel
Cancels the command.
Set Intensifier Gain (Acquire Menu)
Sets the intensifier gain for use of an intensified CCD camera.
Drop-in: SETICCD
You can use this command to set the intensifier gain interactively during acquisition. The same gain is
used for all wavelengths that are acquired. You can change the gain setting by using a slider. Once you
have changed the slider's value, the camera will use that value for all wavelengths acquired, starting with
the next acquisition.
Prior to using this command, you must install its drop-in, SETICCD, using the
Configure Drop-ins/Toolbars command in the Meta Imaging Series Administrator. You must also
configure the intensifier using the Configure Intensifier Gain Control command and specify that the
camera is controlled by the computer.
Setting the Intensifier Gain
To set the intensifier gain, use the following procedure:
Step
Action
1
From the Acquire menu, choose Set Intensifier
Gain. The Set Intensifier Gain dialog box will
appear.
2
If you want a delay to occur after the gain has
been changed, select the desired length using
Delay After Setting Gain.
3
Begin your experiment.
4
To set the gain interactively, select the desired
gain from the Intensifier Gain slider.
5
Choose Close when you have finished.
Set Intensifier Gain - Dialog Box Options
Intensifier Gain
Selects an intensifier gain interactively.
Delay After Setting Gain
Specifies the length of the delay to occur before the next acquisition once the gain is changed. This allows
the intensifier system to lock in to the new setting before acquisition proceeds.
Close
Closes the dialog box.
Set Camera Level and Gain (Acquire Menu)
Sets the intensified CCD camera's black level and video gain.
Drop-in: SETICCD
Use this command to change the ICCD camera's black level and video gain. The control of the black
level and video gain depends on the particular camera used. For some cameras, the black level and
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video gain can be set only by using the computer after control of the camera has been turned over the
computer. In some cases, you may be allowed to change the black level and video gain either manually
or with this command. However, some cameras do not support this command, and you must change the
black level and video gain manually.
Prior to using this command, you must install its drop-in, SETICCD, using the
Configure Drop-ins/Toolbars command in the Meta Imaging Series Administrator. MetaMorph Drop-in
Manager. You must also configure the intensifier using the Configure Intensifier Gain Control command
and specify that the camera is controlled by the computer.
Setting the Camera Level and Gain
To set the ICCD camera black level and gain, use the following procedure:
Step
Action
1
From the Acquire menu, choose Set Camera
Level and Gain. The Set CCD Level and Gain
dialog box will appear.
2
Select the desired black level value from the
Black Level slider.
3
Select the desired video gain value from the
Video Gain slider.
4
Choose Close.
Set Camera Level and Gain - Dialog Box Options
Black Level
Specifies the current black level value used by the intensified CCD camera. For some cameras, the black
level and video gain can be set only by using the computer after control of the camera has been turned over
the computer. In some cases, you may be allowed to change the black level and video gain either manually
or with this command. However, some cameras do not support this command and you must change the
black level and video gain manually.
Video Gain
Specifies the current video used by the intensified CCD camera. For some cameras, the black level and
video gain can be set only by using the computer after control of the camera has been turned over the
computer. In some cases, you may be allowed to change the black level and video gain either manually or
with this command. However, some cameras do not support this command and you must change the black
level and video gain manually.
Close
Closes the dialog box.
PI Video ICCD Settings (Acquire Menu)
Changes the ICCD operating temperature and resets the intensifier if it shuts off due to
overload. If you configured the system for direct gain control from the computer,
additional setting controls that appear on the controller front panel will also be available.
Drop-in: SETICCD
Use this command to reset the intensifier if it shuts itself down due to overload. You can also use the PI
Video ICCD Settings command to specify an operating temperature for the cooled ICCD (the default
setting is -10 degrees Centigrade).
If you specified direct control of the ICCD by the computer with the Configure Intensifier Gain Control
command, a dozen other setting controls will appear in the PI Video ICCD Settings dialog box. For the
most part, these correspond to controls that appear on the ICCD controller box, and include such options
as enabling or disabling Auto-Black Level, Automatic Gain Control, responsiveness to external triggers,
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Micro-Channel Plate (MCP) protection circuitry, and the like.
Prior to using this command, you must install its drop-in, SETICCD, using the
Configure Drop-ins/Toolbars command in the Meta Imaging Series Administrator. You must also
configure the intensifier using the Configure Intensifier Gain Control command.
Specifying the PI Video ICCD Settings
To specify the PI video ICCD settings, use the following procedure:
Step
Action
1
Follow the directions for configuring the
intensifier gain, selecting one of the PI Video
ICCD entries from the Intensifier Model dropdown list.
2
From the Acquire menu, select PI Video ICCD
Settings. The PI Video ICCD Settings dialog
box will appear.
3
If you want to change the operating
temperature of the ICCD, use the Temperature
Set Point spin box to specify the new
temperature. The default setting is
-10 degrees C.
4
If the intensifier has shut down due to
overload, choose Reset Intensifier If It Shut Off
Due to Overload. (Be sure all input to the
camera is off!)
5
If you selected control of the ICCD by the
control box (PI Video ICCD - Control Box)
when you used the Configure Intensifier Gain
Control command, you have finished. Now
skip to Step 9.
OR
If you selected direct control of the ICCD by
the computer (PI Video ICCD - Direct) when
you used the Configure Intensifier gain Control
command, you will see some additional
options in the PI Video ICCD Settings dialog
box. Continue to Step 6.
6
Depending on your experimental conditions,
select or clear the Advanced Settings check
boxes as necessary.
Note: If you are performing quantitative
densitometric or ratiometric analysis of your
images, you should leave Enable Automatic
Gain Control deselected.
7
If you are using an integrating ICCD, you can
specify the number of video frames to be
integrated by using Frames to Integrate.
8
If you need to reset Advanced Settings or
Frames to Integrate to the default values,
choose Reset to Defaults.
9
Choose Close to close the dialog box.
PI Video ICCD Settings - Dialog Box Options
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Temperature Set Point
Specifies the operating temperature of the cooled ICCD. The default setting is -10 degrees C.
Reset Intensifier If It Shut Off Due to Overload
If your ICCD becomes saturated due to an overload of input, it will shut down as a protective measure.
When this happens, you can reset it by choosing this command button.
Advanced Settings
These check boxes can be selected or cleared independently, thereby emulating the controls on the ICCD
controller box. These options will only be displayed if you selected PI Video ICCD - Direct as the Intensifier
Model in the Configure Intensifier Gain Control dialog box. These options include the following:
Enable Micro-Channel Plate Protection Circuitry (default = enabled)
Enable Gamma of 0.45 (default = disabled)
Enable Automatic Gain Control (default = disabled)
Enable Continuous (CW) Intensifier Mode (default = enabled)
Positive Polarity EXT Trigger (default = enabled)
Not EXT Trigger Enabled (default = enabled)
Enable Odd Field for Trigger and Integration (default = enabled)
Enable Any Field for Trigger and Integration (default = enabled)
Turn Off Auto-Black Level (default = enabled)
INVERT Valid Polarity (default = enabled)
Frames to Integrate
If you are using an integrating ICCD, this option specifies the number of video frames to be integrated. A
setting of 0 specifies no integration.
Reset to Defaults
Resets the Advanced Settings check boxes and Frames to Integrate spin box to their default settings.
Close
Closes the dialog box.
Nikon Microscope
The Nikon drop-in controls any Nikon automated microscope that uses the Nikon driver.
Drop-in: NIKON
The Nikon drop-in enables you to control the following:
•
Fluorescence settings
•
Transmitted light settings
•
Light path settings
•
Magnification settings
In addition, the drop-in lets you annotate an image with the Microscope settings, and log current settings.
Notes:
• The XY stage and Z focus are not controlled by this drop-in; they are controlled by the
normal Stage and Focus settings.
Before you install the Nikon drop-in, remove the SCOPE drop-in using the
Configure Drop-ins/Toolbar command in the Meta Imaging Series Administrator. These two drop-ins conflict
with each other.
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Using the Nikon Microscope
To use a Nikon microscope, use the following procedure:
Note: Some dialog box options in this procedure may not be enabled; this depends on
the hardware components you are using. The Record buttons are enabled only when you
are recording a journal.
Step
1
Action
On the Devices menu, select Nikon
Microscope. The Nikon Microscope dialog
box opens.
Note: On the left is a status list
of all your current settings. Use
the different tabs on the dialog
box to change these settings.
2
To configure the Fluorescence settings, click
the Fluor tab.
3
To open/close the fluorescence shutter,
check the Shutter box.
4
In the Filter Block group, select the
fluorescence filter cube.
5
To configure the Transmitted settings,
click the Trans tab.
6
Check the Lamp Shutter box to turn
on/off the Halogen (transmitted) lamp.
7
In the Condenser group, choose a
position for the condenser.
8
Use the Lamp Voltage slider to select
the lamp voltage.
9
Select the position of the Analyzer from
the Analyzer group.
10
To configure light path settings, click the
Light Path tab.
11
Select the position of the optical path
filter from the optical path group.
12
To configure magnification settings, click
the Magnification tab.
13
In the Objective group, select the
objective to use.
14
Use the Z Res slider to select the zoom
level.
15
If you want to annotate your image with
the status information under Current
Settings, use the Image Annotation
group. Select an image from the list
next to Image.
16
Click the Annotate Image with
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Microscope Settings button to annotate
your image.
17
Click the Configure Log button to select
the parameters you want to send to a
data log file.
18
To select a destination for the logged
information, click the Open Log button.
19
If you make any changes to your
microscope or its components, click the
Resync button to update your settings in
MetaMorph.
The Resync button will read the current
microscope settings and reconfigure the
dialog to reflect these settings.
20
Click Close to close the dialog box when
you are finished.
Nikon Microscope - Dialog Box Options
Current Settings
Fluorescence
The parameters in this group are related to the fluorescence shutter (shutter), and the filter block.
Transmitted
This group contains parameters related to the halogen lamp (shutter), condenser, and analyzer.
Light Path
This group contains parameters related to the optical path filter.
Magnification
These parameters represent the objective and the zoom level.
Fluor (Fluorescence) Tab
Trans (Transmitted) Tab
Light Path Tab
Magnification Tab
Image Annotation
Image
Selects an image to annotate with the microscope’s current settings.
Annotate Image with Microscope Settings
Annotate your image with the status information under Current Settings.
ReSync
Reads the current microscope settings and updates the dialog to reflect these settings.
Configure Log
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Allows the selection of image characteristics and data that are to be enabled or disabled from data logging.
Also allows a choice of whether column titles are to be included and if data are to be listed on a single line.
Open Log
Opens a data log file and/or a DDE link to an open spreadsheet application for logging data. This command
will change to F9: Log Data when a log file is open.
Close
Closes the dialog box.
Nikon Microscope Dialog Box Options - Fluorescence Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Shutter
The shutter check box is used to open/close the fluorescence shutter.
Record Shutter
Records the setting to a journal. This option is only enabled when recording a journal.
Filter Block
Selects the active magnification setting for the filter.
Record Filter
Records the setting to a journal. This option is only enabled when recording a journal.
Nikon Microscope Dialog Box Options - Transmitted Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Lamp Shutter
Turns the lamp shutter on/off. The lamp functions as a virtual shutter, and is turned on and off as requrired.
Record Lamp
Records the setting to a journal. This option is only enabled when recording a journal.
Condenser
Selects the active condenser filter.
Record Condenser
Records the setting to a journal. This option is only enabled when recording a journal.
Lamp Voltage
Adjusts the illumination intensity of the transmitted light source. When the shutter is open, changes in
intensity are instaneous; when the shutter is closed, intensity changes are applied when the shutter is
opened. Note: The displayed lamp voltage value provides a reference to which the voltage can be reset
either manually or using a journal.
Record Voltage
Records the setting to a journal. This option is only enabled when recording a journal.
Analyzer
Selects the position of the Analyzer.
Record Analyzer
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Records the setting to a journal. This option is only enabled when recording a journal.
Nikon Microscope Dialog Box Options - Light Path Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Optical Path
Selects the active optical path filter.
Record Default Path
Records the setting to a journal. This option is only enabled when recording a journal.
Nikon Microscope Dialog Box Options - Magnification Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Objective
Selects the active objective.
Record Objective
Records the setting to a journal. This option is only enabled when recording a journal.
Z Res
Selects the zoom level to the camera.
Record Z Res
Records the setting to a journal. This option is only enabled when recording a journal.
Olympus Microscope
The Olympus drop-in controls any Olympus automated microscope that uses the Olympus
driver.
Drop-in: OLYMPUS
The Olympus drop-in enables you to control the following:
•
Fluorescence settings
•
Bright Field settings
•
Transmitted light settings
•
Light path settings
•
Magnification settings
In addition, the drop-in lets you annotate an image with the Olympus settings, and log current settings.
Notes:
• The XY stage and Z focus are not controlled by this drop-in; they are controlled by the
normal Stage and Focus settings.
Before you install the OLYMPUS drop-in, remove the SCOPE drop-in using the
Configure Drop-ins/Toolbar command in the Meta Imaging Series Administrator. These two drop-ins conflict
with each other.
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Using the Olympus Microscope
To use an Olympus microscope, use the following procedure:
Note: Some dialog box options in this procedure may not be enabled; this depends on
the hardware components you are using. The Record buttons are enabled only when you
are recording a journal.
Step
1
Action
On the Devices menu, select Olympus
Microscope. The Olympus Microscope dialog
box opens.
Note: On the left is a status list
of all your current settings. Use
the different tabs on the dialog
box to change these settings.
2
To configure the Fluorescence settings, click
the Fluor tab.
3
To open/close the fluorescence shutter,
check the Shutter box.
4
In the Filter Cube group, select the
fluorescence filter cube.
5
To configure the Bright Field settings,
click the Bright Field tab.
6
In the Top Lens group, select a filter.
7
In the Condenser group, select a filter.
8
Select an aperture location from the
Aperture group.
9
To configure the Transmitted settings,
click the Trans tab.
10
Check the Halogen Lamp box to turn
on/off the halogen (transmitted) lamp.
11
Use the Lamp Voltage slider to select
the lamp voltage.
12
Check the DIA Shutter box to turn on/off
the DIA shutter.
13
Select the position of ND filter wheel 1
from the ND Filter Wheel 1 group.
14
Select the position of ND filter wheel 2
from the ND Filter Wheel 2 group.
15
To configure light path settings, click the
Light Path tab.
16
Select the camera filter to use from the
Camera Port group.
17
To configure magnification settings, click
the Magnification tab.
18
In the Objective Lens group, select the
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objective to use.
19
Use the Z Res slider to select the zoom
level.
20
If you want to annotate your image with
the status information under Current
Settings, use the Image Annotation
group. Select an image from the list
next to Image.
21
Click the Annotate Image with
Microscope Settings button to annotate
your image.
22
Click the Configure Log button to select
the parameters you want to send to a
data log file.
23
To select a destination for the logged
information, click the Open Log button.
24
If you make any changes to your
microscope or its components, click the
Resync button to update your settings in
MetaMorph.
The Resync button will read the current
microscope settings and reconfigure the
dialog to reflect these settings.
25
Click Close to close the dialog box when
you are finished.
Olympus Dialog Box Options - Main
Current Settings
Fluorescence
The parameters in this group are related to the fluorescence shutter (shutter) and the filter cube.
Bright Field
The parameters in this group are related to the top lens, aperture and condenser.
Transmitted
This group contains parameters related to the halogen lamp (shutter), two neutral density filter
wheels, and the DIA shutter.
Light Path
This group contains parameters related to the camera port filter.
Magnification
These parameters represent the objective lens.
Fluor (Fluorescence) Tab
Bright Field Tab
Trans (Transmitted) Tab
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Light Path Tab
Magnification Tab
Image Annotation
Image
Selects an image to annotate with the microscope’s current settings.
Annotate Image with Microscope Settings
Annotate your image with the status information under Current Settings.
ReSync
Reads the current microscope settings and updates the dialog to reflect these settings.
Configure Log
Allows the selection of image characteristics and data that are to be enabled or disabled from data logging.
Also allows a choice of whether column titles are to be included and if data are to be listed on a single line.
Open Log
Opens a data log file and/or a DDE link to an open spreadsheet application for logging data. This command
will change to F9: Log Data when a log file is open.
Close
Closes the dialog box.
Olympus Microscope Dialog Box Options - Fluorescence Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Shutter
The shutter check box is used to open/close the fluorescence shutter.
Record Shutter
Records the setting to a journal. This option is only enabled when recording a journal.
Filter Cube
Selects the active filter cube.
Record Cube
Records the setting to a journal. This option is only enabled when recording a journal.
Olympus Microscope Dialog Box Options - Bright Field Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Top Lens
Selects the active top lens filter.
Record Top Lens
Records the setting to a journal. This option is only enabled when recording a journal.
Aperture
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Adjusts the opening size of the aperture.
Record Aperture
Records the aperture setting to a journal. This option is only enabled when recording a journal.
Condenser
Selects the active condenser filter.
Record Condenser
Records the setting to a journal. This option is only enabled when recording a journal.
Olympus Dialog Box Options - Transmitted Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Halogen Lamp
Turns the halogen (transmitted lamp) on/off.
Record Lamp
Records the lamp selection setting to a journal. This option is only enabled when recording a journal.
Lamp Voltage
Adjusts the illumination intensity of the transmitted light source. When the shutter is open, changes in
intensity are instaneous; when the shutter is closed, intensity changes are applied when the shutter is
opened. Note: The displayed lamp voltage value provides a reference to which the voltage can be reset
either manually or using a journal.
Record Voltage
Records the lamp voltage setting to a journal. This option is only enabled when recording a journal.
DIA Shutter
Opens/Closes the DIA shutter.
Filter Wheel 1
In this group, choose an ND filter for transmitted light from ND wheel 1.
Filter Wheel 2
In this group, choose an ND filter for transmitted light from ND wheel 2.
Record ND1/2
Records the setting to a journal. This option is only enabled when recording a journal.
Olympus Dialog Box Options - Light Path Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Camera Port
Selects the active camera port.
Record Camera Port
Records the setting to a journal. This option is only enabled when recording a journal.
Olympus Dialog Box Options - Mag Tab
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Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Objective Lens
Selects the active objective.
Record Objective
Records the setting to a journal. This option is only enabled when recording a journal.
Zeiss MTB Microscope
The ZEISSMTB drop-in controls any Zeiss automated microscope that uses the Zeiss MTB
driver.
Drop-in: ZEISSMTB
The Zeiss MicroToolBox (MTB) driver and ZeissMTB drop-in enable you to control the following:
•
Fluorescence settings
•
Transmitted light settings
•
Light path settings
•
Magnification settings
In addition, the drop-in lets you annotate an image with the Zeiss settings, and log current settings.
Notes:
• The XY stage and Z focus are not controlled by this drop-in; they are controlled by the
normal Stage and Focus settings.
• The Zeiss driver update enables support for switching between the front and back camera
ports on microscopes that are configured with dual motorized ports.
• The Zeiss MTB device driver optionally auto-lowers the stage before any objective change.
This feature can be enabled from the device configuration dialog for the driver.
• Before you install the ZEISSMTB drop-in, remove the SCOPE drop-in using the Configure
Drop-Ins dialog box in the Meta Imaging Series Administrator. These two drop-ins conflict
with each other.
Using the Zeiss MTB Microscope
To use a Zeiss MTB microscope, use the following procedure:
Note: Some dialog box options in this procedure may not be enabled; this depends on
the hardware components you are using. The Record buttons are enabled only when you
are recording a journal.
Step
1
Action
On the Devices menu, select Zeiss MTB
Microscope. The Zeiss MTB Microscope
dialog box opens.
Note: On the left is a status list of all your
current settings. Use the different tabs on
the dialog box to change these settings.
2
To configure the Fluorescence settings, click
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the Fluor tab.
3
To open/close the fluorescence shutter,
check the Shutter box.
4
In the Reflector Turret group, select the
fluorescence filter cube.
5
In the Exciter Filter group, select the
wavelength from the excitation filter wheel.
6
In the Barrier Filter group, select the
emission filter for fluorescence you wish to
use from the Barrier Filter wheel.
7
To configure the Transmitted settings,
click the Trans tab.
8
Check the Halogen Lamp box to turn
on/off the Halogen (transmitted) lamp.
9
In the Filter Wheel 1 group, choose an
ND filter for transmitted light from ND
wheel 1.
10
In the Filter Wheel 2 group, choose an
ND filter for transmitted light from ND
wheel 2.
11
To configure the light path settings, click
the Light Path tab.
12
In the Lower Prism group, select the
lower prism filter to use.
13
In the Upper Prism group, select the
upper prism filter to use.
14
To configure magnification settings, click
the Magnification tab.
15
In the Objective Nosepiece group, select
the objective you want to use.
16
In the Zoom group, select the zoom
level.
17
If you want to annotate your image with
the status information under Current
Settings, use the Image Annotation
group. Select an image from the list
next to Image.
18
Click the Annotate Image with
Microscope Settings button to annotate
your image.
19
Click the Configure Log button to select
the parameters you want to send to a
data log file.
20
To select a destination for the logged
information, click the Open Log button.
21
If you make any changes to your
microscope or its components, click the
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Resync button to update your settings in
MetaMorph.
The Resync button will read the current
microscope settings and reconfigure the
dialog to reflect these settings.
22
Click Close to close the dialog box when
you are finished.
Zeiss MTB Microscope Dialog Box Options - Main
Current Settings
Fluorescence
The parameters in this group are related to the fluorescence shutter (shutter), the reflector turret
(filter cube), and the excitation and barrier (emission) filter wheels.
Transmitted
This group contains parameters related to the halogen lamp (shutter) and two neutral density filter
wheels.
Light Path
The objects in this group are related to the two prisms (upper and lower) that direct light to either
the documentation port (cameras) or to the eyepieces, and when light is going to the
documentation port, to the appropriate camera port on that module.
Magnification
These parameters represent the objective nosepiece and the zoom.
Fluor (Fluorescence) Tab
Trans (Transmitted) Tab
Light Path Tab
Magnification Tab
Image Annotation
Image
Selects an image to annotate with the microscope’s current settings.
Annotate Image with Microscope Settings
Annotate your image with the status information under Current Settings.
ReSync
Reads the current microscope settings and updates the dialog to reflect these settings.
Configure Log
Allows the selection of image characteristics and data that are to be enabled or disabled from data logging.
Also allows a choice of whether column titles are to be included and if data are to be listed on a single line.
Open Log
Opens a data log file and/or a DDE link to an open spreadsheet application for logging data. This command
will change to F9: Log Data when a log file is open.
Close
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Closes the dialog box.
Zeiss MTB Microscope Dialog Box Options - Fluorescence Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Shutter
The shutter check box is used to open/close the fluorescence shutter.
Record State
Records the setting to a journal. This option is only enabled when recording a journal.
Reflector Turret
Selects a reflector turret.
Record Refractor
Records the setting to a journal. This option is only enabled when recording a journal.
Exciter Filter
Selects the wavelength you want to use from the excitation filter wheel.
Record Exciter
Records the setting to a journal. This option is only enabled when recording a journal.
Barrier Filter
This group is used for selecting the emission filter for fluorescence you want to use from the Barrier Filter
Wheel.
Record Barrier
Records the setting to a journal. This option is only enabled when recording a journal.
Zeiss MTB Microscope Dialog Box Options - Transmitted Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Halogen Lamp
Turns the halogen (transmitted lamp) on/off.
Record Lamp
Records the setting to a journal. This option is only enabled when recording a journal.
Filter Wheel 1
In this group, choose an ND filter for transmitted light from ND wheel 1.
Filter Wheel 2
In this group, choose an ND filter for transmitted light from ND wheel 2.
Record ND1/2
Records the setting to a journal. This option is only enabled when recording a journal.
Zeiss MTB Microscope Dialog Box Options - Light Path Tab
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Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Lower Prism
In the Lower Prism group (the lower prism is the output prism), select where the light should be directed.
For example, 100% doc would send all of the light to the camera, while 100% visual would send it all to the
eyepiece.
Record Lower Prism
Records the setting to a journal. This option is only enabled when recording a journal.
Upper Prism
In the Upper Prism group, select which camera will receive the image.
Record Upper Prism
Records the setting to a journal. This option is only enabled when recording a journal.
Zeiss MTB Microscope Dialog Box Options - Magnification Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Objective Nosepiece
Selects the active objective.
Record Objective
Records the setting to a journal. This option is only enabled when recording a journal.
Zoom
Select the zoom to the camera.
Record Zoom
Records the setting to a journal. This option is only enabled when recording a journal.
Leica DMR Microscope
The LEICADM drop-in controls any Leica automated microscope that uses the Leica DMR
driver.
Drop-in: LEICADM
The Leica drop-in enables you to control the following:
•
Fluorescence settings
•
Transmitted light settings
•
Light path settings
•
Magnification settings
In addition, the drop-in lets you annotate an image with the Leica settings, and log current settings.
Notes:
• The XY stage and Z focus are not controlled by this drop-in; they are controlled by the
normal Stage and Focus settings.
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• Before you install the LEICADM drop-in, remove the SCOPE drop-in using the Configure
Drop-Ins dialog box in the Meta Imaging Series Administrator. These two drop-ins conflict
with each other.
Using the Leica DMR Microscope
To use the Leica DMR, use the following procedure:
Note: Some dialog box options in this procedure may not be enabled; this depends on
the hardware components you are using. The Record buttons are enabled only when you
are recording a journal.
Step
1
Action
On the Devices menu, select Leica DMR
Microscope. The Leica DMR Microscope
dialog box opens.
Note: On the left is a status list of all your
current settings. Use the different tabs on
the dialog box to change these settings.
2
To configure the Fluorescence settings, click
the Fluor tab.
3
To open/close the fluorescence shutter,
check the Shutter box.
4
In the Filter Cube group, select the
fluorescence filter cube.
5
To configure the Transmitted settings,
click the Trans tab.
6
Check the Top Lens box to turn on/off
the top lens.
7
Use the Lamp Voltage slider to select
the lamp voltage.
8
Use the Field Diaphragm slider to select
the field diaphragm position.
9
Use the Aperture Diaphragm slider to
select the aperture diaphragm position.
10
To configure the Light Path settings, click the
Light Path tab.
11
In the Prism #1 group, select a port to
receive light from the prism.
12
In the Prism #2 group, select a port to
receive light from the prism.
13
In the Tube Optic group, select the
position of the tube optic.
14
In the Beam Splitter group, select the
position of the beam splitter.
15
To configure magnification settings, click
the Magnification tab.
16
In the Objective Turret group, select the
objective you want to use.
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17
In the Zoom group, select the zoom
level.
18
If you want to annotate your image with
the status information under Current
Settings, use the Image Annotation
group. Select an image from the list
next to Image.
19
Click the Annotate Image with
Microscope Settings button to annotate
your image.
20
Click the Configure Log button to select
the parameters you want to send to a
data log file.
21
To select a destination for the logged
information, click the Open Log button.
22
If you make any changes to your
microscope or its components, click the
Resync button to update your settings in
MetaMorph.
The Resync button will read the current
microscope settings and reconfigure the
dialog to reflect these settings.
23
Click Close to close the dialog box when
you are finished.
Leica DMR Dialog Box Options - Main
Current Settings
Fluorescence
The parameters in this group are related to the fluorescence shutter (shutter) and the filter cube.
Transmitted
This group contains parameters related to the top lens, lamp voltage, field diaphragm and aperture
diaphragm.
Light Path
This group contains parameters related to the two prisms (upper and lower) that direct light to
either the documentation port (cameras) or to the eyepieces, as well as the tube optic and beam
splitter.
Magnification
These parameters represent the objective nosepiece and the zoom level.
Fluor (Fluorescence) Tab
Trans (Transmitted) Tab
Light Path Tab
Magnification Tab
Image Annotation
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Image
Selects an image to annotate with the microscope’s current settings.
Annotate Image with Microscope Settings
Annotate your image with the status information under Current Settings.
ReSync
Reads the current microscope settings and updates the dialog to reflect these settings.
Configure Log
Allows the selection of image characteristics and data that are to be enabled or disabled from data logging.
Also allows a choice of whether column titles are to be included and if data are to be listed on a single line.
Open Log
Opens a data log file and/or a DDE link to an open spreadsheet application for logging data. This command
will change to F9: Log Data when a log file is open.
Close
Closes the dialog box.
Leica DMR Microscope Dialog Box Options - Fluor Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Shutter
The shutter check box is used to open/close the fluorescence shutter.
Record State
Records the setting to a journal. This option is only enabled when recording a journal.
Filter Cube
Selects the active filter cube.
Record Cube
Records the setting to a journal. This option is only enabled when recording a journal.
Leica DMR Microscope Dialog Box Options - Trans Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Top Lens
Opens and closes the top lens.
Record Top Lens
Records the setting to a journal. This option is only enabled when recording a journal.
Lamp Voltage
In this group, choose an ND filter for transmitted light from ND wheel 1.
Record Voltage
Records the setting to a journal. This option is only enabled when recording a journal.
Field Diaphragm
Adjusts the position of the field diaphragm.
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Drop-in Commands
Record Field
Records the setting to a journal. This option is only enabled when recording a journal.
Aperture Diaphragm
Adjusts the position of the aperture diaphragm.
Record Aperture
Records the setting to a journal. This option is only enabled when recording a journal.
Leica DMR Microscope Dialog Box Options - Light Path Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Prism #1
Selects which port the light should be directed to. For example, select Visual Port to send all of the light to
the eyepiece.
Record Prism #1
Records the setting to a journal. This option is only enabled when recording a journal.
Prism #2
Selects which port the light should be directed to. For example, select Visual Port to send all of the light to
the eyepiece.
Record Prism #2
Records the setting to a journal. This option is only enabled when recording a journal.
Tube Optic
Selects the position of the tube optic.
Record Tube Optic
Records the setting to a journal. This option is only enabled when recording a journal.
Beam Splitter
Selects the position of the beam splitter.
Record Beam Splitter
Records the setting to a journal. This option is only enabled when recording a journal.
Leica DMR Microscope Dialog Box Options - Magnification Tab
Note: The options listed on this tab will vary depending on the configuration of your
microscope.
Objective Turret
Selects the active objective.
Record Objective
Records the setting to a journal. This option is only enabled when recording a journal.
Zoom
Select the zoom to the camera.
Record Zoom
Records the setting to a journal. This option is only enabled when recording a journal.
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Immersion Mode
Immersion Mode
Enables you to select any of the immersion objectives as defined in the Leica software.
Dry Mode
Enables you to select any of the dry objectives as defined in the Leica software.
Record Immersion Mode
Records the setting to a journal. This option is only enabled when recording a journal.
Linkam MDS600/TMS93 Stage
Controls the settings and configuration to create the initial setup to use the Linkam MDS
600 Motor-Driven, Heated Stage, and the Linkam TMS 93 Temperature Programmer, which
provides an interface for programmatic temperature control of the Linkam MDS 600 MotorDriven Stage. The Heating Sequence dialog box enables you to make settings and
schedule journals to control the Linkam TMS 93 Temperature Programmer.
Drop-in: LINKAM
Use this dialog box to make the initial settings for the Linkam MDS 600 Motor-Driven Stage. This dialog
box controls the activation and setting of the stage heating unit and the Liquid Nitrogen Pump (LNP)
stage cooling unit. These two units combine to provide precise stage temperature control and the ability
to quickly and accurately raise or lower the temperature of the stage. The heating unit can be used
without the LNP Pump, but cooling time will be longer. The heating unit can be set to hold a specific
temperature, or to raise or lower the stage temperature. The LNP Pump enables you to more easily
maintain a specific temperature or to raise or lower the temperature at a very precise rate.
Using the Linkam MDS600/TMS93 Stage
To use the controls on the primary area of the Linkam MDS600/TM93 Stage dialog box, complete
the following steps.
Step
Action
1
From the Devices menu, click Linkam
MDS600/TM93 Stage, the Linkam
MDS600/TM93 Stage dialog box opens.
2
Click the appropriate arrow button to change
the stage position, or click Go To Origin to
move the stage to the Origin position set in
either the Move Stage to Absolute Position
or the Move Stage to Relative Position
dialog box.
3
Click the appropriate radio button in the
Heater box. Click On to turn on the heater,
Off to turn off the heater, or click Hold to hold
the currently set temperature.
4
Observe the current stage temperature in
Celsius next to Temp:
5
In the LNP Pump box, click the appropriate
button to either turn on or turn off the LNP
pump.
6
If the dialog box is minimized, click More to
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maximize the dialog box and access the
Settings. Click Less to minimizes the dialog
box.
7
Click Close after all settings are complete or
to close the dialog box.
Setting the Linkam MDS600/TMS93 Stage
To make settings in the Linkam MDS600/TM93 Stage dialog box, complete the following steps.
Step
Action
1
From the Devices menu, click Linkam
MDS600/TM93 Stage, the Linkam
MDS600/TM93 Stage dialog box opens.
2
In the Stage increment box, type or select
the stage increment value to specify the
distance you want the stage to move for a
single click on any arrow button.
3
In the Stage Temperature box, type or select
using the small arrow buttons or the slider
control, the temperature that you want to
achieve or maintain on the stage.
4
In the Heating Rate box, type or select using
the small arrow buttons or the slider control,
the heating rate in degrees Celsius that you
want to achieve or that you want to maintain
on the stage.
5
In the LNP Pump Speed box, type or select
using the small arrow buttons or the slider
control, the pump speed at which you want
to operate the LNP Pump.
6
In the LNP Pump Mode box, click Manual to
operate the LNP Pump manually, or
Automatic to operate the pump
automatically. In manual mode, you must
set the LNP Pump speed to control how
much cooling you are delivering to the stage.
In automatic mode, the controller regulates
the LNP pump speed to maintain or change
the temperature.
Linkam MDS0600/TMS93 Stage - Dialog Box Options
(Arrow Keys)
Moves the stage to the selected position. Use either the Move Stage to Absolute Position or Move Stage to
Relative Position dialog box to specify the limits of movement for this stage.
Go To Origin
Moves the stage directly to the predefined origin position. Use either the Move Stage to Absolute Position or
Move Stage to Relative Position dialog box to set the location of the Origin position.
Heater
Controls the heater unit in the stage. Use these controls to turn the heat on or off, or hold the heat at the
current temperature.
On – Turns on the stage heating unit.
Off – Turns off the stage heating unit.
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Hold – Holds the stage temperature at the current temperature setting.
Temp
Indicates the current stage temperature in degrees Celsius.
LNP Pump
Specifies whether the Liquid Nitrogen Pump is off or on.
Less<</More>>
Minimizes (collapses) or Maximizes (expands) the dialog box.
Close
Closes the dialog box.
Settings
Stage Increment
Specifies the amount of increment for stage movements when using the arrow buttons in this dialog
box. This value is based on values set in either the Move Stage to Absolute Position or Move
Stage to Relative Position dialog box.
Stage Temperature
Specifies the target temperature of the stage in degrees Celsius.
Heating Rate
Specifies the rate in degrees Celsius at which you want the specimen to reach the target
temperature.
LNP Pump Speed
Specifies the pump speed to control the liquid nitrogen’s rate of circulation.
LNP Pump Mode
Specifies the liquid nitrogen pump mode.
Manual – Causes the pump to run continuously when LNP Pump is set to On. To control
the temperature in manual mode, you must adjust the LNP Pump Speed manually to
obtain the correct amount of cooling to maintain the desired temperature or to raise or
lower the temperature.
Automatic – Enables the LNP Pump to be controlled by the program. The controller determines
when the LNP Pump needs to run to maintain the current temperature or to raise or lower the
temperature.
Heating Sequence
Defines one or more heating sequences for the Linkam MDS600 Heated Stage and sends
commands to the TMS93 Stage Controller to complete the sequences.
Drop-in: LINKAM
Use this dialog box to specify sequential heating and cooling sequences for the Linkam MDS600 heated
stage. Sequences that you specify in this dialog box are sent to the TMS93 Stage Controller for
execution. You can create sequences that contain as many as 9,999 steps. Each step can initiate a
journal to run either at the beginning of the ramping of the heating sequence, or at the end of the
temperature holding interval. Journals can be either run once during a heating sequence step, or run
repeatedly during a single step at an interval that you specify as the Journal Rate in seconds for a
maximum 32,767 seconds interval.
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Setting up a Heating Sequence
To configure a heating sequence for the Linkam MDS600 Heated Stage and the TMS93
Stage Controller, complete the following procedure.
Step
Action
1
From the Devices menu, click Heating
Sequence. The Heating Sequence dialog
box opens.
2
If you have a prepared heating sequence
that you want to use, click Load Sequence to
open the Select Sequence dialog box, then
select the sequence that you want to use
and click Open. Use the remaining steps to
modify your sequence as needed. If you are
creating a new sequence, skip this step.
3
In the Loops in Sequence dialog box, type or
select the number of loops that you want to
occur in your heating sequence.
4
In the # of Steps in Sequence box, type or
select the number of steps that you want to
include in your sequence.
5
If you are running one or more journals in
conjunction with any step(s), in the Journal
Acquisition Style box, select the option that
initiates your journal at the appropriate time.
Click Run Journal during Ramping if you
want to start your journal at same time that
ramping starts; click Run Journal after
Holding if you want to start your journal after
the Holding Time is complete.
6
For each sequence step that you have
enabled, ensure that the activation box is
checked.
Note: You can uncheck this box to
temporarily exclude this step from the
sequence, without loosing the setting for that
step.
7
In the Heating Rate box, type or select the
appropriate heating rate in degrees Celsius
per minute.
8
In the Target Temperature box, type or
select the appropriate target temperature
that you want to attain for your sample.
9
In the Holding Time box, type or select the
time in seconds for which you want to hold
the target temperature.
10
Click Select Journal to assign a journal to be
run in conjunction with this step.
11
Click Run Once if you will be running your
selected journal only one time in conjunction
with this step.
12
In the Journal Rate box, type or select the
reoccurrence rate in seconds.
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13
After you have configured your heating
sequence, click Save Sequence to save it to
a Save Sequence file (.hss)
14
Click Run Sequence to run the heating
sequence that you configured or loaded.
Heating Sequence - Dialog Box Options
# of Loops in Sequence
Specifies the number of loops in the sequence that you want to occur during your experiment.
Sequence
Enables you to specify the attributes for the heating sequence.
#Steps in Sequence
Specifies the number of heating steps that you want to occur during each loop in the heating sequence. A
maximum of 9,999 steps can be included in the heating sequence.
Journal Acquisition Style
Specifies whether the journal that you selected to run with this heating sequence step is to be run during the
heating ramp-up or down or once the sequence step has completed the specified holding time.
Run Journal during Ramping
Causes your selected journal to be run in conjunction with ramping from the previous temperature to the
target temperature. The journal is started when ramping begins.
Run Journal after Holding
Causes your selected journal to be run at the end of the specified holding time.
#
Indicates the step number in the sequence.
(checkbox)
When checked, specifies that the step be included in the sequence. Uncheck this box to exclude the step
from the sequence.
Heating Rate [C/min]
Specifies the rate in degrees Celsius per minute at which the temperature of the stage and sample is raised
or lowered.
Target Temperature [C]
Specifies the temperature in degrees Celsius to which you want to raise or lower the temperature of the
sample or stage.
Holding Time
Specifies the amount of time that the sample and stage will be kept at the target temperature.
Select Journal
Selects the journal that you want to run in conjunction with the associated step.
Journal Name
Indicates the name of the journal that you selected to be run in conjunction with this specific heating
sequence step.
Run Once
When checked, specifies that the selected journal is to be run only once in conjunction with the associated
step. Uncheck this box to enable you to specify the number of times to run the journal.
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Journal rate [sec]
Specifies the number of seconds between the repeated initiation of the journal that you selected to run in
conjunction with a specific step in the heating sequence. The repeat interval can be set for a maximum of
32,767 seconds.
Save Sequence
Opens the Save Sequence File dialog box. Use this option to save detailed heating sequences that you
plan to reuse or to save and modify for future use. Type the name that you want to assign to Sequence File
in the file name box, and click Save.
Load Sequence
Opens the Load Sequence File dialog box. Use this option to open and load a previously saved Sequence
File.
Run Sequence
Runs the currently loaded sequence.
Cancel
Closes the Heating Sequence dialog box.
Kodak MotionCorder (Devices Menu)
Acquires and plays back images with the Kodak MotionCorder digital video device.
Drop-in: KODAK
Use this command in either of two ways: Live or Playback. The Live mode configures acquisition for the
Kodak MotionCorder and acquires images using the specified acquisition rate and frame size. Acquired
images can then be played back on the MotionCorder using the Playback buttons in the same manner
as the buttons on a VCR. However, the transfer of images from the camera controller into MetaMorph for
processing and analysis requires that you follow one of two possible additional procedures. In one
method, you first must create a journal to step the controller forward by one frame (see the "Creating a
Journal for Image Transfer" procedure). You will then need to "loop" the journal by a number of iterations
appropriate to the number of frames you want to transfer (see the "Running the Image Transfer Journal"
procedure). In the second method, you will need to use the Kodak Readcam program which is supplied
with your MotionCorder. This program will download the images from the MotionCorder via your
computer's SCSI interface.
Kodak MotionCorder Procedures
Acquiring Images
Playing Back Images
Creating a Journal for Image Transfer
Running the Image Transfer Journal
Configuring Communications
Acquiring Images with the Kodak MotionCorder
To configure acquisition and then acquire images with the Kodak MotionCorder, use the following
procedure. Before you start, however, you should first create a Data Stream setting with a serial
communications component by using the Install and Configure Devices command (Devices
menu).
Step
Action
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1
From the Devices menu, choose Kodak
MotionCorder. The Kodak MotionCorder
dialog box will appear.
2
If you have not already done so, configure
the serial communications between the
camera controller and your computer. You
should only have to perform this routine
once.
3
Select Live from the Playback/Live radio
button group.
4
Use the Acquisition Rate drop-down list to
configure the acquisition rate for time-lapse
recording. This does not need to be the
same as the display rate you intend to use
for playback (the latter typically being the 30
frames/s "video" rate).
5
Use the Acquisition Size drop-down list to set
the frame size for acquisition.
Note: Depending on the acquisition rate you
selected in Step 4, the camera may not be
able to acquire frames above a certain size.
6
If you want to adjust the linearity of the
relationship between pixel intensity and the
video signal amplitude, adjust the gamma
value by selecting from the Gamma
Adjustment drop-down list.
The appropriate value will depend on the
characteristics of the specimen, but, for most
part, a value at or near 1.0 will be sufficient.
7
If you are using an illumination setting ,
select the setting from the Illumination dropdown list. Otherwise, select "[None]."
8
If you want to specify an acquisition region,
choose Set Acq Rgn. The Set Acquisition
Region dialog box will appear, and a fullframe image will be acquired and displayed.
AND
Use the Rectangular Region Tool to draw a
region on the image. Then choose Use
Active Region. This region will be used for all
subsequently acquired image frames.
9
Click the Record Ready button to put the
camera controller into the ready state.
The Record Ready button will become the
Cancel button, which will allow you terminate
the recording.
10
When you are ready to record, click Trigger
Record.
The MotionCorder will acquire frames into
the MotionCorder controller until all memory
is filled or until you choose Cancel.
11
Choose Close to close the dialog box.
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Playing Back Images with the Kodak MotionCorder
To play back the current image data recorded by the MotionCorder, use the following procedure.
Note, however, that this will only display the frames. If you want to save the frames in a stack file
for subsequent processing and analysis, you will need to follow the procedures for creating and
then running an image transfer journal.
Step
Action
1
From the Devices menu, choose Kodak
MotionCorder. The Kodak MotionCorder
dialog box will appear.
2
Select Playback from the Playback/Live radio
button group.
3
Select a playback rate from the Display Rate
drop-down list. Typically, this will be 30fps.
4
Use the buttons in the Playback option group
to control the playback of the image frames.
You can play in reverse, stop, play forward,
step backward, and step forward by clicking
the appropriate button. You can go to a
specific frame by selecting the frame number
with the Go to Frame spin box and clicking
the Go to Frame command button.
Creating a Journal for Image Transfer
To transfer images from the MotionCorder controller to MetaMorph, you will first need to create a
journal that steps the camera controller forward by one step. You will then need to run the journal
in a loop.
To create the stepping journal, use the following procedure:
Step
Action
1
From the Devices menu, choose Kodak
MotionCorder. The Kodak MotionCorder
dialog box will appear.
2
From the Journal menu, choose Start
Recording. The MetaMorph Imaging System
title bar will display the message
"[Recording]."
3
From the first part of the Dest image selector,
choose Add To.
AND
In the second part, select Specified. Then
type a name for the image in the Specify
Image Name dialog box that appears and
choose OK.
4
In the Kodak MotionCorder dialog box,
choose Acquire.
5
Choose the Step Forward button (it will be
marked with an asterisk).
6
From the Journal menu, choose Stop
Recording. The Save Journal As dialog box
will appear.
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7
Type the desired file name for the journal in
the File Name text box, such as "Kodak."
MetaMorph will assign the file extension ".jnl"
to your file name. If necessary, use the Save
In list or Up One Level button to locate the
appropriate drive and folder.
AND
Choose Save.
8
When the Record Journal dialog box
appears, choose No to skip adding the newly
created journal to the current taskbar.
Running the Image Transfer Journal
To run the stepping journal in a loop, use the following procedure:
Step
Action
1
From the Kodak MotionCorder dialog box's
Display Rate drop-down list, select 30fps.
2
Use the Go to Frame spin box and command
button, or the Step Forward button, to go to
the specific frame at which you want to start
image transfer.
3
From the Journal menu, choose Loop a
Journal. The Loop a Journal dialog box will
appear.
4
Select the desired number of loops using the
Number of Loops text box or spin control.
This should correspond to the number of
frames you want to transfer from the
MotionCorder.
5
If you want to confirm each loop during
playback, select Confirm Each Loop.
The message "Continue Looping Journal?"
will appear before each loop during playback,
to which you can choose Yes or No.
6
Choose Press to Select the Journal to Loop
to select the journal that you want looped.
The Select Journal dialog box will appear.
AND
Select the journal file. If necessary, use the
Look In list or Up One Level button to locate
the correct drive and folder. Then choose
Open.
The journal's file name will be displayed in
the Loop a Journal dialog box.
7
If you want to specify an interval between
loops, select Loop on Interval.
AND
Select the length and type of interval using
the text box and drop-down list next to Loop
on Interval.
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8
If you want to specify a prompt on playback
for the number of loops, select On Journal
Playback, Prompt for Number of Loops.
The default prompt, "How many times do you
want to loop the journal?" is displayed in the
text box below this option. You can change
the text if you wish.
9
To loop the stepper journal, choose
Continue. The Loop Control Panel dialog
box will appear while the journal is looping.
10
When you have finished, the MotionCorder
image data will be saved as a MetaMorph
stack file.
Configuring Communications for the Kodak MotionCorder
To configure serial communications between the camera controller and your computer, use the
following procedure. Note, however, that this procedure is best left to your MDC representative,
and, once performed, these settings should not be altered without explicit instruction from your
representative.
Step
Action
1
In the Kodak MotionCorder dialog box,
choose More >> to expand the lower portion
of the dialog box.
2
To select the Data Stream setting for
handling serial communications, choose
Select.
3
To configure the serial port parameters,
choose Configure. The Kodak MotionCorder
Configuration Options dialog box will appear.
4
Make your selections, as required, from the
Comm. Port, Baud Rate, Parity, Data Bits,
and Stop Bits configuration options.
When you have finished, choose OK to
return to the Kodak MotionCorder dialog box.
5
If required, use the Serial Delay (ms) spin
box to configure a delay in the transmission
of commands to the camera controller. This
may be necessary to adjust for overly fast
rates of transmission that might otherwise be
missed by the controller.
6
When you have finished, you can choose
<< Less to condense the Kodak
MotionCorder dialog box.
Kodak - Dialog Box Options
Kodak MotionCorder
Set Acquisition Region
Kodak MotionCorder - Dialog Box Options
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Display Rate
Sets the display rate for playback of the currently acquired images.
Acquisition Rate
Sets the acquisition rate for time-lapse recording with the Kodak MotionCorder.
Acquisition Size
Sets the frame size for acquisition.
Gamma Adjustment
Adjusts the linearity of the response of the camera (i.e., the relationship between the amplitude of the
camera output signal and image pixel intensities).
Illumination
Selects the Illumination setting associated with your external shutter. If you are not using a shutter, select
"[None]."
Playback / Live Selector (radio buttons)
Selects between the playback and acquisition modes. When you select Playback, the video output will
display the current recorded image data, and the Playback option group buttons will be functional. When you
select Live, the video output will display the current live video image at the current frame size and frame
acquisition rate.
Record Ready
Puts the MotionCorder camera controller into the ready state. When you choose this button, it will become
the Cancel button.
Trigger Record
Initiates recording of images to the Kodak MotionCorder controller until all memory is filled, or until you
choose Cancel.
Playback Reverse Button
In Playback mode, this button plays the image frames backwards from the MotionCorder controller.
Playback will continue until the first frame is reached or until you press the Stop Button. If nothing has been
recorded, only "noise" will be displayed.
Stop Button
Stops playback of image frames from the camera controller.
Playback Forward Button
In Playback mode, this button plays the image frames forwards from the MotionCorder controller. Playback
will continue until the last frame is reached or until you press the Stop Button. If nothing has been recorded,
only "noise" will be displayed.
Step Left Button
Jumps the display back by one image frame.
Step Right Button
Jumps the display forward by one image frame.
Go to Frame
Jumps the display to a specified frame. The spin box selects the image frame number, and the command
button jumps the display to that frame.
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Acquire
Initiates image frame transfer from the video output of the camera controller to MetaMorph. This button is
used only during recording of the "stepper" journal, which must be looped to transfer MotionCorder image
data to MetaMorph for processing and analysis (see the "Creating a Journal for Image Transfer" procedure).
Dest
Selects an image window for transfer of the image frames being acquired, played back, or transferred to
MetaMorph.
Set Acq Rgn
Specifies an acquisition subregion. Choosing this button will acquire a full-frame image, on which you can
draw the acquisition subregion with the Rectangular Region Tool.
More >>
Expands the dialog box downwards, revealing the options used for configuring serial communications.
<< Less
Condenses the dialog box.
Select / Unselect
Selects the available Data Stream setting for use in handling serial communications. After you choose this
button, it will become the Unselect button, which you can click to "unload" the setting.
Configure
Opens the Kodak MotionCorder Configuration Options dialog box, from which you can reconfigure your
serial communications parameters (Comm. Port, Baud Rate, Parity, Data Bits, and Stop Bits).
Serial Delay (ms)
Configures a delay in the transmission of commands to the camera controller. This may be necessary to
adjust for overly fast rates of transmission that might otherwise be missed by the controller.
Close
Closes the dialog box.
Set Acquisition Region (Kodak MotionCorder) - Dialog Box Options
Use Active Region
Configures MetaMorph to use the rectangular region you drew in the image window as the acquisition
region.
Cancel
Cancels the command and closes the dialog box.
Auto-Focus via Hardware (Devices Menu)
Emulates triggering the AUTO FOCUS button on the front panel of the Ludl or Prior Zmotor controller.
Drop-in: AUTOFCUS
Use this command to emulate an autofocus button on a stage Z-motor controller. This command can be
used in a journal. A typical journal might include this command to autofocus while scanning each well on
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a multiple-well plate.
You must install and configure a Z-motor device driver before using this command. This command can
only be used Ludl and Prior controllers which have the autofocus card installed in them.
QUICK TIP: When used in a journal, the autofocusing procedure can be greatly improved by using the
Acquire Image command's Record Settings journal function (available only as a command button in the
dialog box), which you can use to fine-tune frame averaging without actually acquiring images.
WARNING:
It is recommended that you set the range of the autofocus Z-motor for the Prior stage with the Configure
Auto-Focus command before you use the Auto-Focus command. The Auto-Focus command should be
used only if an object is guaranteed to be found within the autofocus region. The ending location of the
focus cannot be determined or guaranteed unless an object is inside the autofocus region before
autofocusing.
WARNING:
The use of a CCD camera, rather than a tube camera, is recommended with this command because
tube cameras (Newvicon or SIT) have a substantial lag time that interferes with the autofocus algorithm.
Auto-Focusing the Microscope
To autofocus the microscope using your imaging system, use the following procedure.
Note: If you are using a Prior stage Z-motor, you should first use the Configure AutoFocus command.
Step
1
Action
Install and calibrate a Z-Motor device using
the Meta Imaging Series Administrator.
Note: You must exit MetaMorph
to start the Meta Imaging Series
Administrator.
2
Start MetaMorph and Select the Z-Motor
device in the Device Control dialog box
3
If you are using a Ludl Auto-Focus card,
verify that an object is inside the image
region on the Ludl Controller video output
(the region's size and position is configured
using the front panel of the Ludl autofocus
controller).
4
From the Devices menu, choose Auto-Focus.
MetaMorph will autofocus the microscope.
Configure Auto-Focus via Hardware (Devices
Menu)
Configures the focus range for the Prior stage Z-motor.
Drop-in: AUTOFCUS
Use this command to configure the range of Z-axis movement for the Prior stage during focusing. This
should be done before using the Auto-Focus command (Devices menu). The setting you choose will
depend on the magnification of the microscope objective you are using.
Configuring Auto-Focus via Hardware
To configure the Z-axis focusing range of the Prior stage Z-motor, use the following procedure:
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Step
Action
1
Install and configure a Z-Motor device in the
Meta Series System Administrator.
2
From the Devices menu, choose Configure
Auto-Focus. The Configure Auto-Focus
dialog box will appear.
3
Use the Autofocus Range slider to select a
range of Z-axis movement. The ranges are in
arbitrary units, with higher magnification
requiring a lower number and a lower
magnification requiring a higher number.
4
Choose OK.
Configure Auto-Focus via Hardware - Dialog Box Options
Autofocus Range
Selects a Z-axis range over which focusing movements can be made by the Prior stage Z-motor. Ranges
are given in arbitrary units from 0 to 5. Higher magnifications (for example, 60x and 100x objectives) will
require a lower number, whereas lower magnifications (for example, 5x and 10x objectives) will require a
higher number.
OK
Sets the focusing range and closes the dialog box.
Cancel
Cancels the command.
Auto-Focus via Software (Devices Menu)
Finds the best focal position of the microscope, using a Z-motor and an algorithm to
estimate image resolution.
Drop-in: AUTOFO_S
Use this command to focus the microscope when a previously focused image has become blurred. If you
are just starting an experiment and need to make large adjustments to the focus, you should adjust your
microscope while using the Focus command, or use the Auto-Focus drop-in command.
The Auto-Focus via Software commands rely on the Move Increment (step size) settings configured in
the Focus dialog box. Before you can perform autofocusing with either Auto-Focus via Software
command, you must calibrate and configure your step sizes with the Focus command. This should only
need to be performed once after you install your stage Z-motor hardware.
There are two Auto-Focus via Software commands, each with a slightly different purpose:
Adjust Focus is useful for making minor adjustments when the stage has drifted slightly or the
image has otherwise been put slightly out of focus.
Find Focus is intended for finding the best focus position within a broader Z-axis range but with a
minimum number of acquisitions. This will be particularly useful for acquiring images from a multi-well
or similar application in which there are many stage movements and the focus may change
significantly after each move.
To assess the best focal position, the Auto-Focus via Software commands use the Brenner algorithm to
measure a "focus value" that is based on the sum of squares of the intensity difference between a pixel
and a pixel that is two pixels away from it. This provides a rough estimate of image resolution such that,
the higher the value at a given pixel, the sharper the grayscale transitions must be surrounding that pixel.
When making a comparison of this value for a pixel across several focal planes, the image with the
highest focus value will be the sharpest. When the best focus value has been determined, the focus
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motor will be moved to the corresponding focal plane.
For more information on automated measurement of focus values, see Firestone et al., 1991 or Price
and Gough, 1994.
Firestone, L., Cook, K., Culp, K., Talsania, N.,
and Preston, K., Jr. Comparison of autofocus
methods for automated microscopy. Cytometry
12: 195 - 206, 1991.
Price, J.H. and Gough, D.A. Comparison
of phase-contrast and fluorescence digital
autofocus for scanning microscopy.
Cytometry 16: 283 - 297, 1994.
Configuring Auto-Focus via Software
Adjusting Focus
Finding Focus
Configure Auto-Focus via Software - Dialog Box Options
Adjust Focus
Find Focus
Adjust Focus (Auto-Focus via Software)
Makes fine adjustments to the focal position of the microscope, using a Z-motor and an
algorithm to estimate the sharpest image resolution.
Drop-in: AUTOFO_S
Use this command to make minor adjustments to the focus of the microscope when a previously focused
image has become slightly blurred. If you are just starting an experiment and need to make large
adjustments to the focus, you should adjust your microscope while using the Focus command, or use the
Auto-Focus drop-in command. If you need to find the best focus position within a broader Z-axis range
but with a minimum number of acquisitions, you should instead use the Find Focus command.
The Auto-Focus via Software commands rely on the Move Increment (step size) settings configured in
the Focus dialog box. Before you can perform autofocusing with either Auto-Focus via Software
command, you must calibrate and configure your step sizes with the Focus command. This should only
need to be performed once after you install your stage Z-motor hardware.
The Number of Steps, Increment Step(s), and Range, Current +/- options in the Adjust Focus dialog box
will have an interactive effect on one another. The Number of Steps will always be an odd number, and
will include the current focus position. To use an example, if the Number of Steps has been set to 5,
there will be two focus positions above the current plane and two below it. If you set Increment Step(s) to
3, the Range, Current +/- option will update to reflect the fact that there will be a total range of six steps
above the current focus position and six steps below it. Altering any of these three options will bring
about a change in the setting of one of the other two options.
By default, the Auto-Focus via Software commands uses the Brenner algorithm as the standard method
to assess the best focal position. The Brenner algorithm measures a "focus value" that is based on the
sum of squares of the intensity difference between one pixel and another that is two pixels away from it.
This provides a rough estimate of image resolution such that, the higher the value at a given pixel, the
sharper the grayscale transitions must be surrounding that pixel. When making a comparison of this
value for a pixel across several focal planes, the image with the highest focus value will be the sharpest.
The Find Focus command differs from the Adjust Focus command in that it attempts to use the fewest
possible acquisitions to obtain the optimal focal position. It does so by taking images at the limits of the
selected range and recursively dividing the step size in half.
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An alternative to the Standard algorithm is the Directional Average algorithm. If you are not getting good
results using the Brenner algorithm, try switching to the Directional Average algorithm using the
Algorithm drop-down box. This algorithm gives more accurate focus values with some high magnification
objectives.
For more information on automated measurement of focus values, see Firestone et al., 1991 or Price
and Gough, 1994.
Adjusting Focus (Auto-Focus via Software)
To make minor adjustments to the focal position for the microscope and set the Z-position to that
plane, use the following procedure. (Note: You must first calibrate and configure the Z-motor step
sizes with the Focus command.)
Step
Action
1
In the Devices menu, point to the entry for
Auto-Focus via Software. A secondary menu
will open, displaying two focusing
subcommand entries.
2
Choose Adjust Focus. The Adjust Focus
dialog box will appear.
3
Select an algorithm to use from the Algorithm
drop-down list. Valid choices are Standard
and Directional Avg.
Note: The Standard algorithm will produce
the best results under most conditions and
should be used first.
4
With the Number of Steps spin box, select
the overall number of images to acquire to
test the focus. This will be an odd number
that includes the current focal position.
5
Using the Increment Step(s) spin box, select
the number of steps the Z-motor should be
moved between each acquisition. The size of
these steps will be determined from the
Move Increment setting in the Focus dialog
box. The Range, Current +/- spin box will
update to reflect the Number of Steps and
Increment Step(s) settings you have
selected.
OR
Select an overall range, in Z-motor steps,
from the Range, Current +/- spin box. The
size of these steps will be determined from
the Move Increment setting in the Focus
dialog box. The Increment Step(s) spin box
will update to reflect the Number of Steps
and Range, Current +/- settings you have
selected.
6
If you want to use backlash compensation to
minimize focus position drift due to the effect
of gravity on the Z-motor gears, select the
Backlash Compensation check box. If your
Z-motor hardware has this compensation
already built in, you can leave this check box
cleared.
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7
If you want to see each focus test image as it
is acquired, select the Display Images Being
Acquired check box.
8
When you are ready, choose Auto-Focus.
MetaMorph will acquire the focus test
images, measure a focus value for each,
determine the best value, and move the Zmotor to the corresponding focal plane.
9
When you have finished, choose Close.
Adjust Focus (Auto-Focus via Software) - Dialog Box Options
Algorithm
Specifies the algorithm used to find focus. The options are Standard or Directional Avg. The default is
Standard and uses the Brenner algorithm. Directional Avg gives more accurate focus values with some high
magnification objectives.
Number of Steps
Specifies the number of focus test images to be acquired to determine the best focal position. This will
always be an odd number, and will include the current focus position. Changing this value will change the
total focus range in the Range, Current +/- spin box based on the Z-axis increment selected in the Increment
Step(s) spin box.
Increment Step(s)
Specifies the Z-distance to move between each focus test image. The size of each step will be determined
from the Move Increment setting in the Focus dialog box. The number of steps to move between
acquisitions should be small enough to provide a well-focused image at one of the positions. Changing this
value will change the total focus range in the Range, Current +/- spin box based on the selected Number of
Steps.
Range, Current +/Specifies how far above and below the current Z-position that the Z-motor will be moved while acquiring
focus test images. This distance is specified in steps, as defined by the Focus command. Changing this
value will change the Z-axis increment in the Increment Step(s) spin box, based on the total Number of
Steps.
Backlash Compensation
Selecting this check box will enable a Z-motor movement protocol whereby the focus motor will be moved to
a Z-axis position slightly below the target position, and then moved against gravity to the target position. This
may be desirable so that the Z-motor gears will be fully engaged, thereby avoiding drift due to slippage of
the gears. Some focus devices will have a built-in backlash compensation, but there will be no harm in
leaving this option selected.
Display Images Being Acquired
Displays the focus test images in an image window during autofocusing. The image window will be closed
when autofocusing is complete.
Auto-Focus
Initiates the autofocusing protocol and moves the Z-motor to the best focus position, as determined by the
Auto-Focus algorithm.
Close
Closes the dialog box.
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Find Focus (Auto-Focus via Software) (Devices
Menu)
Finds the optimal focal position of the microscope within a broad Z-axis range, using the
fewest possible acquisitions.
Drop-in: AUTOFO_S
Use this command to find the best focal position of the microscope with the fewest Z-axis steps. This will
be particularly useful for acquiring images from a multi-well plate or similar application in which there are
many stage movements and the focus may change significantly after each move. If you are just starting
an experiment and need to make large adjustments to the focus, you should adjust your microscope
while using the Focus command, or use the Auto-Focus drop-in command. If you need to make minor
adjustments of the focus position within a narrower Z-axis range, you should instead use the Adjust
Focus command.
The Auto-Focus via Software commands rely on the Move Increment (step size) settings configured in
the Focus dialog box. Before you can perform autofocusing with either Auto-Focus via Software
command, you must calibrate and configure your step sizes with the Focus command. This should only
need to be performed once after you install your stage Z-motor hardware.
By default, the Auto-Focus via Software command uses the Brenner algorithm as the standard method
to assess the best focal position. The Brenner algorithm measures a "focus value" that is based on the
sum of squares of the intensity difference between one pixel and another that is two pixels away from it.
This provides a rough estimate of image resolution such that, the higher the value at a given pixel, the
sharper the grayscale transitions must be surrounding that pixel. When making a comparison of this
value for a pixel across several focal planes, the image with the highest focus value will be the sharpest.
The Find Focus command differs from the Adjust Focus command in that it attempts to use the fewest
possible acquisitions to obtain the optimal focal position. It does so by taking images at the limits of the
selected range and recursively dividing the step size in half.
An alternative to the Standard algorithm is the Directional Average algorithm. If you are not getting good
results using the Brenner algorithm, try switching to the Directional Average algorithm using the
Algorithm drop-down box. This algorithm gives more accurate focus values with some high magnification
objectives.
For more information on automated measurement of focus values, see Firestone et al., 1991 or Price
and Gough, 1994.
Finding Focus (Auto-Focus via Software)
To find the best focal position for the microscope within a broad Z-axis range using the least
number of acquisitions, use the following procedure. (Note: You must first calibrate and configure
the Z-motor step sizes with the Focus command.)
Step
Action
1
In the Devices menu, point to the entry for
Auto-Focus via Software. A secondary menu
will open, displaying two focusing
subcommand entries.
2
Select Find Focus. The Find Focus dialog
box opens.
Select an algorithm to use from the Algorithm
drop-down list. Valid choices are Standard
and Directional Avg.
Note: The Standard algorithm
will produce the best results
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under most conditions and
should be used first.
3
Select an overall range, in Z-motor steps,
from the Range, Current +/- spin box. The
size of these steps will be determined from
the Move Increment setting in the Focus
dialog box.
4
With the Accuracy: Step(s) spin box, select
the smallest movement size at which the
focus is to be tested.
5
If you want to use backlash compensation to
minimize focus position drift due to the effect
of gravity on the Z-motor gears, select the
Backlash Compensation check box. If your
Z-motor hardware has this compensation
already built in, you can leave this check box
cleared.
6
If you want to see each focus test image as it
is acquired, select the Display Images Being
Acquired check box.
7
When you are ready, choose Find Focus.
MetaMorph will acquire the focus test
images, measure a focus value for each,
determine the best value, and move the Zmotor to the corresponding focal plane.
8
When you have finished, choose Close.
Find Focus (Auto-Focus via Software) - Dialog Box Options
Algorithm
Specifies the algorithm used to find focus. The options are Standard or Directional Avg. The default is
Standard and uses the Brenner algorithm. Directional Avg gives more accurate focus values with some high
magnification objectives.
Range, Current +/Specifies how far above and below the current Z-position that the Z-motor will be moved while acquiring
focus test images. This distance is specified in steps, as defined by the Focus command.
Accuracy: um(s)
Specifies the Z-distance to move (in ums). Changing this value will change the Number of Z Moves.
Number of Z Moves
Indicates the total number of acquisitions that will be made, based on the settings selected with the Range,
Current +/- and Accuracy: Step(s) spin boxes.
Status
Displays the current status of the Find Focus command. When the command is complete the current Z
position is shown in um(s) — for example, Focused at 5.25.
Backlash Compensation
Selecting this check box will enable a Z-motor movement protocol whereby the focus motor will be moved to
a Z-axis position slightly below the target position, and then moved against gravity to the target position. This
may be desirable so that the Z-motor gears will be fully engaged, thereby avoiding drift due to slippage of
the gears. Some focus devices will have a built-in backlash compensation, but there will be no harm in
leaving this option selected.
Display Images Being Acquired
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Displays the focus test images in an image window during autofocusing. The image window will be closed
when autofocusing is complete.
Find Focus
Initiates the autofocusing protocol and moves the Z-motor to the best focus position, as determined by the
Auto-Focus algorithm.
Close
Closes the dialog box.
Resync Focus Dialog with Olympus Z-Motor
(Devices Menu)
Updates the Focus command's dialog box to reflect the current Z-distance setting on the
Olympus microscope Z-motor. This may be particularly necessary if you make changes
manually to the Z-motor settings.
Drop-in: OLAXRF
Use this command to resynchronize the Focus dialog box settings with the current Z setting on the
Olympus microscope's Z-motor.
Resynchronizing the Focus Dialog with the Olympus Z-Motor
To updates the Focus command's dialog box to reflect the current Z-distance setting on the
Olympus microscope Z-motor, use the following procedure:
Step
Action
1
From the Devices menu, choose Resync
Focus Dialog with Olympus Z Motor. The
Resync Focus Dialog with Olympus Z-Motor
dialog box will appear.
2
Choose Resync. The settings in the Focus
dialog box will be updated.
3
You can leave the Resync Focus Dialog with
Olympus Z-Motor dialog box open if you
expect to make more changes manually to
the Z-motor settings. When you have
finished, choose Close.
Resync Focus Dialog with Olympus Z-Motor - Dialog Box Options
Resync
Updates the Focus command's dialog box to reflect the current Z-distance setting on the Olympus
microscope Z-motor.
Close
Closes the Resync Focus Dialog with Olympus Z-Motor dialog box.
Custom I/O Control (Devices Menu)
Provides control over devices that utilize data streams.
Drop-in: CUSTOMIO
There are four commands in the Custom I/O Control secondary menu: Send Serial Data, Wait for Serial
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Data, Set Digital I/O, and Wait for Digital I/O. A serial communications port must be installed and
configured using the Meta Imaging System Administrator before using Send Serial Data and Wait for
Serial Data.
Send Serial Data (Devices Menu)
Sends a sequential stream of data from the computer to another device via a serial port.
Drop-in: CUSTOMIO
Use this command to send recognized command strings for controlling another device to the device from
the serial port. For example, some intensified CCD cameras have camera gain-switching abilities which
are controlled by two gain (or sensitivity) knobs, and a toggle switch which determines which knob is
active. After you set the gain for each knob, you can instruct the camera to switch between the two
settings by sending the appropriate command string using the Send Serial Data command.
The documentation for the device receiving the data will include the appropriate commands needed to
control the various components of the device. These are typed into a text box in the Send Serial Data
dialog box using the syntax rules followed by MetaMorph. You can use hexadecimal, decimal, or ASCII
Control Codes in your command strings.
Syntax Rules
ASCII Control Code Chart
Sending Serial Data
To send serial data to a peripheral device, use the following procedure:
Step
Action
1
Install and configure a serial communications
port using the Meta Imaging System
Administrator.
2
From the Devices menu, choose Custom I/O.
Then choose Send Serial Data from the
secondary menu that appears. The Send
Serial Data dialog box will appear.
3
Choose Select. This will open the Select
Serial Device dialog box. If there are several
serial devices installed, you can select one
from the drop-down list.
4
If the device receiving data sends back each
character after the device receives it, select
Wait for Echo After Each Character.
AND
Select the amount of time (seconds) that
MetaMorph should wait for the echo before
notifying you about the missing echo using
Timeout.
5
Type the command string(s) to be sent to the
device in the text box at the top of the dialog
box. Your command string(s) must follow the
syntax rules used by MetaMorph.
Syntax Rules
ASCII Control Code Chart
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6
Choose Send.
7
Choose Close to close the dialog box.
Send Serial Data - Dialog Box Options
Device Name
Displays the name of the open serial device. If no serial device is open, "<None>" will be displayed instead.
Command String Text Box
Use this text box to type the command string(s) to be sent to the device. Refer to the device's documentation
to determine the necessary commands to control the device. Your command strings must follow the syntax
rules used by MetaMorph. An ASCII Control Code Chart is provided in this online help for your
convenience.
Wait for Echo After Each Character
Instructs MetaMorph to wait for an echo from the other device before sending the next character and to warn
you if the character is not received. Use this option if the device receiving data sends back each character
after the device receives it.
Timeout
Specifies how long MetaMorph should wait for an echo from the other device before warning you that the
character was not received. You must select Wait for Echo After Each Character before using this option.
Select
Opens the Select Serial Device dialog box. If there are several serial devices installed, you can select one
from the drop-down list.
Send
Sends the command string(s) typed in the Command String Text Box to the device.
Close
Closes the dialog box.
Wait for Serial Data (Devices Menu)
Waits for a sequential stream of data from another device by way of a serial port.
Drop-in: CUSTOMIO
Use this command to specify command strings that MetaMorph should wait for from another device via
the serial port.
The documentation for the device sending the data will include the appropriate commands used by the
device. These are typed into a text box in the Wait for Serial Data dialog box using the syntax rules that
are followed by MetaMorph. You can use hexadecimal, decimal, or ASCII control codes in your
command strings.
Syntax rules
ASCII Control Code Chart
Waiting for Serial Data
To configure the system to wait for serial data from a peripheral device, use the following
procedure:
Step
1
Action
Install and configure a serial communications
port using the Meta Imaging System
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Administrator.
2
From the Devices menu, choose Custom I/O.
Then choose Wait for Serial Data from the
secondary menu that appears. The Wait for
Serial Data dialog box will appear.
3
Choose Select. This will open the Select
Serial Device dialog box. If there are several
serial devices installed, you can select one
from the drop-down list.
4
If you want to log the data received from the
device, select Write Received Data to Log
File and the dialog box expands to include
logging options. (A data log file must be open
to log the data.)
AND
Specify the logging format for the data using
the Log Format text box. You can add text as
desired; the "$" will be replaced by the data
received from the device each time data are
logged.
5
Specify the amount of time MetaMorph
should wait for each character before
warning you about the missing character
using Timeout.
6
Type the command string(s) to be sent by
the device in the text box at the top of the
dialog box. Your command string(s) must
follow the syntax rules used by MetaMorph.
Syntax Rules
ASCII Control Code Chart
7
Choose Wait.
8
Choose Close to close the dialog box.
Wait for Serial Data - Dialog Box Options
Device Name
Displays the name of the open serial device. If no serial device is open, "<None>" will be displayed instead.
Command String Text Box
Use this text box to type the command string(s) to be sent from the device. Refer to the device's
documentation to determine the appropriate commands. Your command strings must follow the syntax
rules used by MetaMorph. An ASCII Control Code Chart is provided in this online help for your
convenience.
Write Received Data to Log File
Instructs MetaMorph to log the received data to the open data log file.
Log Status
Lists the status of the data log file, including the name of the name of log file, if one is open.
Log Format
Specifies the logging format used to log data received from the device. When you have added the desired
text, use the "$" to represent the data that were received from the device. This option will appear when you
select the Write Received Data to Log File check box.
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Timeout
Specifies how long MetaMorph should wait for each character from the device before warning you that the
character was not received.
Select
Opens the Select Serial Device dialog box. If there are several serial devices installed, you can select one
from the drop-down list.
Wait
Instructs MetaMorph to wait for the string specified in the Command String Text Box.
Close
Closes the dialog box.
Set Digital I/O (Devices Menu)
Controls a peripheral digital I/O device by sending signals to it from the computer.
Drop-in: CUSTOMIO
Use this command to send TTL-level voltage signals from MetaMorph by a parallel port or a digital I/O
board. For example, you could use this command to control a valve that opens when the hardware
receives a +5 V signal and closes when the hardware receives a 0 V signal.
Setting Digital I/O
To configure digital I/O communication lines, use the following procedure:
Step
Action
1
Install and configure a digital I/O device
using the Meta Imaging System
Administrator.
2
From the Devices menu, choose Custom I/O.
Then choose Set Digital I/O from the
secondary menu that appears. The Set
Digital I/O dialog box will appear.
3
Choose Configure to set the configuration
options appropriate for the parallel port or the
digital I/O board.
AND
Choose OK when you have finished. The Set
Digital I/O dialog box will update when you
choose OK.
4
Select Continuous Update of Line
Assignments if you want the line states to be
set automatically as you change the status of
the line states in the dialog box. If this option
is deselected, line states will only be
changed when you choose Set Lines.
5
The line numbers and their corresponding
pin position on the parallel port or digital I/O
card will be displayed at the bottom of the
dialog box. The Set State radio buttons next
to each line specify how each line should be
set each time you choose Set Lines.
Select On to set a line to high signal (+5 V).
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Select Off to set a line to low signal (0 V). Or
select Ignore to leave a line set to its present
setting.
6
Repeat Step 5 for each line used by the
device. You may need to use the scroll bar
on the right side of the dialog box to view the
line assignments for some lines.
7
Choose Set Lines.
Note: If you select Continuous Update of
Line Assignments, the digital I/O signal will
be set each time a line state is changed.
8
Repeat Steps 5 - 7 as necessary. Choose
Close when you have finished.
Set Digital I/O - Dialog Box Options
Device Name
Lists the name of the open Digital I/O device. If no Digital I/O device is open, "<None>" will be displayed
instead.
Number of Output Lines
Lists the number of output lines available for the installed digital I/O device.
Continuous Update of Line Assignments
Automatically changes the state of a line whenever its status is changed in the dialog box, rather than
waiting for the user to select Set Lines.
Line #
Lists the line numbers available for the installed digital I/O device.
Pin #
Lists the corresponding pin number for each line used by the digital I/O device.
Set State
Specifies the state for each line. A line can be set to On (high signal, +5 V), Off (low signal, 0 V), or Ignore
(remains in present state).
Slider
Scrolls through the list of available lines.
Set Line
Sets each line to the state (On, Off, or Ignore) specified by the Set State radio buttons.
Configure
Specifies the appropriate configuration options for the parallel port or digital I/O board installed. Use this
option to specify the parallel port.
Close
Closes the dialog box.
Wait for Digital I/O (Devices Menu)
Sends signals to the computer from a digitally-controlled device.
Drop-in: CUSTOMIO
Use this command to receive TTL-level voltage signals from a digitally controlled device by a parallel
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port or a digital I/O board.
QUICK TIP: If you have installed the MMVAR drop-in, you can use the Branch on Variable drop-in
command (Journal menu) to run one journal when the system receives the digital I/O signal or run a
different journal if a timeout occurs. To do so, use the "$Device.DigitalIO.Timeout$" system variable in
the Branch on Variable dialog box. In the absence of a timeout, this variable will be set to a value of 0. If
a timeout occurs, the variable will be reset to 1.
Waiting for Digital I/O Signals
To configure the system to wait for signals from a peripheral digital I/O device, use the following
procedure:
Step
Action
1
Install and configure a digital I/O device
using the Meta Imaging System
Administrator.
2
From the Devices menu, choose Custom I/O.
Then choose Wait for Digital I/O from the
secondary menu that appears. The Wait for
Digital I/O dialog box will appear.
3
Choose Configure to set the configuration
options appropriate for the parallel port or the
digital I/O board.
AND
Choose OK when you have finished. The
Wait for Digital I/O dialog box will update
itself when you choose OK.
4
Use Timeout to specify the amount of time
MetaMorph should wait for signals that
match the selected line states before warning
you.
5
The line numbers and their corresponding
pin position on the parallel port or digital I/O
card will be displayed at the bottom of the
dialog box. The Wait State radio buttons next
to each line specify the expected line signal
that should be received each time you
choose Wait.
Select On to set a line to high signal (+5 V).
Select Off to set a line to low signal (0 V). Or
select Ignore to leave a line set to its present
setting.
6
Repeat Step 5 for each line used by the
device. You may need to use the scroll bar
on the right side of the dialog box to view the
line assignments for some lines.
7
Choose Wait. The Wait for Digital I/O dialog
box will appear, indicating the MetaMorph is
waiting for the digital I/O signals that match
the line states specified the dialog box.
If the signals are not received before the time
specified by Timeout has elapsed, a
message dialog box will appear, stating that
the input lines are not set.
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8
Repeat Steps 5 - 7 as necessary. Choose
Close when you have finished.
Wait for Digital I/O - Dialog Box Options
Device Name
Lists the name of the open Digital I/O device. If there is no Digital I/O device open, "<None>" will be
displayed instead.
Number of Input Lines
Lists the number of input lines available for the installed digital I/O device.
Timeout
Specifies the amount of time MetaMorph should wait for digital I/O signals that match the selected line states
in the dialog box before warning you.
Line #
Lists the line numbers available for the installed digital I/O device.
Pin #
Lists the corresponding pin number for each line used by the digital I/O device.
Wait State
Specifies the state for which to wait for each line. A line can be set to On (high signal, +5 V), Off (low signal,
0 V), or Ignore (remains in present state).
Slider
Scrolls through the list of available lines.
Wait
Instructs MetaMorph to wait for digital signals from the device that match the line states specified by the
Wait State radio buttons (On, Off, or Ignore).
Configure
Specifies the appropriate configuration options for the parallel port or digital I/O board installed. Use this
option to specify the parallel port.
Close
Closes the dialog box.
Syntax Rules
Code
Result
$
"Escape" Character, which is ASCII 27 in
Decimal.
^A thru ^Z
"Control" Character, which is ASCII 1 for
^A through ASCII 26 for ^Z.
\c
Sends the character after the slash. In
this example, the character "c" would be
sent. Useful for sending ^, \, or $
characters.
\ddd
Sends ASCII digits in Decimal.
EXAMPLE: \192.
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\xdd
Sends ASCII digits in Hexadecimal.
EXAMPLE: \x27.
(dddd)
Delays for specified number of
milliseconds.
EXAMPLE: (1000)
Note: If you need to send more than one command at a
time, command strings can be appended serially.
EXAMPLE: "\xdd\xdd\x04".
ASCII Control Code Chart
Hex
Dec
Key
Name
Description
Hex
Dec
Key
Name
Description
00
0
^@
NUL
Null
10
16
^P
DLE
Data Link Escape
01
1
^A
SOH
Start of Header
11
17
^Q
DC1
Device Control 1
02
2
^B
STX
Start of Text
12
18
^R
DC2
Device Control 2
03
3
^C
ETX
End of Text
13
19
^S
DC3
Device Control 3
04
4
^D
EOT
End of Transmission
14
20
^T
DC4
Device Control 4
05
5
^E
ENQ
Inquiry
15
21
^U
NAK
Negative Acknowledge
06
6
^F
ACK
Acknowledge
16
22
^V
SYN
Synchronous Idle
07
7
^G
BEL
Bell
17
23
^W
ETB
End Transmission Block
08
8
^H
BS
Backspace
18
24
^X
CAN
Cancel
09
9
^I
HT
Horizontal Tab
19
25
^Y
EM
End of Medium
0A
10
^J
LF
Line Feed
1A
26
^Z
SUB
Substitute
0B
11
^K
VT
Vertical Tab
1B
27
ESC
Escape
0C
12
^L
FF
Form Feed
1C
28
FS
File Separator
0D
13
^M
CR
Carriage Return
1D
29
GS
Group Separator
0E
14
^N
SO
Shift Out
1E
30
RS
Record Separator
0F
15
^O
SI
Shift In
1F
31
US
Unit Separator
MMKeyPad
Provides a customizable set of program controls on a standard remote keypad device that
can be remotely located from the workstation.
Drop-in: MMKEYPAD
Use the MMKeyPad drop-in to enable you to execute commands for controlling your microscope and
associated devices including Z-motor, filtration, and shutter, and for executing journals and acquiring
images. This drop-in enables you to connect a standard, wired, 17-key keypad to a serial com port, and
to assign a maximum of 16 different journals to the keypad keys, as well as use the pre-assigned
functions. Pressing the Num Lock key switches the keypad from its pre-assigned functions to user
assigned journals. The pre-assigned numeric keys are used to initiate from one to nine user-defined
keys on a single taskbar.
Configuring the MMKeyPad
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To configure and use the MMKeyPad, complete the following procedure.
Step
Action
1
If the Active on COM # check box is on
(checked), click the checkbox to deactivate
the MM Keypad, which enables you to make
configuration settings.
2
If the MMKeyPad dialog box is minimized,
click More>>. The complete MMKeyPad
dialog box opens.
3
In the Serial Port box, select the number of
the Com port to which the keypad is
connected.
Note: To use your keypad, you must first
install the appropriate keypad driver.
4
You can assign journals to any keypad key
except the Num Lock key. Click the key to
which you want to assign the journal. The
Select a Journal to run for each Interval
dialog box opens.
5
Select the folder containing the journal that
you want to associate with the selected key,
then select the journal and click Open.
6
Repeat steps 4 and 5 for each key to which
you want to assign a journal.
7
After you have finished assigning journals to
keys, click Active to activate the keypad.
8
Press the Num Lock key to change from
Journal mode to Standard mode.
9
Open or create a task bar set up with the
dialog boxes that you want to run remotely.
10
To initiate a task on a task bar, press the
remote keypad key associated with the task.
Keys 1 through 9 on the key pad will activate
tasks 1 through 9 on the task bar (top to
bottom).
MMKeyPad - Dialog Box Options
Active
Activates the Keypad on the selected com port. Select the com port that you want to use first, then click this
check box. To configure keypad keys to run specific journals, you must ensure that this "Active" checkbox is
not checked.
More>> / <<Less
Switches the dialog box between its "minimized" and "maximized" format. In minimized format, only the
Active checkbox, the More>> button, and the Close button are available. After all settings have been made,
you can minimize the dialog box to occupy less area on your display.
Close
Closes the MMKeyPad dialog box.
Serial Port
Specifies the Com port to which the Keypad is connected.
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NumLk/(Num Lock)
Switches the keypad between its pre-assigned functions and user assigned journals.
Pop Images (/)
Brings the currently open image window(s) to the front.
Pop Dialogs (*)
Brings the currently open dialog boxes to the front.
DecZMotor (-)
Moves the Z-Motor (Focus) up one step for each key press.
IncZMotor (+)
Moves the Z-Motor (Focus) down one step for each key press.
Tog Shttr (Enter)
Opens and closes the selected shutter device.
Dec Filter (Ins)
Moves the selected filter device (wheel, dichroic, or cube) to the next filter in a negative direction.
Inc Filter (Del)
Moves the selected filter device (wheel, dichroic, or cube) to the next filter in a positive direction.
Taskbar 1-9 (1-9)
Activates the appropriate taskbar command on the active (open) taskbar.
Color Align (Display Menu)
Shifts the red, green, and blue planes of an image independently to bring them into
alignment.
Drop-in: CALIGN
Use this command to shift the color planes in a single 24-bit color image, or in individual 8-bit or 16-bit
images (red, green, and/or blue), to correct for image registration misalignment, such as are caused by
chromatic aberrations or mechanical components such as filter wheels. This feature will be particularly
useful for users of large-chip CCDs who acquire RGB images through separate acquisition steps.
Your method of selecting the source image(s) depends on whether you are working with a single 24-bit
image or with individual "color component" source images. When you first choose the Color Align
command, the Color Align dialog box will be condensed when it appears. If you are using a 24-bit source
image, the Image selector, located in the upper, left corner of the dialog box, is used to select the source
image. If, however, you are using two or three individual "color component" source images, you need to
expand the dialog box by choosing More >>. On the right half of the expanded dialog box, you will see
three Image selectors, which you will use to select the red, green, and/or blue source images.
This command offers two ways to alter alignment values. One method relies on horizontal and vertical
sliders, which you can drag with your pointer to the desired position. The other method involves a table
of values, in which you can enter the number of pixels to move in the horizontal or vertical direction for
each of the color components. A negative value indicates a shift upwards or to the left.
To assist you in the alignment process, a Preview image window is provided so that you can see the
effect of your alignment changes before you apply them permanently. The Preview window centers on a
256 x 256 region in the source image(s), which you can change with a box-in-box region selection
control.
When the alignment operation is performed on 8-bit or 16-bit source images, the results will be saved in
a new 24-bit image. However, when the source image is a single 24-bit color image, the results of the
alignment operation will overwrite the original. Thus, if you want to retain the original 24-bit image, you
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should make a copy of it with the Duplicate Image command (Edit menu), and perform your alignment on
the copied image.
If you are working with 16-bit source images, the Color Align command allows you to adjust the contrast
of the displayed images. You can have MetaMorph adjust the contrast automatically, or you can perform
the adjustments manually. These controls are similar to those found in the Scale 16-Bit Image dialog
box.
Aligning Colors
Three 8-Bit Images
Three 16-Bit Images
A Single 24-Bit Image
Color-Aligning Three 8-Bit Images
To align three 8-bit color component images, use the following procedure:
Step
Action
1
From the Display menu, choose Color Align.
The Color Align dialog box opens.
2
If the dialog box is condensed, choose
More >> to expand it.
3
In the Red, Green, and Blue option groups
on the right side of the dialog box, use the
pertinent Image selectors to select the
source images representing the red, green,
and/or blue component images, respectively.
If you are using only two color source
images, select None Selected for the color
plane that will not be involved.
4
If you wish, use the Destination image
selector to specify a name for the aligned
result image.
5
With the Preview Image box-in-box control,
select the region of the source image to be
displayed in the Preview image window by
dragging the inner box with your pointer.
6
From the Alignment group, select the check
box for the color component image (Red,
Green, or Blue) that you want to shift. You
can select check boxes for two of the color
components if you want to apply the same
shift to both images.
7
To adjust the position of the selected
image(s), drag the sliders in the left half of
the dialog box to the desired location while
watching the movement of the colored
objects in the Preview window.
OR
Click the appropriate cell of the Alignment
Table and type the desired shift, in pixels, in
the cell. Type a negative number in the
H Shift column to specify a shift to the left.
Similarly, type a negative number in the
V Shift column to specify a shift upwards.
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If you want to reset the shift values to zero at
any time, choose Zero Shift.
8
If necessary, repeat Steps 6 and 7 for the
other color components. If you need to bring
one of the source images "to the front" in
your desktop display while doing so, choose
the "S" button in its option group on the right
side of the dialog box.
9
When you are satisfied with the new
alignment, choose Apply.
10
Choose Close.
Color-Aligning Three 16-Bit Images
To align three 16-bit color component images, use the following procedure:
Step
Action
1
From the Display menu, choose Color Align.
The Color Align dialog box opens. If the
dialog box is condensed, choose
More >> to expand it.
2
In the Red, Green, and Blue option groups
on the right side of the dialog box, use the
pertinent Image selectors to select the
source images representing the red, green,
and/or blue component images, respectively.
If you are using only two color source
images, select None Selected for the color
plane that will not be involved.
3
If desired, use the Destination image selector
to specify a name for the aligned result
image.
4
If you need to, you can adjust the contrast of
the source image to assist you with visual
discrimination during the alignment. To do
so, continue to Step 5.
OR
If you do not need to adjust the contrast of
the source images, skip to Step 7.
5
If necessary, you can specify gray levels for
contrast scaling that are outside of the
default range of the image's lowest and
highest gray levels (Image Min/Max). Select
10-Bits (0 - 1023), 12-Bits (0 - 4095), 14-Bits
(0 - 16383), or 16-Bits (0 - 65535) from the
Scale Range list. This will change the range
to include all of the gray values available in
the specified image depth type.
6
If you want MetaMorph to choose the range
of gray levels used for scaling the contrast,
select Auto for the pertinent source image.
OR
To adjust the contrast manually, select the
darkest gray level for the source images
using the pertinent Min sliders or text boxes,
and select the brightest gray level using the
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corresponding Max options.
7
With the Preview Image box-in-box control,
select the region of the source image to be
displayed in the Preview image window by
dragging the inner box with your pointer.
8
From the Alignment group, select the check
box for the color component image (Red,
Green, or Blue) that you want to shift. You
can select check boxes for two of the color
components if you want to apply the same
shift to both images.
9
To adjust the position of the selected color
component image(s), drag the sliders in the
left half of the dialog box to the desired
location while watching the movement of the
colored objects in the Preview window.
OR
Click the appropriate cell of the Alignment
Table and type the desired shift, in pixels, in
the cell. Type a negative number in the
H Shift column to specify a shift to the left.
Similarly, type a negative number in the
V Shift column to specify a shift upwards.
If you want to reset the shift values to zero at
any time, choose Zero Shift.
10
If necessary, repeat Steps 8 and 9 for the
other color components. If you need to bring
one of the source images "to the front" in
your desktop display while doing so, choose
the "S" button in its option group on the right
side of the dialog box.
11
When you are satisfied with the new
alignment, choose Apply.
12
Choose Close.
Color-Aligning a Single 24-Bit Image
To align the color components in a 24-bit color image, use the following procedure:
Step
Action
1
From the Display menu, choose Color Align.
The Color Align dialog box opens. If the
dialog box is expanded, choose
Less << to condense it.
2
If necessary, select the source image with
the Source selector.
3
With the Preview Image box-in-box control,
select the region of the source image to be
displayed in the Preview image window by
dragging the inner box with your pointer.
4
From the Alignment group, select the check
box for the color component (Red, Green, or
Blue) that you want to shift. You can select
check boxes for two of the color components
if you want to apply the same shift to both.
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5
To adjust the position of the selected color
component(s), drag the Alignment Sliders in
the left half of the dialog box to the desired
location while watching the movement of the
colored objects in the Preview window.
Repeat as necessary, for the other color
components.
OR
Click the appropriate cell of the Alignment
Table and type the desired shift, in pixels, in
the cell. Type a negative number in the
H Shift column to specify a shift to the left.
Similarly, type a negative number in the
V Shift column to specify a shift upwards.
If you want to reset the shift values to zero at
any time, choose Zero Shift.
6
When you are satisfied with the new
alignment, choose Apply.
7
Choose Close.
Color Align - Dialog Box Options
Source
Selects a source image for the alignment procedure. This image selector will appear only when you are
using a 24-bit source image. This source image will be overwritten by the alignment operation. Thus, if you
want to retain the original 24-bit image, you should make a copy of it with the Duplicate Image command
(Edit menu), and perform your alignment on the copied image.
Destination
Selects a destination for the alignment result image. This image selector will appear when you are using 8bit or 16-bit source images.
Horizontal and Vertical Alignment Sliders
The sliders shift the selected color component to the left or right (horizontal slider) and up or down (vertical
slider). Changes made with the sliders will be reflected in the Alignment Table. You can apply a shift of up to
10 pixels in any direction with the sliders. Shifts selected with the sliders will be added to any offsets you
have already specified in the Alignment Table.
Alignment (check boxes)
Selects the color component to be shifted. You can select check boxes for two of the color components if
you want to apply the same shift to both.
Alignment Table
Use this table to specify the horizontal (H Shift) and vertical (V Shift) offsets for the red, green, and blue
color components. There is no limit to the size of the shift you can apply (you can even shift a color
component completely out of the image). Type a negative number in the H Shift column to specify a shift to
the left. Similarly, type a negative number in the V Shift column to specify a shift upwards.
Preview Image (box-in-box control)
Allows you to drag the smaller box with the pointer to select a region in the source image to be displayed in
the Preview window.
Apply
Applies the configured alignment shift to the image. If you are using 8-bit or 16-bit color component source
images, a new image will be created with the name you selected with the Destination image selector. If you
are using a 24-bit color source image, this image will be overwritten by the alignment operation.
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Close
Closes the dialog box.
More >>
Expands the dialog box to the right, revealing image selectors for 8-bit and 16-bit source images and
contrast scaling options for 16-bit source images.
Less <<
Condenses the dialog box and reveals the image selector for 24-bit source images.
Zero Shift
Resets the horizontal and vertical shifts to zero for all color component(s).
Image (Red, Green, and Blue option groups)
Selects the 8-bit or 16-bit source images for the red, green, and/or blue components, respectively.
Min (Red, Green, and Blue option groups)
Selects the darkest gray level for the 16-bit color component source image for which you are manually
scaling the contrast. These options (text box and scaling wedges) will be unavailable and will appear
dimmed if you are using 8-bit or 24-bit source images.
Max (Red, Green, and Blue option groups)
Selects the brightest gray level for the 16-bit color component source image for which you are manually
scaling the contrast. These options (text box and scaling wedges) will be unavailable and will appear
dimmed if you are using 8-bit or 24-bit source images.
Auto (Red, Green, and Blue option groups)
Allows MetaMorph to choose the grayscale range to be scaled when you are adjusting the contrast of a 16bit color component source image. This button will be unavailable and will appear dimmed if you are using 8bit or 24-bit source images.
"S" command button (Red, Green, and Blue option groups)
Brings the corresponding 8-bit or 16-bit color component source image to the front on your desktop.
Scale Range
Changes the range of gray levels available for scaling to include the maximum possible number of levels for
the selected image type (10-Bits [0-1023], 12-Bits [0-4095], 14-Bits [0-16383], or 16-Bits [0-65535]), rather
than restricting the range to the values between the image's minimum and maximum gray levels (Image
Min/Max). In journal record/edit mode, a check box will accompany this list box, which you can select to
specify that the recorded 16-bit scaling values are to be used on playback.
Color Mosaic (Display Menu)
Converts color mosaic images to 24-bit color images.
Drop-in: MOSAIC
Use this command to convert color mosaic images to true 24-bit color images. The color mosaic Interline
CCD chip acquires images in which 2 x 2 arrays of pixels encode the red, green, and blue information in
each image. The Color Mosaic command takes the information in these arrays and reencodes it into
single pixels containing 24-bit color information.
The Color Mosaic dialog box can be expanded to display options for configuring the intensity scaling of
the red, green, and blue components of the result image. If you wish, you can direct MetaMorph to
perform the scaling for you automatically. Autoscaling can be applied to any or all of the three color
components. When the dialog box is expanded, the Color Mosaic Preview image window will appear and
the Preview Image box-in-box region selector will become available, which you can use to select a
region on the source image for display in the Color Mosaic Preview window. When you are satisfied with
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your scaling configuration settings, you can condense the dialog box again to save desktop space.
Note: Because of the arrangement of the CCD chip elements and the resulting color
mosaic image information, the conversion process will not work correctly if the source
image has been binned.
Converting a Color Mosaic Image to a 24-Bit Color Image
To converting a Sony Color Mosaic image to a 24-bit color image, use the following procedure:
Step
Action
1
From the Display menu, choose Color
Mosaic. The Color Mosaic dialog box opens.
2
Select the Color Mosaic source image with
the Source image selector. If the source
image is a stack of images, use the
secondary selector to select the Current
plane or All Planes in the stack.
3
Specify a destination for the 24-bit color
image with the Result image selector. You
can Add To or Overwrite the existing image,
or you can specify a New image.
4
If you need to change the mosaic format,
select one from the Mosaic Format dropdown list.
Note: The normal format for raw
mosaic image data is RGGB.
However, if the camera driver is
set to flip the image either
horizontally or vertically, the
format will be different. If the
preview image does not display
as expected, try changing the
Mosaic Format value. Valid
values are: RGGB, GRBG,
BGGR, AND GBRG.
5
If you want MetaMorph to perform intensity
scaling in the result image for you
automatically, select Auto from the Image
Scaling option button group. Then skip to
Step 10.
OR
If you want to specify the intensity scaling
ranges in the result image, select Custom
from the Image Scaling option button group.
6
Choose Preview >> to expand the dialog
box, revealing the color component scaling
options on the right. When you do so, the
Color Mosaic Preview image window will
appear, and the Preview Image box-in-box
region selector will become available.
7
If you have already defined a region of
interest on the Color Mosaic source image,
that region will be displayed in the Color
Mosaic Preview image window. Otherwise,
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drag the inner box in the Preview Image boxin-box control to select a region for display in
the preview window.
8
For each color component option group, use
the slider or text boxes to specify a lower and
upper color intensity value for the scaling
range.
OR
If you want MetaMorph to perform the scaling
for you automatically, based on the minimum
and color maximum intensity values in the
image, select the Autoscale check box.
9
If you need to specify intensity values that
are outside of the default range of the
image's lowest and highest intensity values
(Image Min/Max), select 10-Bits [0-1023], 12Bits [0-4095], 14-Bits [0-16383], or
16-Bits [0-65535] from the Scale Range list.
This will change the range to include all of
the intensity values available for the pertinent
color component image depth. Repeat Step
8 as necessary. When you are satisfied with
the result, you can choose Preview << to
condense the dialog box, if you wish.
10
To create the 24-bit result image, choose
Apply. The new 24-bit image will appear.
11
When you have finished, choose Close.
Color Mosaic - Dialog Box Options
Source
Selects the Sony Color Mosaic source image.
Result
Specifies a destination for the 24-bit color result image. You can Add To or Overwrite the existing image, or
you can specify a New image.
Mosaic Format
Selects the mosaic format to use when processing the image. The normal format for raw mosaic image data
is RGGB. However, if the camera driver is set to flip the image either horizontally or vertically, the format will
be different. If the preview image does not display as expected, try changing the Mosaic Format value. Valid
values are: RGGB, GRBG, BGGR, AND GBRG.
Preview Image (box-in-box control)
Selects a region of interest on the source image for display in the Color Mosaic Preview image window.
Drag the inner box to the desired location, as you would a region of interest that has been defined on an
image. Note: If you have already defined a region of interest on the Color Mosaic source image, that region
will be displayed in the Color Mosaic Preview image window.
Image Scaling
Selects an intensity scaling mode for the result image:
When you select Auto, MetaMorph will select the scaling range for each color component automatically,
based on the minimum and maximum color intensity values already present in the image. Accordingly, the
red, green, and blue component scaling option groups in the expanded right half of the Color Mosaic dialog
box will be unavailable.
When you select Custom, the color component scaling option groups will become available, which you can
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use to select a minimum and maximum intensity value for each of the three scaling ranges. Note: You will
still have the option of autoscaling each color component intensity range individually by selecting the
pertinent Autoscale check box.
Preview >>
Expands the dialog box, revealing the Red Component Scaling, Green Component Scaling, and Blue
Component Scaling option groups. The Color Mosaic Preview image window will also appear, displaying the
region of the source image that has been selected by the Preview Image box-in-box region selector.
Preview <<
Condenses the dialog box. The Color Mosaic Preview image window will also close.
Red Component Scaling
Specifies a minimum and maximum red intensity value for the scaling range in the result image. The
minimum value will be displayed in the result image as intensity (0,X,X), and the maximum value will be
displayed as intensity value (255,X,X). Drag the lower and upper handles of the slider to select a minimum
and maximum value, or type the values in the upper and lower text boxes, respectively. Alternatively, you
can direct MetaMorph to use the minimum and maximum values in the image to define the scaling range.
Green Component Scaling
Specifies a minimum and maximum green intensity value for the scaling range in the result image. The
minimum value will be displayed in the result image as intensity (X,0,X), and the maximum value will be
displayed as intensity value (X,255,X). Drag the lower and upper handles of the slider to select a minimum
and maximum value, or type the values in the upper and lower text boxes, respectively. Alternatively, you
can direct MetaMorph to use the minimum and maximum values in the image to define the scaling range.
Blue Component Scaling
Specifies a minimum and maximum blue intensity value for the scaling range in the result image. The
minimum value will be displayed in the result image as intensity (X,X,0), and the maximum value will be
displayed as intensity value (X,X,255). Drag the lower and upper handles of the slider to select a minimum
and maximum value, or type the values in the upper and lower text boxes, respectively. Alternatively, you
can direct MetaMorph to use the minimum and maximum values in the image to define the scaling range.
Scale Range
Changes the color intensity levels available for scaling to include all of the levels available in the selected
image type (10-Bits [0-1023], 12-Bits [0-4095], 14-Bits [0-16383], or 16-Bits [0-65535]), rather than
restricting the range to the values between the image's minimum and maximum gray levels (Image
Max/Min).
Apply
Creates the 24-bit color image.
Close
Closes the dialog box.
Color Mosaic Preview (image window)
This image window displays a preview of the converted 24-bit result image. If a region of interest has been
defined on the source image, that are will be displayed in the Color Mosaic Preview image window.
Otherwise, the area displayed will be that which has been selected with the Preview Image box-in-box
region selector.
Split View (Display Menu)
Separates multiple wavelength images that have been acquired using an emission splitter
device and a single camera. Acquired images can be either two or four separate
wavelengths of the same sample projected onto a single camera chip.
Dropin: SPLITVIEW
Use this drop-in to separate and organize multiple wavelength images of a single sample originally
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acquired as one image or image stack using a single camera. This dialog can split into two or four
separate images or stacks a single image or stack composed of either two or four discrete image areas
acquired at either two or four discrete wavelengths. When the images are separated, they can be
overlaid and combined into a single image composed of each individual wavelength assigned to a
discrete representative color. The images can be organized into a single stack, or they can be organized
into two or four separate images or stacks.
The advantage of using an image splitter is to enable you to simultaneously acquire two or four images
of the same sample at different wavelengths. Each image is identical to the others and each can be
acquired using a different emission filter. The images can then have appropriate overlay colors assigned
to make it easier to identify the separate wavelengths.
Two different image splitters are available for use with MetaMorph. The Optical Insights splitter can split
an image into either two or four discrete pathways, and direct the individual images onto two or four
separate areas of the camera chip. The Hamamatsu splitter can split the image into only two discrete
pathways.
Procedures:
Splitting Images - Split Tab
Splitting Images - Align Tab
Splitting Images - Configure Tab
Splitting Images - Overlay Tab
Dialog Box Options:
Split View Dialog Box Options - Split Tab
Split View - Dialog Box Options - Align Tab
Split View - Dialog Box Options - Configure Tab
Split View - Dialog Box Options - Overlay Tab
Splitting Images - Split Tab
To begin setting up an image or image stack to be split, complete the following steps, then click
the Configure tab.
Step
1
Action
Ensure that the image or image stack that
you want to split into separate images or
stacks is open.
OR
Open the Acquire dialog and begin acquiring
a Live image.
2
From the Display menu, click Split View. The
Split View dialog box opens.
3
If you are using a live image to align your
views, open the live image in the Source
image selector. If you are splitting a stack,
you can choose whether to split the entire
stack or only a single image in the stack.
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4
On the Split tab, choose whether you want to
organize your destination images as a single
stack, or a single image or a single stack for
each wavelength.
5
Click the Configure tab.
Split View - Dialog Box Options - Split Tab
Source
Selects and opens the image or image stack that you want to split into separate views. When both the
source and destination images are stacks, the wavelengths will be interleaved in the destination stack.
Therefore, when a two wavelength image is saved as a single stack, the image order is Image1, Wave1;
Image1, Wave2; Image2, Wave1; Image2, Wave2, and so on. When selecting images from a stack, you
can select a single image, or the entire stack.
Destination
Designates where the destination images will be placed. The destination image or stack can be organized
into a single stack combining all images and all wavelengths, or they can be organized into two or four
discrete images or stacks.
Stack
Saves the images as a single stack of two or four images or a stack of interleaved wavelength
images. Use the associated image selector to designate an image name.
Separate Images
Saves the separated image or image stack as either two or four separate images or image stacks
with each specific wavelength assigned to a specific stack.
Apply
Applies the settings that you made on the four split view tabs and creates the designated destination images
or image stacks.
Close
Closes the Split View dialog box.
Splitting Images - Align Tab
To make settings on the Align tab in preparation for splitting one or more images, complete the
following steps, then click the Overlay tab if you are using the Color Overlay method to align your
images.
Step
Action
1
Click the Align tab. The Align page is
displayed.
2
In the Image Selector, choose the image that
you want to use to complete your image
alignment steps.
Note: Be sure to select the entire stack or a
single image, as appropriate.
3
In the Alignment Options box, Click
Subtraction to use a grayscale subtraction
alignment image.
OR
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Click Color Overlay to use the color overlay
image alignment method.
4
In the Alignment image box, click Show
alignment image to visually align two or four
images.
5
If you are splitting four images and using the
subtraction alignment method, one
wavelength at a time, choose each of the
image wavelengths that you need to align
with the Wavelength 1 image.
6
In the Region size box, set the size of the
region that you want to include in your
destination image.
7
To help you visually align your images, click
Crosshair. A centered crosshair is placed on
the image.
8
In the image window, you should see either
two or four rectangular regions. These
regions can be resized and moved to
achieve a visual alignment. As you move
the regions, the values for H Shift and V
Shift will change. If you resize the regions,
the Region size values for Height and Width
will change.
9
To reset the values on the Align tab to their
default values, click Reset.
10
If you chose the Color Overlay alignment
method, click the Overlay tab. Otherwise,
once you have finished aligning your
images, Click the Split tab, then click Apply.
Split View - Dialog Box Options - Align Tab
Source Image
Selects the source image to be used for image alignment. If this dialog is being used in conjunction with the
Acquire dialog box, you can use the Live image to ensure that all acquired images are properly aligned.
Alignment Options
Provides two different alignment options that you can use, depending on the bit depth of the image.
Subtraction – Creates a grayscale overlay image in which the selected image is
subtracted from the Wavelength 1 image. You can change the subtraction value from the
default of 128 to improve the visual quality of the subtraction alignment image. When an
image is correctly aligned using the subtraction alignment method, all image details that
are completely identical will cancel each. Therefore, in those areas, only a shade of gray
will be visible.
Color Overlay – Creates an overlay image for images that are less than 24 bits in bit
depth. Use the color overlay option to achieve a precise image alignment.
Alignment Image
Provides settings that enable you to select the images to use for Subtraction alignment and enables you to
display an interactive alignment image.
Show Alignment Image – Overlays the images that you chose as alignment images.
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Wavelength 1 is the reference image; Wavelength 2, 3, or 4 is the subtraction image.
W1 and W2 – Aligns Wavelength 2 image with Wavelength 1
W1 and W3 – Aligns Wavelength 3 image with Wavelength 1
W1 and W4 – Aligns Wavelength 4 image with Wavelength 1
Region Size
Specifies the region size in height and width. Type or select values in the Height and Width boxes to
change the size of the region. This region size is applied equally to all of the image regions (either two or
four).
H Shift
Specifies the left position of the upper left corner of the image.
V Shift
Specifies the top position of the upper left corner of the image.
Multiplier
Multiplies the overall image intensity of the wavelength image with which it is associated.
Subtraction Constant
Specifies a subtraction constant to use when aligning the image. The default constant is 128 for 8-bit
images, and 1000 for 16-bit images.
Crosshair
Places a centered crosshair on the image. This is a centered vertical and centered horizontal line. Use this
option as a visual reference when adjusting and aligning the image regions.
Reset
Resets the region sizes, region positions, multiplier values and subtraction constant to default values.
Splitting Images - Configure Tab
To make settings on the Configure tab in preparation for splitting one or more images, complete
the following steps, then click the Align tab:
Step
Action
1
Click the Configure tab. The Configure page
is displayed.
2
In the Splitter optics box, choose either 2
Wavelenghts or 4 Wavelengths, depending
on the type of splitter.
3
If you chose 2 Wavelengths, choose the
correct image orientation (Left/Right) or
(Top/Bottom).
Note: If your installation uses a twin camera
configuation, check Twin Camera mode.
4
If you want the wavelength values for your
wavelengths annotated into the Image Info
area of each image, type the wavelength
values for each emission filter into the Filters
boxes.
5
Click the Align tab.
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Split View - Dialog Box Options - Configure Tab
Splitter optics
Selects the number of pathways into which the device splits the image. Choose either two or four
wavelengths.
2 Wavelengths – Creates two equal size image areas. The orientation of these images
areas can be either left-to-right (horizontial) or top-to-bottom (vertical).
4 Wavelengths – Creates four equal size image areas. Use the settings on the align tab
to change the size and position of the image areas.
Left/Right – Selects left-to-right (vertical) image orientation.
Top/Bottom – Selects top-to-bottom (horizontial) image orientation.
Filters
Specifies the wavelengths used to acquire the associated image.
Twin Camera mode
Provides a different 2-image configuration from a single source image in which there is no margin between
the two images. This option is intended to be used with image splitters that use two cameras that combine
the two images into a single image. When Left/Right is selected, the images are arranged diagonally (lower
left/upper right). When Top/Bottom is selected, the images are touching.
Splitting Images - Overlay Tab
Complete the following steps to specify the hue to assign to each overlay color applied to individual
wavelengths. You should make these setting before beginning to use the Color Overlay method to align
your images.
Note: Settings on the overlay tab apply only to using the color overlay for image alignment, and have no
effect on the appearance of the destination image.
Step
Action
1
Click the Overlay tab. The Overlay page is
displayed.
2
To change the intensity of any individual hue
relative to the other hues, type or select an
appropriate color in the associated Balance
box.
3
To assign a predefined color, click the Hue
dropdown list, then select the appropriately
named color hue. The Hue indicator box for
the selected wavelength will change to the
newly selected hue.
4
If you want to assign a named color that is
not on the list of predefined colors, click Edit
hue list. The Edit hue list dialog box opens.
5
If you want to assign a color other than those
named in the Hue drop-down list, simply
move the Hue slider control to the location of
the color you want to use.
6
To let MetaMorph automatically control the
intensity of each color, check Auto Balance.
7
If you want to enhance or brighten the areas
where fluorescence probes overlap, check
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Boost Colocalization.
8
To change the overall brightness of the
image, type or select an appropriate value in
the Brightness box. The default value is 50.
9
To use a specific wavelength as the
grayscale "background" (transmitted light)
image in the alignment image, click Gray
adjacent to the wavelength that you want to
assign as grayscale.
10
Click the Align tab to return to the image
alignment setting, and complete the image
alignment.
Split View - Dialog Box Options - Overlay Tab
Hue
Indicates the overlay color assigned to the selected wavelength. The region corresponding to that
wavelength on the source image will be set active. The Hue slider will also indicate the selected Hue.
Balance
Sets a scaling factor for each overlay. The default is 50.
Gray
Sets the wavelength to be used as the grayscale "background" (transmitted-light) image in the alignment
image.
No Gray
Turns of all Gray settings
Hue
Sets the overlay color for the selected wavelength that you are configuring. When a specific wavelength is
selected, move the slider to choose a color to be assigned to the wavelength. If you move the slider to a
position corresponding to one of the default colors, the Hue List will automatically update to display the
name of the color. Otherwise, the list will display "Unnamed." The color box will be updated with the selected
color.
Edit hue list
Opens the Edit Hue List dialog box. Use this to customize and name a hue that is not currently listed.
Auto Balance
Sets the intensity balance between the wavelengths automatically. If you select this option, the Balance
settings will be deactivated .
Boost Colocalization
Enhances the intensity of the areas in which two or more fluorescence probes overlap.
Brightness
Sets the intensity in the alignment image, based on the intensities of the two wavelengths. The default
setting is 50.
Interlace Images (Display Menu)
Creates a single image from the even-field scan lines from one source image and the oddfield scan lines from a second source image.
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Drop-in: ILACE
This command can be used to combine ratio image pairs from Image-1/FL. Images from Image-1/FL are
stored so that the first wavelength will be in the even scan lines and the second wavelength will be in the
odd scan lines of the saved image. Interlace Images can be applied to stacks, provided that both source
stacks have the same number of planes.
Note: This command does not support 24-bit color images.
Interlacing Images
To interlace two images or two stacks, use the following procedure:
Step
Action
1
From the Display menu, choose Interlace
Images. The Interlace Images dialog box
opens.
2
Select the first image using the Numerator
image selector. If the selected image is a
stack, select All (planes) or the Current plane
from the image selector.
3
Select the second image using the
Denominator image selector. If the selected
image is a stack, select All (planes) or the
Current plane from the image selector.
4
Select the desired destination and name for
the interlaced image using the Interlaced
image selector.
5
Choose OK.
7
Choose Close when you have finished.
Interlace Images - Dialog Box Options
Numerator
Specifies the first source image or stack for the interlaced image.
Denominator
Specifies the second source image or stack for the interlaced image.
Interlaced
Specifies the destination and name used for the resulting interlaced image.
OK
Interlaces the selected images or stacks.
Close
Closes the dialog box.
Duplicate as Displayed (Edit Menu)
Creates a 24-bit image from an existing image that includes all graphical elements.
Drop-in: 24BITCPY
Use this command to create 24-bit true color images from binary, 8-, 16-, 24-, or 48-bit images. All
graphical elements will become part of the image: thresholding overlays, object overlays, labels, arrows,
text, region outlines, etc.
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Note: If a region of interest is active in the source image, as indicated by a blinking,
dashed outline, just the area delineated by the region's bounding rectangle (determined
by a rectangular region's outline or by bounding an elliptical or irregularly shaped region
with a rectangular box) will be used to create the new image. If you have drawn regions
on the source image and do not want to restrict the size of the destination image, be sure
to use the Locator Tool to click outside of the regions so that none of the regions is still
active.
QUICK TIP: If you want to specify the colors of region outlines, first convert your source
image to 24 bits using the Color Combine command, then use the Paint Region
command to paint the region outlines, setting the Paint Mode to Region Outline Only and
selecting your preferred color with the Color command button. Be sure to delete the
regions from your source image when you have finished by right-clicking the region
outline and choosing Delete Region from the pop-up menu that appears.
Duplicating a Displayed Image
To create a 24-bit image from an image of another bit-depth, use the following procedure:
Step
Action
1
From the Edit menu, choose Duplicate as
Displayed. The Duplicate as Displayed dialog
box opens.
2
Select the source image with the Source
Image selector.
3
Select the desired destination image using
the Destination Image selector. You can
Overwrite or Add To the existing image, or
you can place the results in a New image
window.
4
When you are ready, choose OK. The 24-bit
image will be created.
5
When you have finished, choose Close.
Duplicate as Displayed - Dialog Box Options
Source Image
Selects the image to be converted to 24 bits.
Destination Image
Selects the destination for the 24-bit image. You can Overwrite the existing image or place the results in a
New image window. Or you can Add To the existing image or stack as a plane.
OK
Creates the 24-bit presentation image.
Close
Closes the dialog box.
Arrow (Display Menu)
Draws an arrow symbol onto the selected image.
Drop-in: ARROW
Use this command to add a graphical arrow symbol to your image to prepare it for publication or for
presentation. You can specify the arrow's shape, size, position, angle of rotation, grayscale value
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(binary, 8-bit, and 16-bit images) or color (24-bit images), and the proportions of the arrow's head and
tail. In addition, you can add a text label to each arrow.
You can choose from four different shapes of arrow. Each can be either single- or double-headed:
Normal
Simple
Simple with Arrow Head
Simple with Triangle Head
WARNING:
The arrow and its label will become a permanent part of the image. If you need to make morphometric or
densitometric measurements of the image, you should do so before drawing an arrow on the image.
Alternatively, you might want to consider making a copy of the original image with the Duplicate Image
command (Edit menu), and draw the arrow on the duplicate image.
For More Information about Images:
Duplicate Image
Stamp Date/Time
Stamp Calibration Bar
Painting an Arrow onto an Image
To paint an arrow symbol onto an image, use the following procedure:
Step
Action
1
From the Display > Graphics menu, choose
Arrow. The Paint Arrow dialog box opens
and an arrow-shaped region in the currently
specified configuration will appear on the
image.
2
If necessary, use the Image selector to select
the image onto which you want to draw the
arrow.
3
Select a shape for the arrow from the Arrow
Type button group.
AND
If you want a double-headed arrow, select
the Double-Headed check box.
4
If you want the arrow to consist of its head
only, with no tail, clear the Include Arrow Tail
check box.
OR
If you want the arrow to have a tail, leave the
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Include Arrow Tail check box selected and
select a size for the tail (8 - 250) with the Tail
Size spin box.
5
To change the overall length of the arrow
(head and tail, combined), select a size
(1 - 100) from the Length spin box or slider.
AND
To change the width of the arrow (head and
tail), select a size (1 - 100) from the Width
spin box or slider.
6
To change the angle of rotation of the arrow,
select an angle (0 - 359) from the Rotation
spin box or slider. An angle of 0 specifies an
arrow pointing to the left.
7
Position the arrow in the image by dragging it
with your pointer, or use the X and Y spin
boxes in the Position option group to select
the X and Y coordinates, respectively.
8
To specify a grayscale value (binary, 8-bit,
and 16-bit images) or a color (24-bit images)
for the border and interior of the arrow,
choose Colors. The Arrow Colors dialog box
will appear.
OR
If you want to use the default color scheme
of a black border and white interior, skip to
Step 12.
9
If you want the image to show through the
interior of the arrow, clear the Paint Arrow
Interior check box.
OR
If you want to specify a "fill" for the arrow,
select the Paint Arrow Interior check box. If
you are drawing the arrow on a grayscale
image (binary, 8-bit, or 16-bit), use the Color
spin box to select a grayscale intensity value
for the interior of the arrow. If you are
drawing the arrow on a 24-bit color image,
choose the Color command button and
select a color from the Color dialog box that
appears and choose OK.
10
If you do not want to draw a border for the
arrow, clear the Paint Arrow Border check
box.
OR
If you want to draw a border for the arrow
that differs in color from its interior, select the
Paint Arrow Border check box. Then specify
a grayscale value (binary, 8-bit, or 16-bit
images) or color (24-bit images) for the
border with the Color option, as in Step 9.
11
Choose OK to return to the Paint Arrow
dialog box.
12
If you want to label the arrow, select the
Paint Region Label check box. Then type the
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desired label in the Label Text box.
13
To apply the arrow to the selected image,
choose Paint.
If you change your mind, you can choose
Undo to remove the arrow. You can also
repaint the removed arrow by choosing
Redo.
14
Choose Close.
Arrow - Dialog Box Options
Image
Selects the image on which you want to draw the arrow.
Arrow Type
Selects a shape for the arrow.
Double-Headed
Specifies that the arrow be double-headed (back-to-back).
Include Arrow Tail
Specifies that the arrow is to be drawn with a tail. If you clear this check box, just the head of the arrow will
be drawn.
Tail Size
Specifies a length for the tail of the arrow. The size can range from 8 to 250.
Position
Specifies the X and Y coordinate of the center of the arrow. If the arrow region is off of the image, you may
need to adjust these settings before you can drag the region with your pointer.
Length
Selects an overall length for the arrow (head and tail, combined). This value can range from 1 to 100. The
default size is 20.
Width
Selects an overall width for the arrow (head and tail). This value can range from 1 to 100. The default size is
20.
Rotation
Selects an angle of rotation for the arrow. This value can range from 0 to 359. An angle of 0 will specify an
arrow that is pointing to the left; an angle of 90 will point the arrow towards the top of the image.
Paint Region Label
Labels the arrow with the text you type in the Label Text box.
Label Text
Specifies a text label for the arrow. To label the arrow, you will also need to select the Paint Region Label
check box.
Paint
Draws the arrow, as configured, onto the image. The arrow will become a permanent part of the image.
Undo/Redo
Undoes or redraws the arrow.
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Colors
Opens the Arrow Colors dialog box, from which you can specify a color for the arrow's interior and border.
Close
Closes the Paint Arrow dialog box. If the Arrow Colors dialog box is open, you will need to close it before
you can close the Paint Arrow dialog box.
Arrow Colors - Dialog Box Options
Paint Arrow Interior
Specifies a "fill" for the interior of the arrow. If you clear this check box, the image will show through the
interior of the arrow.
Color spin box (Paint Arrow Interior)
Selects a grayscale value for the interior of the arrow. If you clear the Paint Arrow Interior check box, this
option will be unavailable. If you are drawing on a binary (1-bit) image, you can select either 0 (black) or 1
(white). If you are drawing on an 8-bit image, you can select an intensity between 0 and 255. If you are
drawing on a 16-bit image, you can select a value between 0 and 65535. If you are drawing on a 24-bit color
image, this spin box will be replaced by a Color command button, which opens the Color dialog box.
Color command button (Paint Arrow Interior)
Opens the Color dialog box, from which you can select a color for the interior of the arrow. This option will be
available only for 24-bit color images.
Paint Arrow Border
Specifies that a border be drawn for the arrow. This allows you to select an intensity value or color for the
outline of the arrow that differs from that of the interior.
Color spin box (Paint Arrow Border)
Selects a grayscale value for the border of the arrow. If you clear the Paint Arrow Border check box, this
option will be unavailable. If you are drawing on a binary (1-bit) image, you can select either 0 (black) or 1
(white). If you are drawing on an 8-bit image, you can select an intensity between 0 and 255. If you are
drawing on a 16-bit image, you can select a value between 0 and 65535. If you are drawing on a 24-bit color
image, this spin box will be replaced by a Color command button, which opens the Color dialog box.
Color command button (Paint Arrow Border)
Opens the Color dialog box, from which you can select a color for the border of the arrow. This option will be
available only for 24-bit color images.
OK
Accepts any changes you made to the settings in the Arrow Colors dialog box and closes the dialog box.
Cancel
Cancels any changes you made to the settings in the Arrow Colors dialog box and closes the dialog box.
Grid (Display Menu)
Draws a grid of a specified gray level on a grayscale or color image in an image window or
on an external video monitor, using the selected number of vertical and horizontal lines.
Drop-in: GRID
Use this command to draw a grid on an image or stack. This command allows you to specify the number
of vertical and horizontal lines, as well as the line color and line width. You can Undo a grid drawn on an
image or the current plane of a stack.
When a video monitor is selected as the location of the grid, this command will draw the grid on a frozen
image displayed on the monitor, not on the live video display. You can use the Live Video dialog box to
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restore live video and clear the grid from the monitor. If you want the grid to be part of the acquired
images, you can draw a grid on a blank image and then use that image as background subtraction
image for live video. (Remember to reverse the colors of the grid lines when you draw the grid--use the
line color 255 on a dark image to create a grid with black lines when using background subtraction.) Or
you can draw the grid on the live video image window and then copy that image.
Drawing a Grid
To draw a grid on an image or video monitor, use the following procedure:
Step
Action
1
From the Display > Graphics menu, choose
Grid. The Draw Grid dialog box opens.
2
If you are drawing the grid on an image in an
image window, select the desired image
using the Image selector. If the selected
image is a stack, select All (planes) or the
Current plane from the image selector.
3
Select whether you want the grid drawn on
the Selected Image or the Video Monitor
from the Draw On group.
4
Select the desired number of vertical and
horizontal lines using Horizontal Lines and
Vertical Lines. The number of boxes that will
be drawn with the current selections will be
displayed next to these options.
5
Select the thickness of the grid lines using
Line Thickness.
AND
Select the grayscale value of the grid lines
using Line Color.
6
Choose Draw Grid.
7
Choose Close when you have finished.
Grid - Dialog Box Options
Image
Specifies the image window for the grid if the image is to be displayed on the computer monitor.
Draw On
Specifies whether the grid will be drawn on the selected image or the external video monitor (if a video
board is being used).
Vertical Lines
Specifies the number of vertical lines for the grid.
Horizontal Lines
Specifies the number of horizontal lines for the grid.
Line Thickness
Specifies the thickness of the grid lines, in pixels.
Line Color
Specifies the grayscale value for the grid lines.
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Draw Grid
Draws the specified grid on the selected image or on the video monitor.
Undo/Redo
Undoes/redoes the last grid drawn on an image or the current plane of a stack.
Close
Closes the dialog box.
Stretch and Mirror (Display Menu)
Resizes an image by rescaling it in the X and Y axis directions. Creates a mirror image by
flipping the image horizontally or vertically.
Drop-in: STRETCH
Use this command when you want to scale an image proportionally to a larger or smaller size, or when
you want to stretch or compress an image using different vertical and horizontal proportions. You can
also flip the image horizontally and/or vertically while stretching it. If a region of interest is active, the
command will resize and/or flip the area in the region of interest.
The Horizontal Mirror and Vertical Mirror options which flip the image are similar to MetaMorph's Flip:
Horizontal and Flip: Vertical commands on the Graphics menu.
Image-1/AT: You can convert Image-1/AT images, acquired using rectangular pixels, for use in
MetaMorph by stretching the image horizontally to 125%.
For More Information about Editing Images:
Duplicate Image with Zoom
Flip
Rotate
Stretching and Mirroring an Image
To stretch and mirror an image, use the following procedure:
Step
Action
1
From the Display menu, choose Stretch and
Mirror. The Stretch and Mirror dialog box
opens.
2
Select the source image using the Source
Image selector. If the selected image is a
stack, select All (planes) or the Current plane
from the image selector.
3
Select the destination image using the
Destination Image selector. You can
overwrite or add to the existing image or you
can place the results in a new image window.
4
Select the desired percentages of horizontal
and vertical stretch or compression using
Stretch Horizontally and Stretch Vertically.
To enlarge or reduce the image
proportionally, select the same value for both
options.
5
You can select Interpolate When Stretching
for images that are stretched greater than
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100 percent.
6
You can select Horizontal Mirror and/or
Vertical Mirror to flip the image in the desired
direction.
7
Choose OK when you have finished.
Stretch and Mirror - Dialog Box Options
Source Image
Selects the source image that you want to stretch, compress, and/or flip.
Destination
Selects the destination for the stretched/compressed image. You can overwrite the existing image or place
the results in a new image window. Or you can add the stretched/compressed image as a plane to an
existing image or stack.
Stretch Horizontally
Specifies the percentage that MetaMorph should stretch or compress the image horizontally.
Stretch Vertically
Specifies the percentage that MetaMorph should stretch or compress the image vertically. To reduce or
enlarge the image proportionally, select the same value for this option as Stretch Horizontally.
Interpolate When Stretching
Select this option when you are stretching an image larger than 100%. Interpolation computes what the
grayscale or color values between the original values should be in the stretched image. Normally, when a
stretch of 400% is applied to an image, sixteen times as many pixels are used to represent each pixel's
value. This creates a chunky, pixelated image that is not as smooth as the original image. Rather than
applying the same value to a larger block of pixels, interpolation computes the intensity values for each new
pixel based on the original values surrounding it.
Horizontal Mirror
Flips the image on its vertical axis, thereby creating a mirror image. You can use Horizontal Mirror and
Vertical Mirror at the same time.
Vertical Mirror
Flips the image on its horizontal axis, thereby turning the image upside down. You can use Horizontal Mirror
and Vertical Mirror at the same time.
OK
Stretches and/or mirrors the selected image.
Cancel
Cancels the command.
Boxes on Binary Image (Display Menu)
Places a fixed grid of boxes of user-specified height, width, and line thickness on a
selected binary image and indicates the image size, the total number of boxes, and total
area in pixels inside each box.
Drop-in: GRIDBIN
Use this command to place a line grid on a binary image. You can set the width, height and line
thickness dimensions in pixels for the grid of boxes, or you can use the default values which are 100 X
100 with a line thickness of 1 pixel. The dimension settings for box width and height control the spacing
between lines on the grid. The image values displayed in the dialog box indicate the image size in
pixels, the area in pixels inside each box, and the number of boxes. The smaller the dimensions for
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width and height, the greater the number of boxes in the image. Depending on the image background,
you can select either white or black lines for the image grid. The default setting is black lines. If your
background is black, select white lines.
You can overlay applications of the grid boxes on a single binary image. This can be useful for creating a
graduated grid with the smallest spacing using the thinnest lines. For example, you might first apply a
grid of 10 X 10 with a line width of 1 pixel, then apply a grid of 20 X 20 with a line width of 2 pixels. Thus,
every second line in the grid is a heavier weight.
Note: Boxes on Binary Image can only be applied to a binary image. If your image is not binary (only
black and white pixels) you can convert it to a binary image using the Binary Image Operations
command.
Note: Boxes on Binary Image makes only complete boxes of the specified dimension in your image. No
partial boxes are created. Any image area not included in a complete box is discarded. Therefore, your
final image with the grid applied might be smaller than the original, and might have areas excluded that
were part of the original image.
Drawing Boxes on a Binary Image
To apply a grid of either black or white boxes to a binary image, complete the following
procedure:
Step
Action
1
Open a binary image to which you want to
apply a box grid.
2
From the Display > Graphics menu, choose
Boxes on Binary Image. The Draw Boxes
on Binary Image dialog box opens.
3
If more than one binary image is open, click
the Image box, and choose the binary image
to which you want to apply the box grid.
4
In the Box Width box, type or select the box
width in pixels.
5
In the Box Height box, type or select the box
height in pixels.
6
In the Line Thickness box, type or select the
line thickness for the grid lines.
7
Click Rename Image if you want the
command to rename your image to a name
composed of the values for the number of
boxes, and the width, height, and the area in
pixels within each box
8
In the Color area, click White for white lines
on a black background, or click Black for
black lines on a white background.
9
Click Draw Boxes to apply the specified grid
to your binary image.
10
Click Revert to undo the application of the
grid to your binary image.
11
Click Close to close the dialog box after you
have completed all steps and are satisfied
with the results.
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Boxes on Binary Image - Dialog Box Options
Image
Indicates the name of the binary image available for application of the grid boxes. Boxes on Binary Image
can only be applied to a binary image. If more that one binary image is open, the Image drop-down list will
contain the names of all open binary images.
Image Size
Indicates the size of the image in pixel width and height.
Box Width
Specifies the width setting to be applied to the box grid.
Box Height
Specifies the height setting to be applied to the box grid.
Line Thickness
Specifies the line thickness to be applied to the box grid.
Rename Image
Indicates that you want to rename the image to a name that incorporates the values for the number of
boxes, the width, height, and the area in pixels within each box.
Box Area
Indicates the area in pixels within each box.
# Boxes
Indicates the total number of boxes within the image.
Color
Specifies whether to apply a box grid of white lines or black lines. Apply white lines to a black background;
apply black lines to a white background.
Draw Boxes
Applies the box grid to the image based on the settings made in the dialog box.
Revert
Removes the most recently applied box grid from the image.
Note: Once you close the dialog box, Revert cannot remove the last grid applied.
Close
Closes the dialog box and permanently applies all grids to the image.
Text (Display Menu)
Draws text of a specified gray value on the selected image.
Drop-in: TEXT
Use this command when you want to place text on an image at a specified location. You can place one
line of text at a time on the selected image. You can erase the image area behind the text and specify a
background color for that area.
You can place the text anywhere on the image prior to drawing. This command creates an active region
to mark the location of the text, so that you can see where the text will be drawn. You can use the
options in the dialog box to change the location of the text or you can move and resize the region, as
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with a rectangular region (the region defines the space for the text). Once you choose Draw, the text will
become part of the image, overwriting any image data under it.
This command can be applied to 1-bit, 8-bit, 16-bit, or 24-bit images. You can use the Undo option in the
dialog box to undo it or you can use the Undo command in the Edit menu.
QUICK TIP: Text boxes can be copied and pasted into successive images in precisely the same location
as in the original image. When you right-click the text region in the source image, a pop-up context menu
will appear, from which you can choose Copy Region. You can then right-click in the destination image
and choose Paste Region from the context menu. The region outline will be pasted into the destination
image, and you can then apply the Draw Text command by selecting the destination image from the
image selector and choosing Draw. You can also use the Arithmetic command to merge text onto an
image.
WARNING:
When you apply the text to an image, the text will become a part of the image itself. If you subsequently
attempt to perform a densitometric or morphometric measurement on the entire image, the grayscale
values and morphometric characteristics of the text will be measured along with the objects in the image.
Be sure to perform your measurements before you apply any graphics to the image. If there is any
chance that you will need to reanalyze the original image, you should make a copy of the original and
apply the text to the copy.
WARNING:
If you are drawing text on a 16-bit image that has already been scaled using the Scale 16-Bit Image
command’s Auto Scale option, you should select grayscale values for the text and background colors
that do not differ greatly from the high and low intensity values in the image itself. Because the text
becomes part of the image, the image intensities will be rescaled when an excessively bright or dark text
label is applied to an autoscaled image. This can have the undesirable effect of severely reducing the
apparent contrast in the image and making it too dark or too bright.
For More Information about Editing Images:
Paint Region
Transfer Regions
Copy
Paste
Arithmetic
Drawing Text on an Image
To draw text on an image or stack, use the following procedure:
Step
Action
1
From the Display > Graphics menu, choose
Text. The Text dialog box opens.
2
Select the desired image using the Image
selector. If the selected image is a stack,
select All (planes) or the Current plane from
the image selector.
3
Select the X and Y coordinates of the desired
location for the text using the X and Y
options.
OR
Click in the middle of the text region and drag
the region outline to the desired location. The
values in the X and Y text boxes will change
accordingly.
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4
Select the desired gray value for the text
color using Text Color.
5
If you want to erase the image area behind
the text, select Erase Image Behind Text.
AND
Select the desired gray value for the
background color behind the text using Back.
Color.
6
Type the desired text in the Text box.
7
Choose Draw.
Note: If you write text that goes beyond the
bounds of the image, an error message will
be displayed.
8
Choose Close when you have finished.
Text - Dialog Box Options
Image
Specifies the image or stack for the drawn text.
X
Specifies the X-coordinate on the image for the location of the text.
Y
Specifies the Y-coordinate on the image for the location of the text.
Text Color
Specifies the grayscale value to be used for the text.
Erase Image Behind Text
Erases the image area behind the text and fills it in with the grayscale value specified by Back. Color.
Back. Color
Specifies the grayscale value used behind the text if you selected Erase Image Behind Text.
Text
Use this text box to type the desired text to be drawn on the image.
Draw
Draws text on the selected image or stack.
Close
Closes the dialog box.
Undo
Undoes the last text drawn on an image.
Gray Wedge (Display Menu)
Draws a reference grayscale "wedge" on the specified image, using your choice of starting
and ending grayscale or color values.
Drop-in: WEDGE
Use this command when you want to place a reference grayscale or color wedge on an image. This
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command places the wedge in the active region in the image, or uses the entire image if there is no
active region. If necessary, you can use the Flip or Rotate commands (Display > Graphics menu) to
change the orientation of the wedge.
Once you have placed a wedge in an image, you can use it to verify that your video monitor is adjusted
correctly by transferring the image to the monitor with the Transfer Image to Video command.
The starting value (gray level or color) will be on the left side of the wedge and the ending value will be
on the right side. If the height is greater than the width, the wedge will be drawn vertically, with the
starting value at the lower edge.
Note: This command does not support binary (1-bit) images.
For More Information about Editing Images:
Flip
Rotate
Transfer Image to Video
Drawing a Gray Wedge on an Image
Drawing a Gray Wedge on a Grayscale Image
Drawing a Color Wedge on a 24-Bit Image
Drawing a Gray Wedge on a Grayscale Image
To draw a reference gray wedge on an 8-bit or 16-bit image or stack, use the following procedure:
Step
Action
1
From the Display > Graphics menu, choose
Gray Wedge. The Gray Wedge dialog box
opens.
2
Select the desired image using the Image
selector. If the selected image is a stack,
select All (planes) or the Current plane from
the image selector.
3
Define an active region on the source image
using a Region Tool.
The width of the active region, in pixels,
should equal the number of gray levels that
you want to represent (for example, the
region should be 256 pixels wide for a wedge
that represents grayscale values 0 - 255).
Otherwise, not all of the grayscale levels in
the specified range can be represented.
4
Select the desired starting gray value for the
left side (or lower edge) of the gray wedge
using Starting Gray Value.
5
Select the desired ending gray value for the
right side (or upper edge) of the gray wedge
using Ending Gray Value.
6
Choose Apply. The reference gray wedge
will be drawn on the image.
If you change your mind, you can choose
Undo to remove the gray wedge. You can
also repaint the removed wedge by choosing
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Redo.
7
Choose Close when you have finished.
Drawing a Color Wedge on a 24-Bit Image
To draw a reference color wedge on a 24-bit color image or stack, use the following procedure:
Step
Action
1
From the Display > Graphics menu, choose
Gray Wedge. The Gray Wedge dialog box
opens.
2
Select the desired image using the Image
selector. If the selected image is a stack,
select All (planes) or the Current plane from
the image selector.
3
Define an active region on the source image
using a Region Tool.
The width of the active region, in pixels,
should equal the number of color levels that
you want to represent (for example, the
region should be 256 pixels wide for a wedge
that represents red values 0 - 255).
Otherwise, not all of the levels in the
specified range can be represented.
4
Choose the Color command button in the
Starting Color row. The Color dialog box will
appear.
5
Select the desired color from the Basic Color
palette displayed at the top of the dialog box,
so that the selected color's outline is
highlighted.
OR
Use the Red, Green, and Blue text boxes to
define the values for a custom color and then
choose Add to Custom Colors.
OR
Click inside the Color Refiner Box to select a
color and then choose Add to Custom
Colors.
6
Choose OK to return to the Draw Gray
Wedge dialog box.
7
Repeat Steps 4 - 6, choosing the Color
command button in the Ending Color row.
8
Choose Apply.
If you need to undo the color wedge you just
created, you can choose Undo. Choose
Redo to reapply the color wedge.
9
When you have finished, choose Close.
Gray Wedge - Dialog Box Options
Grayscale Images
24-Bit Color Images
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Gray Wedge (Grayscale Images) - Dialog Box Options
Image
Specifies the image or stack for the gray wedge.
Starting Gray Level
Specifies the first grayscale level displayed on the left side (or lower edge, depending on its orientation) of
the gray wedge. The default initial value for this option will match the limits of the selected image.
Ending Gray Level
Specifies the last grayscale level displayed on the right side (or upper edge, depending on its orientation) of
the gray wedge. The default initial value for this option will match the limits of the selected image or
grayscale value 255, whichever is lower. This value can be increased for 16-bit images.
Apply
Draws the gray wedge on the specified image in the active region, if available, or on the entire image.
Undo/Redo
Undoes or redraws the gray wedge.
Close
Closes the dialog box.
Gray Wedge (Color Images) - Dialog Box Options
Image
Specifies the image or stack for the color wedge.
Starting Color
Opens the Color dialog box, from which you can select the starting color displayed on the left side (or lower
edge, depending on its orientation) of the color wedge.
Ending Color
Opens the Color dialog box, from which you can select the ending color displayed on the right side (or upper
edge, depending on its orientation) of the color wedge.
Apply
Draws the color wedge on the specified image in the active region, if available, or on the entire image.
Undo/Redo
Undoes or redraws the color wedge.
Close
Closes the dialog box.
Show/Hide Image at Full Screen (Display Menu)
Centers the selected single-plane image and displays it against a black background.
Drop-in: FULLSCR
Use this command to display an image by itself, without any other desktop elements. The image will be
centered in the screen and displayed against a black or white background. The Show Image at Full
Screen command will be particularly useful for preparing images for screen capture or for display during
a presentation. The display will revert back to its original state when you press any keyboard key or the
left mouse button.
You will not be able to use this command simultaneously with the Movie command (Stack menu).
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However, the Movie window has a control to play back at full screen.
QUICK TIP: If you use this command in a journal, you can use the Delay command to specify the length
of time that each image is to be displayed.
Note: This command does not alter the size of the image. If you want to display the
image at its maximum possible size, you will first need to use the Zoom Tool.
Showing an Image at Full Screen
To display an image at full screen, use the following procedure:
Step
Action
1
From the Display > Graphics menu, choose
Show/Hide Image at Full Screen. The
Show/Hide Image at Full Screen dialog box
opens.
2
If necessary, select the image to be
displayed with the Image selector.
3
From the Background Color group, select
whether you would like the image to appear
on a black or white background.
4
Choose OK. The image will be displayed at
full screen.
Note: If the image is already being
displayed at full screen, clicking OK will hide
the image. Conversely, if the image is
currently hidden, clicking OK will show the
image at full screen.
6
If you are performing a screen capture, move
the cursor off-screen and perform your
screen-capture routine.
7
To revert back to the original display state,
press a keyboard key or the left mouse
button.
OR
Select Hide from the Action radio button
group and choose OK.
8
Choose Close.
Note: If you are playing back this command
in a journal, the dialog box will have closed
automatically when you chose OK in Step 4.
Show Images at Full Screen - Dialog Box Options
Image
Specifies the image or stack to be displayed at full screen.
Background Color
Selects whether the image will be shown on a black or a white background.
OK
Displays the selected image
Close
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Closes the dialog box.
Graph Settings
Use the Graph Settings command to configure graphs in MetaMorph.
This command contains settings that affect the features and display of the active graph in MetaMorph.
Opening the Graph Settings Dialog Box
Using the Graph Settings Command
Graph Setting - Dialog Box Options
Opening the Graph Settings Dialog Box
Use one of the following procedures to open the Graph Settings dialog box from any graph in
MetaMorph:
Step
1
Action
Click the Down Arrow button directly below
the left side of the graph and select Graph
Settings from the drop-down menu.
OR
Double-click in any portion of the graph.
OR
Right-click on any portion of the graph that
does not contain data and select Graph
Settings from the drop-down menu.
2
The Graph Settings dialog box opens.
Using the Graph Settings Command
Use the following procedures to configure graph settings:
Configuring a Trace Line
Configuring Graph Titles
Configuring the Background
Configuring a Trace Line
To configure a trace line, use the following procedure:
Step
Action
1
Click the Down Arrow button directly below
the left side of the graph and select Graph
Settings from the drop-down menu. The
Graph Settings dialog box opens.
2
Click the Traces tab.
3
Configure the appearance of the trace line
using the Name, Show, Color, Line Style,
Width, Mark Style, and Size options.
The Name field can be edited. Color displays
the color of the trace line. Line Style specifies
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the type of line used. Width specifies the
pixel width of the line. Mark Style selects the
style of marker to be used. Size selects the
size of the mark style, if applicable
4
If you want to change the color of a trace
line, click the color box for the trace to open
the Color dialog box. Select a color and click
OK.
OR
To make all trace lines the same color, rightclick the color heading and select Change All
Colors to select a color.
5
If you want to change the line style of the
trace, click the drop-down arrow in the Line
Style column and select the desired style.
OR
To make all trace lines have the same line
style, right-click the Line Style heading,
select Change All Line Styles, and choose a
style from the drop-down list.
6
If you want to change the mark style of points
on a trace, click the drop-down arrow in the
Mark Style column and select the desired
style.
OR
To make all trace lines have the same mark
style, right-click the Line Style heading,
select Change All Mark Styles, and choose a
style from the drop-down list.
7
To save the settings you created so they can
be loaded later, click Save to open the Save
Graph Settings dialog box and save your
settings.
8
Click Close when you have finished.
Configuring Graph Titles
To change a graph title, use the following procedure:
Step
Action
1
Click the Down Arrow button directly below
the left side of the graph and select Graph
Settings from the drop-down menu. The
Graph Settings dialog box opens.
2
Click the Graph tab.
3
Type a new title name in the Title text box.
4
To save the settings you created so they can
be loaded later, click Save to open the Save
Graph Settings dialog box and save your
settings.
5
Choose Close when you have finished.
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Configuring the Background
To configure the background, use the following procedure:
Step
Action
1
Click the Down Arrow button directly below
the left side of the graph and select Graph
Settings from the drop-down menu. The
Graph Settings dialog box opens.
2
Click the Appearance tab.
3
Click Background Color to open the Color
dialog box. Select a color and click OK.
4
To save the settings you created so they can
be loaded later, click Save to open the Save
Graph Settings dialog box and save your
settings.
5
Choose Close when you have finished.
Graph Settings – Dialog Box Options
The following tabs are used in the Graph Settings dialog box:
Samples Tab
Graph Tab
Traces Tab
X-Axis Tab
Y-Axis Tab
Legend Tab
Appearance Tab
Undo Changes
Reverses any changes made in the current session.
Load
Opens the Load Graph Settings dialog box. Use this to load previously saved graph settings to apply to the
active graph.
Save
Opens the Graph Settings dialog box. Enables you to save the graph settings of the active graph to a file
(*.cfg) for subsequent re-use using the Load command.
Close
Closes the dialog box.
Graph Settings Dialog Box Options - Samples Tab
Click a button to change the way the graph looks
Enables you to change the appearance of graphs. The following choices, from left to right, are available:
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Trace lines
Trace lines and grid lines
Trace lines and vertical space traces
Trace lines and circle point marks
Trace lines, circle point marks, and grid lines
Trace lines, circle point marks, and vertical space
traces
Plus point marks
Plus point marks and grid lines
Plus point marks and bar markers
Trace lines and bar markers
Trace lines, grid lines, and autoscale X- and Y-axes
Trace lines, bar markers, and vertical space traces
Color scheme
Enables you to change the color scheme of graphs. The following choices, from left to right, are available:
Black background and white foreground.
Black background and colorful outside background.
You can edit the background colors in the
Appearance tab.
White background and black foreground.
White background and colorful outside background.
You can edit the background colors in the
Appearance tab.
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Graph Settings Dialog Box Options - Graph Tab
Title
Default name of graph. This name can be edited.
Graph Type
Selects a graph type to use. The following choices are available: Linear (default), Log x linear y, Linear x log
y, Log x log y, and Bar.
Show Title
Toggles the graph title on and off.
Show Border
Toggles the graph border on and off.
Show Border One Pixel Outside Graph Interior
Ensures the border is outside the graph interior.
Space Traces Vertically
Divides the visible vertical space by each trace (line on graph) and separates the traces so each trace is
clearly visible.
Margins
Top
Specifies the top margin of the graph (in pixels).
Bottom
Specifies the bottom margin of the graph (in pixels).
Left
Specifies the left margin of the graph (in pixels).
Right
Specifies the right margin of the graph (in pixels).
Graph Settings Dialog Box Options - Traces Tab
Key
Displays the color and style of each trace.
Name
This field can be edited.
Show
Toggles the display of the trace.
Color
Displays the color of each trace. Click the color box to edit.
Line Style
Selects the line style of the trace. Solid is the default.
Width
Selects the width (in pixels) of the trace. Click the box to edit.
Mark Style
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Selects the mark style of the trace. None is the default.
Size
Selects the size of the mark style if applicable.
Graph Settings Dialog Box Options - X-Axis Tab
Title
Displays the title of the X-Axis. This field can be edited.
Range
Selects the X-Axis range for the graph. The From and To fields are only enabled when Auto scale range to
encompass the data is not selected.
Auto scale range to encompass the data
Automatically sets the range of the axis to be equal to the minimum and maximum data point values.
Invert range values on axis
Inverts the range value on the X-Axis.
Show
Toggles the following features of the X-Axis:
Title
Major ticks
Axis line
Minor ticks
Bar marker
Tick label
Grid lines
Selects the vertical grid lines displayed on the graph.
Tick Marks
Use automatic placement of ticks
Automatically places ticks on the X-Axis based on the amount and range of data in the graph.
Major ticks
Selects the number of major ticks on the X-Axis. This field is only enabled when Use automatic
placement of ticks is unchecked.
Minor ticks
Selects the number of minor ticks on the X-Axis.
Decimal digits
Selects the number of decimal points to use in the X-Axis values.
Rotate tick labels 90 degrees
Rotates the X-Axis tick labels 90 degrees.
Location of ticks
Selects whether tick marks are located outside or inside the graph, or both.
Graph Settings Dialog Box Options - Y-Axis Tab
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Title
Displays the title of the Y-Axis. This field can be edited.
Range
Selects the Y-Axis range for the graph. The From and to fields are only enabled when Auto scale range to
encompass the data is not selected.
Auto scale range to encompass the data
Automatically sets the range of the axis to be equal to the minimum and maximum data point values.
Invert range values on axis
Inverts the range value on the Y-Axis.
Show
Toggles the following features of the Y-Axis:
Title
Major ticks
Axis line
Minor ticks
Bar marker
Tick label
Grid lines
Selects the vertical grid lines displayed on the graph.
Tick Marks
Use automatic placement of ticks
Automatically places ticks on the Y-Axis based on the amount and range of data in the graph.
Major ticks
Selects the number of major ticks on the Y-Axis. This field is only enabled when Use automatic
placement of ticks is unchecked.
Minor ticks
Selects the number of minor ticks on the Y-Axis.
Decimal digits
Selects the number of decimal points to use in the Y-Axis values.
Rotate tick labels 90 degrees
Rotates the Y-Axis tick labels 90 degrees.
Location of ticks
Selects whether tick marks are located outside or inside the graph, or both.
Graph Settings Dialog Box Options - Legend Tab
Show legend
Toggles the legend on or off.
Columns
Selects the number of columns in the legend.
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Show legend border
Toggles the legend border on or off.
Show legend background
Toggles the legend background on or off.
Key
Trace and marker
Displays the trace in the key as it is represented on the graph—the line width and marker will match
the graph.
Square
Displays each trace as a colored square in the key.
Traces
Key
Displays the color and style of each trace on the legend.
Name
Displays the name of each trace shown on the legend. This field can be edited. You can also drag
a name to rearrange the order shown in the legend.
Graph Settings Dialog Box Options - Appearance Tab
Simple
Enables you to select the foreground color, background color, and font for the active graph.
Custom font and color for each graph item
Opens a table that enables you to select a custom font and color for each graph item.
Foreground Color
Opens the Color dialog box to change the foreground color.
Background Color
Opens the Color dialog box to change the background color.
Font
Opens the font dialog box to change the font and font size for the active graph.
Set Color Threshold (Measure Menu)
Selects a threshold range for 24-bit color images.
Availability: Available for MetaMorph Basic and MetaMorph Premier
Drop-in: CLRTHRES
Use this command to select upper and lower limits for a continuous threshold for a 24-bit color image.
You can select the Red-Green-Blue (RGB) color space model, the Hue-Saturation-Intensity (HSI) color
space model, or the Hue-Saturation-Luminosity (HSL) color space model, and perform thresholding
manipulations through each color component, or "channel," of that model.
The Set Color Threshold command also has an "interactive" Set by Example mode, which allows you to
select a threshold range based on the values of the pixels in the image that you click on with your mouse
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cursor. This is somewhat similar to the use of Color Threshold Mapping Tool in the Region Tools
window. The major difference is that, with the Set Color Threshold command, you can select a pixel that
represents the lower end of the threshold range, and then select a pixel that represents the upper end.
MetaMorph will automatically select all values that are between the values of the two pixels. By contrast,
the Color Threshold Mapping Tool specifies just the values of the selected pixels, providing a
discontinuous threshold range.
Color threshold range settings that are configured with the Set Color Threshold command can be saved
and loaded as color threshold range (*.ctr) state files.
Setting a Color Threshold
To set a color threshold, use the following procedure:
Step
Action
1
From the Measure menu, choose Set Color
Threshold. The Set Color Threshold dialog
box opens.
2
Select the image to be thresholded using the
Image selector.
3
If you want to use a previously saved set of
color threshold settings, choose Load Range.
Otherwise, skip to Step 5.
4
The Load Color Threshold Range dialog box
opens.
AND
Select the icon for the desired color threshold
range (*.ctr) state file. If necessary, use the
Look In list or Up One Level icon button to
locate the correct drive and folder. Then
choose Open. The threshold settings will be
applied to your image. Now skip to Step 11.
5
From the Color Model list, select the color
model you want to use for setting the color
threshold: RGB, HSI, or HSL. Your selection
will determine the options you see in the
lower half of the dialog box.
6
If you selected HSI or HSL as your color
model in Step 5, use the Hue Range radio
button group to select whether the ranges
between the upper and lower limits are to be
included in the threshold range (Inclusive) or
if the ranges outside of the upper and lower
limits are to be thresholded (Exclusive).
7
Use the sliders or the left and right spin
boxes for each of the color channels (RedGreen-Blue, Hue-Saturation-Intensity, or
Hue-Saturation-Luminosity) to select the
lower and upper threshold range values. As
you adjust the settings, the distribution of the
red thresholding overlay that covers pixels
with the selected values will change.
8
If you want to use the interactive "point-andclick" method of selecting the threshold
range, choose Set by Example. The dialog
box will expand, revealing two more options.
If necessary, reset the threshold range by
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selecting the Reset Color Threshold Range
on Next Click check box, so that a check
mark appears in it.
AND
Use the pointer to select pixels in the image
that have the values that you want to include
in the threshold range. As you click the
pixels, they will be covered by the red
thresholding overlay, and the color channel
sliders and spin boxes will update to display
the new values.
If you want to remove the values of the pixels
you selected last, choose Undo Last Click.
9
By default, the thresholding State is in
Inclusive mode, which is to say that the
range that you have selected is included in
the threshold range and will be highlighted by
a red thresholding overlay. If you want to
reverse the selection so that the pixels in the
range you have selected are excluded from
thresholding and all other pixels are included
instead, select Exclusive from the State
option button group.
10
If you want to save the threshold settings,
choose Save Range. The Save Color
Threshold Range dialog box will appear.
Type a name for the color threshold range
(*.ctr) state file in the File Name text box. If
necessary, use the Save In list or Up One
Level icon button to locate the correct drive
and folder. Then choose Save.
11
When you have finished, choose Close.
Set Color Threshold - Dialog Box Options
Image
Selects the image to be thresholded.
Color Model
Selects a color space model (RGB, HSI, or HSL) to use for configuring threshold range settings. The model
you choose will determine the "color channels" that are to be used for selecting the threshold range. The
RGB model will use the Red, Green, and Blue channels. The HSI model will use the Hue, Saturation, and
Intensity channels. The HSL model will use the Hue, Saturation, and Luminosity channels.
Hue Range
Selects whether the ranges between the upper and lower limits are to be included in the threshold range
(Inclusive) or if the ranges outside of the upper and lower limits are to be thresholded (Exclusive). This
option is only available when the HSI or the HSL color space model is selected.
Red
Sets the lower and upper limits for the threshold range values of the red color channel. You can use either
the "arrows" on the slider or the left (lower value) and right (upper value) spin boxes to set these range
limits. This option is available only when the RGB color space model is selected.
Green
Sets the lower and upper limits for the threshold range values of the green color channel. You can use either
the "arrows" on the slider or the left (lower value) and right (upper value) spin boxes to set these range
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limits. This option is only available when the RGB color space model is selected.
Blue
Sets the lower and upper limits for the threshold range values of the blue color channel. You can use either
the "arrows" on the slider or the left (lower value) and right (upper value) spin boxes to set these range
limits. This option is only available when the RGB color space model is selected.
Hue
Sets the lower and upper limits for the threshold range values of the hue channel. The colors on the slider
indicate which hues in the image will be selected. You can use either the "arrows" on the slider or the left
(lower value) and right (upper value) spin boxes to set these range limits. The black bar under the slider
indicates the color range that will be thresholded. If you selected an Inclusive range, the bar will span
between the slider's lower and upper arrows. If you selected an Exclusive range the bar will be outside of
one of the arrows. This option is available when either the HSI or the HSL color space model is selected.
Saturation
Sets the lower and upper limits for the threshold range values of the saturation channel. This determines
how "undiluted" the selected colors will be. Colors with lower saturation will be paler, with a pastel
appearance. Colors with higher saturation will be purer and more vivid. The "gray wedge" on the slider
indicates which saturation values in the image will be selected. You can use either the "arrows" on the slider
or the left (lower value) and right (upper value) spin boxes to set these range limits. This option is available
when either the HSI or the HSL color space model is selected.
Intensity
Sets the lower and upper limits for the threshold range values of the intensity channel. This determines how
bright or dark the selected colors will be. Colors with lower intensity will be darker, while those with higher
intensity will be brighter. The "gray wedge" on the slider indicates which intensity values in the image will be
selected. You can use either the "arrows" on the slider or the left (lower value) and right (upper value) spin
boxes to set these range limits. This option is available when either the HSI or the HSL color space model is
selected.
Luminosity
Sets the lower and upper limits for the threshold range values of the luminosity channel. As with the Intensity
value, the Luminosity value determines the brightness of the selected colors, but the associated HSL color
space model takes into account the eye's differential sensitivity to light of different wavelengths. The "gray
wedge" on the slider indicates which luminosity values in the image will be selected. You can use either the
"arrows" on the slider or the left (lower value) and right (upper value) spin boxes to set these range limits.
This option is available when either the HSI or the HSL color space model is selected.
Load Range
Opens the Load Color Threshold Range dialog box, from which you can select a color threshold range (*.ctr)
state file. When you load the settings file, the threshold settings will be applied automatically to the selected
image.
Save Range
Opens the Save Color Threshold Range dialog box, which you can use to specify a name for a color
threshold range (*.ctr) state file and save your current threshold settings.
State
Selects a thresholding state for the image:
Inclusive thresholds pixels with color values that have been selected by the three range sliders,
Exclusive reverses the thresholding, such that pixels with color values that have been selected by the sliders
are excluded, and all other pixels are included in the thresholding, and
Off disables thresholding.
Set by Example >>
Places the Set Color Threshold command into an interactive mode. The values of image pixels that you click
with the pointer will be added to the threshold range. If you select a pixel that represents the lower end of the
threshold range, and then select a pixel that represents the upper end, MetaMorph will automatically select
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all values that are between the values of the two pixels for the three color channels that make up the color
space model. When you choose Set by Example >>, the Set Color Threshold dialog box will expand,
revealing the Reset Color Threshold on Next Click check box and the Undo Last Click command button.
Set by Example <<
Condenses the Set Color Threshold dialog box and takes the command out of interactive mode.
Reset Color Threshold on Next Click
When this check box is selected, the next mouse click in the image window will clear the threshold range.
Undo Last Click
Removes the values of the pixel that was last selected from the threshold range.
Close
Closes the dialog box.
Combine into B&W + Color (Display Menu)
Combines two grayscale images (8-bit or 16-bit) into a single 8-bit image containing both
gray values and color values. Converts a single grayscale image into an 8-bit image
containing both grayscale and color values. This command can also be applied to a single
plane or to all planes in an image stack.
Drop-in: COMB_BWC
Use this command when you want to add color to a specific range of gray values in a grayscale image to
make them stand out, or when you want to combine two grayscale source images into a single image
which displays the contribution from one source image in color. You can specify that the colored regions
be transparent (that is, will show brightness variations) or be opaque (that is, will be at a uniformly
maximum intensity level).
You can select a palette for the colored region from an available list. The list includes color palettes of a
particular color (Red, Green, or Blue), a palette that varies from white (highest intensity) to red (RedWhite), a palette that varies from red to blue (Blue-Red), and a "pseudocolor" palette that comprises the
entire color spectrum (RainBow). Pseudocolor (Display Mode Tool). Alternatively, you can select a userdefined palette that you have saved previously.
The resulting image will have a custom look-up table with 127 palette levels, a portion of which are
grayscale and the remainder of which are in color. By default, these are roughly evenly divided between
grayscale and color, but the relative proportions can be changed to suit your needs.
Note: This command does not support 24-bit color images.
Using Combine into B&W + Color
To convert one or two grayscale images into a single image containing both grayscale and color
values, use the following procedure:
Step
Action
1
From the Display menu, choose Combine
into B&W + Color. The Combine 2 Images
into B&W + Color dialog box opens.
2
Select the desired destination image using
the Destination image selector. You can
overwrite or add to the existing image, or you
can place the results in a new image window.
AND
From the Source for Color image selector,
select a source image or stack to serve as
the source for the creation of the color
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portion of the destination image.
3
If necessary, use # Color Values to specify
the number of color values into which the
color portion of the result image is to be
divided. This number, plus the number in #
Gray Values, will always equal 127.
Alterations of one will produce automatic
reciprocal changes in the other.
4
Use Clear Level to enter a threshold value
below which the result pixels are to be
displayed in grayscale.
AND
Use Saturation to enter a value above which
result pixels are to be displayed at the
maximum color value. This will also specify
the maximum color value.
5
Select a color palette (RainBow, Blue-Red,
Red-White, Red, Green, or Blue) from the
Look Up Table list.
OR
Select the Use .LUT File check box and
choose Select LUT. Select an icon for a
previously stored look-up table file (*.lut) from
the Select a LUT File dialog box that appears
and choose Open.
6
From the Source for Gray Scale image
selector, select a source image or stack to
serve as the source for the creation of the
grayscale portion of the destination image. If
you are converting a single source image
into an image with grayscale and color
values, this will be the same as the image
selected in the Source for Color image
selector.
7
Use Black Level to enter a threshold value
below which the result pixels are to be
displayed as black.
AND
Use White Level to enter a value above
which the result pixels are to be displayed as
white.
8
If you wish, select the Auto Scale check box
to have MetaMorph automatically scale the
display of gray levels.
9
Choose OK.
10
Choose Close when you have finished.
Combine into B&W + Color - Dialog Box Options
Destination
Selects a destination for the result image. You can add to or replace the existing image or stack of images,
or you can place the results in a new image window.
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Source for Color
Selects the image or stack of images that will serve as the source for the creation of the color portion of the
resulting image.
# Color Values
Specifies the number of values into which the color portion of the look-up table is divided. The total number
of values in the color and grayscale portions will equal 127. Changing the number of values in one portion
will automatically cause a reciprocal change in the other.
Clear Level
Specifies a threshold value for the color source below which the result pixel will be displayed in grayscale.
This value is also used to specify the minimum color value in the color portion of the result image.
Saturation
Specifies a value for the color source above which the result pixel will be displayed at the maximum color
value. This value is also used to specify the maximum color value in the color portion of the result image.
Look Up Table
Selects the palette to be used to create the color range of the result image. You can select from the
following:
RainBow: a "pseudocolor" palette that includes the entire color spectrum.
Blue-Red: a palette that varies from red (highest intensity) to blue.
Red-White: a palette that varies from white (highest intensity) to red.
Red: a palette entirely of red values.
Green: a palette entirely of green values.
Blue: a palette entirely of blue values.
Use .LUT File
Allows you to load a previously stored look-up table (*.lut) file to apply to the color portion of the new image
when you choose Select LUT.
Select LUT
Opens the Select a LUT File dialog box, from which you can load a previously stored look-up table to apply
to the color portion of the new image. This option will be unavailable and will appear dimmed unless Use
.LUT File is selected.
Source for Gray Scale
Selects the image or stack of images that will serve as the source for the creation of the grayscale portion of
the resulting image.
# Gray Values
Specifies the number of values into which the grayscale portion of the look-up table is divided. The total
number of values in the grayscale and color portions will equal 127. Changing the number of values in one
portion will automatically cause a reciprocal change in the other.
Black Level
Specifies a threshold value for the grayscale source below which the result pixel will be displayed as black.
White Level
Specifies a value for the grayscale source above which the result pixel will be displayed as white.
Auto Scale
Automatically expands the range of grayscale values in the new image.
OK
Executes the command.
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Close
Closes the dialog box.
Log Color Threshold (Log Menu)
Exports the 24-bit color threshold settings as text to a log file or by Dynamic Data
Exchange to another application, such as a spreadsheet. The color values of the upper
and lower threshold range limits will be stored for each color channel (red, green, and
blue).
Drop-in: EXTHRESH
Use this command to save a text-based record of the threshold settings for a 24-bit color image. This
command will save the settings for the threshold in "bins," the number of which is determined by the
binning sensitivity used by the Threshold Image command.
Logging a Color Threshold
To save a 24-bit color threshold as text, use the following procedure:
Step
Action
1
From the Log menu, choose Log Color
Threshold. The Log Color Threshold dialog
box opens.
2
Select a source image from the Image
selector.
3
Choose Open Log. The button title will
change to F9: Log Data, and the Open Data
Log dialog box will open.
4
Determine whether you want to save the
thresholding information to a text file, by DDE
link to an open spreadsheet, or both, by
selecting the Dynamic Data Exchange (DDE)
and/or A Text File check boxes.
Note: If you are opening a DDE link, you
must already have the spreadsheet
application running and the desired
worksheet open.
5
If you selected A Text File in Step 4, the
Open Data Log File dialog box will appear.
Select the icon for the desired log file or type
a new file name in the File Name text box. If
the desired folder is not listed at the top of
the dialog box, use the Save In list or Up
One Level icon button to change to the
correct location. Then select a file name. If
you select an existing log file name, the Log
File Exists dialog box will appear. You can
Overwrite the contents of the file, Append
new data, or Cancel.
AND
Choose Save.
6
If you selected Dynamic Data Exchange
(DDE) in Step 4, the Export Log Data dialog
box will appear.
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Select the desired spreadsheet program from
the Application list. Choose Default to use
the default settings for the selected
application.
Choose OK to open the DDE link. Once the
application is open, MetaMorph will
reappear.
7
To configure the data log file for logging,
choose Configure Log from the Export 24-Bit
Threshold dialog box. The Configure Log
dialog box will appear.
AND
From the Configuration list, select the
parameters you want to log so that each is
marked by a check mark next to its name
(you can choose Enable All or Disable All if
you want to include or exclude all of the
parameters listed). Then choose OK to return
to the Export 24-Bit Threshold dialog box.
8
Choose F9: Log Data to save the threshold
settings.
9
Choose Close.
Log Color Threshold - Dialog Box Options
Image
Selects the 24-bit color source image whose threshold settings you want to save.
Open Log
Opens a data log and/or a DDE link to an open spreadsheet for logging data. This command changes to F9:
Log Data when a log file is open.
F9: Log Data
Sends the color threshold settings to an open data log.
Configure Log
Allows the selection of image and threshold bin data that are to be included or omitted from data logging.
Also allows a choice of whether column titles are to be included and if data are to be listed on a single line.
Close
Closes the dialog box.
Save Original and Result Loop (Journal Menu)
Applies a journal to each image in a selected directory, saving each original image and its
corresponding result image in a separate directory, using a user-supplied base file name
and numeric suffix. A second journal is then applied to perform measurements on the
result images and log data automatically.
Drop-in: SAVELOOP
Use this command to loop through all images in a directory, applying a journal to each image in turn and
saving both the original image and its corresponding result image in another separate directory.
The first original image will be saved in the selected directory as Basename0001.xxx and its
corresponding result image will be saved in the same directory as Basename0002.xxx. The next original
image will be saved as Basename0003.xxx, and so on. The base name is specified in the Save Original
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and Result Loop dialog box in the Subject ID text box, which can accommodate up to four characters.
Each original image in the specified directory will be displayed in turn in an image window that receives a
window title which you supply with the Open into Image Named text box. This is intended to simplify
journal writing so that the journal can be configured to use the specified image window name. The image
will be processed by the journal you select using the Select Journal One command button, and the result
image will be displayed in a second image window that receives a window title which you supply with the
Save Image Named text box.
A second journal is then invoked with the Select Journal Two command button. This journal can be
applied to either the original image or the result image, depending on how you configure the journal. A
typical use might be to perform thresholding and measurement of the result image and to log the
measured data to a data log or an object log. The effects of this second journal on the images
themselves will not be saved.
Saving Original and Result Images from a Journal Loop
To apply the Save Original and Result Loop command, use the following procedure.
Step
Action
1
From the Journal menu, choose Save
Original and Result Loop. The Save Original
and Result Loop dialog box opens.
2
To select the directory containing the original
images to be processed, choose Set Open
Directory. The Set Open path dialog box will
appear.
AND
Select an icon for any image that is in the
source image directory. If necessary, use the
Save In list or Up One Level icon button to
select the correct drive and folder. Then
choose Save.
3
To select the folder where the both the
original images and the processed result
images are to be saved, choose Set Save
Directory. The Set Save Path dialog box will
appear.
AND
Use the Save In list or Up One Level icon
button to select the correct drive and folder. If
necessary use the Create New Folder icon
button to create a new folder under the
currently selected one. Then choose Save.
WARNING:
Be sure not to specify the original source
folder for saving the result images. If you do,
the journal may continue to operate on the
newly saved images, resulting in an infinite
loop routine as the source images continue
to be loaded from the folder in which result
images are being stored.
4
In the Subject ID text box, type a base name
that is to be applied to all images (original
and result) that will be saved in the result
directory. The base name can be any
combination of alphanumeric characters.
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5
In the Open into Image Named text box, type
a name for the image window that will be
used to display the original source images.
6
Choose Select Journal One. The Select
Journal One dialog box will appear.
AND
Select the icon for the journal that is to be
applied to each original source image. This
journal should already exist, and should be
written so as to operate on the image window
specified by the Open into Image Named text
box. If necessary, use the Look In list or Up
One Level icon button to select the correct
drive and folder for the desired journal. Then
choose Open.
7
In the Save Image Named text box, type a
name for the image window that will be used
to display the processed image that results
from the actions of the journal selected in
Step 6.
8
A second journal is then applied to either the
original or result images, depending on how
you write the journal. A typical use might be
to open a data log or object log, perform
thresholding and measurement on each
image, and then log the data that is obtained.
To select the second journal, choose Select
Journal Two. The Select Journal Two dialog
box will appear.
AND
Select the icon for the journal that is to be
applied to each image. If necessary, use the
Look In list or Up One Level icon button to
select the correct drive and folder for the
desired journal. Then choose Open.
9
When you are ready to apply the journal
loop, choose Run. Each original image will
be opened and displayed in an image
window, the selected journal will be applied
to it, and the result image will be displayed in
a second image window. Both the original
and result images will then be saved to the
new folder. The process will repeat until all
images in the source folder are used.
10
When you have finished, choose Close.
Save Original and Result Loop - Dialog Box Options
Set Open Directory
Selects the folder that contains the original source images. Once selected, the path for the folder will be
displayed in the text box to the right. If you wish, the path can be typed directly into this text box, and the text
can be edited.
Set Save Directory
Selects the folder into which both source images and result images will be saved, using the base file name
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specified in the Subject ID text box. If you wish, the path can be typed directly into this text box, and the text
can be edited.
WARNING:
Be sure not to specify the original source folder for saving the result images. If you do, the journal may
continue to operate on the newly saved images, resulting in an infinite loop routine.
Subject ID
Sets the base file name that will be used for storing the original and result images in the new folder. This
base can be any combination of alphanumeric characters. The first original image will be saved in the
selected folder as Basename0001.xxx and its corresponding result image will be saved in the same folder
as Basename0002.xxx. The next original image will be saved as Basename0003.xxx, and so on.
Open into Image Named
Specifies the name for the image window that will be used to display the original source image. The journal
that is to be applied should be configured so as to use this window's name.
Select Journal One
Selects the journal to be applied in turn to each source image. The name of the selected journal will be
displayed in a status line to the right of this command button.
Save Image Named
Specifies the name for the image window that will be used to display the processed result image.
Select Journal Two
Selects the second journal, to be applied to either the original or the result images (depending on how you
write the journal). If you want to apply the journal to all of the result images, the journal should reference the
name of the window as specified by the Save Image Named option. A typical use of a second journal might
be to threshold and measure each image and save the measurement data in a log file. Any effects of this
second journal on the images themselves will not be saved.
Run
Starts the journal loop for all images in the selected folder, saving the original and result images in the new
folder.
Close
Closes the dialog box.
Set Image Zoom (Display Menu)
Applies a selected magnification level to an image that is between 1 and 800 percent of the
original. Zooms a region of the image so that it fills the image window.
Drop-in: SETZOOM
Use this command to specify a precise zoom level for an image between 1 and 800 percent of the
original magnification, or to zoom a region of the image so that it fills the entire image window. This
command, which is fully journalizable, allows you to set a zoom level that is not available in the Zoom
Tool pop-up menu.
Note: If you want to save a copy of the resized image, use the Edit menu's Duplicate Image with Zoom
command.
Setting an Image Zoom Level
To specify and apply a zoom level to an image with the Set Image Zoom command, use the
following procedure:
Step
1
Action
From the Display menu, choose Set Image
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Zoom. The Set Image Zoom dialog box
opens.
2
Use the Image selector to select the image
for which you want to change magnification.
3
If you want to change the image
magnification to a specific zoom level, use
the Zoom spin box to select a value for the
new zoom level, expressed as a percent of
the original magnification. Then choose Set
Zoom. The image will be displayed at the
new zoom level. Now skip to Step 5.
OR
If you want to zoom in on a region of the
image, continue to Step 4.
4
To zoom in on a region of the image, use the
Rectangular Region Tool to draw the
region. Then choose Zoom to Fit. The region
will be zoomed to fill the image window.
5
Choose Close to close the dialog box.
Note: If you want to save a copy of the
image at the new zoom level, choose
Duplicate Image with Zoom from the Edit
menu.
Set Image Zoom - Dialog Box Options
Image
Selects the source image for which you want to change the zoom level.
Zoom
Specifies a zoom level to be applied to the selected image. The values are expressed as a percent of the
original magnification. You can select any integer between 1 and 800.
Set Zoom
Applies the specified zoom level to the selected image.
Zoom to Fit
Zooms in on a defined rectangular region, filling the image window.
Close
Closes the dialog box.
FFT (Process Menu)
Performs fast Fourier transform filtering of images in the frequency domain, rather than in
the spatial domain.
Drop-in: FFT
Use this command to remove or enhance patterns of periodic "noise" in an image. This command can
also be used to apply a Blur, High Pass, or Homomorphic Fourier filter to images in the frequency
domain. When you apply the filter, the source image will first be transposed from the spatial domain into
the frequency domain using the Fast Fourier Transform. The filter will then be applied to the image.
Finally, the image will be processed using an inverse fast Fourier transform that returns it back to the
spatial domain.
Because images processed with this command are filtered in the frequency domain and not the spatial
domain (the way the Sharpen and Low Pass filters function), you don't need to correct for problems with
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pixels that are within a distance of half the width of the kernel from the edge of the image. This avoids
the problem with convolution filters in which a bordering band of pixels remains unprocessed around the
periphery of the image.
The Remove Patterns filter is particularly useful for removing periodic noise from an image, such as
might be caused by the photomultiplier tube in an intensifier. Enhance Patterns accentuates such
patterns. Click here for a pair of "before" and "after" images which illustrate the effects of the Remove
Patterns filter.
The effects of the Blur filter are similar to those of the Low Pass filter in that high spatial frequency
elements in the image will be removed, leaving a hazy image containing low frequency information only.
This operation attenuates image information above a selected spatial frequency. The cutoff frequency is
selected with the Radius slider. Click here for an example showing the effects of the filter.
The High Pass filter attenuates the low frequency information in the source image. The result of this
operation is an image that retains much of its edge information, and the resulting images can resemble
those that result from application of an edge-detection convolution. As with the Blur filter, the Radius
slider is used to select the degree of filtering. Click here for a figure which illustrates the effects of the
filter.
The Homomorphic filter simultaneously performs a contrast enhancement and a compression of the
dynamic range of intensities. The resulting effect is somewhat similar to that of the Unsharp Mask
command. This figure illustrates the effects of the filter.
Applying a Fast Fourier Transform Filter
To apply an FFT filter to an image, use the following procedure:
Step
Action
1
From the Process menu, choose FFT. The
FFT dialog box will appear.
2
From the Source image selector, select a
source image to be filtered.
3
Select a destination for the filtered image
with the Result image selector.
4
From the Operation group, select the FFT
filter you want to apply: Blur, High Pass,
Homomorphic, Remove Patterns, or
Enhance Patterns.
5
Use the Radius slider to specify the cutoff
frequency (percent) for the filter.
6
Choose Apply.
7
When you have finished, choose Close.
FFT - Dialog Box Options
Source image
Selects the source image you want to filter.
Result image
Selects the destination for the filtered image. You can place the results in a new image window or in a new
plane appended to an existing image or stack, or you can overwrite an existing image.
Operation
Selects the filter to be applied to the source image:
Blur selects the image smoothing operations that attenuate high-frequency components.
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High Pass attenuates the low-frequency components.
Homomorphic simultaneously performs a contrast enhancement and a compression of the
brightness dynamic range (similar to Unsharp Mask).
Remove Patterns removes periodic patterns of "noise" from the image.
Enhance Patterns accentuates periodic patterns in the image.
Radius
Specifies the radius, expressed as a percentage, of the filter cutoff frequency.
Apply
Applies the selected filter operation to the source image.
Undo/Redo
Undoes or redoes the last applicable command that did not create a new result image. (The Undo/Redo
buttons in MetaMorph apply to any previously applied command, as if you selected it from the Edit menu,
not just to the last command in the dialog box where the button was chosen.)
Close
Closes the dialog box.
The FFT Pattern Removal Filter
Before:
After:
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The Blur FFT Filter
H(u,v)
1
.5
0
1
2
3
D(u,v)
D0
The equation for the Blur (Butterworth Low Pass) filter is:
1
H ( u, v ) =
2n
1 + [D(u, v )/ D0 ]
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The High Pass FFT Filter
H(u,v)
1
.5
0
1
2
3
D0
D(u,v)
The equation for the High Pass (Butterworth High Pass) filter is:
1
H (u, v ) =
2n
1 + [D0 / D(u, v )]
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The Homomorphic FFT Filter
H(u,v)
3
.5
D0
D(u,v)
The process applied by the Homomorphic filter is:
Source
Dest
i
n
at
i
o
n
I
n
ver
s
e
exp
H(u,v)
FFT
l
n
+
Image
Image
FFT
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Convolve with Image Kernel (Process Menu)
Allows you to define and/or convolve an image with a custom convolution kernel. You can
create a custom image kernel using part of another image as a template for the new image
kernel. You can also use this command to load kernels from a file on disk.
Drop-in: KERNEL
Use this command when you want to use or define the values to be used in a custom convolution kernel
for processing images. This command allows you to define a region on an image and then copy its
values into a table of values for a kernel that is the same size as the region.
When you open this dialog, the drop-in will create its own region upon the image to be used to create the
image kernel (the default will be the active image when the dialog was opened). The size of this region is
determined by the Width and Height defined in the dialog box. Width and Height also define the size of
the image kernel, so that kernel size and region size are identical. The custom kernel can range in size
from 2x2 to 128x128 elements. Each element can be individually set to a value between -32,768 and
32,767. The Convolve with Image Kernel dialog box includes a table of the element values, which can be
edited by right-clicking on the desired element in the table. You can use the scroll bars to examine the
parts of kernels that are larger than the table window.
Once you have defined your custom image kernel, you can apply it to an image specified by the Source
image selector and/or Save the new kernel for future use. You can also Print a copy of the image kernel.
When a custom convolution kernel is applied to a selected image, the result image is formed by
convolving the image with the kernel divided by the Element scale. Then the result image is divided by
the Result scale factor. In some cases, you may want specify an Offset value to be added to all pixel
gray values after the convolution is completed, so that all pixel values remain within the image's
grayscale range (0 - 255 for 8-bit images, or 0 - 32767 for 16-bit images).
Absolute Value allows you to determine how negative gray level results will be dealt with. For example, a
kernel that would create a value of -50 in an 8-bit image would set the pixel's gray level value to zero if
Absolute Value were set to No, or would set the value to 50 ( |150-200| = 50 ) if Absolute Value were set
to Yes.
Image-1/AT: This drop-in can be used to load and apply kernels created with Image-1/AT.
Convolving with an Image Kernel
To define a custom convolution and apply it to an image, use the following procedure.
Note: Once you have defined a custom kernel, you can
skip steps 4 - 10 if you want to apply that kernel to an image
later during the same session.
Step
Action
1
From the Process menu, choose Convolve
with Image Kernel. The Convolve with Image
Kernel dialog box will appear.
2
Select the desired source image using the
Source image selector.
AND
If you plan to define the kernel by copying
values from a region to the kernel editor,
select the image you want to use from the
Image with Region to Copy image selector.
3
Select the desired destination using the
Result image selector.
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4
If the dialog box is condensed, choose
More >> so that you can see the kernel size
options. A region will appear that that is the
same size as the kernel size.
5
To change the size (pixels) of the kernel,
type the desired dimensions in the Width and
Height text boxes. You can also change the
size of the kernel by resizing the region.
The kernel must be at least 2x2 elements,
but cannot be larger than 128x128 elements.
6
Once you have the region positioned over
the part of image you want to use for the
kernel, choose Copy to copy its values into
the kernel table.
To see the kernel table, choose View. The
dialog box will expand to include a table. You
can edit the kernel elements by clicking
inside the table cells.
7
If necessary, use Element to change the
element scaling from the default value of 1.
The result of the kernel element divided by
the Element scale should not be much less
than 1.0. Otherwise, significant numerical
inaccuracies can occur.
8
If necessary, use Result to change the result
scaling from the default value of 1. The result
of the convolution will be divided by this
value. The value of the Result scale must be
set so that the sum of elements becomes as
close to 1 as possible, otherwise the image
values will overflow the frame buffer.
OR
Select the Autoscale Result check box so
that the best Result value can be determined
for you automatically.
9
If you want an intensity offset value to be
added to the final image pixel values after
the convolution, select the desired value
using Offset.
10
Select Yes in the Absolute Value group to
use the absolute value of each resulting
pixel. Select No to clip any negative values to
zero.
11
Choose Apply to apply the custom kernel.
Convolve with Image Kernel - Dialog Box Options
Source image
Selects the source image to which you want to apply the convolution kernel.
Result image
Selects a destination for the result image. You can overwrite the existing image or place the results in a new
image window. Or you can add the resulting image as a plane to an existing image or stack.
Image with Region to Copy
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Select the image for the region used by the Copy command. Whenever the Convolve with Image Kernel
dialog box is in its expanded state, a region for the kernel will appear on this image.
More >>
Expands the dialog box to include the kernel element options.
Less <<
Condenses the dialog box.
Width
Specifies the width of the kernel. The width can range from 2 to 128 elements.
Height
Specifies the height of the kernel. The height can range from 2 to 128 elements.
Element
Specifies the number by which each kernel element should be divided before the convolution is to be
performed. The total of the kernel elements divided by the Element scale should not deviate much from 1.0.
Otherwise, the resulting image will be too dark or too light.
Result
The result of the convolution is divided by this value. The value of the Result scale must be set so that the
sum of elements becomes as close to 1.0 as possible. For example, if the sum of the elements is 8, set
Result to 8 so that the value of sum of the elements becomes 1.
Offset
Specifies the intensity offset value that is to be added to the final pixel values in the image after the
convolution is completed so that all gray values will remain within the image's range (for example, 0 - 255 for
8-bit images).
Absolute Value
Specifies whether absolute values of each resulting pixel will be used or whether negative values are to be
clipped to zero. Select No to clip the negative values to zero.
Autoscale Result
Instructs MetaMorph to determine the best Result value automatically, so that the sum of elements becomes
as close to 1 as possible.
Copy
Copies the values in the region created by Convolve with Image Kernel to the kernel table. You should use
this command after you specify the kernel's size and other elements.
Load
Loads a previously saved kernel so that you can edit it or apply it to the selected image.
Save
Saves the current kernel to disk so that you can use it during future sessions.
Print
Prints a copy of the Kernel Table to the default printer.
View
Displays the Kernel Table.
Kernel Table
Displays the elements for the current kernel. You can edit these values by clicking inside the desired table
cell. Each element can be individually set to any value between -32,768 and 32,767. The size of the kernel
is governed by the Width and Height options.
Apply
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Applies the current convolution kernel to the specified image.
Close
Closes the dialog box.
2D Deconvolution (Process Menu)
Removes image haze from a stack by performing either No Neighbors or Nearest
Neighbors deconvolution on a plane in a stack of images.
Drop-In: 2DDECON
Use this command when you want to reduce the effects of out-of-focus haze from a stack. This
command can be applied to 8-bit or 16-bit images.
The No Neighbors command performs the operation without considering out-of-focus information
contained in adjacent planes. Instead, an unsharp mask operator that utilizes a convolution kernel
based on the properties of the imaging system is used to blur the image plane. This kernel is an
estimation of the 3D PSF. The blurred input is then subtracted from the original input, removing the
added blurred component along with the original out of focus information.
The Nearest Neighbors command also uses an estimation of the three-dimensional Point Spread
Function (PSF) to compute the contributions from out-of-focus planes, and uses this to generate an
image in which the effects of out-of-focus information are reduced.
Stacks that lend themselves well to haze-removal will have planes that were acquired with the same
analog adjustment settings, and will have a maximum dynamic range. The command is useful for
stacks where planes are not spaced closely together. If the planes are spaced closely together (for
example, less than one-half micron for a stack imaged with an oil objective) the results obtained using
the Nearest Neighbors and No Neighbors commands will be very similar.
MetaMorph enables you to control how much out-of-focus information (Scaling Factor) is removed and
how much scaling is performed on the output image (Result Scale). The greater the Scaling Factor, the
greater the amount of haze that is removed from the original, and thus the lower the amount of
brightness from the original will be retained. If you select Auto Result Scale, MetaMorph selects a Result
Scale that matches the selecting Scaling Factor so that the brightness reduction is automatically
minimized.
MetaMorph also enables you to enter experimental settings that help specify the PSF (filter kernel) used
to approximate the contribution of out-of-focus information from adjacent planes. As a general rule,
these settings are determined by the data collection and are not typically altered to change the quality of
results.
Note: Results obtained from the Nearest Neighbors command are not suitable for use in
quantitative gray level analysis.
The Process of Nearest Neighbors Deconvolution
The process that MetaMorph uses to remove haze from a plane can be broken down into four steps:
Note: This table describes the Nearest Neighbors deconvolution process that MetaMorph
carries out. It is not a procedure for you to follow and complete.
Step
Result
1
Convolving the adjacent planes with the PSF
approximates the out-of-focus haze in the
desired plane. To speed up computations,
MetaMorph averages the adjacent planes
before the convolution operation.
2
Before MetaMorph subtracts the estimate of
the haze from the target plane; it first
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decreases the intensity range of haze
estimate so that the result of the subtraction
operation will be greater than zero (if the
result was zero, the image would be
uniformly black). Thus, rather than:
100% - 100% = 0 intensity range, the
subtraction operations will look similar to the
following if a Scaling Factor of 0.75 is
applied:
100% -75% = 25% intensity range
(approximately).
3
Next, MetaMorph subtracts the scaled haze
estimate from the target plane to remove the
out-of-focus information.
4
After the out-of-focus haze estimate has
been subtracted, the contrast of the result
plane can be "stretched" by multiplying the
output by the Result Scale. This will not
restore the number of gray levels to that of
the original image, but the range can be
approximately restored.
2D Deconvolution Procedures
No Neighbors
Nearest Neighbors
2D Deconvolution - No Neighbors
To perform no neighbors deconvolution on an image stack, use the following procedure:
Step
Action
1
From the Process menu, choose 2D
Deconvolution->No Neighbors. The No
Neighbors dialog box opens.
2
If the desired source stack is not displayed in
the Source image selector, select it using the
image selector.
3
If the desired destination image is not
displayed in Result image selector, select it
using the image selector. You can place the
results in a new image window.
4
Select Auto Result Scale if you want to
attempt to automatically select an
appropriate result scale that matches the
scaling factor.
5
Use Filter Size to select the spatial size of
the kernel. Larger kernel sizes lead to less
sever subtraction of hazy features.
6
Select a Scaling Factor. This selects how
much out-of-focus information will be
subtracted from the target image. The larger
the Scaling Factor, the more information is
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subtracted from the target image.
7
Use the Result Scale option to increase the
contrast lost during the subtraction of the
blur estimate from the original image. Note:
Skip this step if you selected Auto Result
Scale in Step 5. Changing the Result Scale
setting will disable Auto Result Scale.
8
Choose Apply.
9
Choose Close when you have finished.
2D Deconvolution - Nearest Neighbors
To perform nearest neighbors deconvolution on an image stack, use the following procedure:
Step
Action
1
From the Process menu, choose 2D
Deconvolution->Nearest Neighbors. The
Nearest Neighbors dialog box will appear.
2
If the desired source stack is not displayed in
the Source image selector, select it using the
image selector. Note: If the source stack
contains only one plane the Nearest
Neighbors operation will still operate, but will
produce the same output as the No
Neighbors command.
3
If the desired destination image is not
displayed in the Result image selector,
select it using the image selector. You can
overwrite or add to the existing image or you
place the results in a new image window.
4
Select Auto Result Scale if you want to
attempt to automatically select an
appropriate result scale that matches the
scaling factor.
5
Use Filter Size to select the spatial size of
the low pass filter. Larger kernel sizes lead
to less sever subtraction of hazy features.
6
Select a Scaling Factor. This selects how
much out-of-focus information will be
subtracted from the target image. The larger
the Scaling Factor, the more information is
subtracted from the target image.
7
Use the Result Scale option to increase the
contrast lost during the subtraction of the
blur estimate from the original image. Note:
Skip this step if you selected Auto Result
Scale in Step 5. Changing the Result Scale
setting will disable Auto Result Scale.
8
Choose Apply.
9
Choose Close when you have finished.
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2D Deconvolution Dialog Box Options
The following options are used for both the No Neighbors and Nearest Neighbors versions of the 2D
Deconvolution command.
Main tab
Select the primary 2D Deconvolution dialog box options.
Settings tab
Selects the 2D Deconvolution settings dialog box options.
Source image
Selects the source stack to deconvolve.
Result image
Selects the destination for the result image. You can place the results in a new image window or you can
add the resulting image as a plane to an existing image or stack.
Apply
Applies the selected filter to the source image.
Close
Closes the dialog box.
2D Deconvolution Dialog Box Options – Main tab
The following options are used for both the No Neighbors and Nearest Neighbors versions of the 2D
Deconvolution command.
Note: Filter Size, Scaling Factor, and Result Scale are preset to recommended default
settings appropriate for most images. The recommended values are noted for each
option.
Filter Size
Specifies the size of the filter applied the stack. Larger filter sizes lead to the detection of larger, lower
intensity areas and thus less severe intensity subtraction. (Recommended minimum Filter (Kernel) Size is
equal to 7)
Scaling Factor
Specifies the scaling factor applied to the stack after the filter is applied. Smaller numbers allow more of the
original image features to be retained in the result image. (Recommended Scaling Factor should be equal
to or greater than .9)
Result Scale
Restores the contrast lost during the subtraction of the haze estimate from the original image.
(Recommended Result Scale should be 2 or 3.) Note: If you type a fractional value in the Result Scale box,
it will automatically be rounded to the nearest integer when the entry is processed.
Auto Result Scale
Enables MetaMorph to select a Result Scale value that matches the selected Scaling Factor.
Supress Noise
Activates background noise suppression.
Set To Defaults
Resets all settings to the original values.
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2D Deconvolution Dialog Box Options – Settings tab
The following options are used for both the No Neighbors and Nearest Neighbors versions of the 2D
Deconvolution command.
Distance
Selects whether the dimensions of the image (XY Spacing and Z Spacing) are taken from MetaMorph’s
calibrations (Calibration), or set manually (User Specified) in the dialog.
XY Spacing
Sets, in microns, the size of a pixel laterally. Editable only if Distance is set to User Specified.
Z Spacing
Sets, in microns, the distance between adjacent planes. Editable only if Distance is set to User Specified.
Numerical Aperture
Sets the numerical aperture of the objective lens used to collect the image.
Refractive Index
Sets the refractive index of the medium in which the object lens is dipped. Typically, this is 1.0 (air), 1.3333
(water), or 1.515 (oil). Note: You must type or select a refractive index value greater than 1. This option will
not accept a refractive index value of less than 1.
Wavelength
Sets the peak wavelength of the detected intensity spectrum (in nanometers).
Measured PSF Decon (Process Menu)
Deconvolves image stacks using a measured point spread function (PSF) obtained from a
stack containing fluorescent microspheres.
Availability: Available for MetaMorph Basic and MetaMorph Premier
Drop-in: MEASPSF
Use the Measured PSF Decon dialog to deconvolve image stacks distorted by blur due to a Point
Spread Function (PSF). This PSF is a characteristic of the microscope’s optics. The optical properties
are such that individual points of light (fluorescent intensity) originating in the sample cannot be resolved
onto the camera chip without some amount of blur being introduced into the image by the microscope
optics. The amount of blur is determined, measured, and corrected using the Measured PSF
deconvolution algorithm.
The Measured PSF Decon dialog uses a reference stack of either a single bead or multiple beads to
determine the PSF required for effective deconvolution of an image. To use this dialog you must open
both the stack that you want to deconvolve and the bead stack.
Once both stacks are opened and selected, chose the algorithm that you want apply, the number of
iterations that you want to occur, and where applicable, the typical values for Sigma and Frequency that
you want to use.
You must chose whether you are using a single bead or multiple beads specified by regions. If your bead
stack has multiple beads, regions should be drawn around one or more of the beads and Extract PSF
from one or more regions should be selected. Whereas, if you have only one bead that has already
been processed by the Measured PSF Decon dialog, select Use PSF stack directly for deconvolution.
If you want to view the processed bead stack used as the PSF for deconvolution in addition to your
deconvolved stack, click Display PSF stack result.
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Note: The Measured PSF Decon command might have difficulty processing very large
images. If you attempt to process images that are too large, the program displays the
message "Maximum Image Limits Exceeded."
Deconvolving an Image using the PSF
Use the following typical procedure to guide you in using the Measured PSF Decon
dialog box for deconvolving images:
Step
Action
1
Open the source image stack that you want
to deconvolve.
2
Open the bead stack that you want to use for
the point spread function.
3
Click Source image and select the source
image from the list of open images.
4
Click Bead Stack and select the bead stack
from the list of open images.
5
In the Algorithm box, select the Fast
algorithm.
6
In the Iterations box, type or select an
iteration number from two to ten. A typical
iteration value is six or seven.
7
In the Sigma box, type or select a value at or
close to 0.7.
8
In the Frequency box, type or select an
appropriate value for how often you want
smoothing to run. A typical value is 4 or 5.
This causes the smoothing function to run
during the fourth or fifth iteration.
9
In the Bead Stack Process area, click Use
PSF stack directly for deconvolution if you
have a PSF stack previously created by the
Measured PSF Decon operation.
OR
Click Extract PSF from one or more regions
if you created one or more regions to identify
the bead or beads on which to base the
Measured PSF Decon operation. Choose
this option even if you have a bead stack
containing only a single bead that is not
centered.
10
If you chose Extract PSF from one or more
regions, you can click Display PSF stack
result to create and view the stack used as a
PSF in the deconvolution performed by the
Measured PSF Decon function.
11
Click Apply to process your source image
using the settings that you made.
12
Click Close to close the Measured PSF
Decon dialog box.
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Measured PSF Decon - Dialog Box Options
The following are the three image selectors:
Source image
Displays the name of the image to be deconvolved.
Bead Stack
Indicates the name of the image that will be used for the Point Spread Function (PSF) bead stack or image
stack.
Result image
Specifies the name of the image that will be created during deconvolution. This is the standard destination
image selector. The destination specifies location of the deconvolved image result.
Algorithm
Specifies the algorithm to be used for the deconvolution. Algorithms are Fast, Medium, or Slow (Most
Robust). The slow algorithm is the most robust.
The following three settings are configurations for each algorithm.
Iterations
Specifies the number of times that the algorithm is to be repeated during the deconvolution process.
Suggested values when using the Fast algorithm are between 5 and 7. Suggested values when using the
medium algorithm are between 10 and 20, and for the slow, most robust algorithm are between 20 and 100.
Sigma
Sets the smoothing factor for the deconvolution process. This is how much smoothing is applied to the
image during deconvolution to prevent noise build-up. Sigma is used for only the fast and medium
algorithms; and is not visible when Algorithm is set to Slow (Most Robust). Typical value is 0.7 for both the
fast and medium algorithms.
Frequency
Specifices how often the Sigma smoothing factor will be applied during the deconvolution process. For
example a frequency of 2 indicates that smoothing will be applied every other iteration. This option is used
for only the fast and medium algorithms. Typical values are either 4 or 5 for both the fast and medium
algorithms. This value can be used to control how often and/or how soon you want to apply smoothing
during deconvolution. Thus, it might be better to apply smoothing later rather than earlier during
deconvolution.
Bead Stack Processing
Controls how the PSF or bead stack is used.
Use PSF stack directly for deconvolution
Processes the PSF "as-is" (single bead, no regions in the latest image window.)
Extract PSF from one or more regions
For regions placed around some or all beads in the bead stack, a PSF is created by averaging
intensities from all regions using the location (x, y, and z) of the each region’s maximum intesnity
as the center averaging coordinate.
Display PSF stack result
Produces an additional stack along with the deconvolved result. The second stack is the PSF used
for the deconvolution operation, and is called, "PSF Result."
Note: This control is available only when Extract PSF from one or more regions is
enabled.
Apply
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Applies the selected settings and creates the specified deconvolved image and optional PSF result.
(Status)
Indicates the current status of the Measured PSF Deconvolution process.
Close
Closes the Measured PSF Decon dialog box.
Batch Deconvolution (Process Menu)
Provides Measured Point Spread Function (PSF) 3D deconvolution processing of multiple
image stacks both on a single system or on multiple systems in a shared processing
environment using network connections.
Drop-in: 3DDECON
Use this dialog box when you want to process more than a single image stack using the Measured Point
Spread Function (PSF) deconvolution method. Each image stack can be processed for multiple
wavelengths. You can choose the most appropriate algorithm as defined by its processing speed and
processing iterations. In addition, you can specify a smoothing factor (Sigma) and how often in the
iterations loop it should be applied. When processing multiple stacks, you need to specify the first stack
in a folder and the total number of stacks to be processed.
Batch Deconvolution requires PSF image stacks of microspheres (beads). These stacks can be with or
without square regions surrounding the microspheres. For each wavelength that you are processing,
you need one PSF stack.
If you are processing a large number of stacks and your system is part of a network with other
MetaMorph systems with the same software version, you can initiate the distributed processing feature
that is part of this command and share the processing capabilities of several processors in your network.
Using this feature enables you to deconvolve more image stacks in the same time as you would
deconvolve one stack on a single system. When using this feature, you have the option of doing all of
your image stack deconvolution processing on server systems only, using the client system only for
managing server processing tasks.
This dialog box is divided into the following four areas:
•
Algorithms Tab – Contains the settings for selecting one of three PSF Deconvolution
algorithms and the most appropriate settings for the number of iterations to run and the amount
of smoothing to apply.
•
Directories Tab – Contains the settings for the location and names of the source stacks,
destination for deconvolved stacks, and number of source stacks to be processed.
•
Wavelengths Tab – Enables you to specify a maximum of four wavelengths. The
Wavelengths tab is used to specify the relevant microsphere (bead) stack PSF image for the
selected wavelength.
•
Networking Tab – Enables you to establish network connections to other systems in your
network in order to create a distributed or shared system environment.
Summary Procedure
1. Select a deconvolution method (Fast, Medium, or Slow).
2. Select the directories containing your stacks for deconvolution.
3. Designate PSF stacks with or without square regions surrounding microspheres. Select one PSF for
each wavelength.
4. If you are using distributed batch deconvolution, configure your network settings. This includes ensuring
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that a copy of MetaMorph is installed on each server system that will process image stacks, and that
you have started hmagserv.exe on each system.
5. Process your image stacks.
Configuring Batch Deconvolution
To configure Batch Deconvolution to process your image stacks, complete the following
procedure:
Step
Action
1
In the Batch Deconvolution dialog box, click
the Algorithms tab.
2
In the Algorithm box, select the speed of the
algorithm that you want to use. Choose the
Fast algorithm for fastest processing.
Obtaining the best results from this algorithm
requires that your image quality is the best
possible. For more robust processing,
choose the Medium algorithm. For the best
quality and most robust processing, choose
the Slow algorithm.
3
In the Iterations box, type or select an
interaction number from two to ten. A typical
iteration value is six or seven.
4
In the Sigma box, type or select a value at or
close to 0.7.
5
In the Frequency box, type or select an
appropriate value for how often you want
smoothing to run. A typical value is 4 or 5.
For example, a value of 4 causes smoothing
to run during the fourth iteration.
6
Click the Directories tab, then click Select
First File. The Select Base File dialog box
opens.
7
In the Select Base File dialog box select the
folder containing the image stacks that you
want to process, then click the first stack file
that you want to process, and click Open.
8
Click Destination. The Browse for Folder
dialog box opens. Choose an existing
folder, or create a new folder in which to
store your processed images.
9
In the Number of Source Stacks dialog box,
type or select the total number of stacks in
your selected directory that you want to
process.
10
Click the Wavelengths tab.
11
In the Number of Wavelengths box, type or
select the number of wavelengths in the
stacks. All stacks should have the same
number of wavelengths and the wavelength
frequency values for all stacks should match.
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12
For each wavelength that you are going to
process, click Select Psf for Wavelength n.
The Select Base File dialog box opens.
13
Select the directory folder where your PSF
stacks that you want to use are stored, then
choose the appropriate PSF stack for the
wavelength number to which you are
assigning it.
14
If you are using distributed Batch
Deconvolution processing, click the
Networking tab, otherwise, skip to Step 17.
15
Before adding Server IP addresses to the
client system, make sure that each system
that will be included in the Batch
Deconvolution distributed processing
network has a copy of the same version of
MetaMorph that is on the client sytem
installed.
Note: The MetaMorph sofware only needs to
be installed, but not running. A memory key
is needed only when installing or running
MetaMorph. A memory key is not needed on
remote servers that are processing image
stacks using Batch Deconvolution.
16
Before adding server IP addresses to the
Client system, run hmagserv.exe on each
server system that will be processing image
stacks. Note: Click Start, then choose Run
and type hmagserv.exe in the Run box,
and click OK.
17
On the Networking tab, in the New IP
Address box, type a static IP address for
each system that you want to add to the
Server IP Addresses list, then click Add
Server IP.
Note: All IP addresses added to the Server
IP Addresses list must be static IP
addresses. Ask you system administrator to
be sure that all IP addresses that you want
to use are Static.
18
If you do not want to use the Client system
to process image stacks, uncheck Use this
computer for processing.
19
After all configuration steps have been
completed on all tabs, click Apply to begin
processing your image stacks.
20
To close the Batch Deconvolution dialog box
and discontinue processing, click Close.
Batch Deconvolution - Dialog Box Options
Batch Deconvolution - Dialog Box Options - Algorithms
Batch Deconvolution - Dialog Box Options - Directories
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Batch Deconvolution - Dialog Box Options - Wavelengths
Batch Deconvolution - Dialog Box Options - Networking
Batch Deconvolution - Dialog Box Options - Algorithms
Algorithm
Specifies the algorithm to be used for the deconvolution. Algorithms are Fast, Medium, or Slow (most
robust). The slow algorithm is the most robust.
The following three settings are configurations for each algorithm.
Iterations
Specifies the number of times to repeat the algorithm during the deconvolution process. Suggested values
when using the Fast algorithm are between 5 and 7. Suggested values when using the medium algorithm
are between 10 and 20, and for the slow, most robust algorithm are between 20 and 100.
Sigma
Sets the smoothing factor for the deconvolution process. This is how much smoothing is applied to the
image during deconvolution to prevent noise build-up. Sigma is used for only the fast and medium
algorithms; it is not visible when Algorithm is set to Slow (most robust). Typical value is 0.7 for both the fast
and medium algorithms.
Frequency
Specifies how often the Sigma smoothing factor will be applied during the deconvolution process. For
example, a frequency of 2 indicates that smoothing will be applied every other iteration. This option is used
for only the fast and medium algorithms. Typical values are either 4 or 5 for both the fast and medium
algorithms. This value can be used to control how often and/or how soon you want to apply smoothing
during deconvolution. Thus, it might be better to apply smoothing later rather than earlier during
deconvolution.
Batch Deconvolution - Dialog Box Options - Directories
Select First File
Opens the Select Base File dialog box. Use this dialog box to select the first stack from your batch for
deconvolution. The number of stacks processed is determined by the value in the Number of Source Stacks
box. All stack files to be processed must be located in the same folder.
Destination
Selects the directory where the stacks from batch deconvolution will be saved. A message on this tab
indicates: Output filenames will be the input filenames with a prefix of "decon_".
Number of Source Stacks
Specifies the total number of source stacks to process from the selected folder.
Batch Deconvolution - Dialog Box Options - Wavelengths
Select Psf for Wavelength #
Selects the file corresponding to the processed PSF (bead stack) to use in the batch deconvolution. Text
below the button indicates either <None> or the filename of the PSF stack.
Number of Wavelengths
Sets the number of wavelengths, and thereby the number of PSF’s, to be used for batch deconvolution.
This setting determines how many Select PSF for Wavelength #... buttons appear in the dialog. The
minimum value is 1 and the maximum value is 4.
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Batch Deconvolution - Dialog Box Options - Networking
Note: When running batch processing in a distributed processing configuration, systems
in the configuration can process only complete image stacks. Therefore, the best
processing performance is achieved when you have a large number of stacks for
processing. There is no benefit in processing single stacks in this configuration.
New IP Address
Accepts a numeric IP address in the format of uint.uint.uint.uint, where the 4-numbered string designates the
static IP address of a machine to be used to batch deconvolution.
Note: You must configure each system used for distributed batch deconvolution
processing to a static IP address. You can include any system accessable to your
system by way of an IP address, as long as the address is static.
Add Server IP
Adds the current value of New IP Address to the Server IP Addresses list.
Remove Server IP
Removes the currently selected IP address from the Server IP Addresses list.
Server IP Addresses
Displays a list of IP address of machines that can be used for batch deconvolution.
Use this computer for processing
Controls whether this system (the client computer on which MetaMorph is running, and which is controlling
the selection of server systems), is used for processing.
Apply
Applies all settings that you have made and initiates Batch Deconvolution processing.
Close
Closes the Batch Deconvolution dialog box. If Batch Deconvolution is currently processing, clicking close
will discontinue processing.
3D Deconvolution (Process Menu)
Provides Several 3D Deconvolution methods using the AutoQuant 3D Deconvolution
Algorithms.
Availability: Available for MetaMorph Basic and MetaMorph Premier
Drop-in: 3DDECON
Use this drop-in to deconvolve image stacks acquired from Widefield, Confocal, and Two-Photon
microscopes. This dialog controls the AutoQuant 3D Deconvolution software and takes into
consideration a variety of factors including objective lens parameters such as Numerical Aperture and
Refractive Index. It also enables you to limit and control your degree of participation in making optional
settings.
This dialog box contains the following four tabbed pages:
Settings – Accepts basic algorithm settings and shows the current microscope settings.
Optics – Specifies settings relative to the optical properties of the microscope.
PSF – Provides options and settings for choosing the PSF processing method.
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Expert – Provides options and setting for applying advanced PSF Deconvolution techniques. (Not
recommended for normal use.)
3D Deconvolution Procedures
The AutoQuant 3D Deconvolution Procedures are divided into five parts. The first part is for the
non-tabbed area of the dialog box. The remaining four parts correspond to the dialog box tabs:
Settings, Optics, PSF, and Expert. Complete these procedures in sequence. For additional
information, refer to the corresponding dialog box options.
To deconvolve an image stack using either an associated PSF bead stack or a calculated PSF,
complete the following procedures:
Step
Action
1
From the Process menu, choose 3D
Deconvolution>AutoQuant Decon. The 3D
Deconvolution dialog box opens.
2
Open the image stack that you want to
deconvolve; or if you have more that one
image open at the time, click the Source
image selector in the 3D Deconvolution
dialog box to choose the image that you
want to deconvolve.
3
If you want to designate a unique name for
the destination image, click the Result image
selctor. Use the standard MetaMorph image
selector conventions to rename your
destination image.
4
If you have a saved State File of a previous
3D Deconvolution dialog box state that you
want to use or modify, click Load State and
choose the appropriate state file.
5
Go to the procedure for the Settings Tab.
Setting Parameters - Settings Tab
To configure the Settings Tab, complete the following procedure:
Step
Action
1
On the Settings tab, in the Algorithms
Settings area, in the Total Iterations box,
type or select the number of iterations that
you want to run. To determine the
appropriate number of iterations, refer to the
tables for Total Iterations on the Dialog Box
Options page.
2
If you want to use the default Algorithm
settings, click Set Defaults and proceed to
step 5.
3
Determine the noise level present in your
image stack, and choose the appropriate
noise level setting in the Noise Level box.
The choices available are Low, Medium, and
Other. Low and Medium set predefined
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default values. Other enables you to type a
custom value in the Noise Value box. The
default value for Low is 2; the default value
for Medium is 20.
4
If you chose Other as the Noise Level, type
an appropriate custom value in the Noise
Value box.
5
If you need to have faster processing, and
you are willing to accept some amount of
reduced resolution, click the performance
check box to place a check for Faster
Processing/Reduced Resolution.
6
It is recommended that you initially use the
expert setting built into the software.
However, if you decide that you want to
manually designate the expert-level PSF
settings, uncheck Use Recommended
Expert Settings. Otherwise, leave this
checkbox checked.
7
Go to the procedure for the Optics Tab.
Setting Parameters - Optics Tab
To configure the Optics Tab, complete the following procedure:
Step
Action
1
Click the Optics tab. The optics tab page is
displayed.
2
In the Modality box, choose the appropriate
microscope modality that corresponds to the
type of microscope that was used to acquire
your image stack. Refer to Modality in the
3D Deconvolution Dialog Box Options.
3
In the Objective lens area, choose whether
you want to enable the program to obtain the
values for Numerical Aperture (NA) and
Refractive Index from the image data or
whether you want to set these values
manually.
4
If you chose Manual for the Objective Lens
settings, in the Lens NA and Refractive
Index boxes select the Numeric Aperture
and Refractive Index for the lens that was
used to acquire the image stack.
5
In the Image spacings area, choose whether
you want to enable the program to obtain the
values for X, Y, and Z image spacing from
the image calibration data or whether you
want to manually specify these values in
microns.
6
If you choose Manual for Image spacings,
type or select the correct image spacing
values for X, Y, and Z. For additional
information about making these settings
refer to Image Spacings on the Dialog Box
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Options topic page.
7
In the Emissive Wavelength area, choose
whether you want to enable the program to
obtain the values for each channel from the
image data, or whether you want to manually
specify these values for Channels 1, 2, and
3.
8
If you choose Manual for Emissive
Wavelength, in the Ch(x) boxes, type or
select the appropriate wavelength value in
nanometers for each active channel.
9
Go to the procedure for the PSF Tab.
Setting Parameters - PSF Tab
To configure the PSF Tab, complete the following procedure:
Step
Action
1
Click the PSF tab. The PSF tab page is
displayed.
2
For the PSF Starting Point, click Theoretical
PSF if you do not have a PSF image of a
bead plate. If you have a bead plate PSF
image, click Measured PSF.
3
If you chose Theoretical PSF, you can
correct for spherical aberrations. Click the
Spherical Aberrations check box, then
choose either Detect SA or Calculate SA.
4
Detect SA determines a Spherical Aberration
correction value from the image. Click
Detect SA to enable the software to
determine the SA correction value to apply;
OR click Calculate SA to open the Spherical
Aberration dialog box.
5
If you chose Calculate SA, the Spherical
Aberration dialog box opens.
6
In the Spherical Aberration dialog box, in the
Sample embedding medium RI box, type or
select the Refraction Index (RI) value for the
embedding medium you are using.
7
In the Spherical Aberration dialog box, in the
Depth from coverslip(um) box type or select
the value in microns for your coverslip
thickness, then click OK.
8
If you chose Measured PSF as your PSF
Starting Point, in the Measured PSF area on
the PSF tab click the Select PSF button for
each active channel and select the
appropriate PSF file for channel.
9
If you chose Measured PSF as your PSF
Starting Point, in the Measured PSF area on
the PSF tab you can specify whether you
want to use PSF spacings from your image
data or whether you want to manually
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specify PSF spacing values.
10
To use PSF spacing values from the image
data, under PSF Spacings(um), click From
Image; to manually specify your image
spacing values, click Manual, then type or
select your image spacing values in the X, Y,
and Z boxes.
11
If the Use Recommended Expert Settings
checkbox is not checked, go to the
procedure for the Expert tab.
Setting Parameters - Expert Tab
Notes:
• This tab will be available only if you have not checked Use recommended Expert Settings on
the Settings tab.
• If you make any setting changes on the Expert tab, you must leave the setting Use
Recommended expert Settings on the Settings tab checked. If you uncheck this setting, all
your settings on the Expert tab will be reset to their default values.
Hint: Once you have made settings on the Expert tab that you want to recall and reuse,
click Save State and save a state file with a name that indicates that it contains a unique
group of expert settings.
To modify the settings on the Expert tab for their default values, complete the following
procedure:
Step
Action
1
Click the Expert tab. The Expert tab page is
displayed.
2
Uncheck the XY Montage box if your
deconvolution results are creating rigid, boxlike artifacts (pixelation) in your images. The
Default for this setting is On (checked).
3
Check the Z Montage box to divides your
dataset into subsections along the optical
axis and deconvolve these subsections
separately. This option should only be
turned On for image stacks with a large
Depth setting, such as a stack containing
more than 100 planes. The Default for this
setting is Off (unchecked).
4
Uncheck Dynamic subvolumes if you do not
want the available system memory to
determine the largest subvolume allowable.
This option is enabled only when XY
Montage is checked. The Default for this
setting is On (checked).
5
If you need to define a different value for
Subvolume overlap, type the new value in
the Subvolume overlap box. This value
specifies the amount of pixel seam overlap
in a montage. Increase this value to reduce
artifacts. The default value is 10.
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6
To change the amount of padding around
image borders, type a new value in the XY
Guardband box. Larger borders decrease
the amount of edge and ringing artifacts.
The default value is 10.
7
To change the number of planes that will be
added to the top and bottom of the
subvolume, type a new value into the Z
Guardband box. This guardband prevents
artifacts at the seams of these subvolumes.
The default value is 6.
8
Uncheck the Intensity Correction box to turn
off intensity correction. Intensity Correction,
when turned on, prevents abrupt intensity
changes from one image plane to another.
Turn this option off if it is interfering with the
accuracy of your image data. The default for
this option is On (checked).
9
Uncheck Minimum Intensity Removal to turn
this option off. Leaving this box checked
removes the erroneous background intensity
level from the image data. Uncheck this box
if your image stack has no erroneous image
data. The default for this option is On
(checked).
10
Check Accelerated SA Detection if you want
to reduce the time it takes to detect and
correct for Spherical Aberration (SA). If you
check this box, binning is applied to the
image during SA detection. Using
Accelerated SA Detection can influence the
accuracy of the correction. The Default for
this setting is Off (unchecked).
11
Check Pre-condition Imported PSF to
precondition the bead stack image whenever
the measured PSF algorithm is applied. The
default, and recommended setting for this
option is Off (unchecked).
12
In the Object First Guess box Choose the
appropriate setting: Flat Sheet, Linear
Filtered Data, or Original Data. See the
detailed explanation of these choices on the
Expert Tab – Dialog Box Options page. This
option is used to select the starting point for
the iterative estimation process of the object.
13
In the PSF Processing area, type a new
value for the Axial Stretch Factor. This
applies a stretch factor to the Z axis when
Theoretical PSF is selected. See the
detailed explanation of this setting on the
Expert Tab – Dialog Box Options page.
14
In the PSF Processing area, type a new
value for the PSF Waist, measured in Airy
Discs. The PSF waist is the narrowest part
of the PSF measurement area. See the
detailed explanation of this setting on the
Expert Tab – Dialog Box Options page.
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15
Click disable PSF Constraints to discontinue
PSF limitations that are set on the PSF tab.
16
In the Result Save Interval box, type or
select a higher or lower value than the
default value. This value must be a multiple
of the Total Iterations value. See the
detailed explanation of this setting on the
Expert Tab – Dialog Box Options page.
17
After all settings on all the tabs have been
completed, Click OK; your selected source
image stack will be processed accord to the
settings you made in this dialog box.
18
Click Cancel to discontinue using the
AutoQuant 3D Deconvolution and close the
dialog box.
3D Deconvolution - Primary Dialog Box Options
Source image
Opens the image selector for the Source image. This is the image that you want to deconvolve. Select the
name of the image that you want to deconvolve from the list of images that are open.
Result image
Indicates the name of the Destination image. By default, this name is "Deconvolved." Click on the
"Specified" image name(deconvolved) to rename the destination image name or to select and overwrite an
existing image name.
OK
Runs the AutoQuant 3D Deconvolution command using the settings that you made.
Load State
Opens the Load State dialog box. This option enables you to load a previously saved state file and apply
one or more of the saved settings to the option settings in this dialog box. The settings of controls in the
main and settings dialogs can be all loaded from a state file.
Save State
Opens the "Save State file as" dialog box. Use this option to store the current AutoQuant settings for this
dialog box.
Cancel
Closes the 3D Deconvolution Options dialog box.
3D Deconvolution - Dialog Box Options - Settings Tab
Microscope Settings
Summarizes the current microscope-related settings that have been made in the dialog box .
Modality – Indicates the type of microscope that was used to acquire the image(s).
Lens NA – Indicates the Numerical Aperture (NA) of the objective used to acquire the image.
Refractive Index – Indicates the refractive index value for the objective used to acquire the image.
Image Spacings (um) – Indicates the assigned image spacing values microns. These values are
based on either the image calibration values if the image is calibrated and Calibrated is selected for
Image spacings on the Optics tab, or the values specified for X, Y and Z if Manual is selected for
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Image spacings on the Optics tab.
Wavelengths (nm) – Indicates the selected wavelength values for each channel in nanometers.
PSF – Indicates that the Point Spread Function will be used to deconvolve the image.
Algorithm Settings
Provides the primary settings that you can use to optimize the performance of your selected PSF algorithm.
You can use the default values as a starting point. By combining different setting values from the available
settings, you can determine settings that are most ideal for your images.
Set Defaults
Resets all values in the Algorithm Settings box to their original, default values. These values are
different when Performance: Faster Processing/Reduced Resolution is checked.
Total Iterations
Specifies the number of times that you want the algorithm to run to complete the deconvolution
process. A greater number of iterations can improve the resolution of the result image. The default
value for this setting changes when Faster Processing/Reduced resolution is selected.
Different modalities (microscope configurations) can require different iteration values in order to
optimize the deconvolution image processing process. Values set higher than the recommended
setting might not provide any additional improvement to the image.
If you choose standard performance, (Faster Processing/Reduced Resolution is not checked), the
following recommended Total Iterations setting values apply:
For Widefield Data:
Total Iterations
1 – 80
80 – 100
100 – 350
350 – 500
Determining Factors
Minimum range of values; specify a value in this
range if speed is the most important consideration.
Recommended range for an optimal choice between
processing speed and processing accuracy and as
an initial range of values.
Maximum recommended range under normal
conditions; specify a value in this range if resolution
is the most important consideration.
Typically not recommended; these higher iteration
settings might provide better resolution, but require
images with very low noise levels.
For Confocal Data:
Total
Iterations
1 – 40
40 – 60
60 – 100
Determining Factors
Minimum range of values; specify a value in this
range if speed is the most important consideration.
Recommended range for an optimal choice between
processing speed and processing accuracy and as
an initial range of values.
Maximum recommended range under normal
conditions; specify a value in this range if resolution
is the most important consideration.
If you check Faster Processing/Reduced Resolution, the following recommended Total Iterations
setting values apply:
For Widefield Data:
Total
Iterations
1 – 40
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40 – 70
70 – 100
Recommended range for an optimal choice between
processing speed and processing accuracy and as
an initial range of values.
Maximum recommended range under normal
conditions; specify a value in this range if resolution
is the most important consideration.
For Confocal Data:
Total Iterations
1 – 10
10 – 20
20 – 40
Determining Factors
Minimum range of values; specify a value in this
range if speed is the most important consideration.
Recommended range for an optimal choice between
processing speed and processing accuracy and as
an initial range of values.
Maximum recommended range under normal
conditions; specify a value in this range if resolution
is the most important consideration.
Noise Level
Specifies the amount of noise suppression to apply to your image. Choose the amount of
noise suppression that you want to apply based on the amount of noise that you visually
detect. Depending on the setting that you choose, the program will apply the specified
amount of smoothing during image processing to suppress the noise in your image.
Choose from the following settings to specify the amount of noise suppression that you
want to apply.
Low – specifies that noise is minimally detected in you image. Choose this
setting to apply a noise suppression value of 2. typically use this setting for
Widefield images.
Medium – specifies that moderate amounts of noise are visible in your image.
Choose this setting to apply a noise suppression value of 20. Typically use this
setting for Confocal images.
Other – specifies a noise-suppression value different than the available preset
values.
Guidelines:
• Widefield images usually contain lower noise levels.
• Confocal images usually contain moderate noise levels.
Noise Value
Indicates or specifies a value that represents the amount of noise suppression that will be applied
to your image. If you choose a noise suppression level of Low, a value of 2 is displayed in this
settings box; if you choose a noise suppression level of Medium, a value of 20 is displayed in this
settings box. To choose a different noise suppression level, choose Other as the Noise Level
setting, and type an appropriate value in this box.
Performance: Faster Processing/Reduced Resolution
Improves the processing time, while slightly reducing the resolution. When it is necessary to use a
high number of iterations or when this dialog will be used to process a large number of images, you
can decrease the total processing time by checking this box.
Note: It is recommended that you leave this box unchecked when you are processing
images from thick samples or noisy data sets.
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Use Recommended Expert Settings
Applies the recommended expert settings and hides the Expert page tab. It is
recommended that you leave this checkbox checked. Uncheck this checkbox and click
the Expert tab to modify the default expert settings.
Notes:
• Before making any modifications to the settings on the Expert tab, you should become
familiar with all of the settings on this tab and how each setting affects your results.
Generally, you should not change the Expert settings from their default values. Change
these settings only for special situations.
• To reset the settings on the Expert tab to their default values, uncheck this checkbox,
then check it again. When you re-select the Expert tab, any values or settings that you
made on the Expert tab will have been reset to their original default values.
3D Deconvolution - Dialog Box Options - Optics Tab
Microscope
Specifies the type of microscope from which your image was acquired.
Modality
Specifies the imaging method used to acquire your images based on the microscope configuration.
Modality choices are the following:
•
Confocal – Point Scan
•
Confocal – Spinning Disk
•
Two photon
•
Widefield – Fluorescence
•
Widefield – Transmitted
Objective Lens
Accepts optical properties information about the objective in use.
Image – Specifies that the values for Numerical Aperture and Refractive Index will be obtained
from the data stored with the image, if available. The Numerical Aperture and Refractive Index
boxes are deactivated.
Manual – Specifies that the values typed or selected in the Numerical Aperture and Refractive
Index boxes will be used.
Lens NA – Specifies the numerical aperture (NA) of the objective that you used to acquire your
image.
Refractive index – Specifies the refractive index of the objective that you used to acquire your
image.
Image spacing
Specifies whether a calibrated or user-defined spacing will be used.
Calibrated– Specifies that the program will use the calibration values for X, Y and Z stored with the
image.
Manual (um)– Enables you to specify the calibration settings for Z that you want to use, but uses
the values for X and Y stored with the image.
X, Y, and Z
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Indicates the X, Y, and Z spacing of the image to which the source image selector points.
X Spacing
Defines the X dimension of a single pixel in microns. Calculate X Spacing by dividing the
width of the image in microns by its width in pixels. Type or select the X Spacing width in
microns. Decimal values can be typed in. The value you enter should be accurate to
within 3% of the total width.
Y Spacing
Defines the Y dimension of a single pixel in microns. Calculate Y Spacing by dividing the
height of the image in microns by its height in pixels. Type or select the Y Spacing height
in microns. Decimal values can be typed in. The value you enter should be accurate to
within 3% of the total height.
Z Spacing
Defines the calibrated vertical, Z-distance between the acquisition points in an image
stack. Calculate Z Spacing by dividing the total Z-distance of the image stack by the total
number of image planes in the stack. The value you enter should be accurate to within
3% of the total Z-distance.
Emissive Wavelength
Specifies the emissive wavelength of the fluorescent dye used to stain the sample. This value
should be the wavelength at which the image was acquired. Use a value of 520 for brightfield data.
Default value is 520.
Specifies whether the image wavelength values used will be the values stored in the image data or
manually entered values.
Image
Specifies that the image processing will use the wavelength values stored in the image.
Manual (um)
Specifies that the image processing will use the wavelength values that you enter or select in the
separate boxes for each channel.
Ch1
Accepts the wavelength value for Channel 1 in nanometers.
Ch2
Accepts the wavelength value for Channel 2 in nanometers.
Ch3
Accepts the wavelength value for Channel 3 in nanometers.
3D Deconvolution - Dialog Box Options - PSF Tab
PSF Starting Point
Chooses either a theoretical PSF or a measured PSF to complete your image deconvolution.
Theoretical PSF
Selects the theoretical PSF. Use the Theoretical PSF settings to complete your PSF configuration.
This is a Blind deconvolution process.
Measured PSF
Selects the measured PSF. Use the Measured PSF settings to complete your PSF configuration.
This is a Non-Blind deconvolution process.
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Theoretical PSF
Specifies settings for theoretic PSF deconvolution processing.
Spherical Aberrations
Indicates the amount of spherical aberration present in the objective used to acquire the image. If
you know the value, you can type it into the box.
Detect SA
Detects the amount of spherical aberration present in the image you are processing.
Calculate SA
Calculates the amount of spherical aberration based on the values you enter in the Spherical
Aberration dialog box. Click Calculate SA to open the Spherical Aberration dialog box.
Measured PSF
Specifies settings for measured PSF deconvolution processing.
Ch1, Ch2, Ch3 – Select PSF
Specifies and selects the PSF files for each channel that will be used for deconvolution.
PSF Spacings
Specifies whether the PSF spacing values will be from the image data or manually typed into the
provided boxes for X, Y, and Z.
From Image
Specifies that the PSF spacing values will be derived from the image data.
Manual
Specifies that the PSF spacing values will be manually typed into the boxes for X, Y, and Z.
X, Y, Z
Accepts image pixel spacing values for X, Y, and Z.
3D Deconvolution - Dialog Box Options - Expert Tab
Subvolume
XY Montage
Divides the data into subvolumes along the XY dimensions. This option reduces the amount of
RAM required by the deconvolution application. The default value for this setting is On (checked).
Note: The default for this setting is On. It should be turned Off only if the deconvolution
application is producing rigid, box-like artifacts in your data.
Z Montage
Divides the dataset into subsections along the optical axis and deconvolves these subsections
separately.
Note: The default for this option is Off (unchecked). It should only be turned On for image
stacks with a large Depth setting, such as a stack containing more than 100 planes. This
option reduces the amount of RAM required by the deconvolution process. It might also
be useful in instances where the sample thickness causes the PSF to change along the Z
axis. In these cases, Z Montage enables blind deconvolution to find different PSF
solutions for different depths.
Dynamic subvolumes
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Specifies that the size of the subvolume will be determined based on the available memory, if this
is checked. This option uses the available system memory to determine the largest subvolume
allowable. This will be enabled only if XYMontage is on. The default value, if XY Montage is on is
True (checked).
Subvolume overlap (Pixels)
Specifies the number of pixels by which the adjacent sub volumes will overlap. Larger values result
in longer processing but reduce edge and seaming artifacts, should they occur. The default value
is 10.
XY Guardband (Pixels)
Specifies the amount of padding around image borders. The XY Guardband provides a region of
pixels around the border of the processed image stack or subvolume, but disregards this region
before the results of deconvolution are returned. Larger values increase processing time, but
decrease edge and ringing artifacts. The default value is 10.
Z Guardband (Pixels)
Specifies the number of planes that will be added at the top and bottom of the subvolume. This
guardband prevents artifacts at the seams of these subvolumes. The possible values are integers
from 0 to N/2, where N is the depth of the XZ or YZ field. The default value is 6.
Note: The Z-Guardband should never be larger than the subvolume overlap region. A
value of 6 is appropriate for most image stacks.
Pre-processing
Intensity Correction
Applies correction to fluctuations in the image intensity values across the depth of an image stack.
This option should be checked if the image intensity changes abruptly from one image plane to
another in areas where image intensity should be constant from one image plane to the next.
Fluctuations can result from minor variations in illumination intensity. The default value is On
(checked).
Minimum Intensity Removal
Specifies that the program will automatically calculate and remove the erroneous background
intensity level in the data. The most common cause of this erroneous background intensity is the
electronic dark current (background electrical signal level) of the photodetector or CCD camera.
Other causes are the bias voltage of amplifiers in the camera, back-scattered light that penetrates
the emission filter and nonspecific dye that can leak into the embedding medium, among other
causes. The default for this option is On (checked).
Accelerated SA Detection
Reduces the time required to detect Spherical Aberrations in your image stack by applying binning
to the image data. In this instance, each group of four pixels is treated as one. Applying
acceleration might influence the calculated spherical aberration correction value to not represent
the true amount of spherical aberration present in the objective used to acquire the image stack.
The default for this option is Off (unchecked).
Pre-Condition Imported PSF
Applies a preconditioning algorithm to the PSF image whenever you have selected Measured PSF
and are using a bead stack. The default, and recommended setting for this option is Off
(unchecked).
Object First Guess
Specifies the most appropriate deconvolution method for the 3D Deconvolution algorithm to use to
produce the initial guess to restore the image. This option is used to select the starting point for the
iterative estimation process of the object. Choose one of the following options:
Flat sheet – Provides a constant-valued volume; use this setting for extremely noisy data.
Original data – Specifies the default value; use this setting for the majority of your images.
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Linear filtered data – Runs the source image through a linear filter to remove some blur; use this
setting for images containing a very strong fluorescent signal.
PSF Processing
Contains the expert level settings used to configure PSF processing.
Axial Stretch Factor
Specifies the factor by which the theoretical (calculated) PSF is lengthened along the Z axis to
more accurately initiate the first guess, particularly for a confocal data set. The default Axial Stretch
Factor is 1 for Widefield data and 3 for Confocal.
PSF Waist (Airy Disc’s)
Specifies size of the narrowest part of the PSF, usually measured in Airy Disc diameters. The
default setting is 1 for both Widefield and Confocal.
Disable PSF Constraints
Removes the limitations placed on the Point Spread Function (PSF).
Result Save Interval
Specifies the number of iterations that will occur between storage of intermediate deconvolution results on
disk. For example, if the number of Total Iterations is 20 and the Save Interval is 5, the deconvolution
results will be saved at 5, 10, 15 and 20 iterations. The program automatically checks for available disk
space before beginning deconvolution. This setting is available only when the Expert tab is active.
Sub-Pixel Shift (Process Menu)
Shifts an image by a selected sub-pixel distance in the horizontal and/or vertical direction.
Drop-in: SUBSHIFT
Use this command to shift an image by a non-integer number of pixels. This function may useful in cases
in which your regions of interest, which are defined over precise pixels, do not line up exactly in register
with objects you want to measure. This command is also helpful in aligning planes in a stack when they
are out of register with one another by a fraction of a pixel. Shifts are measured in one-twentieths of a
pixel.
Applying a Sub-Pixel Shift
To apply a shift to an image or stack plane by a non-integer number of pixels, use the following
procedure:
Step
Action
1
From the Process menu, choose Sub-Pixel
Shift. The Sub-Pixel Shift dialog box will
appear.
2
With the Source image selector, select the
image or stack plane you want to shift.
3
Select a destination for the result image with
the Result image selector. You can add to or
overwrite the existing source image or stack,
or you can specify a new image.
4
Select a whole number for the offset,
measured in one-twentieths of a pixel, with
the Horizontal Shift and/or Vertical Shift spin
boxes.
5
Choose Apply to apply the shift.
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When you have finished, choose Close.
Sub-Pixel Shift - Dialog Box Options
Source image
Selects the image or stack plane to be shifted.
Result image
Selects a destination for the shifted result image. You can add to or overwrite the existing source image or
stack, or you can specify a new image.
Horizontal Shift
Selects a shift in the X-axis direction, measured in one-twentieths of a pixel. The number you enter should
be an integer.
Vertical Shift
Selects a shift in the Y-axis direction, measured in one-twentieths of a pixel. The number you enter should
be an integer.
Apply
Applies the shift to the selected image or stack plane.
Close
Closes the dialog box.
Optical Density (Scaled) (Process Menu)
Performs quantitative densitometry on an entire image.
Drop-in: ODSCALE
Use this command to display the optical densities of a brightfield source image in a scaled 8-bit or 16-bit
image. You will need both a "raw" source image and a bright shading-correction reference image. A
background reference image is optional. The grayscale value of each pixel in the output image will be
determined by the following formula:
Result = ( -Log10 [ { Source - Background + Offset } / Shading ] ) * (Scaling Factor)
The Scaling Factor option will be useful for adjusting the brightness of the resultant image, so as to allow
the optical densities to be displayed at image intensities that are more easily discerned by eye.
Adjustment of the Scaling Factor will have a reciprocal effect on the Max OD Result control--a lower Max
OD Result will be correlated with a higher Scaling Factor and a brighter result image.
The optical density scaling operation can also be performed on 24-bit color source images. The intensity
values from color images are weighted equally (Intensity = [R + G + B] / 3).
Note: The Auto Calibrate option will set the MetaMorph system calibration to use optical density (OD)
units. The Calibrate Gray Levels data table will contain two entries, one for an OD of zero and one for an
OD of 1, along with their corresponding gray values. This calibration will affect measurements of all
images, and involves all densitometric commands, such as Show Region Statistics, Integrated
Morphometry Analysis, Morphometry Histogram, Measure Pixel, and Linescan. If you subsequently need
to disable the optical density system calibration, you must open the Calibrate Gray Levels dialog box
(Measure menu) and clear the Use Calibration check box.
Image-1/AT: The Optical Density (Scaled) command is similar to the Optical Density operation in the
MVPMATH program from Image-1/AT.
Using Optical Density (Scaled)
To perform quantitative densitometry on an entire image, use the following procedure.
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Note: You must have both a source image and a shading image. The source image
should be a brightfield image.
Step
Action
1
From the Process menu, choose Optical
Density (Scaled). The Optical Density
(Scaled) dialog box will open.
2
Select a source image from the Source
image selector.
3
Select an image for shading using the
Shading image selector.
Note: Before applying the Shading Image,
using the Background Image selector, select
a background image to be subtracted from
the source image.
4
Use the Result Image selector to select a
destination image.
5
If you wish, specify an offset value in the
Offset spin box. This value will be added to
the grayscale value of each pixel before its
optical density is calculated.
6
In the Scaling Factor spin box, select a value
by which the optical density of each pixel is
multiplied before being displayed in the
destination image.
OR
Use the Max OD Result spin box to select
what you expect to be the maximum optical
density in the measured image, so that an
appropriate range of intensities will be used
in the displayed image.
Note: Adjustment of the Scaling Factor will
have a reciprocal effect on the Max OD
Result control--a lower Max OD Result will
be correlated with a higher Scaling Factor
and a brighter result image.
7
Choose between an 8-bit and a 16-bit output
from the Result Depth group.
8
If you want to use the scaled optical density
calibration for other images, select the Auto
Calibrate check box to set the MetaMorph
system calibration to use the optical density
units.
9
Choose Apply. The scaled optical density
result image will be displayed. If you selected
the Auto Calibrate check box in Step 8, the
system will also be set to use the measured
optical density units.
10
When you have finished, choose Close to
close the dialog box.
Optical Density (Scaled) - Dialog Box Options
Source image
Selects the source image. This should be a brightfield image.
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Background image
Selects a background reference image. Use of a background image is optional.
Shading image
Selects a shading correction image. This image is required. Note: Before applying the shading image, it
should have the background subtracted from it.
Result image
Selects the destination for the scaled optical density image. You can create a new image or you can
overwrite or add to an existing image.
Offset
Specifies an offset value for the equation if there are pixels in the source image that have a lower gray value
than the pixels in the shading image.
Scaling Factor
Specifies a scaling factor to be applied to the optical density value of each pixel in the destination image.
Max OD Result
Selects the maximum optical density in the measured image, so that an appropriate range of intensities will
be used in the displayed image. Note: The Scaling Factor and the Max OD Result options have a reciprocal
effect on one another--a lower Max OD Result will be correlated with a higher Scaling Factor and a brighter
result image.
Auto Calibrate
Sets the MetaMorph system calibration to use the measured optical density (OD) units. This will affect
measurements of all images, and will involve all densitometric commands. If you need to disable the optical
density system calibration, you will need to open the Calibrate Gray Levels dialog box (Measure menu) and
clear the Use Calibration check box.
Result Depth
Specifies the bit-depth of the Destination Image.
Apply
Executes the quantitative densitometry calculations and displays the scaled optical density image.
Close
Closes the dialog box.
Ratio Images (Process Menu)
Computes a ratio image from two images (or stacks).
Drop-in: RATIO
Use this command when you want to analyze a ratio image in MetaMorph or when you want to build a
ratio image but do not have access to MetaFluor. It can also be used simply to correct shading errors in
images.
You can specify the minimum and maximum permissible ratio.
This command also allows you to specify use of the IMD (Intensity-Modulated Display) mode palette for
the ratio image, as well as which source image to use for the intensity. The IMD mode is an alternative to
the Pseudocolor palette. The IMD palette uses a custom look-up table that associates color hues with
the ratio values, and the intensities of each hue with source image brightness.
Note: For some images, you may find it desirable to threshold the denominator and
numerator images before building the ratio image.
Note: This drop-in does not support the active region.
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IMD Display
The Intensity-Modulated Display (IMD) display mode, devised
by Dr. Roger Tsien, is an alternative to the Pseudocolor display
palette. The IMD palette associates color hues with the ratio image,
and the intensity of each hue with wavelength intensity.
Building a Ratio Image
To build a ratio image, use the following procedure:
Step
Action
1
From the Process menu, choose Ratio
Images. The Ratio Images dialog box will
appear.
2
Select the desired image for the numerator
using the Numerator image selector.
EXAMPLE:
If you have images from 340 and 380 nm
wavelengths, you would select the 340 nm
wavelength image with the Numerator image
selector.
3
Select the desired image for the denominator
using the Denominator image selector.
EXAMPLE:
If you have images from 340 and 380 nm
wavelengths, you would select the 380 nm
wavelength image with the Denominator
image selector.
4
Select the destination image for the ratio
using the Ratio Image selector.
5
Select the desired IMD display using the IMD
Display list.
Your choice will depend mostly upon whether
the ratio image's values are expected to be
evenly distributed throughout the ratio range
or clustered around one ratio value. If most
of the values are clustered, the 64 Ratios
with 4 Intensities selection will produce the
best results.
OR
If you want to use a pseudocolor display
instead of the IMD display, select None from
the list and skip Step 6.
6
Select the desired source for the IMD
intensity from the IMD Intensity list. You
should select the intensity from the brighter
image. If you are using this command in a
journal and do not know which image will be
brighter, you can select Average Num. and
Denom. to use an average from the two
images.
7
Select the minimum and maximum values for
the ratio using the Min Ratio and Max Ratio
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options.
8
Choose Apply to create the ratio image.
9
Choose Close when you have finished.
Ratio Images - Dialog Box Options
Numerator
Selects the numerator image of the pair of images used to build the ratio image.
Denominator
Selects the denominator image of the pair of images used to build the ratio image.
Ratio Image
Selects the destination for the ratio image. You can create a new image or you can overwrite or add to an
existing image.
IMD Display
Selects the desired IMD display for the ratio image. The IMD display will use a custom look-up table that
consists of hues corresponding to the selected number of ratios, with each hue having its own range of
intensities. For example, a ratio image that was built using the 8 Ratios with 32 Intensities option will have
eight distinct hues, each with 32 intensities visible in its contrast/threshold slider (as opposed to the
continuous range of values visible for a pseudocolor image).
IMD Intensity
Selects the source for the intensity values. Select the brighter image. If you are using a journal and do not
know which image will be brighter, select Average Num. and Denom. instead.
Min Ratio
Selects the minimum ratio value for the ratio image.
Max Ratio
Selects the maximum ratio value for the ratio image.
Apply
Builds the ratio image.
Close
Closes the dialog box.
Use Region for Background (Legacy)
Note: The Use Region for Background command is now available through the
Background and Shading Correction command in the Process menu. The stand alone
Use Region for Background command is no longer available from the MetaMorph
desktop and can only be accessed through the Journal Editor.
Takes the maximum, minimum, or average intensity value of a selected region of interest
and subtracts it from each pixel in the current image plane or in an entire stack of images.
Drop-in: SUBRGN
Use this command to perform background subtraction based on the intensity levels in a selected region
of interest. Once you have delineated a region, you can subtract the maximum, minimum, or average
intensity value in that region from each pixel in the image. If you have defined several regions of interest
in the image, you can determine the one that gives the best results by selecting from among them and
observing the result images. You can add to or replace the existing image or stack of images, or you can
place the results in a new image window.
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Note: If your source image is a stack and you choose All Planes, the calculated
background value from this current plane will be subtracted from all of the planes of the
stack. To calculate a different background value for each plane, record this command
into a journal using the current plane in the image selector. Then run the journal using
the Loop for All Planes command.
Using a Region's Intensity Value for Background Subtraction in an
Image
To subtract a region's intensity value from each pixel in an image or stack of images, use the
following procedure:
Step
Action
1
From the Process menu, choose Use Region
for Background. The Use Region for
Background dialog box will appear.
2
Select the desired image from the Source
image selector. If the source image is a stack
of image planes, select either the Current
plane or All Planes.
3
Selects a destination for the processed
image with the Result image selector. You
can place the results in a new image window
or in a new plane appended to an existing
image or stack, or you can overwrite the
existing image.
4
If there is more than one region of interest
defined in the image, select the desired
region with the Region to Be Used as
Background spin box.
5
Select which intensity value in the region that
you want to subtract: Average, Minimum, or
Maximum.
6
Choose Apply.
7
When you have finished, choose Close.
Use Region for Background - Dialog Box Options
Source
Selects the image or stack of images that you wish to process. If the source image is a stack, you can select
either the Current plane or All Planes in the stack.
Note: If your source image is a stack and you choose All Planes, the calculated
background value from this current plane will be subtracted from all of the planes of the
stack. To calculate a different background value for each plane, record this command
into a journal using the current plane in the image selector. Then run the journal using
the Loop for All Planes command.
Result
Selects a destination for the result image. You can add to or replace the existing image or stack of images,
or you can place the results in a new image window.
Region to Be Used as Background
Selects the region of interest to use for determining the intensity value to be subtracted. The region number
may not match the region label. See Sequence Region LabelsSequence Region Labels.
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Region Measurement to Use
Select which intensity value in the selected region that you want to subtract: Average, Minimum, or
Maximum.
Apply
Applies the intensity value subtraction process to the Source image.
Undo / Redo
Undoes or redoes the last applicable command that did not create a new result image. (For this command, it
undoes the results of overwriting the source image with the background-subtracted result image.) Undo /
Redo buttons in MetaMorph apply to any previously applied command that did not create a new result
image, not just the last use of the command in the dialog box from which the button was chosen.
Close
Closes the dialog box.
Overlay Images (Display Menu)
Overlays up to seven images, assigning a different color to each. Typically, one of the
images is a grayscale transmitted-light image, and the other overlay images are
fluorescence images.
Drop-in: OVERFLUO
Use this command to combine a "background" image, such as a brightfield transmitted-light image, with
up to six fluorescence "probe" images. The images will be combined in such a way that the information
available in the background image can be seen through the fluorescence images. Each fluorescence
image can be assigned a different color in the result image, and the relative contribution of each image
("balance") to the final result can be adjusted. The source images can be either 8-bit or 16-bit images,
and can be individual, single-plane images or they can be selected planes in an image stack. The result
image will be a 24-bit color image. The Overlay Preview image window displays a 256x256 preview of
the overlay result.
The resulting overlay image will, to a large extent, have the image characteristics of the source images.
For example, if you have significant contribution from background in your fluorescence images, this may
show up in the final image as an unwanted, diffuse coloration of the entire image. To avoid such an
effect, you should be sure to threshold the image so as to include only the objects of interest in the
threshold range. Overlay Images will take thresholding into account, adding only the thresholded areas
of interest in the source images to the final result. Similarly, if the contrast is too low in the source
images, you should perform any appropriate enhancements to the source images, such as increasing
contrast or scaling of the intensity range, before combining them in the final overlay image.
This command does not directly handle binary (1-bit) images. If you want to use a binary source image,
you must first convert it to an 8-bit mask image with the Threshold Image command (Process menu).
Note: The Overlay Images command is somewhat similar to Combine into B&W + Color
in that it creates a combined color and B&W image. However, Overlay Images allows you
to create images in which the colored regions that represent the dye probes are
transparent, and the Black & White "background" image can be seen "through" the
fluorescence images. Combine into B&W + Color, on the other hand, is useful for
combining one or two grayscale images into a single color/B&W image with a complex
custom palette.
Overlay Images - Procedures
Overlaying Images
Editing the Hue List
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Overlaying Images
To create a fluorescence "overlay" image, use the following procedure. Be sure to have all
images already open in the application workspace, and perform any thresholding or contrast
enhancements before proceeding.
Step
Action
1
From the Display menu, choose Overlay
Images. The Overlay Images dialog box
opens.
2
With the # Images spin box, select the
number of images that you will be combining,
including the transmitted light image
(optional), if any.
3
If you have a stack of fluorescent images of
different wavelength, select the Images from
Stack check box. Otherwise, leave the check
box cleared.
Note: If your stack contains more image
planes than you want to include in the
overlay, make sure that the images you do
intend to include are in the topmost planes of
the stack.
4
If your source images are individual, singleplane images, use the first image selector in
the Source column to select the grayscale
"background" (transmitted light) image. If you
are not using a "background" image, select
[None].
OR
If your source images are planes in an image
stack, select the source stack with the
Source image selector. Then select the radio
button that indicates which plane will be the
"background" image. If you are not using a
"background" image, select No Plane Is Gray
Image.
5
For each color overlay image, select the
image or plane you are about to configure by
clicking its Hue color box. If the image is a
single-plane image, use the corresponding
Source image selector to select the desktop
image.
AND
Use the Hue slider or the drop-down Hue List
in the Hue Selection option group to select a
color to be assigned to the image.
If you move the slider to a position
corresponding to one of the default colors,
the Hue List will automatically update to
display the name of the color. Otherwise, the
list will display "Unnamed." Conversely, if you
select a different color from the Hue List, the
slider will move to the corresponding location
on the Hue color bar.
Note: If necessary, you can edit the default
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list of available colors.
6
If you want MetaMorph to set the intensity
balance between the images automatically,
select Auto Balance.
OR
If you want to adjust the intensity balance
between the images manually, clear the Auto
Balance check box and use the Bal spin
boxes to adjust the relative contribution from
each of the images.
7
If necessary, use the Overlay Brightness spin
box to change the overall brightness of the
result image.
This may be necessary because the
algorithm used to encode the intensity in the
result image derives its values from the
grayscale "background" image, and any pixel
with an intensity value of 0 (black) will be
assigned an intensity of 0 in the result image,
resulting in loss of overlay image information.
8
If you want to enhance the areas in which the
fluorescent probes overlap, select Boost
Colocalization.
9
Select a destination for the result image with
the Dest image selector.
10
A 256x256 Overlay Preview image window
will display a preview of the overlay result. If
your source images are larger than 256x256,
use the box-in-box control in the upper right
of the Overlay Images dialog box to select a
different region to display in the preview
window, as desired.
11
When you are ready, choose Apply to create
the new overlay image.
Note: If you want to hide the Image Window
Tools, right-click in the overlay image window
and choose Hide Image Window Toolbar
from the pop-up context menu that appears.
12
Choose Close to close the dialog box.
Editing the Overlay Images Hue List
To edit, add to, or remove a color from the hue list, use the following procedure.
Step
Action
1
From the Overlay Images dialog box, choose
Edit Hue List. The Edit Hue List dialog box
will appear.
2
To add a new color to the Hue List, drag the
handle of the Hue slider to the desired
position. The color box in the upper right
corner will update as you do so.
AND
Type the name for the new color in the Name
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of Hue text box. You may want to use the
name of the fluorescent dye to which the
assigned image corresponds (for example,
"DAPI" or "Rhodamine"). Then choose Add.
3
If you want to remove an entry in the Hue
List, highlight the entry, and then choose
Remove.
4
If you want to edit the name or hue of an
existing entry, double-click the entry's name
in the Hue List. The Hue slider and Name of
Hue text box will update.
AND
Make your changes to the Hue slider setting
or the name in the Name of Hue text box.
Then choose Add.
5
When you have finished, choose OK to
return to the Overlay Images dialog box.
Overlay Images - Dialog Box Options
Overlay Images
Edit Hue List
Overlay Images - Dialog Box Options
# Images
Selects the number of images, including any grayscale "background" (i.e., transmitted-light) image, that you
want to include in the overlay image. This number will determine how many image configuration rows
(Hue/Bal/Source) are displayed in the dialog box.
Auto Balance
Sets the intensity balance between the source images automatically. If you select this option, the Bal and
Overlay Brightness spin boxes will be unavailable.
Images from Stack
Reconfigures the Overlay Images dialog box for use with a stack source image. When you select this check
box, a single Source image selector will be displayed, and each image configuration row will be labeled for
its corresponding stack plane. The Plane N Is Gray Image radio button group will also be displayed.
Boost Colocalization
Enhances the intensity of the areas in which two or more fluorescence probes overlap.
Show Preview
Select this option to view an overlay image preview window. The overlay section displayed can be
manipulated using the Preview Image control.
Overlay Brightness
Sets the intensity in the result image, based on the intensities of the overlay source. the default setting is 50.
Increasing this value will produce a brighter result image. This may be necessary because the algorithm
used to encode the intensity in the result image derives its values from the grayscale "background" image,
and any pixel with an intensity value of 0 (black) will be assigned an intensity of 0 in the result image,
resulting in loss of overlay image information.
Hue (colored boxes)
Displays the hue currently assigned to the image or plane. The image being configured is selected by
clicking its corresponding Hue box. An arrow at the left indicates which image or plane is the one currently
being configured. If the source images are single-plane images, the first (topmost) box will always be gray,
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because this configuration row is reserved for the grayscale (transmitted-light) image.
Bal
Sets a scaling factor for each overlay, relative to the Bal factors of the other overlay images. The default
setting is 50.
X Align
Changes the X position of the source image or plane.
Y Align
Changes the Y position of the source image or plane.
Source
If the source images are single-plane images, this column of image selectors selects the source images for
the final overlay image. By default, the first (topmost) selector selects the grayscale image. If the source
images are planes in a stack, just one Source image selector will be displayed, which will be used for
selecting the source image stack.
Plane N Gray
Selects a stack plane to be used as the grayscale "background" (transmitted-light) image in the final overlay
image. If you are incorporating only fluorescence images into a color overlay image, and do not want a
"background" image, select No Gray. This column of radio buttons will appear only when you have selected
the Images from Stack check box.
Destination
Specifies the result image.
Preview Image (Box-in-Box Control)
Selects a subregion of the source images to display in the 256x256 Overlay Preview image window. This
option will be disabled if the source images do not have a width or height greater than 256 pixels across.
Hue (slider)
Selects a color to be assigned to the image or plane currently being configured. If you move the slider to a
position corresponding to one of the default colors, the Hue List will automatically update to display the
name of the color. Otherwise, the list will display "Unnamed." Conversely, if you select a different color from
the Hue List, the slider will move to the corresponding location on the Hue color bar.
Hue List
Displays the currently selected color. Clicking the "down arrow" will open the drop-down list, from which you
can select a different color to be assigned to the image or plane currently being configured. If you select a
different color, the Hue slider will move to the corresponding location on the color bar. Conversely, if you
move the Hue slider to a position corresponding to one of the default colors, the Hue List will automatically
update to display the name of the color. Otherwise, the list will display "Unnamed."
Edit Hue List
Opens the Edit Hue List dialog box, from which you can change a Hue List entry's name or assigned color,
add a new entry, or remove an existing entry.
Apply
Creates the overlay image.
Close
Closes the dialog box.
Edit Hue List - Dialog Box Options
Hue List
Displays the current set of entries in the Hue List. (Note: This list does not have an interaction with the Hue
slider or entry name text box in the manner seen in the Overlay Images dialog box.)
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Hue (slider)
Selects a color for a new entry that you want to add to the Hue List.
Name of Hue
Specifies a name for a new entry that you are adding to the Hue List. By default, this text box displays "New
Hue."
Remove
Removes the entry currently highlighted in the Hue List. You can remove any entry, with the exception of the
Unnamed entry.
Add
Adds a new entry to the Hue List, using the color selected with the Hue slider and using the name specified
in the Name of Hue text box. If you assign a different color to an entry whose name is already in use, a
message box will appear, asking if you wish to overwrite the existing entry.
OK
Accepts the currently configured Hue List and closes the Edit Hue List dialog box.
Produce Background Correction Image (Legacy)
Note: The Produce Background Correction Image filter is now available through the
Basic Filters command in the Process menu. The stand alone Background Correction
Image command is no longer available from the MetaMorph desktop and can only be
accessed through the Journal Editor.
Creates a background correction image for a source image or stack. The background
image is generated from the source through a series of user-specified operations. This
function helps to remove noise and unwanted fluorescence from large objects
(background) in an image.
Dropin: BACKCORR
Background Correction is similar to the Median Filter in that it works by selecting the median pixel value
of the pixels in an NxM pixel area and replacing the center pixel with this value. This process is repeated
for every pixel in the image unless the user specifies a sub-set ratio. The resulting background image is
then subtracted from the source image to produce a new image with less background noise and
unwanted fluorescence. While the Median Filter uses only the true median pixel at the 50% mark, the
background correction process may be set to use an arbitrary percentage by the user.
The filter works to 12-bit accuracy. If the original image is a true 16 bits deep, the least significant 4 bits
will be ignored. The function lets the user specify that the operation be performed on a lower bit range to
accommodate cameras that use only 12 or 14 bits.
Note: This function operates exclusively on the full image, ignoring any active region on
the source image.
The following images show the effects of producing a background image and subtracting it from a source
image, using a kernel size of 3 x 3:
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Original Image
Correction Image
Image after
Subtraction
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Producing a Background Correction Image
To create a background correction image:
Step
Action
1
From the Process menu, choose Produce
Background Correction Image. The Background
Correction dialog box will appear.
2
Select the desired source image or stack using
the Source image selector.
3
Select the desired destination image using the
Dest image selector. You can overwrite or add to
the existing image or you can place the results in
a new image window.
4
Use Size to select the size of the convolution
kernel.
5
If you want to speed up processing by using a
sub-sample, select the desired value using SubSample Ratio.
6
Select the pixel intensity (p%) to be used in
creating the background. A percentage of 100
will produce a background of maximum intensity,
while a percentage of 0 will produce a
background based on minimum intensity.
7
Under True Depth of 16 it Image select which bits
of the image will be used to determine the median
(or p%) pixel.
8
Click apply to create the background image.
Note: When performing background
correction on a large stack, the
progress of the operation is
indicated in the MetaMorph status
bar. You can cancel the operation by
pressing the <Esc> key.
9
From the Process menu, choose Arithmetic. The
arithmetic dialog box will appear.
10
Select your source image as Source 1 and the
background image as Source 2, then select a
destination for the new image.
11
Select the subtract operation and click apply.
12
Click close to close the dialog box.
Produce Background Correction Image - Dialog Box Options
Source
Selects the image from which the background will be produced.
Destination
Selects a destination for the resulting background image.
Size (pixels n x n)
Specifies the size (in pixels) of the regions (kernels) used to determine each median (or p%) pixel.
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Sub-sample Ratio
Speeds up the processing of large kernels by limiting the calculations to a sub-sample, rather than every
pixel. For example, if 2 is selected as the sub-sample, the pixels in columns/rows 1, 3, 5, 7, etc. will be used
(1/4th the pixels). If 3 is selected, the pixels in columns/rows 1, 4, 7, 10, etc. will be used (1/9th). Likewise, if
8 is selected, the pixels in columns/rows 1, 9, 17, etc. will be used (1/64th). The maximum sub-sample value
is 16 (samples 1 in 256 pixels).
%(50 = median)
Determines which pixel's intensity value will be used in each kernel to create the background. A value of 50
indicates the median pixel will be used. Other values indicate that the pixel at the given percentage in the list
will be used.
True depth of 16-bit image:
Lets you select which bits of the image will be used for determining the median (or p%) pixel. The
calculations are only done to 12-bit precision, so it is preferable to perform the calculations on the most
significant bits in the image. Since some cameras use only the lowest 12 or 14 bits for the data, which can
not be determined by MetaMorph, the user can direct the procedure to operate on the proper set of 12 bits.
Apply
Performs the operations used to produce the background image.
Close
Closes the dialog box.
Log Image Annotation (Log Menu)
Saves an annotation, along with the data from the current image or stack plane, to a data
log.
Drop-in: LOGAN
Use this command to save image data with an annotation to a data log for the current plane of a selected
image. You must first create the annotation with the Annotate Image command from the File menu. The
Log Image Annotation command will allow you to open and configure the data log file. This command is
fully journalizable, and can be particularly useful when used in combination with the Loop for All Planes
command from the Journal menu.
For More Information about Logging Images:
Annotate Image
View Current Data Log
Close Data Log
Loop for All Planes
Logging Image Annotations
To save image annotations to a data log, use the following procedure:
Step
Action
1
If you have not yet created an annotation for
the image plane being measured, create one
with the Annotate Image command from the
File menu.
2
From the Log menu, choose Log Image
Annotation. The Log Image Annotation dialog
box will appear.
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3
Use the Image selector to select the image
whose data and annotation are to be logged.
4
If necessary, choose Configure Log. The
Configure Log dialog box will appear.
AND
From the Configuration list, select the items
that you want to be logged. Then choose OK.
The Log Image Annotation dialog box will
reappear.
5
Choose Open Log and open a text-based
data log, a DDE link to an open spreadsheet,
or both. The button's title will change to "F9:
Log Data."
6
Choose F9: Log Data to log your data.
7
Choose Close.
Log Image Annotation - Dialog Box Options
Image
Selects the image whose data and image annotation are to be logged.
Data Log
Indicates the file name of the data log to which the annotation and data are to be logged. If no log file is
open, the line will read "Data log not open." After logging the data and annotation, the line will read "Logged
to Filename.xxx."
Open Log
Opens a data log file and/or a DDE link to an open worksheet for logging data. This command changes to
F9: Log Data once a log file is open.
F9: Log Data
Sends data measurements to an open data log.
Configure Log
Allows the selection of image characteristics and data that are to be enabled or disabled from data logging.
Also allows a choice of whether column titles are to be included and if data are to be listed on a single line.
Close
Closes the dialog box.
Log Image Histogram (Log Menu)
Stores grayscale or color value data from the Histogram Tool's histogram in a data log.
Drop-in: LOGHISTO
Use this command to save grayscale or color value histogram data from an entire image. The number of
pixels in the image at each grayscale level or RGB color value will be stored. You have the option of
defining the range of grayscale or color value bins on the basis of the minimum and maximum values in
the image (Image Min/Max), an 8-bit range (0 - 255 for the grayscale values or for each of the color
channels), a 10-bit range (0 - 1023), a 12-bit range (0 - 4095), a 14-bit range (0 - 16383), or a 16-bit
range (0 - 65535). If you select one of the bit-defined ranges, you can fine-tune the minimum and
maximum bin values using the Start and End spin boxes, respectively.
Logging Image Histogram Data
To save grayscale or color value data, on a bin-by-bin basis, to a data log, use the following
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procedure:
Step
Action
1
From the Log menu, choose Log Image
Histogram. The Log Image Histogram dialog
box will appear.
2
With the Image selector, select the image
from which you want to save grayscale or
color value bin data.
3
From the Range drop-down list, select the
range of grayscale or color bin values for
which you want to log data. Select
Image Min/Max if you want to restrict the
range to those values actually present in the
image,
8 Bit or RGB if you want to use a range of 0 255 (for 8-bit images or for each color
channel in a 24-bit image),
10 Bit if you want to use a range of 0 - 1023,
12 Bit if you want to use a range of 0 - 4095,
14 Bit if you want to use a range of 0 16383, or
16 Bit if you want to use a range of 0 65535.
4
If you selected one of the bit-defined ranges
in Step 3, you can fine-tune the range further
by selecting a minimum and maximum bin
value with the Start and End spin boxes,
respectively.
5
If you want to select the parameters to be
logged to the log file, choose Configure Log.
The Configure Log dialog box will appear.
AND
From the Configuration table, click the
entries for the parameters to be logged, so
that a check mark appears next to them.
Then choose OK to return to the Log Image
Histogram dialog box.
6
Open a data log by clicking the Open Log
command button.
Once the data log is open, the text on the
Open Log button will change to F9: Log
Data.
7
When you are ready to save the grayscale or
color value data, choose F9: Log Data, or
press the [F9] function key on your keyboard.
The data will be sent to the open data log.
If you sent your data to a text-based data log,
you can inspect the data immediately by
choosing View Current Data Log from the
Log menu.
8
When you have finished, choose Close.
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Log Image Histogram - Dialog Box Options
Image
Selects the image from which you want to store grayscale or color value pixel count (binned) data.
Range
Select the range of grayscale or color bin values for which you want to log data:
Image Min/Max restricts the range to those values actually present in the image.
8 Bit or RGB uses a range of 0 - 255 (for 8-bit images or for each color channel in a 24-bit image).
10 Bit uses a range of 0 - 1023.
12 Bit uses a range of 0 - 4095.
14 Bit uses a range of 0 - 16383.
16 Bit uses a range of 0 - 65535.
Start
Selects the minimum (starting) value for the range of bins to be logged. This option becomes available when
you select one of the bit-defined ranges from the Range drop-down list.
End
Selects the maximum (ending) value for the range of bins to be logged. This option becomes available when
you select one of the bit-defined ranges from the Range drop-down list.
Configure Log
Opens the Configure Log dialog box, from which you can select the parameters to be logged to the data log
file.
Open Log
Opens a data log and/or a DDE link to an open spreadsheet application for logging data. This command will
change to F9: Log Data when a log file is open.
F9: Log Data
Sends the binned data to the open data log.
Close
Closes the dialog box
Open Object Log (Log Menu)
Opens an existing or new object log for storing morphometric measurement data.
Drop-in: AUTOMEAS or IMA
Use this command to open an object log for logging morphometric measurement data. Measure Objects,
Measure Objects with Mask, Recalculate Object Parameters, Measure Single Object, Integrated
Morphometry Analysis, and Show Individual Object Data are commands whose measurements are
logged to an object log. You can log the data to a text file, by Dynamic Data Exchange (DDE) to an open
worksheet in a spreadsheet program, or to both.
For MetaMorph to log measurement data, it must know where you want the data stored, that is, which
text file or open, DDE-linked spreadsheet to use. This information is supplied by the Open Object Log
command. What is logged will be based on the types of measurement data selected using the Configure
Object Measurements command. Which object measurements are logged will be based on the filters you
defined using the Configure Object Classifiers command, excluding or including objects based on
selected parametric criteria. For commands which log individual object data, even if a log file is open and
configured, nothing will be logged until you choose Log Data from the Log menu. This command allows
you to log measurement data selectively when you need it. For morphometric measurement of many
objects, logging occurs when the measurement is performed.
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You can view the logged data by
(1) Opening the current text-based object log using View Current Object Log,
(2) Opening a comma-delimited text file in a text editor, or
(3) Switching to an external DDE-linked spreadsheet application.
Opening an Object Log File
To open an object log, use the following procedure. If you want to log data to an external
spreadsheet application, you must first start the spreadsheet program and open the desired
worksheet.
Step
Action
1
From the Log menu, choose Open Object
Log. The Open Object Log dialog box will
appear.
2
Select Dynamic Data Exchange (DDE) to log
directly to an open spreadsheet program.
Select A Text File to log the data to a text
file.
Note: You can select both options.
3
If you selected A Text File in the previous
step, the Open Object Log File dialog box will
appear.
Select an icon for an existing log file or type
a new file name in the File Name text box. (If
necessary, use the Look In list or Up One
Level icon button to change the current drive
and folder to the correct location.) Then
choose Open.
4
If you selected an existing log's file name in
Step 3, the Log File Exists dialog box will
appear. You can Overwrite the contents of
the file, Append new data, or Cancel.
5
If you selected Dynamic Data Exchange
(DDE) in Step 2, the Export Log Data dialog
box will appear.
Select the desired application from the
Application list. Choose Default to use the
default settings for the selected application.
Choose OK to open the DDE link.
Additional Information About DDE:
Using a New Microsoft Excel Worksheet
Using a Microsoft Excel Worksheet Other Than the Default
Creating a DDE Link to Lotus 1-2-3, Borland Quattro Pro, or MicroCal Origin
Linking to Another Application
Open Object Log - Dialog Box Options
Open Object Log File
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Export Log Data
Open Object Log File - Dialog Box Options
File Name
Lists the name of selected file.
Files of Type
Determines the file format of the files displayed in the File Name list. For opening log files, the default is
*.LOG. Select All Files (*.*) to display all file names.
Save In
Displays the currently selected folder. Click the icon for the desired folder to display its files. Click the Up
One Level icon button to go up one level in the directory structure.
Save
Opens the log file.
Cancel
Cancels the command.
Close Object Log (Log Menu)
Closes the current object log (text file) or the dynamic data exchange (DDE) link to a
running spreadsheet program.
Drop-in: AUTOMEAS or IMA
Use this command to close the current object log when you have finished logging data or before you
open a new object log. All log files will be closed automatically by MetaMorph upon exiting.
Note: This command closes the DDE link to the spreadsheet program--it does not close
or save worksheet files. You must switch to the spreadsheet program to perform these
tasks.
Closing an Object Log File
To close an object log, use the following procedure:
Step
Action
1
Select the Log menu.
2
Choose Close Object Log.
Pause Object Logging (Log Menu)
Pauses logging of data to the current object log.
Drop-in: AUTOMEAS or IMA
Use this command before you make measurements that you do not want to log. Use the Resume Object
Logging command when you want to continue logging data to the current log file.
Pausing Object Data Logging
To pause object data logging, use the following procedure:
Step
1
Action
Select the Log menu.
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Choose Pause Object Logging.
Resume Object Logging (Log Menu)
Resumes logging of data to the current object log.
Drop-in: AUTOMEAS or IMA
Use this command when you want to continue logging data to an object log after using the Pause Object
Logging command.
Resuming Object Data Logging
To resume object data logging, use the following procedure:
Step
Action
1
Select the Log menu.
2
Choose Resume Object Logging.
View Current Object Log (Log Menu)
Displays the contents of the current object log (text file) in table format using the Viewer
window.
Drop-in: AUTOMEAS or IMA
Use this command to view the current object log within MetaMorph. If the current object log contains no
data, a message dialog box will appear, stating "The file Filename.log could not be viewed because it
has no columns or rows." Empty log files can occur if you open an existing log file and then close it
without actually logging any data or if you log to a new log file with all of the logging parameters disabled.
The Viewer window has adjustable column widths so that you can resize the columns to best fit on your
screen. (Drag the vertical cell borders between the column labels.) The maximum width of a column is
about 1/4 the width of the screen. As with any other window, you can also adjust the Viewer window's
size by dragging its borders.
If you want to print a table in the Viewer, there is a Print Table command in the Viewer's Control Menu
(click the icon in the window's upper left corner). This command will print the text that is visible within the
each column's width. Thus, you should adjust the columns before printing. The size of the window and
the position of the scroll bars will have no effect on what is printed; if the data can fit in the column width,
it will be printed.
You can also view a log file by using a spreadsheet program or a text editor, such as the Windows
WordPad program.
Note: If you are currently logging to an open, DDE-linked spreadsheet application, you will need to
switch to that application to view the data. View Current Object Log displays text files only.
Viewing the Current Object Log
To view the current object log, use the following procedure:
Step
Action
1
From the Log menu, choose View Current
Object Log. The Viewer window will open.
2
The Viewer window can be resized by
dragging its borders, as with other windows.
If the labels or data are not entirely visible,
you can adjust the width of the columns in
the table by dragging the vertical cell border
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between the labels until the columns are the
desired width. The maximum width of a
column is 1/4 of the width of the screen.
3
To close the Viewer window, click the Close
button in its upper right corner.
Log All Object Data (Log Menu)
Logs all object data from an image after it has been measured.
Drop-in: AUTOMEAS
Use this command to log object data from a measured image if an object log was not open at the time
you measured the image. If, after you have measured the image, you decide that you want to log its
object data, you can open the log file and choose Log All Object Data without measuring the image
again. This command does not apply to measurements made by single measurement commands.
Logging All Object Data
To log all object data, use the following procedure:
Step
Action
1
Measure the desired image using the
Measure Objects, Measure Single Object, or
Measure Objects with Mask command.
2
Open the object log file using the Open
Object Log command in the Log menu.
3
From the Log menu, choose Log All Object
Data.
4
If the data have already been logged, the
message, "You have already logged this
measurement set. Do you really want to write
another set to this log?" will appear.
Choose Yes if you want to log the
measurement set again, or choose No to
cancel the logging.
Open Summary Log (Log Menu)
Opens an existing or new summary log for storing summaries of morphometry statistics.
Drop-in: AUTOMEAS or IMA
Use this command to open a summary log for logging summary data of morphometric measurements
collected with the Show Classifier Statistics command. You can log the data to a text file, by Dynamic
Data Exchange (DDE) to an open worksheet in a spreadsheet program, or to both.
For MetaMorph to log measurement data, it must know where you want the data stored, that is, which
text file or open, DDE-linked spreadsheet to use. This information is supplied by the Open Summary Log
command. What is logged will be based on the types of measurement data selected using the Configure
Object Measurements command. Which object measurements are logged will be based on the filters you
defined using the Configure Object Classifiers which exclude or include objects based on selected
parametric criteria. Logging occurs when the measurement is performed.
You can view the logged data by
(1) Opening the current text-based summary log using View Current Summary Log,
(2) Opening a comma-delimited text file in a text editor, or
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(3) Switching to an external DDE-linked spreadsheet application.
Opening a Summary Log File
To open a summary log, use the following procedure. If you want to log data to an external
spreadsheet application, you must first start the spreadsheet program and open the desired
worksheet.
Step
Action
1
From the Log menu, choose Open Summary
Log. The Open Summary Log dialog box will
appear.
2
Select Dynamic Data Exchange (DDE) to log
directly to an open spreadsheet. Select A
Text File to log the data into a text file.
Note: You can select both options.
3
If you selected A Text File in the previous
step, the Open Summary Log File dialog box
will appear.
Select an icon for an existing log or type a
new file name in the File Name text box. (If
necessary, use the Look In list or Up One
Level icon button to change the current drive
and folder location to the correct location.)
Then choose Open.
4
If you selected an existing log file name in
Step 3, the Log File Exists dialog box will
appear. You can Overwrite the contents of
the file, Append new data, or Cancel.
5
If you selected Dynamic Data Exchange
(DDE) in Step 2, the Export Log Data dialog
box will appear.
Select the desired application from the
Application list. Choose Default to use the
default settings for the selected application.
Choose OK to open the DDE link.
Additional Information about DDE:
Using a New Microsoft Excel Worksheet
Using a Microsoft Excel Worksheet Other Than the Default
Creating a DDE Link to Lotus 1-2-3, Borland Quattro Pro, or MicroCal Origin
Linking to Another Application
Open Summary Log - Dialog Help
Open Summary Log File
Export Log Data
Open Summary Log File - Dialog Box Options
File Name
Lists the name of the currently selected file.
Files of Type
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Determines the file format of the files displayed. For opening log files, the default is *.LOG. Select All Files
(*.*) to display all file names.
Save In
Displays the currently selected folder. Click the icon for the desired folder to display its files. Click the Up
One Level icon button to go up one level in the directory structure.
Save
Opens the log file.
Cancel
Cancels the command.
Close Summary Log (Log Menu)
Closes the current summary log (text file), or closes the dynamic data exchange (DDE) link
to a running spreadsheet program.
Drop-in: AUTOMEAS or IMA
Use this command to close the current summary log when you have finished logging data or before you
open a new summary log. All log files will be closed automatically by MetaMorph upon exiting.
Note: This command closes the DDE link to the spreadsheet program--it does not close or save
worksheet files. You must switch to the spreadsheet program to perform these tasks.
Closing a Summary Log File
To close a summary log, use the following procedure:
Step
Action
1
Select the Log menu.
2
Choose Close Summary Log.
Pause Summary Logging (Log Menu)
Pauses logging of summary data to the current summary log.
Drop-in: AUTOMEAS or IMA
Use this command before you make measurements that you do not want to log. Use the Resume
Summary Logging command when you want to continue logging data to the current log file.
Pausing Summary Data Logging
To pause summary data logging, use the following procedure:
Step
Action
1
Select the Log menu.
2
Choose Pause Summary Logging.
Resume Summary Logging (Log Menu)
Resumes logging of summary data to the current summary log.
Drop-in: AUTOMEAS or IMA
Use this command when you want to continue logging data to a summary log after using the Pause
Summary Logging command.
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Resuming Summary Data Logging
To resume summary data logging, use the following procedure:
Step
Action
1
Select the Log menu.
2
Choose Resume Summary Logging.
View Current Summary Log (Log Menu)
Displays the contents of a current summary log (text file) in table format using the Viewer
window.
Drop-in: AUTOMEAS or IMA
Use this command to view the current summary log within MetaMorph. If the current summary log
contains no data, a message dialog box will appear, stating "The file Filename.log could not be viewed
because it has no columns or rows." Empty log files can occur if you open an existing log file and then
close it without actually logging any data or if you log to a new log file with all of the logging parameters
disabled.
The Viewer window has adjustable column widths so that you can resize the columns to best fit on your
screen. (Drag the vertical cell borders between the column labels.) The maximum width of a column is
about 1/4 the width of the screen. As with any other window, you can also adjust the Viewer window's
size using its borders.
If you want to print a table in the Viewer, there is a Print Table command in the Viewer's Control Menu
(click the icon in the window's upper left corner). This command will print the text that is visible within the
each column's width. Thus, you should adjust the columns before printing. The size of the window and
the position of the scroll bars will have no effect on what is printed; if the data can fit in the column width,
it will be printed.
You can also view a log file by using a spreadsheet program or a text editor, such as the WordPad
program.
Note: If you are currently logging to a DDE-linked spreadsheet application, you will need to switch to that
application to view the data. View Current Summary Log displays text files only.
Viewing the Current Summary Log
To view the current summary log, use the following procedure:
Step
Action
1
From the Log menu, choose View Current
Summary Log. The Viewer window will open.
2
The Viewer window can be resized by
dragging its borders, as with other windows.
If the labels or data are not entirely visible,
you can adjust the width of the columns in
the table by dragging the vertical cell border
between the labels until the columns are the
desired width. The maximum width of a
column is 1/4 of the width of the screen.
3
To close the Viewer window, click the Close
button in its upper right corner.
Open EdgeList Log (Log Menu)
Opens an existing or new edgelist log for storing each object's centroid and vertex X,Y-
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coordinate data in an image.
Drop-in: AUTOMEAS or IMA
Use this command to open an edgelist log for logging edgelist data. Data will logged to an open edgelist
log when any of the following measurement commands are used: Measure Objects, Recalculate Object
Parameters, and Log All Object Data. Measure Single Object, and Show Individual Object Data. You can
log the data to a text file, by Dynamic Data Exchange (DDE) to an open worksheet in a spreadsheet
program, or to both.
For MetaMorph to log measurement data, it must know where you want the data stored, that is, which
text file or open, DDE-linked spreadsheet to use. This information is supplied by the Open EdgeList Log
command. Which object measurements are logged will be based on the filters you defined using the
Configure Object Classifiers which exclude or include objects based on selected parametric criteria.
Unlike other log files, the edgelist log is always configured to log the same measurement data (object
number, number of vertices, centroid X, centroid Y, and then on following lines, the vertex X and Y
coordinates, starting with the pixel at the top, leftmost point and moving clockwise around the object).
Data in an edgelist log are always reported in pixel values, even if the image has been calibrated.
There are two options in the Preferences dialog box (Measure Objects tab page) that will help you when
you are working with edgelist logs. By selecting both Draw Object Border and Draw Centroid Mark, you
will see a visual representation on the measured image of the centroid and object borders being logged.
You can view the logged data by
(1) Opening the current text edgelist log using View Current EdgeList Log,
(2) Opening a comma-delimited text file in a text editor, or
(3) Switching to an external DDE-linked application.
Opening an EdgeList Log File
To open an edgelist log, use the following procedure. If you want to log data to an external
spreadsheet application, you must first start the spreadsheet program and open the desired
worksheet.
Step
Action
1
From the Log menu, choose Open EdgeList
Log. The Open EdgeList Log dialog box will
appear.
2
Select Dynamic Data Exchange (DDE) to log
directly to an open spreadsheet. Select A
Text File to log the data into a text file.
Note: You can select both options.
3
If you selected A Text File in the previous
step, the Open EdgeList Log File dialog box
will appear.
Select an icon for an existing log or type a
new file name in the File Name text box. (If
necessary, use the Look In list or Up One
Level icon button to change the current drive
and folder to the correct location.) Then
choose Open.
4
If you selected an existing log file name in
Step 3, the Log File Exists dialog box will
appear. You can Overwrite the contents of
the file, Append new data, or Cancel.
5
If you selected Dynamic Data Exchange
(DDE) in Step 2, the Export Log Data dialog
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box will appear.
Select the desired application from the
Application list. Choose Default to use the
default settings for the selected application.
Choose OK to open the DDE link.
Additional Information about DDE:
Using a New Microsoft Excel Worksheet
Using a Microsoft Excel Worksheet Other Than the Default
Creating a DDE Link to Lotus 1-2-3, Borland Quattro Pro, or MicroCal Origin
Linking to Another Application
Open EdgeList Log - Dialog Box Options
Open EdgeList Log File
Export Log Data
Open EdgeList Log File - Dialog Box Options
File Name
Lists the name of the selected file.
Files of Type
Determines the file format of the files displayed in the File Name list. For opening log files, the default is
*.LOG. Select All Files (*.*) to display all file names.
Save In
Displays the currently selected folder. Click the icon for the desired folder to display its files. Click the Up
One Level icon button to go up one level in the directory structure.
Save
Opens the log file.
Cancel
Cancels the command.
Close EdgeList Log (Log Menu)
Closes the current edgelist log text file, or closes the dynamic data exchange (DDE) link to
a running spreadsheet program.
Drop-in: AUTOMEAS or IMA
Use this command to close the current edgelist log when you have finished logging data or before you
open a new edgelist log. All log files will be closed automatically by MetaMorph upon exiting.
Note: This command closes the DDE link to the spreadsheet program--it does not close or save
worksheet files. You must switch to the spreadsheet program to perform these tasks.
Closing an EdgeList Log File
To close an edgelist log, use the following procedure:
Step
Action
1
Select the Log menu.
2
Choose Close EdgeList Log.
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Pause EdgeList Logging (Log Menu)
Pauses logging of data to the current edgelist log.
Use this command before you make measurements that you do not want to log. Use the Resume
EdgeList Logging command when you want to continue logging data to the current log file.
Pausing EdgeList Data Logging
To pause edgelist data logging, use the following procedure:
Step
Action
1
Select the Log menu.
2
Choose Pause EdgeList Logging.
Resume EdgeList Logging (Log Menu)
Resumes logging of edgelist data to the current edgelist log.
Drop-in: AUTOMEAS or IMA
Use this command when you want to continue logging data to an edgelist log after using the Pause
EdgeList Logging command.
Resuming EdgeList Data Logging
To resume edgelist data logging, use the following procedure:
Step
Action
1
Select the Log menu.
2
Choose Resume EdgeList Logging.
View Current EdgeList Log (Log Menu)
Displays the contents of the current edgelist log (text file) in table format with the Viewer
window.
Drop-in: AUTOMEAS or IMA
Use this command to view the current edgelist log within MetaMorph. If the current edgelist log contains
no data, a message dialog box will appear, stating "The file Filename.log could not be viewed because it
has no columns or rows." Empty log files can occur if you open an existing log file and then close it
without actually logging any data or if you log to a new log file with all of the logging parameters disabled.
The Viewer window has adjustable column widths so that you can resize the columns to best fit on your
screen. (Drag the vertical cell borders between the column labels.) The maximum width of a column is
about 1/4 the width of the screen. As with any other window, you can also adjust the Viewer window's
size using its borders.
If you want to print a table in the Viewer, there is a Print Table command in the Viewer's Control Menu
(click the icon in the window's upper left corner). This command will print the text that is visible within the
each column's width. Thus, you should adjust the columns before printing. The size of the window and
the position of the scroll bars will have no effect on what is printed; if the data can fit in the column width,
it will be printed.
You can also view a log file by using a spreadsheet program or a text editor, such as the WordPad
program.
Note: If you are currently logging to a DDE-linked spreadsheet application, you will need to switch to that
application to view the data. View Current EdgeList Log displays text files only.
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Viewing the Current EdgeList Log
To view the current edgelist log, use the following procedure:
Step
Action
1
From the Log menu, choose View Current
EdgeList Log. The Viewer window will open.
2
The Viewer window can be resized by
dragging its borders, as with other windows.
If the labels or data are not entirely visible,
you can adjust the width of the columns in
the table by dragging the vertical cell border
between the labels until the columns are the
desired width. The maximum width of a
column is 1/4 of the width of the screen.
3
To close the Viewer, click the Close button in
its upper right corner.
Display EdgeList Log as Image (Log Menu)
Creates a binary stack of the edgelists and an 8-bit pseudocolored image of the centroids
stored in the selected edgelist log.
Drop-in: EDGELIST
Use this command to create a binary stack of the edgelist data stored in an edgelist log file so that you
can view the data in a graphical representation. This command is useful for timelapses of single objects.
The binary stack has a default size of 512 x 512 pixels (which you can alter), and consists of one
edgelist per plane.
The 8-bit centroid image marks the center of mass for each object. Each centroid in this image is
assigned a gray value between 1 and 255 to represent its object number. If there are more than 255
centroids, the gray value will loop back to 1 and continue incrementing. If you have trouble viewing this
image, you may want to Binarize the image.
You may use this command to load arbitrary data into a MetaMorph image window, provided that the
data uses the format of an edgelist log file. This may be of particular use in photon-counting experiments
conducted over time. The edgelist log file format is
Object number, number of points, centroid X, centroid Y
X1, Y1
(X and Y vertices in pixels of the edgelist points)
....
Xn, Yn
Note: The edgelist log must be closed before using this command.
If you want to view the edgelists for all of the objects in a single image, you may perform a Stack
Arithmetic operation (select Maximum) on the stack of edgelists to create one image.
Displaying an EdgeList Log File as an Image
To display an edgelist file as an image, use the following procedure:
Step
1
Action
If the edgelist log is open, close it before
using the Display EdgeList Log as Image
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command.
2
From the Log menu, choose Display
EdgeList Log as Image. The Display
EdgeList Log as Image dialog box will
appear.
3
Choose Set Filename and select the icon for
the desired edgelist log file from the Select
Log File dialog box that appears. (If
necessary, use the Look In list or Up One
Level icon button to locate the appropriate
folder.) Then choose Open.
4
From the Draw EdgeList Points As group,
select Vectors to display the edgelists as
connected outlines.
OR
If you are working with data that has been
converted from a photon-counting
experiment, select Points to display the
edgelists as single pixels.
5
Image Width and Image Height each display
the default value of 512 pixels as the
dimensions used for the destination images.
You will only need to change these default
values if the data in the edgelist log cannot
be displayed within that range.
If the data from the edgelist log cannot be
displayed using the specified dimensions, an
error message will appear. This error
message will suggest the minimum values
needed to display the edgelist log data.
6
Choose OK. MetaMorph will create the
binary stack of the edgelists and the 8-bit
image of the centroids stored in the selected
edgelist log.
Display EdgeList File as Image - Dialog Box Options
Draw EdgeList Points As
Selects between Vectors (the default), which displays the edgelists as connected dots, and Points, which
displays the edgelists as single pixels. Generally, there will be no difference in the displays for most edgelist
data. However, if you are using a text file of converted "photon-counting" data as your source file, you
should select Points.
Image Width
Specifies the width of the edgelist and centroid image windows. This value must be large enough to
accommodate all of the data in the edgelist log. If the data from the edgelist log cannot be displayed using
the specified dimensions, an error message will appear. This error message will suggest the minimum
values needed to display the edgelist log data. The default value is 512.
Image Height
Specifies the height of the edgelist and centroid image windows. This value must be large enough to
accommodate all of the data in the edgelist log. If the data from the edgelist log cannot be displayed using
the specified dimensions, an error message will appear. This error message will suggest the minimum
values needed to display the edgelist log data. The default value is 512.
Filename
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This status line lists the currently loaded edgelist log.
Set Filename
Opens the Select Log File dialog box, from which you may select the edgelist log that you want to use.
OK
Creates the binary edgelist stack and the 8-bit centroid image.
Cancel
Cancels the command.
Configure Object Classifiers (Measure Menu)
Configures measurement filters for measuring objects by defining value ranges that either
include or exclude certain objects.
Drop-in: AUTOMEAS
Use the Configure Object Classifiers command when you want to measure certain objects in the image
while excluding other classes of objects from measurements.
You can measure all of the objects in the image first by using the Measure Objects command and then
using the Show Individual Object Data command to determine measurement parameters and values that
you want to use to define the filters in the Configure Object Classifiers dialog box. After you define the
filters, you can then remeasure the objects. If you know how you want to define the filters, you can
configure them in the Configure Object Classifiers dialog box first and then measure the objects. You
can save and load classifier sets for future measurements.
Measurement Term Definitions
Statistical Term Definitions
Configuring Object Classifiers
Overview of Configuring Object Classifiers
Configuring Object Classifiers
Creating New Object Classifier Sets
Loading and Saving Object Classifier Sets
Overview of Configuring Object Classifiers
The Configure Object Classifiers command has a complicated dialog box. To help you use it in
the most efficient manner, a basic overview of the procedure is presented in this table while
separate steps are explained in separate procedure tables.
Step
Action
1
From the Measure menu, choose Configure
Object Classifiers. The Configure Object
Classifiers dialog box will appear.
2
When you first start MetaMorph, the Default
classifier set current listed in the Classifiers
list will be the only available set.
You can create a new classifier set using the
New Classifier command (refer to Creating
New Object Classifier Sets), or load a
previously saved classifier set using the Load
Set command (refer to Loading and Saving
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Object Classifier Sets).
3
Configure each classifier set as necessary
for measurement (refer to Configuring
Object Classifiers).
4
Select the Active option above the Classifier
list for each classifier set that you want to
use for measurements and logging.
5
Measure the objects using the desired
measurement command from the Measure
menu.
6
If necessary, use the Classifier Statistics
dialog box to view the classifier statistics
generated from the active classifier set(s)
and/or log the classifier statistics generated
from the active classifier sets.
7
If necessary, you can reconfigure existing
active classifier sets or select new sets as
the active classifier sets.
Choose Recalc to update the measurements
whenever you want to use a revised or new
classifier set. Recalc will update the
measurement data. (The Recalc button
replaces the Measure button once an image
has been measured.)
8
Choose Close when you have finished.
Configuring Object Classifiers
To configure object classifiers for the image you want to measure, use the following procedure
(refer to Overview of Configuring Object Classifiers for an overview of this command).
Step
Action
1
Select the classifier set you want to configure
so that it is visible at the top of the Classifiers
list in the Configure Object Classifiers dialog
box.
2
Select the desired filters for this set from the
Filters list so that each is marked by a check
mark. Deselect any previously selected filters
that are unnecessary.
Note: You can select Show Descriptive Text
to display a description of each filter. You
can also select Active from the Show Filters
group if you want only the selected filters
displayed in the Filters list.
3
Select Inclusive from the Filter Values group
if you want to include only those objects
whose measurements fall inside the filter
range when recording measurement data.
OR
Select Exclusive to exclude all objects whose
measurements fall inside the filter range
when recording measurement data.
4
Type the desired filter range values in the
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Filter Range text boxes (the text boxes allow
you to set the range by completing the
equation shown in the dialog box).
5
If you want to change the default color set
(Random) to another color for the selected
classifier set, select a new color set from the
drop-down color list located next the Reset
command. This color set will be used to paint
the measured objects.
6
Repeat Steps 1 - 5 for each classifier set that
you want to configure.
Creating New Object Classifier Sets
To create a new classifier set, use the following procedure (refer to Overview of Configuring
Object Classifiers for an overview of this command).
Note: Before creating a new classifier set using the Point to Object option, you must
threshold and measure the image with the Measure Objects command. You should also
confirm that you have selected a filter from the Filter list to use for the initial values and
that this filter is also selected in the Preferences dialog box by using the Preferences
command button in the Configure Object Classifiers dialog box.
Step
Action
1
From the Configure Object Classifiers dialog
box, choose New Classifier. The New
Classifier dialog box will appear.
2
Type a name for the new classifier in the
Classifier Name text box.
3
Select the desired option from the Initial Filter
Values group:
Duplicate Current Classifier uses the current
classifier values.
Use Default Filter Values uses MetaMorph's
stored values.
Point to Object uses the selected object
within the image window.
4
If you selected Point to Object, position the
pointer over the desired object and click the
left mouse button. (Point to Object finds the
nearest centroid when you select the object.)
The New Classifier dialog box will display the
selected object number.
5
Choose OK. MetaMorph will create the new
classifier and close the New Classifier dialog
box.
Loading and Saving Object Classifier Sets
To load a previously saved object classifier set, use the following procedure (refer to Overview of
Configuring Object Classifiers for an overview of this command).
Step
1
Action
From the Configure Object Classifiers dialog
box, select Load Set File. The Load
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Classifier Set dialog box will appear.
2
Select the icon for the desired file. If
necessary, select the correct folder or drive
using the Look In list or Up One Level button.
3
Choose Open. MetaMorph will load the
classifier set file, adding its name to the
bottom of the Classifier list. MetaMorph will
also close the Load Classifier Set dialog box.
4
Open the Classifier list to select the newly
added classifier for configuring.
To save a new object classifier set, use the following procedure:
Step
Action
1
Select the desired object classifier set, if it is
not currently displayed at the top of the
Classifiers list.
2
From the Configure Object Classifiers dialog
box, select Save Set File. The Save
Classifier Set As dialog box will appear.
3
Type the desired file name in the File Name
text box. (MetaMorph will assign the
appropriate file name extension.) If
necessary, select the correct folder or drive
using the Save In list or Up One Level
button.
4
Choose Save. MetaMorph will save the
classifier set and close the Save Classifier
Set As dialog box.
Configure Object Classifiers - Dialog Box Options
Classifier Set File
Indicates the currently loaded classifier filter set file, if any.
Classifiers
Selects the current classifier set. The text box below the drop-down list can be used to edit the name of the
currently selected classifier (except Default).
Active
Enables or disables the selected classifier set for use during measurements. Sets which are noted as
"(active)" in the Classifiers list will be used for measurements.
New Classifier
Opens the New Classifier dialog box, which is used to create new classifier sets. You can create a new
classifier set by duplicating the current classifier by using default filter values or by using the pointer to
"point" to a measured object that has the desired classifier characteristics. The last option is not available
until you have thresholded the image and then measured it using the Measure Objects command.
Remove Classifier
Removes the current classifier from the Classifiers list.
Load Set File
Loads a previously saved classifier set (*.cls) file.
Save Set File
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Saves the currently selected classifier set as a .cls file.
Reset
Resets the current classifier set to the "factory" settings.
Color Drop-Down List Box ("Random")
Selects the color used for painting the items that pass the selected classifier set. The default is Random.
Filters
Selects the filters that make up each classifiers set. The Filter Values option is used in conjunction with the
Filter Range to select the limiting conditions for screening objects through each filter.
Show Descriptive Text
Displays a description of each filter at the bottom of the dialog box.
Show Filters
Displays all filters in the Filters list if the All option is displayed. Otherwise only the active filters are
displayed.
Filter Values
If Inclusive is selected, this option includes only those objects whose measurements fall inside the filter
range when recording measurement data. If Exclusive is selected, this option excludes all objects whose
measurements fall inside the filter range when recording measurement data.
Filter Range
Specifies the filter range of acceptable values used to determine if an object passes the filter.
Measure/Recalc
Measures or recalculates the objects in the current image which pass the selected object classifiers. Recalc
is faster than Measure because the objects do not have to be traced. The Recalc command is the same as
Recalculate Object Parameters in the Measure menu.
Preferences
Opens the Preferences dialog and displays the New Classifier tab page. The New Classifier preferences
allow you to select which classifier parameters you want used in the "point to object" mode of the Create
New Classifier command. Any parameter that you select (by double-clicking on the parameter name so that
a check mark appears next to it) from the Auto Classifier Definition list will be used to define a new classifier
when using the "point to object" mode.
Description
Provides a brief description of the selected parameter in the Filters table. This status box will be displayed
when you select the Show Descriptive Text check box.
Closes
Closes the dialog box.
Configure Object Measurements (Measure Menu)
Selects which object measurements will be recorded in log files and displayed in the Show
Individual Object Data dialog box and the Show Classifier Statistics dialog box.
Drop-in: AUTOMEAS
Use this command before measuring data that will be logged to object and/or summary logs. This
command selects which type of object measurements will be logged and displayed. MetaMorph
completes and stores all object measurements in its temporary buffer, whether or not you configure them
for your use. This allows you to log only a selected set of measurements, but use other measurements
as object classifiers to filter out ranges of objects. You can save and load object measurement sets for
future use.
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The Configure Object Measurements dialog box includes a list of measurements that you can enable or
disable. Descriptions of these measurements can be seen by enabling Show Descriptive Text. You can
save and load object measurement sets (*.mes). The summary log is configured separately using the
Configure Summary Log command in the Configure Object Measurements dialog box.
Measurement Term Definitions
Statistical Term Definitions
Configuring Object Measurements - Procedures
Configuring Object Measurements
Creating New Measurement Sets
Loading and Saving Measurement Sets
Configuring Object Measurements
To configure object measurements, use the following procedure:
Step
Action
1
From the Measure menu, choose Configure
Object Measurements. The Configure Object
Measurements dialog box will appear.
2
Select the measurement set you want to
configure from the Measurement Sets list so
that its name appears in the text box below
Measurement Sets.
If you do not want to use the Default set, you
can create a new measurement set using the
New Set command or load a previously
saved set using the Load Set command.
3
Double-click each measurement in the
Measurements to Log list that you want to
include in the current set. Those included will
be marked with a check mark. Deselect any
measurements marked with a check mark
that you do not want to include.
You can use All On/All Off to enable or
disable all measurements. You can also use
the Reset button to reset the measurement
values to "factory" settings.
4
While you are selecting measurements, you
can enable Show Descriptive Text if you
want to read a short description of each
measurement. You can also select Active
from the Show Log Measurements group box
to limit the measurements listed in the
Measurements to Log list to those which
have been selected for the current set.
5
Select the numeric format for the
measurement data from the drop-down list
below Measurements to Log.
6
Repeat Steps 2 - 5 for each measurement
set you want to configure.
7
To configure a summary log for logging,
choose Configure Summary Log. The
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Configure Log dialog box will appear.
8
Once you have configured the desired
measurement set(s) and the summary log,
you can select the measurement set you
want to use for the next group of
measurements so that it is displayed in the
Measurement Sets list.
AND
Choose Close to close the dialog box.
Creating New Measurement Sets
To create a new measurement set, use the following procedure:
Step
Action
1
Select New Set from the Configure Object
Measurements dialog box. The New
Measurement Set dialog box will appear.
2
Type the desired name in the Set Name text
box.
3
Select Duplicate Current Set from the Initial
Logging Values group to use the current set's
values as the initial values for the new set.
OR
Select Default Logging Values to use the
MetaMorph's stored values as the initial
values.
4
Choose OK. The new set will be created and
added to the bottom of the Measurements
Set list in the Configure Object
Measurements dialog box.
Loading and Saving Measurement Sets
To load a previously saved measurement set, use the following procedure:
Step
Action
1
Select Load Set File from the Configure
Object Measurements dialog box. The Load
Measurement Set dialog box will appear.
2
Select the icon for the desired file. If
necessary, select the appropriate drive or
folder with the Look In list or Up One Level
icon button.
3
Choose Open. The measurement set will be
loaded and its name will be added to the
bottom of the Measurement Sets list.
4
If you want to configure the new set, open
the Measurement Sets list and select the set
so that its name appears in the text box
below Measurements Sets.
To save the measurement sets currently listed in the
Measurement Sets list together in one file, use the following
procedure:
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Step
Action
1
Select Save Set File from the Configure
Object Measurements dialog box. The Save
Measurement Set As dialog box will appear.
2
Type the icon for the desired file name. If
necessary, select the appropriate drive or
folder with the Save In list or Up One Level
icon button.
3
Choose Save to save the measurement set.
Configure Object Measurements - Dialog Box Options
Measurement Set File
Lists the measurement set file currently loaded and displayed in the Measurement Sets list.
Measurement Sets
Lists of the measurement sets that have created during the current work session or loaded with the
current measurement set file.
Measurement Sets Text Box
Allows you to rename a user-created measurement set. You may not rename the Default set.
New Set
Creates a new measurement set that uses either current set's values or the default logging values as the
initial values for the new set.
Remove Set
Removes the current set from the Measurement Sets list.
Load Set File
Loads a measurement set file (*.mes), consisting of one or more measurement sets, into the Measurement
Sets list.
Save Set File
Saves a measurement set file consisting of all of the measurement sets currently listed in the Measurement
Sets list.
Reset
Resets the Measurements to Log selections to the factory default settings.
All On/All Off
Enables or disables the selection of all measurements in the Measurements to Log list.
Show Descriptive Text
Displays a description at the bottom the dialog for the currently selected measurement in the Measurements
to Log list.
Show Log Measurements
Displays all available measurements in the Measurements to Log list when All is selected. Displays only
those which are marked with a check mark if Active is selected.
Configure Summary Log
Opens the Configure Log File dialog box so that you can select the parameters to be logged to the log file.
Measurements to Log
Specifies which measurements will be included in the current set and used for logging by marking them with
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a check mark.
Numeric Format
Specifies the numeric format used to log and data measurement data.
Close
Closes the dialog box.
Annotate Measured Objects (Measure Menu)
Adds annotations to measured objects in an image.
Drop-in: ANMEAS
Use this command to label individual measured objects in an image with a selected annotation. You can
simply identify objects with a character such as an asterisk, or label them with an ID number, or you can
label each object with such measurement parameters as its area, centroid X and Y coordinates, average
gray value, shape factor, etc. Alternatively, you can add your own annotation to each object. More than
one parameter or comment can be used in the annotation.
WARNING:
When you apply the annotation to an image, the text will become a part of the image itself. If you
subsequently attempt to perform a densitometric or morphometric measurement on the entire image, the
grayscale values and morphometric characteristics of the text will be measured along with the objects in
the image. Be sure to perform your measurements before you apply any graphics to the image. If there
is any chance that you will need to reanalyze the original image, you should make a copy of the original
and apply the annotation to the copy.
Note: To annotate objects with their measurement parameters, you must first set the image threshold
and measure the objects with the Measure Objects command (Measure menu). This command can be
also used to annotate images that have not been measured, but any measurement parameters that are
specified in the annotation will be read as "0".
Measurement Term Definitions
Annotating Measured Objects
To annotate measured objects in an image, use the following procedure. To include
measurement parameters in the annotation, you must first set the image threshold and measure
the objects with the Measure Objects command (Measure menu).
Step
Action
1
From the Measure menu, choose Annotate
Measured Objects. The Annotate Measured
Objects dialog box opens.
2
Select the image to be annotated with the
Image to Annotate image selector.
3
Choose Show Codes. The Annotate
Measured Image Codes dialog box will
appear, displaying the object parameter
codes that you will use to specify what will
appear in the object annotations.
4
In the Annotation Text box, enter the code for
the measurement parameter you want to
appear in each measured object's
annotation. For example, if you want to label
objects with their area, type "$02". Be sure to
include the dollar sign.
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Note: You can specify several parameters to
be used as object labels, separating the
codes that you type into the Annotation Text
box with a comma, space, or hyphen. The
default entry for Annotation Text is "$*",
which labels objects with an Object ID
number. If you delete the dollar sign, this
command will label each object with the
asterisk that remains. You can change the
label to any keyboard character, such as a
plus sign (+).
5
You can specify the number of decimal
points to be displayed in the annotation. For
example, to display Total Area to one
decimal point, you would set the Annotation
Text field to "$00.1". The "$00" will specify
the display of the variable "Total Area," while
the ".1" restricts the output of this variable to
one decimal place. Each variable can have
its own precision.
6
If you want to change the color of the
annotation label, enter the value in the
Foreground Color spin box. If your selected
image is one that has been measured, the
values will correspond to the Pseudocolor
scale associated with the image. Thus, for
example, a Foreground Color value of 200
will produce a red annotation.
7
If you want the areas behind the annotations
to be opaque, select the Fill Background
check box, so that a check mark appears in
it. If your selected image has been
measured, the values will again correspond
to the pseudocolor scale associated with the
image. Thus, for example, a Background
Color value of 150 will produce a yellow
background.
8
Choose Annotate. The annotations will
appear in the image window.
9
To undo the annotation, click Undo.
10
Choose Close.
Annotate Measured Objects - Dialog Box Options
Image to Annotate
Selects the image to be annotated.
Annotation Text
Enter the code here for the measurement parameter you want to appear in the annotation.
Foreground Color
Selects a color for the annotation label text. If your selected image is one that has been measured, the
values will correspond to the pseudocolor scale associated with the image. For example, a value of 200 will
produce a red annotation.
Fill Background
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Fills the annotation label background with the color corresponding to the value entered in the Background
Color spin box.
Background Color
Selects a color for the annotation label background. If your selected image is one that has been measured,
the values will correspond to the pseudocolor scale associated with the image. For example, a value of 150
will produce a yellow background.
Annotate
Labels the image objects with the selected annotation.
Show Codes
Displays a list of the object measurement parameters and their codes. The code for your selected
measurement parameter is entered in the Annotation Text box.
Undo/Redo
Removes/replaces the annotation.
Close
Closes the dialog box.
Measure Objects (Measure Menu)
Compiles selected measurements about objects in a binary image or a thresholded image
using the thresholded or binary values to distinguish objects from the background.
Drop-in: AUTOMEAS
Use this command to measure all objects in a selected image or region and display them in a new 8-bit
image window using a pseudocolor display. The selected image must be a binary image or must be
thresholded first. This command ignores single pixel objects.
When an image is measured, MetaMorph stores the measurements in a temporary buffer until they are
replaced by the results from the next measurement command. There are two ways to use the
measurements compiled by Measure Objects: (1) View them immediately using Show Individual Object
Data or Show Classifier Statistics before performing another measurement command, or (2) Store them
permanently in an object log or summary log.
Prior to using Measure Objects you will need to select the desired types of measurements using
Configure Object Measurements. You may also want to configure filters to include or exclude objects
from measurements using Configure Object Classifiers.
WARNING:
The maximum number of objects that can be measured in an image is 16,300. If there are more objects
in an image than this maximum, MetaMorph will display an error message dialog box and abort the
Measure Objects command.
Shortcut: CTRL + M
An Introduction to Automatic Object Analysis
Creates a boundary between the objects and the background (non-object
image information) of the image on the basis of the image's gray values. When
an image is thresholded, all of its objects can be measured using an automatic
object analysis command such as Measure Objects.
Measuring Objects
To measure objects, use the following procedure:
Step
Action
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1
If you want to save the measurement data
generated by the Measure Objects
command, open an object, summary, or
edgelist log from the Log menu.
2
If the image is an 8-bit, 16-bit, or 24-bit
image, threshold it using the Threshold Tool
or the Threshold Image command so that
the threshold values clearly delineate the
desired objects from the background.
OR
If the image is a binary image, continue to
Step 3.
3
From the Measure menu, choose Measure
Objects.
Note: If you have already applied an
automatic measurement command to any
image during the current work session,
MetaMorph will display a message dialog
box to remind you that it will erase the data in
the buffer with the new measurements. (The
display of this prompt can be turned off using
the Preferences command.)
4
When MetaMorph has finished measuring
objects, it will display the objects it found in a
new image window using a pseudocolor
display.
WARNING:
The maximum number of objects that can be
measured is 16,300. If there are more
objects that this maximum, MetaMorph will
display an error message and abort the
measurement command.
Recalculate Object Parameters (Measure Menu)
Recalculates the objects in the current image which pass the selected object classifier
filters defined by Configure Object Classifiers and previously calculated with the Measure
Objects command.
Drop-in: AUTOMEAS
An image must be measured with an automatic measurement command such as Measure Objects
before Recalculate Object Parameters can be used. Because this command recalculates objects and
then redraws the image without tracing the objects, Recalculate Object Parameters should be used
when defining classifiers to determine if the classifiers are defined properly. Since Recalculate Object
Parameters does not trace the objects, it is faster than using the original measurement command again.
This command is the same as the Recalc command button found in the Configure Object Classifiers
dialog box.
Shortcut: CTRL + SHIFT + M
An Introduction to Automatic Object Analysis
Recalculating Object Parameters
To recalculate object parameters, use the following procedure:
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Step
Action
1
Reconfigure the object parameters you want
to change using the Configure Object
Classifiers command.
2
From the Measure menu, choose
Recalculate Object Parameters.
3
The objects that pass the active classifiers
will be recalculated and then redrawn in the
image window.
Measure Objects with Mask (Measure Menu)
Compiles selected measurements about objects in an image using a matching 1-bit or 8bit mask image (consisting only of gray values 0 and 255) to distinguish objects (gray
value 255) from the background (gray value 0) in the image. After MetaMorph measures the
source image's objects, it will display the objects that were defined by the matching mask
image in a new 8-bit image window using a pseudocolor display.
Drop-in: AUTOMEAS
Use this command to measure objects in a source image (8-bit, 16-bit, or 24-bit) using a matching mask
image, and display the measured image using a pseudocolor display. The power of this command
comes from its ability to use mask images, such as those created from the source image using the
Binary command (Process menu). For example, you could use the Binary command’s Segment option
to apply a "watershed" segmentation of objects in the image prior to measuring them with the Measure
Objects with Mask command.
Once an image is measured, MetaMorph stores the measurements in a temporary buffer until they are
replaced by the results from the next measurement command. There are two ways to use the
measurements compiled by Measure Objects with Mask: (1) View them immediately using Show
Individual Object Data or Show Classifier Statistics before performing another measurement command,
or (2) Store them permanently in an object log.
Prior to using the Measure Objects with Mask command you will need to select the desired types of
measurements using Configure Object Measurements. You may also want to configure object classifier
filters to include or exclude objects from measurements using Configure Object Classifiers.
WARNING:
The maximum number of objects that can be measured in an image is 16,300. If there are more objects
in an image than this maximum, MetaMorph will display an error message dialog box and abort the
Measure Objects with Mask command.
An Introduction to Automatic Object Analysis
Measuring Objects Using a Mask Image
To measure objects in an image using a matching mask image, use the following procedure:
Step
Action
1
Threshold the image you want to measure, to
separate the objects from their background.
2
Create a binary mask image with the
Process menu's Binary command.
3
Open an object, summary, or edgelist log
from the Log menu if you want to save the
measurement data generated by the
Measure Objects with Mask command.
4
From the Measure menu, choose Measure
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Objects with Mask. The Measure Objects
with Mask dialog box will appear.
Note: If you have already applied an
automatic measurement command to any
image during the current work session,
MetaMorph will display a message dialog
box to remind you that it will erase the data in
the buffer with the new measurements. (The
display of this prompt can be turned off using
the Preferences command.)
5
Select the image to be measured (8-bit,
16-bit, or 24-bit) from the Gray Image
selector.
6
Select the desired 1-bit or 8-bit binary mask
image from the Mask Image selector.
7
If the desired destination image is not
displayed in the image selector next to
Destination, select it using the image
selector. You can overwrite or add to an
existing 8-bit image or you can place the
results in a new 8-bit image window.
8
Choose OK.
9
When MetaMorph has finished measuring
the thresholded objects in the Gray Image,
based on the mask overlay in the Mask
Image, it will display the objects it found in
the selected destination image window using
a pseudocolor display.
WARNING:
The maximum number of objects that can be
measured is 16,300. If there are more
objects that this maximum, MetaMorph will
display an error message and abort the
measurement command.
Measure Objects with Mask - Dialog Box Options
Gray Image
Selects the source image (8-bit, 16-bit, or 24-bit) for measuring objects with a matching mask image. This
image should be thresholded before measuring, so that objects can be separated from background.
Mask Image
Selects the 1-bit or 8-bit binary mask image. This will determine where on the Gray Image objects will be
measured.
Destination
Selects the destination image for the pseudocolor measured result image. You can place the results in a
new 8-bit image window or overwrite/append to an existing 8-bit image.
OK
Measures the selected image.
Cancel
Cancels the command.
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Measure Single Object (Measure Menu)
Compiles selected measurements about a single object selected with the Locator Tool in a
binary image or a thresholded image (8-bit, 16-bit, or 24-bit).
Drop-in: AUTOMEAS
Use this command when you want to measure (and log data from) one or more objects in the image.
When you apply Measure Single Object to an image or active region, MetaMorph will find all of the
objects in the image using the threshold values. It will then display the objects it found, using a
pseudocolor display in a new image window. All of the objects will be marked with the same
pseudocolor.
MetaMorph will also open the Measure Single Object dialog box, which includes a list of the current
measured object's data. An object can be measured by positioning the pointer over the object and
clicking the left mouse button. The object's data will appear in the Measure Single Object dialog box and
you can then log the data. After an object is measured, MetaMorph will paint the object a color based on
the classifier sets you are currently using.
Once an image is measured, MetaMorph stores the measurements in a temporary buffer until they are
replaced by the results from the next measurement command. There are two ways to use the
measurements compiled by Measure Single Object: (1) View them immediately using the command's
dialog box or using the Show Classifier Statistics command before performing another measurement
command, or (2) Store them permanently in an object log or summary log.
Prior to using Measure Objects you will need to select the desired types of measurements using
Configure Object Measurements. You may also want to configure object classifier filters to include or
exclude objects from measurements using Configure Object Classifiers.
WARNING:
The maximum number of objects that can be measured in an image is 16,300. If there are more objects
in an image than this maximum, MetaMorph will display an error message dialog box and abort the
Measure Single Object command.
Shortcut: CTRL + SHIFT + M
Configuring Drop-ins/Toolbars
An Introduction to Automatic Object Analysis
Measurement Term Definitions
Statistical Term Definitions
Measuring a Single Object
To measure a single object, use the following procedure:
Step
1
Action
If the image is an 8-bit, 16-bit, or 24-bit
image, threshold it using the Threshold Tool
or Threshold Image command so that the
threshold values clearly delineate the desired
objects from the background.
OR
If the image is a binary image, continue to
Step 2.
2
From the Measure menu, choose Measure
Single Object.
Note: If you have already applied an
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automatic measurement command to any
image during the current work session,
MetaMorph will display a message dialog
box to remind you that it will erase the data in
the buffer with the new measurements. (The
display of this prompt can be turned off using
the Preferences command.)
3
MetaMorph will find all of the objects in the
image using the threshold values. When
MetaMorph has finished, it will display the
objects it found in a new pseudocolor image
window. All of the objects will share the
pseudocolor. It will also open the Measure
Single Object dialog box.
WARNING:
The maximum number of objects that can be
measured is 16,300. If there are more
objects that this maximum, MetaMorph will
display an error message and abort the
measurement command.
4
If you want to log the measurement data,
open an object log or edgelist log using the
corresponding Open Log command button in
the dialog box or the Open Object Log or
Open EdgeList Log command from the Log
menu.
Open Log will be replaced by the F9: Log
Data command button.
5
Select the object you want to measure from
the pseudocolored image window by clicking
in the center of the object. MetaMorph will
paint it a color based on the classifier filter
sets you are currently using and then will
number it.
The Measure Single Object dialog box
displays the measured object data.
6
Choose F9: Log Object or press the [F9] key
to log the displayed object or edgelist data.
MetaMorph will log the data and gray out the
F9: Log Object command button until you
select a different object.
7
To review the data for a previously measured
object, use the Object spin box to select the
object. MetaMorph will display the selected
object's data in the dialog box and its object
number stamp in the pseudocolor image
window.
8
Choose Close when you have finished.
Measure Single Object - Dialog Box Options
Open Log
Opens an object log or edgelist log for logging data if desired. Changes to "F9: Log Data" when a log file is
open.
F9: Log Data
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Logs the currently selected object or edgelist data to the open object log or edgelist log. You can also press
the [F9] function key to log these data. After the current data have been logged, the command button will be
unavailable and will appear dimmed until you select another object.
Object
Specifies the object ID number of a previously measured object and displays that object's data in the dialog
box. Also marks the object number stamp on the selected object in the pseudocolor image window.
Morphometry Histogram (Measure Menu)
Displays a histogram of measurement data from the last automatic measurement in bar
graph form.
Drop-in: AUTOMEAS
Use this command to display a histogram of data from a selected measurement parameter. This
command allows you to see a visual representation of the data from automatic measurements in the
form of a bar graph. This command is similar to Image-1/AT's Data Histogram command or MetaMorph's
Integrated Morphometry Analysis command. You can configure autoscaling and the number of bins in
the graph.
Prior to using this command, you must threshold and measure an image to provide data for the
histogram. The menu item for this command will remain disabled until you have measured an image.
You can log data from this command to an open data log. First use the Open Data Log command to
open a data log. You can use the Log Data command or its keyboard shortcut, the [F9] function key, to
log the data.
An Introduction to Automatic Object Analysis
Measurement Term Definitions
Displaying a Morphometry Histogram
To display a morphometry histogram, use the following procedure:
Step
1
Action
If you have not measured the image,
threshold it using the Threshold Tool or the
Threshold Image command.
AND
Measure the image using the desired
automatic measurement command.
2
From the Measure menu, choose
Morphometry Histogram. The Morphometry
Histogram dialog box will appear.
3
If you want to log data, open a data log using
the Open Log command.
Once the data log is opened, the text on the
Open Log button will change to F9: Log
Data, and you can then log measurement
parameter data whenever you desire.
4
To configure data logging, choose Configure
Log.
5
If you want to autoscale the data displayed in
the graph so that plot's X and Y axis is based
on the range of the measured object data,
select Auto-Scale Graph.
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6
Select the number of bins you want to
display in the graph using Number of Bins.
7
To display the data from a measurement
parameter, select its name from the
Measurement list.
When you select a different measurement
parameter, MetaMorph will update the graph
to display the applicable data.
8
Choose Close when you have finished.
Morphometry Histogram - Dialog Box Options
Measurement
Selects the measurement parameter data to be displayed in the graph. You can select from any of the
parameters that are currently selected in the Configure Object Measurements dialog box. The data in the
graph are automatically updated when you select a new measurement parameter.
Number of Bins
Specifies the number of bins of data displayed in the graph.
Auto-Scale Graph
Automatically displays the data in the graph so that the plot's X and Y axis are based on the range of the
measured object data.
Open Log
Opens a data log and/or a DDE link to an open spreadsheet application for logging data. This command
changes to F9: Log Data once a log file is open.
F9: Log Data
Logs the currently displayed data from the dialog box to an open data log or to an open spreadsheet
application via a DDE link. To assist you in logging the appropriate data when several measurement dialog
boxes are open, "F9" will be added to the name of this option in the active dialog box to indicate which data
will be logged when you press [F9].
Configure Log
Opens the Configure Log dialog box so that you can select the parameters to be logged to the log file.
Close
Closes the dialog box.
Cut Objects (Measure Menu)
Cuts an area of an image thresholded as "one object" into two or more objects based on
the placement of a region, so that the thresholded "object" will be counted and measured
as multiple objects.
Drop-in: CUTJOIN
This command can be used in situations where two or more objects are thresholded as one object
because they overlap or are otherwise not well-defined. This command cuts by "painting" a "cut" line in
the segmentation overlay, which MetaMorph will sense while performing image segmentation.
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To apply the Cut Objects command more than once to an image, you can write a journal, using the Loop
for All Regions command, that allows you to threshold the image and place regions on the all of the
desired cut mark locations before the marked objects are cut.
Shortcut: [F7]
Cutting Objects
To separate overlapping objects prior to image segmentation and measurement, use the
following procedure:
Step
Action
1
Threshold the image for measurement using
the Threshold Image command (Process
menu).
2
Draw a region through the desired cut mark
location using a Line Region Tool. The
region must completely bisect the edges of
the object.
3
From the Measure menu, choose Cut
Objects. The object marked with the active
region will be "cut."
4
Repeat Steps 2 and 3 for each "cut" you
want to create.
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5
Turn off the active region indicator by
choosing the Selector Tool and right-clicking
with the mouse.
6
Measure the image as desired.
Join Objects (Measure Menu)
Joins areas of an image, thresholded as multiple objects, into a single object, so that the
thresholded "objects" will be counted and measured together.
Drop-in: CUTJOIN
This command can be used in situations where a single object is thresholded as multiple "objects"
because it is not well-defined. This command joins by "painting" a "join" line between the objects in the
segmentation overlay, which MetaMorph will sense while performing image segmentation.
To apply the Join Objects command more than once to an image, you can write a journal, using the Loop
for All Regions command, that allows you to threshold the image and place regions on all of the desired
join mark locations before the marked objects are joined.
Shortcut: CTRL + [F7]
Joining Objects
To join objects prior to image segmentation and measurement, use the following procedure:
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Step
Action
1
Threshold the image for measurement using
the Threshold Image command.
2
Link the separated parts of an object by
using a Line Region Tool to draw a line
region that starts inside the first part and
ends inside the second part .
3
From the Measure menu, choose Join
Objects. The objects marked with the active
region will be "joined."
4
Repeat Steps 2 and 3 for each "join" you
want to create.
5
Turn off the active region indicator by
choosing the Selector Tool and right-clicking
with the mouse.
6
Measure the image as desired.
Integrated Morphometry Analysis (Measure
Menu)
Opens the Integrated Morphometry Analysis (IMA) interface for the measurement, display,
and logging of morphometric data.
Drop-in: IMA
Measurement Term Definitions
Statistical Term Definitions
Use this command when you want to perform morphometric measurements of objects in your image.
You can select parameters for measurement or define classifier filters which restrict your measurements
to just those objects that meet your set criteria.
You can select any of four ways to display morphometric data:
(1) In a table showing the data for each measured object,
(2) In a table showing a statistical summary of the data collected from all of the objects,
(3) In a histogram showing distribution of the data in a bar graph, or
(4) In a scatterplot showing the relationship between any two parameters that you have measured.
The IMA interface has four interactive modes that allow you to "point-and-click" as you work back and
forth between the objects in the image window and the data being displayed in the IMA data table,
histogram, or scatterplot:
(1) None allows you to use region tools or the Cut and Join Drawing Tools, change image
magnification, set thresholding, or apply graphics stamps to the image without altering the current
set of measurements.
(2) Single allows you to add or remove individual objects from the current set of measurements by
clicking them in the image window.
(3) Teach allows you to use the parameters of objects you click in the image window to configure the
classifier filter criteria.
(4) Find allows you to find an object's entry in the object measurement data table, its bin in the
histogram, or its data point on the scatterplot by clicking the object in the image window.
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Conversely, an object can be found in the image window by clicking its entry in the data table, its
bin in the histogram, or its data point in the scatterplot.
State files contain your Integrated Morphometry Analysis dialog box configuration settings, such as the
threshold levels of your color images, classifier filter settings, interactive mode, and the list of parameters
you have selected for measurement and/or classification. These files can be saved to or loaded from
disk. Additionally, you can configure log files and perform data logging from within the Integrated
Morphometry Analysis dialog box.
Using Integrated Morphometry Analysis
Overview of Morphometric Analysis
Performing Measurements in IMA
Performing Measurements Interactively in IMA
Setting the Classifier Filter Range
Setting the Classifier Filter Range Interactively
Relating Data Table Entries to Image Objects
Logging IMA Data
Saving a State File
Loading a State File
Overview of Morphometric Analysis
To perform a morphometric analysis with the Integrated Morphometry Analysis drop-in, use the
following procedure.
Note: Unless your image is a binary image or a binarized "mask" image (i.e., an 8-bit
image that has only two pixel intensity values, such as 0 and 255), it must first be
thresholded before conducting your analysis.
Step
Action
1
From the Measure menu, choose Integrated
Morphometry Analysis. The Integrated
Morphometry Analysis dialog box will appear.
2
Select a source image or stack plane from
the Image selector.
3
Select your parameters for measurement
from the Parameters List Box.
4
If desired, you can restrict your analysis to
image objects that meet a set of specific
quantitative criteria by using classifier filters.
This can be performed either by setting the
classifier filter ranges manually or
interactively.
5
Perform your measurements. Measurements
can be performed for the entire image
automatically, or you can select image
objects for measurement interactively.
6
You can use the Display drop-down list to
select between
(1) A view of the morphometric data
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associated with each thresholded image
object (Objects),
(2) A view of the statistical data for the entire
group of objects (Summary),
(3) A view of a histogram of the object
measurements (Histogram), or
(4) A scatterplot showing the relationship
between any two parameters that you have
measured (Scatterplot).
7
If you want to relate the data displayed in the
object measurement data table, histogram,
or scatterplot more clearly to an individual
object in the image window, you can "find"
objects interactively.
8
When you are satisfied with your data, you
can log the data.
9
If desired, you can save and load the
Integrated Morphometry Analysis dialog box
settings or Color Threshold level settings (if
a 24-bit image was being analyzed) by
choosing Save State and Load State,
respectively.
10
When you have finished, choose Close.
Performing Measurements in IMA
To measure objects automatically throughout your image, use the following procedure. Be sure
that your image has been thresholded first.
Step
Action
1
If the data region at the right of the IMA
dialog box is hidden, choose Show Data to
expand the dialog box. If you later want to
hide the data table, you can choose Hide
Data.
2
Select Measuring from the Setup Parameters
For group.
3
In the Parameters List Box, select which
measurement parameters you want to
analyze by double-clicking the desired
entries. A check mark will appear next to
each selected entry.
4
If desired, you can change the number of
significant digits in your data from the Format
drop-down list box. This can be done for
each parameter you have selected from the
Parameters List Box.
5
Choose Measure. A green object overlay will
appear over the measured objects in the
source image, and the morphometric
measurements will appear in the object
measurement data table, histogram, or
scatterplot.
6
If you need to redo the measurement,
choose Reset Current, make any necessary
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changes in the Parameters List Box and the
Format or Filter Range boxes, and then
choose Measure again.
7
If you perform another measurement, the
most recently measured data set will be
displayed in the data table and histogram. If
you want to view or log all of the data from
successive measurements, select
Accumulated from the Show/Log Data group.
(The Reset Accumulated button will replace
the Reset Current button.) Each new data set
will be added to the previously displayed set,
and will appear in the data table or
histogram.
8
If you need to undo the last measurement,
select Current from the Show/Log Data
option button group and choose Reset
Current.
OR
If you want to undo all measurements, select
Accumulated from the Show/Log Data option
button group and choose Reset
Accumulated.
Performing Measurements Interactively in IMA
To measure objects interactively with the Integrated Morphometry Analysis interface, use the
following procedure. Be sure that your image has been thresholded first.
Step
Action
1
In the Parameters List Box, select the
parameter that you want to measure by
double-clicking its entry. A check mark will
appear next to the entry.
2
Select Single from the Image Interactive
Modes section.
3
In the image window, click the object you
want to measure. A green object overlay will
appear over the measured object, and its
measurements will appear in the object
measurement data table and histogram.
4
Repeat Step 3 for all objects in the image
window that you want to measure. As each
object is selected, the data table, histogram,
or scatterplot will display the additional
measurement for each of the newly selected
objects.
Setting the Classifier Filter Range
When classifying objects, a range of acceptable values for each classifier filter needs to be defined. Only
objects which pass the criteria set by the filter range will be included when classifying objects. Each
classifying parameter has its own Filter Range. This option will appear only when the Classifying radio
button is selected in the Setup Parameters For group.
A range can be defined to include objects whose measurements fall within a specified range. To do so,
select < = N < = from the Filter Range list. However, if you want to analyze all objects outside a particular
range, select > N or N > instead. Set the limits of your range by entering desired values in the text boxes on
either side of the drop-down list box.
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Setting the Classifier Filter Range Interactively
To add an object’s parameters to the Integrated Morphometry Analysis classifier filter range
interactively, use the following procedure. Be sure that your image has been thresholded first.
Step
Action
1
In the Parameters List Box, select the
parameter that you want to use as a
classifier filter by double-clicking its entry. A
check mark will appear next to the entry.
2
Select Teach from the Image Interactive
Modes section.
3
In the image window, click the object whose
measurements you want to use to define the
classifier filter criteria. A yellow object overlay
will appear over each selected object, and its
measurements for the currently highlighted
parameter will appear in the Filter Range text
boxes. Simultaneously, all of the selected
classifier filters will be updated. Other image
objects with measurements that "pass" the
updated classifier filters will appear with a
green object overlay in the image window.
Note: If you have selected Draw Failed
Classifier Objects in the Measure Objects
page of the Preferences dialog box (Edit
menu), objects that fail the classifier filter will
be displayed with a blue object overlay. If this
option has not been selected, failed objects
will simply show the red thresholding overlay.
4
Repeat Step 3 for all objects in the image
window that you want to use to update the
classifier filter criteria. As each object is
selected, the Filter Range text boxes will
reflect the measurements of the selected
objects for the currently highlighted
parameter.
Relating Image Objects to Their Data
The Find interactive mode allows you highlight an object's entry in the data table, its bin in the
histogram, or its data point on the scatterplot by selecting the object in the image with your
pointer. Conversely, you can click a data entry, histogram bin, or scatterplot data point, and the
associated object(s) will become marked in the image window with a yellow object overlay.
To "find" the object associated with a data entry in the IMA data table, or to "find" the data
associated with a particular object in the image window, use the following procedure. Be sure that
your image has first been thresholded and measured.
Step
Action
1
Select Find from the Image Interactive
Modes section.
2
To find the data entry for an object in the
image window, use the pointer to select the
object. A yellow object overlay will appear
over the selected object, and the data table
will "scroll" to the object's data entry, which
will be highlighted.
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3
To find an object associated with an entry in
the data table or point on the scatterplot, or
all objects associated with a particular bin in
the histogram, use your pointer to select the
data table entry, histogram bin, or data point.
A yellow object overlay will appear in the
image window over the object(s) associated
with the selected table entry, histogram bin,
or data point.
Note: You can select a range of adjacent
table entries or histogram bins by clicking the
first entry or bin, holding down the [SHIFT]
key, and selecting the ending table entry or
bin. Similarly, you can select an assortment
of table entries, histogram bins, scatterplot
points, or image objects by holding down the
[CTRL] key while you make your selections.
Logging IMA Data
To log data from the IMA interface, use the following procedure. IMA will automatically select the
data to be logged from the list of parameters you have selected from the Parameters List Box.
Accordingly, Steps 1 - 4 in the following table are optional.
Step
Action
1
Choose Configure Log. The Configure Log
dialog box will appear.
2
From the list of parameters, select the
parameters you want to log by doubleclicking their entries so that each is marked
by a check mark.
3
You can choose Enable All or Disable All if
you want to select or deselect all of the
parameters listed.
4
Choose OK to return to the IMA dialog box.
5
Choose Open Log. The button's title will
change to "F9: Log Data." Each of the
displays available in the Display group has a
different type of data associated with it.
Therefore, depending on the Display option
currently selected, you will be specifying a
log file for logging either object, summary,
or data measurement data. Switch between
the displays and specify the log file names
for the other two data types after you have
selected the first one.
6
Choose F9: Log Data to log your data. If you
have selected different file names for the
different data types, you will need to switch
between the displays and log the data
separately for each.
Saving a State File
To save the settings you used in your Integrated Morphometry Analysis dialog box, use the
following procedure:
Step
Action
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1
Select Save State. The Integrated
Morphometry: Save State dialog box will
appear.
2
If you want to save your Integrated
Morphometry Analysis dialog box settings
(for example, the Setup Parameters For and
Display modes, interactive mode, parameter
selections, classifier filters, etc.), select the
check box next to Integrated Morphometry
State. A check mark will appear in the box.
These settings will be saved in an .ima file.
3
If you are working with a 24-bit color image,
the 24 Bit Image Threshold check box will be
available for selection. Otherwise the check
box and title will be unavailable and will
appear dimmed. To save color image
threshold level settings and their associated
image overlays, select the 24 Bit Image
Threshold check box. These settings will also
be saved in the .ima file.
4
If you want to save the criterion settings for a
classifying filter range separately from the
.ima file, select the Classifier Filters in a *.cls
File check box.
(Although these settings can also be saved
in the .ima file along with the dialog box and
color threshold settings, saving the classifier
filter settings to a .cls file allows you to use
them independently of the parameter
measurement settings.)
5
Finally, if you want to save the settings for
selection of measurement parameters
separately from the .ima file, select the
Parameter Measurements in a *.mes File
check box.
(These settings, too, can also be saved in an
.ima file, but saving them to an .mes file
allows you to use them independently of the
classifier filter settings.)
6
When you have finished, choose OK.
Loading a State File
To load a previously saved Integrated Morphometry Analysis (IMA) State File (*.ima, *.mes, *.cls),
use the following procedure:
Step
Action
1
From the Integrated Morphometry Analysis
dialog box, select Load State. The Load
State File dialog box will appear.
2
Select the icon for the desired state file. If the
desired folder is not currently displayed, use
the Look In list or Up One Level icon button
to change the current folder.
Note: The dialog box defaults to a display of
files with the .ima file name extension. If you
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want to select an .mes or .cls file, you will
need to select All Files (*.*) from the Files of
Type list.
3
Choose Open.
Integrated Morphometry Analysis - Dialog Box Options
Image
Lists the images available to be selected for analysis by the Integrated Morphometry Analysis interface.
Setup Parameters For
Selecting Measuring from this option allows you to pick parameters from the Parameters List Box that are to
be measured. Selecting Classifying allows you to pick parameters that are to be used for applying a
classification filter to the displayed data. Superficially, the Measuring and Classifying parameter lists that
appear in the Parameters List Box as a result of this selection will look identical. However, they serve very
different functions. When you select Measuring, items selected in the Parameters List Box will be measured,
and the Format list will appear. When you select Classifying, items selected in the Parameters List Box will
be used to define object classifier filters, using the values entered in the Filter Range boxes that appear.
Parameters List Box
Lists the morphometric parameters available for either measurement or for defining an object classifier filter
(depending on the selection made in the Setup Parameters For option button group). (Click here for
Measurement Term Definitions.)
Format
Specifies the number of decimal places to be displayed in the morphometry data. You can specify that
figures be carried out to anywhere between zero and eight decimal places. This option is only visible when
you select Measuring from the Setup Parameters For group.
Filter Range
Sets quantitative criteria for objects, filtering out those objects that do not have morphometric parameters
that fall within the specified range from those that do. This option is available only when Classifying is
selected from the Setup Parameters For group.
Note: If the range set in the Filter Range field filters out all objects, or there are no
legitimate objects to measure, summary variables are set to 0 when logged.
Show/Log Data
Determines whether the data to be displayed or logged are from the current measurement set (Current) or
from the accumulation of measurement sets (Accumulated).
Reset Current/Reset Accumulated
Removes measurements from the active image and clears the data display. When Show/Log Data is set to
Current, the button will be labeled Reset Current, and only the most recent measurement will be removed.
When Show/Log Data is set to Accumulated, the labeling will switch to Reset Accumulated, and all
measured data will be cleared when you click the button. This option is journalizable.
Image Interactive Modes
This option allows you to pick one of the following four modes for manipulation of morphometric data:
None allows you to use Region Tools such as the Cut and Join Drawing Tools, to change zoom
magnification, or to apply graphics stamps to the image without affecting measurements.
Single allows you to add or remove individual objects from the current measurement set by clicking
them in the image window.
Teach allows you to add the parameters of objects you click in the image window to the classifier filter
criteria.
Find allows you to find objects in the object measurement data table, or find their histogram bin, by
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clicking an object in the image window, and vice versa.
Measure
Measures all objects in the active image window, applying any specified classifier filters, and displays the
specified measurement parameters in the data display at the right of the dialog box. This option is
journalizable.
Open Log
Initiates the process of logging data to a text file, via Dynamic Data Exchange (DDE) to an open
spreadsheet, or to both. When you choose Open Log, its title will change to "F9: Log Data." The data that
will be logged is determined by the Display mode:
When you select Objects or Scatterplot, an object log file will be opened, and the data associated with
each object will be logged to this file.
When you select Summary, a summary log file will be opened, and the statistical data for the entire
group of objects will be displayed.
When you select Histogram, a data log file will be opened, and the histogram will be displayed.
F9: Log Data
Logs the data for all selected parameters. (See preceding entry for Open Log.) This option is journalizable.
Configure Log
Selects the image characteristics and data to be logged.
Show Data
Expands the dialog box to the right to reveal the IMA data.
Hide Data
Condenses the dialog box to hide the IMA data.
Reset Filters
Unchecks all selected items in the Classifying parameters list box and returns the Filter Range values to
default.
Save State
Saves the Integrated Morphometry dialog box settings as state files. When you choose this command, the
Integrated Morphometry: Save State dialog box will appear.
Selecting one of the upper two check boxes (Integrated Morphometry State or 24 Bit Image Threshold) will
specify whether you want to save the dialog box settings, the 24-bit color image threshold level settings, or
both, to an .ima file. (The associated image must be a 24-bit color image for you to be able to use the 24 Bit
Image Threshold option.)
The lower two check boxes (Classifier Filters in a *.cls File and Measurement Parameters in a *.mes File)
can be selected if you want to save your measurement parameter choices to a measurement set (*.mes) file
or if you want to save classifier filter settings to a classifier set (*.cls) file, respectively.
Load State
Loads previously saved state files that specify Integrated Morphometry Analysis dialog box settings (*.ima),
color image threshold level settings (*.ima), measurement parameter choices (*.mes), and/or classifier filter
settings (*.cls) for a particular image. This option is journalizable.
Preferences
Displays the MetaMorph Preferences dialog box with the Measure Objects page selected. This is the same
page as is displayed when you choose Preferences from the Edit menu.
Display
Selects between the following choices:
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Objects – a view of a table of morphometric data for each thresholded image object
Summary – a table of the statistics regarding the entire group of objects
Histogram – a histogram of the object data. Contains the following fields:
X
This drop-down box will appear when Histogram or Scatterplot has been selected from the Display
list. When more than one object parameter has been measured, this drop-down box allows the
selection of the measurement parameter to be graphed on the X-axis. Only measurement
parameters activated in the Parameters List Box will be displayed here.
# of Bins
Changes the number of bins (and the width of the bins) that are displayed in a histogram. This spin
box will appear when Histogram has been selected from the Display list.
Set Filter Range from Histogram Calipers
This command button will appear when Histogram has been selected from the Display list. Use this
command after moving the histogram calipers to the desired location in the histogram display to
interactively reset the classifier Filter Range for a measurement parameter. Objects that fall outside
of the filter range will revert to the red thresholding overlay (or will change to a blue object overlay if
you have selected Draw Failed Classifier Objects in the Measure Objects page of the Preferences
dialog box); objects that stay within the filter range will retain the green object overlay.
Scatterplot – a scatterplot showing the relationship between any two parameters that you have
measured. Contains the following fields:
X
This drop-down box will appear when Histogram or Scatterplot has been selected from the Display
list. When more than one object parameter has been measured, this drop-down box allows the
selection of the measurement parameter to be graphed on the X-axis. Only measurement
parameters activated in the Parameters List Box will be displayed here.
Y
This drop-down box will appear when Scatterplot has been selected from the Display list. When
more than one object parameter has been measured, this drop-down box allows the selection of
the measurement parameter to be graphed on the Y-axis. Only measurement parameters activated
in the Parameters List Box will be displayed here.
Use X Calipers
This command button will appear when Scatterplot has been selected from the Display list. Use this
command after moving the scatterplot calipers to the desired location on the X-axis of the
scatterplot display to interactively reset the classifier Filter Range for the measurement parameter
represented on the X-axis. Objects that fall outside of the filter range will revert to the red
thresholding overlay (or will change to a blue object overlay if you have selected Draw Failed
Classifier Objects in the Measure Objects page of the Preferences dialog box); objects that stay
within the filter range will retain the green object overlay.
Use Y Calipers
This command button will appear when Scatterplot has been selected from the Display list. Use this
command after moving the scatterplot calipers to the desired location on the Y-axis of the
scatterplot display to interactively reset the classifier Filter Range for the measurement parameter
represented on the Y-axis. Objects that fall outside of the filter range will revert to the red
thresholding overlay (or will change to a blue object overlay if you have selected Draw Failed
Classifier Objects in the Measure Objects page of the Preferences dialog box); objects that stay
within the filter range will retain the green object overlay.
Close
Closes the dialog box.
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Internally Threshold Objects (Measure Menu)
Thresholds objects so that pixels below a selected percentage of the maximum intensity in
each object are removed, and then measures the processed objects.
Drop-in: THRESHOB
Use this command during morphometric analysis to separate fluorescent objects that overlap. This
command uses the internal intensity maximum of each object as initially thresholded, and then sets a
new threshold for each object based on a percentage of its maximum. Pixels that fall below the selected
percentage are omitted from the new threshold range. The objects are then measured as delineated by
the new threshold.
CAUTION: This method for splitting objects will shrink object sizes and alter many morphological
measurements. Measurements made after internal thresholding will be made on the basis of the
resulting objects after processing, not on the original objects as depicted in the original source image.
All single-pixel objects are ignored by MetaMorph during measurement. If the original thresholding of the
source image produces single-pixel objects, these objects will be ignored. As an added feature, the
dialog box for this command contains an option that allows you to perform a dilation convolution prior to
measurement, which will "grow" single-pixel objects and thereby prevent their loss. You should be
aware, however, that this convolution will expand the boundaries of other objects, as well, and your data
may become skewed as a result.
Internally Thresholding Objects
To threshold and measure objects on the basis of their individual intensity maxima, use the
following procedure:
Step
Action
1
Threshold the image by applying the
Threshold Image command from the
Measure menu or by using the Threshold
Tool.
2
Select the desired source image using the
Source image selector.
3
Select the desired destination image using
the Dest image selector. You can overwrite
or add to the existing image, or you can
place the results in a new image window.
4
With the Percent of Objects' Internal Max
spin box, select a percentage of the
maximum intensity in each object below
which pixels will be removed from the final
thresholding range.
5
If you want to carry out a dilation convolution
prior to measurement, select Dilate Objects
to Save Those Reduced to Single Pixel.
6
When you are ready, choose Measure.
The internal thresholding will be carried out
and a new set of object measurements will
be made. When MetaMorph has finished
remeasuring the objects, it will display the
objects in a new image window using a
pseudocolor display.
7
When you have finished, choose Close.
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Internally Threshold Objects - Dialog Box Options
Percent of Objects' Internal Max
Selects a percentage of the maximum intensity in each object below which pixels will be removed from the
final thresholding range. Pixels that fall below the selected percentage will be omitted from the new
threshold range.
Dilate Objects to Save Those Reduced to Single Pixel
Performs a dilation convolution prior to measurement. This will "grow" single-pixel objects and thereby
prevent their loss during subsequent measurement.
Source
Selects the image to be processed and measured.
Dest
Selects the destination for the pseudocolor measurement image. You can overwrite the existing image or
place the results in a new image window. Or you can add the measurement image as a plane to an existing
image or stack.
Measure
Carries out the internal thresholding procedure, measures the objects as delineated by their respective new
threshold ranges, and displays the measured objects in a new pseudocolor image.
Close
Closes the dialog box.
Create Regions Around Objects (Regions Menu)
Draws regions around objects in the currently active image or plane in a stack. You must
first threshold the image or apply the Measure Objects or Integrated Morphometry
Analysis commands.
Drop-in: TRACEOBJ
Use this command to trace the outlines of objects automatically in the current image or the current plane
of an image stack. MetaMorph will determine the edges of objects based on the distribution of the
thresholding overlay and will draw a region of interest around each object. This feature can be useful for
embellishing an image in preparation for presentation.
Note: You must first threshold the image or apply the Measure Objects or Integrated Morphometry
Analysis commands before you can apply the Create Regions Around Objects command. Consequently,
object tracing with this command requires that either the AUTOMEAS or IMA drop-in also be installed.
One particularly powerful application of this command is in the analysis of subregions within regions,
such as in the study of subnuclear components within a cell. This can be accomplished with a combined
approach involving use of the Configure Object Classifiers and Integrated Morphometry Analysis
commands.
Note: The regions that are drawn are "true" regions that can be moved and resized as with any other
region. You can move and modify these regions using the Region Toolbar .
Automatically Tracing Regions Around Objects
To create regions automatically around all objects in an image, use the following procedure:
Step
1
Action
Threshold the image by applying the
Threshold Image command from the
Measure menu or by using the Threshold
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Tool.
2
If you want to number the regions that will be
drawn, select the Draw Labels Next to
Regions check box from the Region Label
tab page of the Preferences dialog box (Edit
menu).
Note: This step can also be performed after
the Create Regions Around Objects
command has been applied.
3
From the Regions menu, choose Create
Regions Around Objects. MetaMorph will
determine the locations of the edges of each
object and will draw a region automatically
around each object. The region numbers will
correlate with the object numbering system
that MetaMorph uses, starting in the upper
left corner.
Show Classifier Statistics (Measure Menu)
Displays measurement statistics based on the objects in a measured image that pass the
selected classifier filter.
Drop-in: AUTOMEAS
Use this command when you want to display or log measurement data from objects that pass a
particular classifier filter. The image used for this command can be one that was created when
measurements were made with the Measure Objects or Measure Objects with Mask command.
The Classifier Statistics dialog displays the following statistics for each type of measurement: Count,
Average, Standard Deviation, Minimum, Maximum, and Total.
The Show Classifier Statistics dialog box can display statistics from the current measured image or an
accumulated total from all measured images. The accumulated total will include statistics compiled from
all measured images created since the beginning of the worksession or after the last time the Reset
command button was chosen.
The statistics are compiled from the selected classifier or from all classifiers combined together if the All
Classifiers option is selected. All Classifiers should be used only when the active classifier sets do not
have overlapping filters that would cause an object to be counted more than once. Data for Std. Dev.
(standard deviation) will not be available when All Classifiers is selected.
An Introduction to Automatic Object Analysis
Statistical Term Definitions
Showing Classifier Statistics
To show classifier statistics, use the following procedure:
Step
Action
1
From the Measure menu, choose Show
Classifier Statistics. The Classifier Statistics
dialog box will appear.
2
If you plan to log data, choose Open Log to
open a log file or DDE link. When you have
opened a log file. the Open Log command
will be change to "F9: Log Data."
3
Select the desired classifier set from the
Classifier list. You can select All Classifiers if
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you want to view statistics compiled from all
of the classifier sets.
MetaMorph displays the statistics for the
selected classifier set(s) at the top of the
Classifier Statistics dialog box. Only objects
that pass the classifier filters in the classifier
sets will be included the statistics.
4
If you only want to display statistics from the
current measured image, select Current from
the Show group.
OR
If you want to display an accumulated total
from all measured images, select
Accumulated from the Show group. The
accumulated total will include statistics
compiled from all measured images created
since the beginning of the work session or
after the last time the Reset command button
was chosen.
5
Choose F9: Log Data or press the [F9]
function key if you want to log the data
displayed in the dialog box. If the selected
classifier set has already been logged, the
command button will be unavailable until you
select another classifier set.
6
Repeat Steps 3 - 5 for each classifier set you
want to view.
The image window and the data in the
Classifier Statistics dialog box will be
automatically updated whenever you select a
new set from the Classifier list or modify the
classifier filters using the Configure Object
Classifiers command.
7
Choose Close when you have finished.
Show Classifier Statistics - Dialog Box Options
Classifier Statistics Table
Displays the following statistics for each type of measurement: Count, Average, Standard Deviation,
Minimum, Maximum, and Total. The width of each column can be adjusted by dragging the column dividers
to the desired width.
Open Log
Opens a summary log and/or a DDE link to an open spreadsheet application for logging data. This
command will change to F9: Log Data when a log file is open.
F9: Log Data
Logs the currently displayed data from the dialog box to an open summary log or open spreadsheet
application by way of a DDE link. To assist you in logging the appropriate data when several measurement
dialog boxes are open, "F9" will be added to the name of this option in the active dialog box to indicate
which data will be logged when you press [F9].
Classifier
Selects the classifier set for display in the Classifier Statistics Table. The statistics are compiled from the
selected classifier, or from all classifiers combined together if you select All Classifiers. You should use All
Classifiers only when the active classifier sets do not have overlapping filters that would cause an object to
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be counted more than once.
Note: Data for Std. Dev. (standard deviation) will not be available when you select All
Classifiers.
Show
Displays statistics from the current measured image or an accumulated total from all measured images. The
accumulated total will include statistics compiled from all measured images created since the beginning of
the work session or after the last time the Reset command button was chosen.
Reset
If you selected Accumulated from the Show group, this button resets the accumulated total for the statistics
from all measured images.
Close
Closes the dialog box.
Show Individual Object Data (Measure Menu)
Displays measurement data for an individual object in the last measured image created by
the Measure Objects or Measure Objects with Mask commands.
Drop-in: AUTOMEAS
Use this command when you want to display or log data for one or more objects in a measured image.
An object's data can be viewed by positioning the pointer over the object and pressing the left mouse
button, or you can select the object ID number from the Object Number spin box. The data will appear in
the Individual Object Data dialog box and an object ID number stamp will label the object in the image.
When an object's data are displayed, the measurements can be logged to an object log or an edgelist
log.
Note: The Show Individual Object Data dialog box will close if you reset the object measurements with
the Reset Object Measurements command, since there will be no object data to display in the Individual
Object Data dialog box.
An Introduction to Automatic Object Analysis
Measurement Term Definitions
Statistical Term Definitions
Showing Individual Object Data
To show individual object data, use the following procedure:
Step
Action
1
Measure the desired image using the
Measure Objects or Measure Objects with
Mask command.
2
From the Measure menu, choose Show
Individual Object Data. The Show Individual
Object Data dialog box will appear.
An object ID number stamp will appear on
the image. (The first object is determined by
finding the topmost object, left to right.)
3
If you plan to log data, choose Open Log to
open an object log or DDE link to an open
spreadsheet application.
Once you have opened a log file, the Open
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Log command will be replaced by the F9:
Log Data command.
4
To display an object's data in the Show
Individual Object Data dialog box, select its
object number using Object and press the
[TAB] key.
If you do not know the object's number, you
can position the pointer over the desired
object in the measured image and click the
left mouse button. MetaMorph will display the
object ID number and data for the closest
object (based on the position of its centroid).
The object ID number for the displayed
object will be stamped on the measured
image window for identification purposes.
5
Choose F9: Log Data to log the data
displayed in the Show Individual Object Data
dialog box. If the data for the selected object
have just been logged, the command button
will be unavailable until you select another
object.
6
Choose Close to close the Show Individual
Object Data dialog box.
Show Individual Object Data - Dialog Box Options
Individual Object Data Table
Displays the selected object's measurement values. The width of each column can be adjusted by dragging
the column dividers to the desired width.
Open Log
Opens an object log and/or a DDE link to an open spreadsheet application for logging data. This command
will change to F9: Log Data when a log file is open.
F9: Log Data
Logs the currently displayed data from the dialog box to an open object log or DDE-linked spreadsheet. To
assist you in logging the appropriate data when several measurement dialog boxes are open, "F9" will be
added to the name of this option in the active dialog box to indicate which data will be logged when you
press [F9].
Object
Selects and lists the object whose data is displayed in the Individual Object Data Table at the top of the
dialog box. Objects can also be selected for display by positioning the pointer over the object in the
measured image and pressing the left mouse button.
Reset Object Measurements (Measure Menu)
Clears the classifier statistics summaries derived from the last measured image.
Drop-in: AUTOMEAS
Use this command when you want to clear the temporary buffer that MetaMorph uses to store classifier
statistics summaries from the last measured image.
Note: This command is independent of the Integrated Morphometry Analysis command functions.
Choosing this command will not reset the IMA measurements, and choosing the IMA Reset button will
not reset measurements made with the Measure Objects command.
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An Introduction to Automatic Object Analysis
Resetting Object Measurements
To reset object measurements, use the following procedure:
Step
Action
1
From the Measure menu, choose Reset
Object Measurements.
2
If there are measurements stored in
MetaMorph's buffer the Object
Measurements dialog box will appear.
Note: This dialog box will not appear unless
you select the Warn User When
Measurement Data Will Be Erased check
box in the Preferences dialog box's Measure
Objects tab page.
3
Choose Yes if you want to reset the object
measurements.
OR
Choose No to cancel the command.
Create Classifier Stack (Measure Menu)
Creates a stack from the results of the Measure Objects command, displaying objects that
match each classifier filter in separate planes.
Drop-in: MSTACK
Use this command after you have applied the Measure Objects command to an image and want to
display the objects that pass each filter in separate planes of an image stack.
Before using this command, you must configure the classifier filters to include or exclude objects from
measurements using the Configure Object Classifiers command, and then measure the objects using the
Measure Objects command.
Creating a Classifier Stack
To create a classifier stack, use the following procedure:
Step
Action
1
Configure the desired classifier filters using
the Configure Object Classifiers command.
2
Measure the objects using the Measure
Objects command.
3
Select the measured image so that it is the
active image.
4
From the Measure menu, choose Create
Classifier Stack. A stack will be created,
consisting of the objects that match each
filter displayed in a separate plane.
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Measure Distance with Annotation (Measure
Menu)
Displays the distance of a line drawn with a Line Region Tool and logs the current text
annotation whenever the distance measurement data are logged.
Drop-in: ANDIST
Use this command to determine the distance of an object or area of interest within an image, and to log
an annotation each time a distance measurement is logged with the Log Data command.
The distance measurement can be displayed in units which have been calibrated with the Calibrate
Distances command. If the units have not been calibrated, MetaMorph will display the distance in pixels.
You can log each measurement to an open data log if desired. First, use the Open Data Log command
to open a data log. You can use Log Data or its keyboard shortcut, the [F9] function key, to log the data.
Measuring Distance with Annotation
To measure the distance of a line and make an annotation, use the following procedure:
Step
Action
1
From the Measure menu, choose Measure
Distance with Annotation. The Measure
Distance with Annotation dialog box will
appear.
2
Select the desired image using the Image
selector.
3
Choose Open Log to open a data log.
4
To configure the data log for logging, choose
Configure Log. The Configure Log dialog box
will appear.
AND
From the Configuration list, select the
parameters you want to enable for logging,
so that each is marked by a check mark next
to its name (you can choose Enable All or
Disable All if you want to select or deselect
all of the parameters listed).
Choose OK to return to the Measure
Distance with Annotation dialog box.
5
Draw a line of the desired distance using the
Single Line Tool, Multi-Line Tool, or Traced
Line Tool.
6
Select the desired line so that it is the active
region. MetaMorph will measure the distance
of the line and display it in the dialog box.
7
Type the desired annotation in the Log
Annotation text box. You can delimit entries
that are to be sent to separate columns in the
data log by separating them with commas
when you enter them into the annotation text
box.
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8
When you want to log the measured distance
and annotation, choose F9: Log Data.
9
To measure another distance, create and
select another line using the process
described in Steps 5 - 8.
Note: You can edit the distance of the line by
double-clicking the line using the left mouse
button and dragging the round handles that
appear.
10
Choose Close when you have finished
measuring the distances.
Measure Distance with Annotation - Dialog Box Options
Image
Selects the image for measuring distances and annotating.
Distance
Displays the data from the current distance measurement.
Log Annotation
Specifies the annotation to be logged when you choose F9: Log Data. You can delimit entries that are to be
sent to separate columns in the data log by separating them with commas when you enter them into the
annotation text box.
Open Log
Opens a data log and/or a DDE link to an open spreadsheet program for logging data. This command
changes to F9: Log Data when a log file is open.
F9: Log Data
Logs the currently displayed data from the dialog box to an open data log or to an open spreadsheet by way
of a DDE link. To assist you in logging the proper data when several measurement dialog boxes are open,
"F9" will be added to the name of this option in the active dialog box to indicate which data will be logged
when you press [F9].
Configure Log
Opens the Configure Log dialog box so that you can select the parameters to be logged to the data log.
Parameters marked with a check mark will be logged for subsequent measurements.
If you select Log Column Titles, a line listing the measurement titles will be logged (1) the first
time you use the configured measurement, (2) whenever you enable/disable measurement
parameters, or (3) whenever the logged measurement is different from the previous measurement
in the log file.
If you select Place Log Data on Current Line, subsequently logged data will be appended to the
current line in the log file, rather than on a new line. Log Column Titles will be unavailable when you
select this option.
Close
Closes the dialog box.
Graph Intensities (Apps Menu)
Measures and logs intensity data from selected regions in an image stack or in a series of
live video images.
Availability: Available for MetaVue; included in MetaMorph Premier and MetaMorph Basic
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Drop-in: BTIME
Use this command to measure intensity values
(1) Over time from a series of live video images,
(2) Over time, plane number, Z-axis distance, or wavelength from a stack of images.
Intensity data that can be measured, graphed, and logged include such parameters as are displayed in
the Show Region Statistics dialog box. These include the average intensity, standard deviation of the
intensity, integrated intensity (summed over all pixels in the region), maximum and minimum grayscale
levels, thresholded area (expressed as either numbers of pixels or as a percent of total), and the like.
The HSI intensity value is used for logging color images.
For a stack, intensity measurements over time are determined by the acquisition time of each plane in
the stack. Alternatively you can measure the intensity values over plane number. Intensity
measurements can be made over wavelength for stacks acquired using the Acquire Spectral Scan
command.
If you are using a digital camera, Measure Brightness uses the acquisition settings from the Acquire from
Digital Camera command.
Graphing Intensities
To measure brightness over time, plane, or frame, use the following procedure:
Step
Action
1
From the Apps menu, choose Graph
Intensities. The Configure Graph Intensities
dialog box opens.
2
From the Measure From group, select Live
Video, Disk, or Stack.
3
From the Measurement drop-down list, select
the intensity statistic you want to measure
and graph.
4
If you want to measure only those pixels that
fall within the threshold settings, select Use
Threshold for Intensity Measurements.
5
If you selected Live Video in Step 2, choose
Acquisition to open the Configure Acquisition
dialog box and chose acquisition options.
Additional Acquisition Options
6
If you selected Stack in Step 2, select the
desired stack from the Image selector.
AND
From the Measure Regions Over group,
select Time, Plane Number, Z Distance, or
Wavelength.
7
If you selected Disk in Step 2, choose Select
Files and select the image files you want to
measure.
8
Select X Axis to configure the X-axis. Select
the desired Range, Number of Planes, or
Number of Frames (as applicable). Select
the number of Tick Marks for the axis. The
number should be divisible by the range or
number of tick marks.
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If you are measuring regions over time,
select the unit of time using Time Scale.
Then choose OK.
9
Choose Y Axis to configure the Y-axis.
Select the Minimum Gray Level and
Maximum Gray Level values. Then select the
desired number of Tick Marks.
If you want to use calibrated values, select
Use Calibrated Gray Values. Then choose
OK.
10
To configure an open data log for logging,
choose Configure Log.
11
Choose OK to continue to the Measure
Regions dialog box.
After you have completed the preceding steps, use the following procedure to measure region
intensity values.
Note: If you are working with a stack, you need to have the Select Plane dialog box
opened and positioned in an accessible location during brightness measurements.
Step
1
Action
If you are working with live video, choose
Refresh Video. This will update the image
presented in the Live Video image window
with the most recently acquired frame. It is
recommended that you do this before
creating new regions.
Note: You must choose Refresh Video to
update the video window, or else have open
the live video window for the acquisition
command appropriate for your video device
(Live Video, Acquire from Digital Camera,
Acquire from Flashbus, etc.).
2
Create region(s) in the live video or stack
image window using a Region Tool. You can
modify or add regions at any time during the
measurements.
Note: Regions drawn with a two-dimensional
Region Tool (Rectangular, Ellipse, Trace, or
Auto-Trace) must be at least 2x2 in size to
be valid in MetaMorph.
3
To log data to an open data log, choose F9:
Log Data.
4
Choose Begin to start measuring brightness
and graphing the intensity values for the
regions in the Measure Regions graph.
If you are using a stack, the Select Plane
dialog box will appear. Begin will change to
Play Stack after it has been selected. You
can use the options in the Select Plane
dialog box to start and stop the stack.
Choosing Play Stack resets the stack to the
first plane and plays it.
OR
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If you are using live video, the Begin button
will change to a Pause/Resume button.
5
You can attach event notes at any time by
typing a short text description in the text box
at the bottom of the dialog box and choose
New Event.
A line marker will be placed inside the graph
at the appropriate spot along the X-axis.
6
If you want to change the X-axis or Y-axis
configuration settings during acquisition,
choose Change X-Axis or Change Y-Axis.
When you have adjusted the options as
desired in the appropriate dialog box, choose
OK to begin acquisition again.
7
When you have graphed the data, you can
click and hold the pointer over any point
within the graph to view a summary of that
point's data. The scroll bar will change to a
data display. The bullets preceding the data
and the trace line in the graph are colorcoded to match the region outline.
For more information about configuring
graph, refer to Working with Graphs .
You can choose Clear if you want to clear
the data from the graph.
8
Choose Close to close the dialog box and
graph when you have finished.
Selecting Files from Disk for Measuring Regions
Selection Type
Selects the method by which you specify which files to measure:
Quick Select will allow you to pick the first file in a series that is numbered by file name or by
extension, and will automatically select all other files in that series.
Numbered Names and Numbered Extensions allow you to select the first and last numbered
files in their respective series.
Select File
This button will appear when Quick Select has been selected as the Selection Type. Choose this to select
the first file in a series of files that have numbered file names or extensions. MetaMorph will automatically
select all subsequent files in that series.
Select First
This button will appear when either Numbered Names or Numbered Extensions is selected as the Selection
Type. Choose this to select the first file in a series of files that have numbered file names or extensions.
Select Last
This button will appear when either Numbered Names or Numbered Extensions is selected as the Selection
Type. Choose this to select the last file in a series of files that have numbered file names or extensions.
Graph Intensities - Options
Configure Graph Intensities
Configure Acquisition
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Graph Intensities
Configure Graph Intensities - Dialog Box Options
Measure From
Specifies whether the brightness measurements will be made from a stack, from image files residing on
disk, or from live video.
Measure Regions Over
Specifies whether intensity values will be measured over Time, Plane Number, Z Distance, Wavelength or
Frame. Time is the only option available for live video.
Measurement
Selects an intensity statistic to be measured, graphed, and logged over image frames. The selections
include: Thresholded Area, Average Intensity (which, in 24-bit color images, takes the arithmetic mean for all
three channels), Standard Deviation, Signal to Noise, Integrated, Minimum, Maximum, % Thresholded Area,
Average Red, Average Green, Average Blue, and Average (Red, Green, Blue) (which gives the mean for
each of the three channels simultaneously).
Use Threshold for Region Measurements
Specifies that only pixels whose gray values fall within the threshold settings will be included in the intensity
measurements. For example, if you want view the average gray value of all non-zero pixels in an 8-bit
image, set the image's threshold range from 1 - 255 and select this option.
Image
Specifies the stack to measure if Stack is selected in the Measure From group.
Select Files
Opens the Select Files dialog box which is used to select which files on disk are to be measured.
Acquisition
Opens the Configure Acquisition dialog which is used to select the desired acquisition interval and additional
acquisition options.
X-Axis
Configures the X-axis. If you are measuring brightness over time, the unit of time is selected with the Time
Scale option. For all measurements, the desired range or number of planes and the number of tick marks for
the axis are selected. You can select a tick mark value between the range of 2 to 1000.
Y-Axis
Configures the Y-axis. The minimum and maximum gray values and the tick marks for the axis are selected.
The use of calibrated gray values is also enabled using this dialog box. You can select a tick mark value
between 2 and 1000.
Configure Log
Opens the Configure Log File dialog box so that you can select the parameters to be logged to the log file.
Note:
The New Event button in the Measure Regions dialog box will only be enabled if you turn
on "Event" in the list of parameters to measure in the Configure Log File dialog box.
OK
Sets the configuration options for measuring brightness and opens the Measure Regions dialog box.
Cancel
Cancels the command.
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Configure Log - dialog box options
Configuration
Specifies the parameters to be logged. Parameters marked with a check mark will be logged in subsequent
measurements. Double-click the desired parameter to mark it with a check mark.
Enable All
Enables all parameters in the Configuration list.
Disable All
Disables all parameters in the Configuration list.
Log Column Titles
If Log Column Titles is selected, a line listing the measurement titles will be logged (1) the first time you use
the configured measurement, (2) whenever you are enable/disable measurement parameters, or (3)
whenever the logged measurement is different from the previous measurement in the log file.
Place Log Data on Current Line
When you select this option, subsequently logged data will be appended to the current line in the log file,
rather than to a new line. The Log Column Titles option will become unavailable when you select this option.
OK
Reconfigures the log file.
Cancel
Cancels the Configure Log command.
Configure Acquisition (Graph Intensities) - Dialog Box Options
Measure Every
Selects the number and unit (Frames, Milliseconds, Seconds, Minutes, or Hours) for the time interval
between measurements from Live Video.
Illum
Selects the illumination setting associated with the shutter. If you are not using a shutter, leave this selection
as "[None]."
Force Image Acquisition
Acquires an image into the frame buffer and then measures the brightness from the acquired image, rather
than directly from the Live Video image. This is useful when using a digital camera or when acquiring over a
very long time interval. Measure Regions uses the settings from Acquire Image, rather than from Live Video,
if you are not using a digital camera.
Force Image Update
Redraws the image window for each measurement taken on the graph. Checking this box can slow down
the Graph Intensities command.
Use Journal for Acquisition
Runs the selected journal instead of the Acquire Image command. The journal must include an image
acquisition command.
OK
Accepts the current acquisition configuration .
Cancel
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Cancels the acquisition configuration and closes the Configure Acquisition dialog box.
Graph Intensities - Dialog Box Options
Log Data
Enables/disables logging of data to the open data log.
Begin
Starts measuring brightness and graphing intensity values for the selected region to the Measure Regions
graph. If you are measuring from a stack, the Select Plane dialog box will appear and Begin will change to
Play Stack. If you are measuring from live video, Begin will change to Pause/Resume.
Pause/Resume
If you are measuring from live video, the Begin button changes to a Pause/Resume button. When live video
is paused using Pause, the Refresh Video command will be carried out for you automatically.
Change X Axis
Changes the X-axis configuration options. If you are measuring brightness from a stack, you will need to
choose Stop in the Select Plane dialog box to pause the playing of the stack before choosing this command.
Change Y Axis
Changes the Y-axis configuration options. If you are measuring brightness from a stack, you will need to
choose Stop in the Select Plane dialog box to pause the playing of the stack before choosing this command.
Clear Graph
Clears the data from the Measure Regions graph.
Refresh Video
Updates the image in the live video image window with the most recently acquired frame.
Note:
You must choose Refresh Video to update the video window, or else have open the live
video window for the acquisition command appropriate for your video device (Live Video,
Acquire from Flashbus, etc.).
New Event
Records the event text in the text box next to the command in the Measure Regions graph and in the log file,
if applicable. A marker is placed at the appropriate spot along the graph's X-axis.
Note:
The New Event button in the Measure Regions dialog box will only be enabled if you turn
on "Event" in the list of parameters to measure in the Configure Log File dialog box.
Close
Closes the Measure Brightness dialog box and the graph.
Calipers (Measure Menu)
Measures the distance between a pair of movable "caliper" lines.
Drop-in: CALIPERS
Use this command to measure the distance between a pair of parallel lines that can be dragged
displayed on an image. This may be particularly useful for making quick determinations of distances in
live images during focusing or acquisition. The length of the two caliper lines can be resized, and the
location, distance between, and angle of the calipers can be altered by dragging various points on the Hshaped caliper display with your pointer. Both the caliper lines and their measured distance values can
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be stamped directly onto the image. Distance measurements can be logged to a data log, which you can
open and configure from within the Calipers dialog box.
Note: To express the distance measurement in "real" units, be sure to apply the Calibrate Distances
command before stamping the image or logging the measurement.
Performing Distance Measurements with Calipers
To use the calipers for distance measurements, use the following procedure:
Step
Action
1
From the Measure menu, choose Calipers.
The Calipers dialog box will appear, and an
H-shaped set of caliper lines will appear in
the active image window.
2
If necessary, use the Image selector to select
the image to be measured.
3
If desired, you can select the color in which
the calipers and the measured distance
value are displayed by using the Color dropdown list. This will affect only the color of the
caliper display, not the stamp (see Step 7).
4
The calipers can be moved by single-clicking
the cross-bar so that it is displayed as a
blinking line, indicating that it is active, and
then dragging the cross-bar to the desired
location with your pointer.
5
You can adjust the distance between the two
caliper edge lines in one of three ways. The
distance between the calipers will be
displayed on the image, and the value in the
Distance spin box will be updated:
(1) Single-click one of the caliper edge
lines so that it is displayed as a blinking
line. Then drag the line to the desired
distance. The other caliper line will
remain anchored.
(2) Double-click the caliper cross-bar so
that "nodes" appear at each end. With
your pointer, drag one of nodes away
from the other to the desired distance.
(3) Use the Distance spin box to specify
the distance between the caliper lines.
6
Similarly, you can change the angle of the
calipers in one of three ways. The value in
the Angle spin box will be updated:
(1) Double-click one of the caliper edge
lines so that nodes appear at each end.
With your cursor, drag one of nodes up
or down until the desired angle is
achieved. The other node will act as a
pivoting anchor. Note: You can extend
the length of the end lines simultaneously
while you adjust the angle of the calipers
by dragging the edge line node.
(2) Double-click the caliper cross-bar so
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that nodes appear at each end. With your
pointer, drag one of nodes up or down
until the desired angle is achieved. The
other node will act as a pivoting anchor.
Note: You can lengthen the cross-bar
simultaneously while you adjust the angle
of the calipers by dragging the cross-bar
node.
(3) Use the Angle spin box to specify the
angle of the calipers. Angle values can
range from -180 to 180 degrees, rotating
counterclockwise from the horizontal.
7
If you wish to stamp the caliper lines and the
measured distance value directly on the
image, you can specify a color for the stamp
that differs from the color of the caliper
display. This will be particularly useful if you
plan to make several measurements.
To specify a color for the stamp, select the
desired color from the Overlay Color dropdown list.
8
If you do not wish to log the measurement
values, you can stamp the values and the
caliper lines directly on the image by
choosing Stamp. If necessary, you can
remove the stamps by choosing Clear
Overlay. (Alternatively, you can choose Clear
Measurement Stamps from the Graphics
menu, or use its keyboard shortcut, [ALT] +
[C].) Repeat Steps 6 - 8 as desired for
additional measurements and stamps. Then
skip to Step 11.
OR
If you plan both to log the measurement data
and to stamp the image, you have the option
of stamping the image automatically when
you log the data. To configure the Calipers
command to do so, select the Stamp as Data
Is Logged check box.
9
Open a data log using the Open Log
command button. When the data log is open,
the text on the Open Log button will change
to F9: Log Data.
AND
To configure the data log for logging, choose
Configure Log. Select the parameters you
want to log by double-clicking their entries in
the Configuration list and choose OK to
return to the Calipers dialog box.
10
When you are ready, choose F9: Log Data,
or press the [F9] function key.
11
When you have finished, choose Close.
Calipers - Dialog Box Options
Image
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Selects the image to be measured.
Distance
Specifies a distance between the two caliper edge lines. This spin box will update when the arrangement of
the caliper lines is changed in the image window.
Angle
Specifies an angle for the caliper lines. Angle values can range from -180 to 180 degrees, rotating
counterclockwise from the horizontal. This spin box will update when the arrangement of the caliper lines is
changed in the image window.
Color
Selects the color in which the calipers and the measured distance value are to be displayed. This will not
affect the color of the overlay stamp.
Open Log
Opens a data log and/or a DDE link to an open spreadsheet application for logging data. This command will
change to F9: Log Data when a log file is open.
F9: Log Data
Logs the currently displayed data from the dialog box to an open data log or to an open spreadsheet
application via a DDE link. To assist you in logging the appropriate data when several measurement dialog
boxes are open, "F9" will be added to the name of this option in the active dialog box to indicate which data
will be logged when you press [F9].
Configure Log
Opens the Configure Log dialog box so that you can select the parameters to be logged to the log file.
Overlay Color
Specifies a color for the stamp. This will be particularly useful if you plan to make several measurements.
Stamp as Data Is Logged
Configures the Calipers command to stamp the image automatically when you log distance measurement
data.
Clear Overlay
Removes all stamps from the image.
Stamp
Stamps the image with the current arrangement of caliper lines and the measurement value. This stamp will
become a permanent part of the image overlay unless you click the Clear Overlay button or choose Clear
Measurement Stamps from the Graphics menu.
Close
Closes the dialog box.
Count Cells (Apps Menu)
Counts single cells automatically in a clear 8-bit, 16-bit, or 24-bit image.
Availability: Available for MetaVue and MetaMorph Basic; included in MetaMorph Premier
Drop-in: CELLCNT
Use this command for simple cell-counting based on
(1) Threshold-based detection of discrete cell boundaries,
(2) Use of an interactively selected Standard Area, or
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(3) Use of an interactively selected integrated pixel intensity value.
If you need to perform more complicated morphometric measurements, it may be better to do so by
using standard morphometric methods, such as the Integrated Morphometry Analysis command
(Measure menu). To separate two types of cells from each other based on their staining, such as when
measuring live vs. dead cells, you should use the Count 2 Types of Cells command, also located in the
Measure menu.
Note: Because of its reliance on threshold ranges, Count Cells is not appropriate for use
with binary images.
Boundary-based detection uses the fact that objects in an image that are of importance tend to have
pixel intensities that are significantly different from background. When you define a threshold range, the
Count Cells command can measure the number of cells in your image automatically, based on the
boundaries of what MetaMorph perceives as discrete, single objects.
Counting objects is often complicated by the fact that objects overlap or clump together when
thresholded, resulting in counts that are lower than the actual number of objects present. Using the
second cell-counting method allows you to define a Standard Area. This is a value that will be used to
represent the size of a typical cell, based on the assumption that the cells you want to count are of fairly
uniform size (area). You can select a Standard Area interactively with the Count Cells command by
clicking objects in the image window. MetaMorph will calculate an average size for all of the cells you
click. Objects will then be counted by dividing the total thresholded area of each clump by the Standard
Area. Clumps that are less than half the Standard Area will be omitted from the final cell count.
The third method is in some ways similar to the second in that a standard is selected interactively by
clicking objects in the image. This method, however, is based on the integrated pixel intensity, that is, the
arithmetic sum of the intensity values of all pixels in the thresholded area. This value will be used as a
standard for the subsequent measurement and counting. This may be useful under conditions in which
moderately fluorescent cells are clumped together, resulting in a region that is smaller than in size than
the number of cells present, but the fluorescence of the clumped cells is noticeably additive. As with the
use of a Standard Area, thresholded objects that have an integrated intensity less than half the standard
value will be omitted from the final cell count.
Integral to each of these methods is the selection of a threshold range. Regardless of the method you
choose, you will need to define a threshold range to separate the objects of importance from their
background. Count Cells keeps its own threshold, ignoring any other current thresholding in the image.
To set or adjust the thresholding used by Count Cells, use the Set Threshold button in the Count Cells
dialog box for 24-bit images. Use the Image Window Threshold Slider to adjust the thresholding used in
8- or 16-bit images. The Measure button in the Count Cells dialog box will then apply the thresholding
automatically during the measurement process. If you need to change the thresholding, you can use the
dialog box's Set Threshold command button. When you have finished, the measurement will proceed
automatically.
Counting Cells
Overview of Single-Cell Counting
Configuring Measurement Based On…
Object Boundaries
Standard Area
Integrated Intensity
Setting Color Thresholding
Configuring Data Logging
Overview of Single-Cell Counting
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To perform simple single-cell counting, use the following general procedure:
Step
Action
1
From the Apps menu, choose Count Cells.
The Count Cells dialog box opens.
2
If necessary, select the image containing the
cells you want to count with the Source
Image selector.
3
If you want to view an image that highlights
the counted cells and displays the number of
cells in each cluster next to it, select the
Display Result Image check box. This may
be useful for comparison with the original
source image to determine how good your
configuration and thresholding was.
4
Configure your measurement by choosing
the Configure button. Your configuration
procedure will depend on whether you want
to base your measurement on
Detection of cell boundaries,
Use of a Standard Area, or
Use of a standard integrated intensity.
5
If the image is 24-bit and you need to adjust
the thresholding, click Set Threshold to open
the Set Color Threshold dialog box.
6
Click Use Images’s Threshold to use the
image’s own threshold and automatically run
the measure command. The number of cells
that are detected will appear in the Count
status line of the Count Cells dialog box.
7
When you are ready to carry out the
measurement, choose Measure. The number
of cells that are detected will appear in the
Count status line of the Count Cells dialog
box.
8
If you want to save the measurement data,
you can open a data log. The Open Log
button will become a Log Data button. When
you are ready to log the count data, choose
Log Data.
9
When you have finished counting cells,
choose Close.
Configuring Cell-Count Measurement Based on Object Boundaries
To configure cell-counting based on the boundaries of thresholded objects, use the following
procedure:
Step
Action
1
From the Count Cells dialog box, choose
Configure. The Set Counting Method dialog
box will appear.
2
From the Counting Method radio button
group, select Count Each Discrete Object
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as 1.
Configuring Cell-Count Measurement Based on Standard Area
To configure cell-counting based on a Standard Area, use the following procedure:
Step
Action
1
From the Count Cells dialog box, choose
Configure. The Set Counting Method dialog
box will appear.
2
From the Counting Method radio button
group, select Use Standard Area to Estimate
Objects in a Cluster.
3
Choose Next >. The Set Counting Method
dialog box will close, and the Set Standard
dialog box will appear. Simultaneously, a
blue overlay will appear over any thresholded
regions of the image.
5
As necessary, use the Source Image
selector to select the image containing
objects that you want to use to define the
standard. This does not need to be the same
image as the one containing the cells you
want to count. For example, you could use
an image containing objects of a known size,
such as microspheres.
6
If you need to adjust the thresholding for the
image, click Set Threshold. The Set Color
Threshold dialog box opens if the image is in
color; if not, a red overlay is placed over the
image and you can adjust the threshold
using the Image Windows threshold slider.
After you finish selecting the threshold range
and have closed the Set Threshold dialog
box, you will return to the Set Standard
dialog box..
7
In the image window, click several objects
that are of a "typical" size. As you select the
objects, a yellow overlay will appear over
them in the image window, and the Total
Area, Count, and Standard Area spin boxes
will update, displaying the total area
selected, the number of selected cells, and
the average computed area, respectively.
You can modify these values directly, if
desired.
8
When you have finished, choose Next >. The
Set Standard dialog box will close, and the
Count Cells dialog box will reappear.
Configuring Cell-Count Measurement Based on Integrated Intensity
To configure cell-counting based on a standard integrated intensity value, use the following
procedure:
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Step
Action
1
From the Count Cells dialog box, choose
Configure. The Set Counting Method dialog
box will appear.
2
From the Counting Method radio button
group, select Use Integrated Intensity to
Estimate Objects in a Cluster.
3
Choose Next >. The Set Counting Method
dialog box will close, and the Set Standard
dialog box will appear. Simultaneously, a
blue overlay will appear over any thresholded
regions of the image.
4
As necessary, use the Source Image
selector to select the image containing
objects that you want to use to define the
standard. This does not need to be the same
image as the one containing the cells you
want to count. For example, you could use
an image containing objects of a known
brightness, such as fluorescent beads of a
specific diameter.
5
If you need to adjust the thresholding for the
image, click Set Threshold. The Set Color
Threshold dialog box opens if the image is in
color; if not, a red overlay is placed over the
image and you can adjust the threshold
using the Image Windows threshold slider.
After you finish selecting the threshold range
and have closed the Set Threshold dialog
box, you will return to the Set Standard
dialog box.
6
In the image window, click several objects
that are of a "typical" brightness and size. As
you select the objects, a yellow overlay will
appear over them in the image window, and
the Total Intensity, Count, and Unit Intensity
spin boxes will update, displaying the total
integrated intensity, the number of selected
cells, and the average integrated intensity,
respectively. You can modify these values
directly, if desired.
7
When you have finished, choose Next >. The
Set Standard dialog box will close, and the
Count Cells dialog box will reappear.
Configuring Data Logging for Cell-Count Measurement
To configure logging of your single-cell count data, use the following procedure. You can follow
this procedure either before or after you perform the actual measurement.
Step
Action
1
From the Count Cells dialog box, choose
Open Log. The Open Data Log dialog box
will appear.
2
Select Dynamic Data Exchange (DDE) to log
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directly to an open spreadsheet program.
Select A Text File to log the data to a text
file.
Note: You can select both options.
3
If you selected A Text File in the previous
step, the Open Data Log File dialog box will
appear.
Select an existing log file or type a new file
name in the File Name text box. (If
necessary, use the Look In list or Up One
Level button to change the current drive and
folder to the correct location.)
AND
Choose Open.
4
If you selected an existing log's file name in
Step 3, the Log File Exists dialog box will
appear. You can Overwrite the contents of
the file, Append new data, or Cancel.
5
If you selected Dynamic Data Exchange
(DDE) in Step 2, the Export Log Data dialog
box will appear.
Select the desired application from the
Application list. Choose Default to use the
default settings for the selected application.
Choose OK to launch the external
application. After the application opens, click
the MetaMorph Imaging System button in the
Windows Taskbar to return to MetaMorph.
6
If necessary, choose Configure Log from the
Count Cells dialog box to select which data
to log. The Configure Log dialog box will
appear.
AND
From the Configuration table, select the data
types that you want to log. Then choose OK.
7
The Open Log button will have changed to a
Log Data button. To log your cell count data,
choose this button after you have performed
your measurement.
8
If necessary, click the Back button at the top
of this Help window to return to the
procedure you were just reading.
Count Cells - Dialog Box Options
Configure
Opens the Set Counting Method dialog box, from which you can select the counting method (boundarybased, Standard Area-based, or integrated intensity-based).
Set Threshold (24-bit Images Only)
Opens the Set Color Threshold dialog box, from which you can change the thresholding range. This should
only be necessary for images in which the thresholding has never been set, or in cases where you want to
change the thresholding.
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Source Image
Selects the image containing the cells you want to count.
Use Image’s Threshold
Applies the image’s own threshold and automatically run the measure command. The number of cells that
are detected will appear in the Count status line of the Count Cells dialog box.
Open Log
Opens a text-based data log file or an OLE connection to an open spreadsheet program for logging count
data if desired. Changes to "Log Data" when a log file is open.
Log Data
Sends your cell-count measurement data to the open data log or spreadsheet.
Configure Log
Opens the Configure Log dialog box, from which you can select the types of data to be stored in the data
log.
Display Result Image
When selected, displays a measurement image in a separate window, showing only the objects that have
been counted, along with a number next to each cluster which indicates the number of cells in the cluster.
Count
This status line indicates the number of cells that have been counted.
Measure
Carries out the measurement, based on the configuration of the counting method and the current threshold
state of the image. (Note: If you change the threshold range with the Set Threshold command button,
measurement will proceed automatically when you have finished.)
Close
Closes the Count Cells dialog box.
Set Counting Method (Count Cells) - Dialog Box Options
Counting Method
Selects a cell-counting method:
Count Each Discrete Object as 1 - Bases the cell-count measurement on what MetaMorph perceives to be
the object boundaries, as defined by thresholding. When you select this option, choosing Next > will return
you to the Count Cells dialog box, from which you should then select a thresholding range. After you select
the threshold range, measurement will proceed automatically.
Use Standard Area to Estimate Objects in a Cluster - Bases the cell-count measurement on a selected
Standard Area. When you select this option, choosing Next > will open the Set Standard dialog box, which
you will use to select the Standard Area.
Use Integrated Intensity to estimate Objects in a Cluster - Bases the cell-count measurement on a standard
integrated intensity value. When you select this option, choosing Next > will open the Set Standard dialog
box, which you will use to select the standard integrated intensity.
Cancel
Cancels your selection of cell-counting method and closes the Set Counting Method dialog box, returning
you to the Count Cells dialog box.
Next >
Accepts your selection of cell-counting method and closes the Set Counting Method dialog box. If you
selected Count Each Discrete Object as 1, you will return to the Count Cells dialog box. If you selected
either of the other two methods, the Set Standard dialog box will appear.
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Set Standard (Count Cells) - Dialog Box Options
Source Image
Selects the image containing the cells you want to use to define the standard area or integrated intensity.
This does not need to be the same image as the one containing the cells you want to count. For example,
you could use an image containing objects of a known size or brightness, such as microspheres or
fluorescent beads of a specific diameter.
Set Threshold
24-bit Images:
Opens the the Set Color Threshold dialog box, from which you can change the thresholding range
for the image you are using to define your standard (which could be the same as the image
containing the cells you want to count).
8- or 16-bit Images:
Places a red overlay over the image. You can adjust the thresholding using the Image Windows
threshold slider.
Clear Counts
Clears the Total Area or Total Intensity settings and the Count setting.
Total Area
Indicates the summed area, in pixels, of all objects that have been selected. This option is seen when you
select the Standard Area-based measurement method.
Total Intensity
Indicates the summed intensity values of all pixels in the objects that have been selected. This option is
seen when you select the integrated intensity-based measurement method.
Count
Indicates the number of objects that have been selected for defining the standard value.
Standard Area
Indicates the size of the Standard Area, based on the Total Area and the Count. This option is seen when
you select the Standard Area-based measurement method.
Unit Intensity
Indicates the unit intensity value, based on the Total Intensity and the Count. This option is seen when you
select the integrated intensity-based measurement method.
< Back
Cancels the standard selection process and returns you to the Set Counting Method dialog box.
Next >
Accepts the standard selection and returns you to the Count Cells dialog box.
Cancel
Cancels the standard selection process and returns you to the Count Cells dialog box.
Count 2 Types of Cells (Apps Menu)
Counts two types of cells automatically in separate 8-bit or 16-bit images or in a single 24bit image.
Drop-in: CELLCNT
Use this command to count cells that have been labeled with two different fluorescent dyes. In particular,
this command has been optimized to distinguish between live and dead cells based on their appearance
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after staining with a Live/Dead or bacterial staining kit. Measurement of the two types of cells will then
proceed based on
(1) Threshold-based detection of discrete cell boundaries,
(2) Use of an interactively selected Standard Area, or
(3) Use of an interactively selected integrated pixel intensity value.
If you need to perform complicated morphometric measurements, it may be better to do so by using
standard morphometric methods, such as the Integrated Morphometry Analysis command (Measure
menu). To perform simple single-cell counting, you should use the Count Cells command, also located in
the Measure menu.
Note: Because of its reliance on threshold ranges, Count 2 Types of Cells is not
appropriate for use with binary images.
Boundary-based detection uses the fact that objects in an image that are of importance tend to have
pixel intensities that are significantly different from background. When you define a threshold range for
one of your cell types, the Count 2 Types of Cells command can measure the number of those cells in
your image automatically, based on the boundaries of what MetaMorph perceives as discrete, single
objects.
Counting objects is often complicated by the fact that objects overlap or clump together when
thresholded, resulting in counts that are lower than the actual number of objects present. Using the
second cell-counting method allows you to define a Standard Area. This is a value that will be used to
represent the size of a typical cell, based on the assumption that the cells you want to count are of fairly
uniform size (area). You can select a Standard Area interactively with the Count 2 Types of Cells
command by clicking objects in the image window. MetaMorph will calculate an average size for all of
the cells you click. Objects will then be counted by dividing the total thresholded area of each clump by
the Standard Area. Clumps that are less than half the Standard Area will be omitted from the final cell
count.
The third method is in some ways similar to the second in that a standard is selected interactively by
clicking objects in the image. This method, however, is based on the integrated pixel intensity, that is, the
arithmetic sum of the intensity values of all pixels in the thresholded area. This value will be used as a
standard for the subsequent measurement and counting. This may be useful under conditions in which
moderately fluorescent cells are clumped together, resulting in a region that is smaller than in size than
the number of cells present, but the fluorescence of the clumped cells is noticeably additive. As with the
use of a Standard Area, thresholded objects that have an integrated intensity less than half the standard
value will be omitted from the final cell count.
Counting 2 Types of Cells
Overview of Two Cell Type Counting
Changing the Threshold Range:
8-Bit or 16-Bit Images
24-Bit Images
Configuring Data Logging
Changing the Threshold Range
8-Bit or 16-Bit Images
24-Bit Images
Overview of Two Cell Type Counting
To perform counting of two types of cell, use the following general procedure:
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Step
Action
1
From the Apps menu, choose Count 2 Types
of Cells. The Count 2 Types of Cells dialog
box opens.
2
If you are using a single, 24-bit source
image, select the image containing the cells
you want to count with the Source Image
selector, if necessary.
OR
If you are using separate 8-bit or 16-bit
source images, select the appropriate
images using the respective image selectors
in the LIVE Cells and DEAD Cells option
groups. (You will be able to change these
default names in Step 6, if desired.) If you
only see a single image selector, the
command is still in a single-image
configuration. The appropriate image
selectors will appear when you finish the
configuration process (Steps 3 - 10).
3
If you want to view an image that highlights
the counted cells and displays the number of
cells in each cluster next to it, select the
Display Result Image check box. This may
be useful for comparison with the original
source image to determine how good your
configuration and thresholding was.
4
Configure your measurement by choosing
the Configure button. The Configure page of
the Count 2 Types wizard will appear. This
page will display a message informing you of
the purpose of the Count 2 Types of Cells
command. Choose Next >. The Set Kit page
of the wizard will appear.
5
From the Kit drop-down list, select the
staining kit that was used for staining the
cells in your image. Then choose Next >. The
Set Names page of the wizard will appear.
6
Depending on the kit you selected in Step 5,
default names will be assigned to the two
cells types. If you want to change these
names, type the desired names in the Type 1
Name and Type 2 Name text boxes. Then
choose Next >. The Set Sources page of the
wizard will appear.
7
If you are using a single, 24-bit source
image, select One 24 Bit Image for Both
Types of Cells from the Source Images
option button group.
OR
If you are using separate 8-bit or 16-bit
source images, select Separate Images for
Each Type.
When you have finished, choose Next >.
8
If you selected [Other] when you picked a
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staining kit in Step 5, the Staining page of the
wizard will appear. This page is used for
informing MetaMorph whether one of the two
stains used is binding non-specifically to all
cells or if two independent stains are binding
to mutually exclusive sets of cells.
From the Staining option button group, select
Type1/Type2, All Cells/Type1, or All
Cells/Type2. Then choose Next >.
9
The Set Counting Method page will appear.
Select a cell-counting method from the
Counting Method group:
Count Each Discrete Object as 1
configures cell-counting based on the
boundaries of thresholded objects,
Use Standard Area to Estimate
Objects in a Cluster configures cellcounting based on a Standard Area, and
Use Integrated Intensity to Estimate
Objects in a Cluster configures cellcounting based on a standard integrated
intensity value.
10
Your configuration procedure will proceed
depending on the counting method you
selected in Step 9. If you selected
Count Each Discrete Object as 1, you will
return to the Count 2 Types of Cells dialog
box. Proceed to Step 11.
Use Standard Area to Estimate Objects in a
Cluster, the Set Standard page of the wizard
for Standard Areas will appear. You will next
need to configure use of a Standard Area
for each type of cell.
Use Integrated Intensity to Estimate Objects
in a Cluster, the Set Standard page of the
wizard for Standard Areas will appear. You
will next need to configure use of a
standard integrated intensity for each type
of cell.
11
When you are ready to carry out the
measurement, choose Measure. The number
of cells that are detected will appear in the
Count status line of the Count 2 Types of
Cells dialog box.
12
If you are using separate 8-bit or 16-bit
source images, select Use Image Threshold
for each image to use the image’s threshold
to determine the cell count. You can then
adjust the thresholding using the image
window toolbar or the Threshold Image
command and select Use Image Threshold
again to recount the cells, if necessary.
OR
If you are using a single, 24-bit source
image, select Set Threshold to open the Set
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Color Threshold dialog box and set the
threshold for the image.
13
If you want to save the measurement data,
you can open a data log. The Open Log
button will become a Log Data button. When
you are ready to log the count data, choose
Log Data.
14
When you have finished counting cells,
choose Close.
Configuring Cell-Count Measurement Based on Standard Area
To configure cell-counting based on a Standard Area, use the following procedure:
Step
Action
1
From the Set Standard page of the Count 2
Types of Cells wizard, use the Source Image
selector to select the image containing
objects that you want to use to define the
standard. This does not need to be the same
image as the one containing the cells you
want to count. For example, you could use
an image containing objects of a known size,
such as microspheres.
2
If you need to adjust the thresholding for the
image you are using to select a standard,
choose Set Threshold and select an
appropriate threshold range from the Set
Threshold dialog box which appears. You
may find that adjusting the threshold is easier
if you select the Use Transparent Thresholds
check box from the Windows tab page of the
Preferences dialog box (Edit menu). After
you finish selecting the threshold range and
have closed the Set Threshold dialog box,
you will return to the Set Standard dialog
box.
3
In the image window, click several objects
that are of a "typical" size. As you select the
objects, a yellow overlay will appear over
them in the image window, and the Total
Area, Count, and Standard Area spin boxes
will update, displaying the total area
selected, the number of selected cells, and
the average computed area, respectively.
You can modify these values directly, if
desired.
4
When you have finished, choose Next >. The
Set Standard dialog box will close, and the
Count 2 Types of Cells dialog box will
reappear.
Configuring Cell-Count Measurement Based on Integrated Intensity
To configure cell-counting based on a standard integrated intensity value, use the following
procedure:
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Step
Action
1
From the Set Standard page of the Count 2
Types of Cells wizard use the Source Image
selector to select the image containing
objects that you want to use to define the
integrated intensity standard. This does not
need to be the same image as the one
containing the cells you want to count. For
example, you could use an image containing
objects of a known brightness, such as
fluorescent beads of a specific diameter.
2
If you need to adjust the thresholding for the
image you are using to select a standard,
choose Set Threshold and select an
appropriate threshold range from the Set
Threshold dialog box which appears. You
may find that adjusting the threshold is easier
if you select the Use Transparent Thresholds
check box from the Windows tab page of the
Preferences dialog box (Edit menu). After
you finish selecting the threshold range and
have closed the Set Threshold dialog box,
you will return to the Set Standard dialog
box.
3
In the image window, click several objects
that are of a "typical" brightness and size. As
you select the objects, a yellow overlay will
appear over them in the image window, and
the Total Intensity, Count, and Standard
Intensity spin boxes will update, displaying
the total integrated intensity, the number of
selected cells, and the average integrated
intensity, respectively. You can modify these
values directly, if desired. Clicking a selected
object will undo the selection.
4
When you have finished, choose Next >. The
Set Standard dialog box will close, and the
Count 2 Types of Cells dialog box will
reappear.
Changing the Threshold Range for 8-Bit or 16-Bit Images (Count 2 Types
of Cells)
To adjust a threshold range for 8-bit or 16-bit source images, use the following procedure. If your
image has never been thresholded, a red overlay will initially be displayed over the entire image.
Step
Action
1
In the Count 2 Types of Cells dialog box,
choose the Use Image’s Threshold button for
the first cell type.
2
Adjust the image’s threshold using the image
window toolbar or the Threshold Image
command in the Measure menu.
3
Repeat Steps 1 - 2 for the second cell type.
4
If necessary, click the Back button at the top
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of this Help window to return to the
procedure you were just reading.
Changing the Threshold Range for 24-Bit Images (Count Cells)
To adjust a threshold range for a single 24-bit color source image, use the following procedure:
Step
Action
1
In the Count 2 Types of Cells dialog box,
choose the Set Threshold button. The Set
Color Threshold dialog box opens.
2
If you want to use a previously saved set of
color threshold settings, choose Load Range.
Otherwise, skip to Step 4.
3
The Load Color Threshold Range dialog box
opens.
AND
Select the icon for the desired color threshold
range (*.ctr) state file. If necessary, use the
Look In list or Up One Level icon button to
locate the correct drive and folder. Then
choose Open. The threshold settings will be
applied to your image. Now skip to Step 10.
4
From the Color Model list, select the color
model you want to use for setting the color
threshold: RGB, HSI, or HSL. Your selection
will determine the options you see in the
lower half of the dialog box.
5
If you selected HSI or HSL as your color
model in Step 4, use the Hue Range radio
button group to select whether the ranges
between the upper and lower limits are to be
included in the threshold range (Inclusive) or
if the ranges outside of the upper and lower
limits are to be thresholded (Exclusive).
6
Use the sliders or the left and right spin
boxes for each of the color channels (RedGreen-Blue, Hue-Saturation-Intensity, or
Hue-Saturation-Luminosity) to select the
lower and upper threshold range values. As
you adjust the settings, the distribution of the
red thresholding overlay that covers pixels
with the selected values will change.
7
If you want to use the interactive "point-andclick" method of selecting the threshold
range, choose Set by Example. The dialog
box will expand, revealing two more options.
If necessary, reset the threshold range by
selecting the Reset Color Threshold Range
on Next Click check box, so that a check
mark appears in it.
AND
Use the pointer to select pixels in the image
that have the values that you want to include
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in the threshold range. As you click the
pixels, they will be covered by the red
thresholding overlay, and the color channel
sliders and spin boxes will update to display
the new values.If you want to remove the
values of the pixels you selected last, choose
Undo Last Click.
8
By default, the thresholding State is in
Inclusive mode, which is to say that the
range that you have selected is included in
the threshold range and will be highlighted by
a red thresholding overlay. If you want to
reverse the selection so that the pixels in the
range you have selected are excluded from
thresholding and all other pixels are included
instead, select Exclusive from the State
option button group.
9
If you want to save the threshold settings,
choose Save Range. The Save Color
Threshold Range dialog box will appear.
Type a name for the color threshold range
(*.ctr) state file in the File Name text box. If
necessary, use the Save In list or Up One
Level icon button to locate the correct drive
and folder. Then choose Save.
10
When you have finished, choose Close.
11
If necessary, click the Back button at the top
of this Help window to return to the
procedure you were just reading.
Configuring Data Logging for Cell-Count Measurement
To configure logging of your single-cell count data, use the following procedure. You can follow
this procedure either before or after you perform the actual measurement.
Step
Action
1
From the Count 2 Types of Cells dialog box,
choose Open Log. The Open Data Log
dialog box will appear.
2
Select Dynamic Data Exchange (DDE) to log
directly to an open spreadsheet program.
Select A Text File to log the data to a text
file.
Note: You can select both options.
3
If you selected A Text File in the previous
step, the Open Data Log File dialog box will
appear.
Select an existing log file or type a new file
name in the File Name text box. (If
necessary, use the Look In list or Up One
Level button to change the current drive and
folder to the correct location.)
AND
Choose Open.
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4
If you selected an existing log's file name in
Step 3, the Log File Exists dialog box will
appear. You can Overwrite the contents of
the file, Append new data, or Cancel.
5
If you selected Dynamic Data Exchange
(DDE) in Step 2, the Export Log Data dialog
box will appear.
Select the desired application from the
Application list. Choose Default to use the
default settings for the selected application.
Choose OK to launch the external
application. After the application opens, click
the MetaMorph Imaging System button in the
Windows Taskbar to return to MetaMorph.
6
If necessary, choose Configure Log from the
Count Cells dialog box to select which data
to log. The Configure Log dialog box will
appear.
AND
From the Configuration table, select the data
types that you want to log. Then choose OK.
7
The Open Log button will have changed to a
Log Data button. To log your cell count data,
choose this button after you have performed
your measurement.
8
If necessary, click the Back button at the top
of this Help window to return to the
procedure you were just reading.
Count 2 Types of Cells - Dialog Box Options
Count 2 Types of Cells - Dialog Box Options
Configure
Opens the Set Counting Method dialog box, from which you can select the counting method (boundarybased, Standard Area-based, or integrated intensity-based).
Source
Selects the single 24-bit source image that contains the cells you want to count. If you have configured the
cell count to use two separate 8-bit or 16-bit images, two unlabeled image selectors will appear in the Count
2 Types of Cells dialog box, one in each cell type's option group (image selector, Use Image’s Threshold
button, and Count status line).
Use Image’s Threshold
Uses the image’s current threshold settings when counting cells. You can adjust thresholding for the image
using the Threshold button on the image’s tool bar or by using the Threshold Image command in the
Measure menu.
Count
These status lines indicate the numbers of each type of cell that have been counted.
Set Threshold
Opens the Set Color Threshold dialog box. Use this command to change the threshold on a 24-bit image.
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Open Log
Opens a text-based data log file or an OLE connection to an open spreadsheet program for logging count
data and threshold values if desired. Changes to "Log Data" when a log file is open.
Log Data
Sends your cell-count measurement data to the open data log or spreadsheet.
Configure Log
Opens the Configure Log dialog box, from which you can select the types of data to be stored in the data
log.
Measure
Carries out the measurement, based on the configuration of the counting method and the current threshold
state of the image.
Close
Closes the Count 2 Types of Cells dialog box.
Set Kit (Count 2 Types of Cells) - Dialog Box Options
Kit
Selects the staining kit that was used to stain the cells in the source image. This will affect the way Count 2
Types of Cells expects cells to be stained.
< Back
Cancels the kit selection process and returns you to the Configure message page of the wizard.
Next >
Accepts the kit selection and forwards you to the Set Names page of the wizard.
Cancel
Cancels the kit selection process and returns you to the Count 2 Types of Cells dialog box.
Set Names (Count 2 Types of Cells) - Dialog Box Options
Type 1 Name
Specifies a name for the type 1 cells. This name will be used for both the counting result image and for the
pertinent labels in the Count 2 Types of Cells dialog box and wizard pages, as well as data logging.
Type 2 Name
Specifies a name for the type 2 cells. This name will be used for both the counting result image and for the
pertinent labels in the Count 2 Types of Cells dialog box and wizard pages, as well as data logging.
< Back
Returns you to the Set Kit page of the wizard.
Next >
Accepts the source image format selection and forwards you to the Set Sources page of the wizard.
Cancel
Cancels the source image format selection process and returns you to the Count 2 Types of Cells dialog
box.
Set Sources (Count 2 Types of Cells) - Dialog Box Options
Source Images
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Selects the source image format. Select
One 24 Bit Image for Both Types of Cells if you are using a single 24-bit source image, or
Separate Images for Each Type if you are using separate 8-bit or 16-bit source images.
< Back
Returns you to the Set Names page of the wizard.
Next >
Accepts the source image format selection. If you select [Other] as the staining kit, you will be forwarded to
the Staining page of the wizard. If you selected one of the staining kits, you will be forwarded to the Set
Counting Method page of the wizard.
Cancel
Cancels the source image format selection process and returns you to the Count 2 Types of Cells dialog
box.
Staining (Count 2 Types of Cells) - Dialog Box Options
Note: This page of the configuration wizard will appear only if you have selected "[Other]"
as the staining kit.
Staining
This option informs MetaMorph whether one of the two stains is binding non-specifically to all cells or if two
independent stains are binding to mutually exclusive sets of cells. Select Type1/Type2, All Cells/Type1, or
All Cells/Type2.
< Back
Returns you to the Set Sources message page of the wizard.
Next >
Accepts the staining format selection and forwards you to the Set Counting Method page of the wizard.
Cancel
Cancels the staining format selection process and returns you to the Count 2 Types of Cells dialog box.
Set Counting Method (Count 2 Types of Cells) - Dialog Box Options
Counting Method
Selects a cell-counting method:
Count Each Discrete Object as 1 - Bases the cell-count measurement on what MetaMorph
perceives to be the object boundaries, as defined by thresholding. When you select this option,
choosing Next > will return you to the Count 2 Types of Cells dialog box.
Use Standard Area to Estimate Objects in a Cluster - Bases the cell-count measurement on a
selected Standard Area. When you select this option, choosing Next > will open the Set Standard
page of the configuration wizard, from which you can select the Standard Area.
Use Integrated Intensity to estimate Objects in a Cluster - Bases the cell-count measurement
on a standard integrated intensity value. When you select this option, choosing Next > will open the
Set Standard page of the configuration wizard, from which you can select the standard integrated
intensity.
Cancel
Cancels your selection of cell-counting method and closes the Set Counting Method dialog box, returning
you to the Count 2 Types of Cells dialog box.
Next >
Accepts your selection of cell-counting method and closes the Set Counting Method dialog box. If you
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selected Count Each Discrete Object as 1, you will return to the Count 2 Types of Cells dialog box. If you
selected either of the other two methods, the Set Standard page of the configuration wizard will appear.
Set Standard (Count 2 Types of Cells) - Dialog Box Options
Source Image
Selects the image containing the cells you want to use to define the standard area or integrated intensity.
This does not need to be the same image as the one containing the cells you want to count. For example,
you could use an image containing objects of a known size or brightness, such as microspheres or
fluorescent beads of a specific diameter.
Set Threshold
Opens the Set Threshold dialog box, from which you can change the thresholding range for the image you
are using to define your standard (which could be the same as the image containing the cells you want to
count).
Clear Counts
Clears the Total Area or Total Intensity settings and the Count setting.
Total Area
Indicates the summed area, in pixels, of all objects that have been selected. This option is seen when you
select the Standard Area-based measurement method.
Total Intensity
Indicates the summed intensity values of all pixels in the objects that have been selected. This option is
seen when you select the integrated intensity-based measurement method.
Count
Indicates the number of objects that have been selected for defining the standard value.
Standard Area
Indicates the size of the Standard Area, based on the Total Area and the Count. This option is seen when
you select the Standard Area-based measurement method.
Integrated Intensity
Indicates the average integrated intensity value, based on the Total Intensity and the Count. This option is
seen when you select the integrated intensity-based measurement method.
< Back
Cancels the standard selection process and returns you to the Set Counting Method dialog box.
Next >
When determining the standard for the first type of cell, this button leaves up the Set Standard dialog box so
that you can proceed to select the standard for the second type of cell. When you have selected the second
standard, Next > accepts the standard selection and returns you to the Count 2 Types of Cells dialog box.
Cancel
Cancels the standard selection process and returns you to the Count 2 Types of Cells dialog box.
Measure Colocalization (Apps Menu)
Provides quantitative data regarding regions of overlap of two fluorescent probes in an
image. The area, average intensity, and integrated intensity in the region of overlap can be
measured and saved to a log file.
Availability: Available for MetaVue and MetaMorph Basic; included in MetaMorph Premier
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Drop-in: COLOCAL
Use this command to display and save measurements of the area of overlap between two fluorescent
probes, or the average or integrated intensities in the region of overlap. The intensity of pixels in 24-bit
color images is calculated as the mean of the red, green, and blue intensities. Typically, two source
images are used. The same view of the preparation must be used for both images, but they should be
acquired at different excitation or transmission wavelengths, as appropriate for the respective probes
being used. If desired, you can use a Region Tool to define and select a specific region of interest for
measurement. This should be defined in the Source A image. Both source images must be thresholded
prior to performing the measurement.
Note: Areas of overlap will be expressed in pixels. To express the area in "real" units
(square microns, millimeters, etc.), use Calibrate Distances before using this command.
Measuring Colocalization
To measure the region of overlap between two fluorescent probes, use the following procedure.
The two source images must first be thresholded.
Step
Action
1
From the Apps menu, choose Measure
Colocalization. The Measure Colocalization
dialog box opens.
2
If desired, use a Region Tool to define a
region of interest for the measurement. This
must be defined in the source image that will
be selected with the Source A image
selector.
Note: Regions drawn with a two-dimensional
Region Tool (Rectangular, Ellipse, Trace, or
Auto-Trace) must be at least 2x2 in size to
be valid in MetaMorph.
3
Use the Source A and Source B image
selectors to select the two views of the
preparation. These will correspond to the
wavelength image for each of the two
respective fluorescent probes.
4
In the Method group, select the parameter
you want to measure. Select
Area if you want to measure the area of
overlap,
Average if you want to measure the average
pixel intensity, or
Integrated if you want to measure the
integrated pixel intensity.
5
If you want to log the measurement data,
open a data log file. Then, if desired,
choose Configure Log from the Measure
Colocalization dialog box and configure your
data logging. Otherwise continue to Step 6.
6
Choose Measure. Once measured, the data
will be displayed in the dialog box. If you
have opened a data log, the data will also be
logged automatically.
7
Choose Close to close the dialog box.
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Measure Colocalization - Dialog Box Options
Source A
Selects the image for the first probe. If you want to define regions of interest for the measurement, they must
be drawn on this image. This image must be thresholded prior to measurement.
Source B
Selects the image for the second probe. This image must be of the same sample as in the Source A image,
differing only in the wavelength used for imaging the second dye. Any regions of interest drawn on this
image will be ignored. As with the Source A image, the image for Source B must be thresholded prior to
measurement.
Value for All A
Displays the value of the parameter selected in the Method group (Area, Average pixel intensity, or
Integrated pixel intensity) for the entire thresholded region in the Source A image. The intensity of pixels in
24-bit color images is calculated as the mean of the red, green, and blue intensities.
A Overlapping B
Displays the value of the selected parameter for that part of the thresholded Source A image that is also
thresholded in the Source B image. This may not necessarily be the same as the B Overlapping A
measurement for average or integrated pixel intensity because the B Overlapping A measurement is derived
from the Source B image, not the Source A image, and may therefore have different intensity values in the
regions of interest.
A Not Overlapping B
Displays the value of the selected parameter for that part of the thresholded Source A image that is outside
of the thresholded region in the Source B image.
Value for All B
Displays the value of the parameter selected in the Method group (Area, Average pixel intensity, or
Integrated pixel intensity) for the entire thresholded region in the Source B image.
B Overlapping A
Displays the value of the selected parameter for that part of the thresholded Source B image that is also
thresholded in the Source A image. This may not necessarily be the same as the A Overlapping B
measurement for average or integrated pixel intensity because the A Overlapping B measurement is derived
from the Source A image, not the Source B image, and may therefore have different intensity values in the
regions of interest.
B Not Overlapping A
Displays the value of the selected parameter for that part of the thresholded Source B image that is outside
of the thresholded region in the Source A image.
Method
Selects the parameters to be measured. The intensity of pixels in 24-bit color images is calculated as the
mean of the red, green, and blue intensities.
Area measures the area of overlap between the two probes. This will be expressed in pixels unless
you apply the Calibrate Distances command (Measure menu) prior to using the Measure
Colocalization command.
Average measures the average pixel intensity in the regions of overlap.
Integrated measures the integrated pixel intensity in the regions of overlap (equivalent to the sum
of all grayscale values for every pixel in the region).
Configure Log
Displays the Configure Log dialog box, which allows you to select the image parameters to be saved in an
open data log. You must use the Open Data Log command (Log menu) to log Measure Colocalization data.
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Measure
This command performs and displays the measurement of the colocalization data that you specified in
Method. The data will be displayed in the Measure Colocalization dialog box. If a data log is open, the data
will be logged automatically when you choose the Measure command.
Close
Closes the dialog box.
Morphology Filters
Provides a set of tools that can be used to transform binary and grayscale images through
morphology filtering and analysis.
Drop-in: MORPHOLOGY
Use this drop-in to smooth noisy images or to simplify overly complex images. Both of these processes
can make the data that you need to extract from your images more accessible.
Morphology analysis also provides the ability for you to extract specific features from images.
Morphology operations rely on processes based on shape.The filters are interrelated, with most
constructed as combinations of the simplest pieces: Dilate and Erode. Dilate acts by growing bright
pixels, while erode does the opposite by growing dark pixels, functioning as the morphological
complement of dilate.
Several of the image filters use dilate or erode as their foundation, but add more generalized
functionalities to achieve more powerful filtering abilities.
Application Note PDF — Using the MetaMorph Morphology Filters
Note: Morphology Filters can be used individually. However, complex tasks, such as
segmentation, are often best performed through a combination of several steps. For
example, you can combine filters to remove noise, estimate and remove background, and
extract features. You can benefit most by creating journals containing several
Morphology filters.
Note: Morphology Filters can be used on either an entire image, or an active region of
interest.
Definition:
Neighborhood – A region surrounding each pixel in the source image defined by the
filter shape and diameter, width, or area, and centered on the pixel on which the filter is
acting.
Applying Morphology Filters
To process an image using one or more morphology filters, complete the following procedure.
Step
Action
1
From the Process Menu, click Morphology
Filters. The Morphology Filters dialog box
opens.
2
Open one or more images that you want to
process.
3
If more that a single image is open, click
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Source Image to select the image that you
want to process.
4
Using the Result Image selector, choose
whether you want to Overwrite, Add to, or
create a New destination image. The
destination image name by default is the
name of the filter used to process the image.
5
Click the image name button, then click
specified to open the Specify Image Name
dialog box. Type an image name, then click
OK.
6
Using the Morphology Filters Use
Comparison Chart, determine the filter or
filters that are best suited for the image(s)
that you need to process.
7
Click the filter that you want to use.
8
If there are parameter settings associated
with the filter, set these parameters
according to the guidelines in the
Morphology Image Filters Use
Comparison Chart and in the Dialog Box
Options. Where applicable, choose the most
appropriate filter shape. The circle is well
suited for most biological samples.
9
Set the most appropriate filter size. Refer to
the guidelines in the Morphology Image
Filters Use Comparison Chart.
10
If applicable to your selected filter and your
image(s), choose Use sequential filtering
and/or Use reconstruction.
11
Click Apply to process your image(s) with
the Image Filter and settings that you have
selected.
12
Click Undo to undo the last filter that was
applied to the image.
13
Click Close to close the Morphology Filters
dialog box.
Morphology Filters - Dialog Box Options
Source Image
Opens the Image Selector for the source image. Morphology Filters can process images of any grayscale
bit depth, including binary, but not color images. When processing image stacks, each image in the stack is
processed before proceeding to the next image, until all images in the stack have been processed.
Result Image
Opens the Image Selector for the destination image.
Operation
Defines the action of the parameters and the filter.
Image Filters
Lists the available morphology filter types for smoothing and simplifying images. Click to select the filter that
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you want to use.
Dilate
Performs morphological dilation (neighborhood max). This is the complementary operation of
erode. This filter grows bright blobs and shrinks dark blobs. Applicable Parameters: Filter shape
and size.
Erode
Performs morphological erosion (neighborhood min). Complementary operation of dilate. This filter
grows dark blobs and shrinks light blobs. Applicable Parameters: Filter shape and size.
Close
Performs morphological close. Filters out dark blobs by first applying dilate then erode.
Complementary operation of open. Applicable Parameters: Filter shape and size, reconstruction
on/off if circle or square filter shape.
Open
Performs morphological open. Filters out bright blobs by first applying erode then dilate.
Complementary operation of close. Applicable Parameters: Filter shape and size, reconstruction
on/off if circle or square filter shape.
Close-open
Performs morphological close-open. Filters out dark then light blobs by first applying close then
open. Complementary operation of open-close. Applicable Parameters: Filter shape and size,
sequential filtering on/off, reconstruction on/off if circle or square filter shape.
Open-close
Performs morphological close-open. Filters out light then dark blobs by first applying open then
close. Complementary operation of close-open. Applicable Parameters: Filter shape and size,
sequential filtering on/off, reconstruction on/off if circle or square structuring element.
Center filter
Performs morphological center filter, a pointwise median of 3 images: the original, its close-openclose, and its open-close-open. This filter combination simultaneously smoothes both dark and
bright blobs. Applicable Parameters: Filter shape and size, sequential filtering on/off,
reconstruction on/off if circle or square structuring element.
Lomo filter
Performs morphological lomo filter, a pointwise mean of 2 images: the close and the open of the
original. This filter automatically repeats itself until smoothness is achieved. This filter combination
simultaneously smoothes both dark and bright blobs. Applicable Parameters: Filter shape and
size, sequential filtering on/off, reconstruction on/off if circle or square filter shape.
Reconstruct from below
Performs a grayscale reconstruction of original from the Marker image. The Marker image expands
intensities from below the original intensity signal, retaining only the bright blobs into which they
grow, filling the remaining area with darkness. Complementary operation of reconstruct from
marker above. Applicable Parameters: Marker Image.
Reconstruct from above
Performs a grayscale reconstruction of original from Marker image. The Marker image expands
from above the original intensity signal, retaining only the dark blobs into which they grow, filling the
remaining area with brightness.. Complementary operation of reconstruct from marker below.
Applicable Parameters: Marker Image.
Extract Features
Lists the available image analysis tools for enhancing and extracting visually important features. Click to
select the analysis tool that you want to use. For guidelines on using these features, refer to the
Morphology Extract Features Use Comparison Chart.
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Gradient
Performs morphological gradient for edge detection: dilate minus erode. Applicable Parameters:
Filter shape (not area) and size.
Top hat
Detects bright spots that are removed by open filtering by subtracting the open of original from the
original. Complementary operation of bottom hat. Applicable Parameters: Filter shape and size,
reconstruction on/off if circle or square filter shape
Bottom hat
Detects dark spots that are removed by close filtering by subtracting the original from the close of
original. Complementary operation of top hat. Applicable Parameters: Filter shape and size,
reconstruction on/off if circle or square filter shape.
Regional max
Detects local maxima points and plateaus and returns them white on black background.
Applicable Parameters: none.
Regional min
Detects local minima points and plateaus and returns them white on black background.
Applicable Parameters: none.
H-dome
Detects bright spots using a gray level threshold relative to each pixels local surroundings. The
absolute difference between the original and its reconstruction by the marker image created by
subtracting a constant intensity from the original. Applicable Parameters: threshold.H is for Height
threshold.
H-basin
Detects dark spots using a gray level threshold relative to each pixels local surroundings, but
returns them as bright on zero background. The absolute difference between the original and its
reconstruction by the marker image created by adding a constant intensity to the original.
Applicable Parameters: threshold. H is for Height threshold.
Watershed lines
Performs a watershed transform and returns the thin watershed boundaries as white and the region
interiors as black. Useful for segmentation problems. The grayscale input should represent the
boundaries between objects to be segmented, so edge detectors (including morphological gradient
on this same dialog) are commonly used for this purpose. Note: Only very smooth or simple input
images give a small number of basins without the use of a marker image. With a marker image,
each non-black marker blob becomes a region in the final result. Applicable Parameters: Marker
Image if checkbox selected.
Watershed basins
Performs watershed transform, resulting in each pixel given an integer label (may not be unique if
more region labels than can be represented at your destination image’s bit-depth) Note: Only very
smooth or simple input images give a small number of basins without the use of a marker image.
With a marker image, each non-black marker blob becomes a region in the final result. .
Applicable Parameters: Marker Image if checkbox selected.
Parameters
Defines the available options.
Filter Shape – Indicates the available filter shapes. Click to select the filter shape that you want to
use.
Filter Shape defines the spatial neighborhood around each pixel within which calculations are
performed to locally smooth and simplify the image. Each filter has an associated settable
parameter that defines its relative size. For example, for the Circle filter, you can set the diameter
of the circle in pixels.
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Circle – Applies a filter in the shape of a circle. The dimension of the circle is defined by
the Diameter parameter.
Square – Applies a filter in the shape of a square. The dimension of the square is defined
by the Width parameter.
Diamond – Applies a filter in the shape of a diamond. The dimension of the diamond is
defined by the Width parameter.
Area – Specifies a threshold value in adjacent pixels (squared) that make up an area of
contiguous pixels, commonly referred to as a blob. Depending on the filter using this
value, blobs of fewer pixels than specified are removed from the image.
Diameter – Specifies the diameter of the circle filter shape.
Width – Specifies the width of a square or diamond filter shape.
Use sequential filtering – Applies a sequence of progressively larger filter shapes, until the userspecified size is reached. Produces visually better smoothing results than single application of the
user-specified filter shape, however, more processing time is required. This option can be used
with close-open, open-close, center, and lomo filters only.
Use reconstruction – Removes blobs without distorting and rounding off the edges of those blobs
that remain. After applying a fixed-shape filter (circle, diamond, or square) to remove blobs, retains
or "reconstructs" the original shape of remaining blobs. This option can be used with open, close,
top hat, bottom hat, close-open, open-close, center, and lomo filters only. This option is inactive if
the "area" filter shape, which already includes reconstruction.
Threshold – Edit box for graylevel height offset threshold. Hidden except for use with H-dome/Hbasin.
Morphology Image Filters Use Comparison Chart
Filter Name
Best for…
Use for…
Best Filter Shape
Filter Size
Cautions
Dilate
Growing bright
objects
Enhancing bright
features or shrinking
dark features
Usually you should use
circles since biological
applications tend not to
have right angles or
any particular
orientation.
Spatial extent to
which bright
pixels expand.
Similar to
selecting a
paintbrush size.
Diamond or square of
width 3 gives some
precise control over
very small
neighborhoods
Repeated
application of a
small structuring
element is done
more easily with
a single large
one.
Dilate grows bright pixe
of noise as well as the
bright objects your
interested in, so use
other filters (median, lo
pass, other morphology
filters such as closeopen, etc.) to remove
noise and small
unwanted bright spots
prior to dilating.
Shrinking dark
objects
Joining nearby bright
blobs or separating
dark blobs
Expanding a binary
mask to include a
larger masked region
Erode
Growing dark
objects
Shrinking
bright objects
Enhancing dark
features or shrinking
bright features
Joining nearby dark
blobs or separating
bright blobs
Shrinking a binary
mask to include a
smaller masked region
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Usually you should use
circles since biological
applications tend not to
have right angles or
any particular
orientation.
Diamond or square of
width 3 gives some
precise control over
very small
Spatial extent to
which dark pixels
expand. Similar
to selecting a
paintbrush size.
Repeated
application of a
small structuring
element is done
more easily with
Version 7.0
Dilate moves the spatia
location of object edge
so if edge localization i
important, use one of t
edge-preserving
morphological filters
(open, close, lomo,
center…).
Erode grows dark pixe
of noise as well as the
dark objects your
interested in, so use
other filters (median, lo
pass, other morphology
filters such as closeopen, etc.) to remove
noise and small
unwanted dark spots
Drop-in Commands
neighborhoods
Open
Functions like
an enhanced
erode
Bettter edge
localization
than erode.
Splitting connected
bright blobs (or
connecting nearby dark
blobs)
Need to determine
which blobs that you
want to mark for
deletion
Removing unwanted
bright blobs and noise.
The shape is required
to "fit" entirely within a
bright blob to prevent
its removal.
Does not
expand noise
as much as
erode.
Close
Functions like
an enhanced
dilate.
Growing bright objects
Better edge
localization
than dilate.
Moving edges (object
boundaries) and
Expanding bright noise
and small features.
Combines
Close and
Open
Determine filter
size according to
the demension
required to "fit"
the filter element
entirely within a
bright blob to
prevent its
removal.
Use
reconstruction
for retaining
detail of the
remaining blobs.
Shrinking dark objects
Does not
expand noise
as much as
dilate.
Close-Open
a single large
one.
Need to determine
which blobs that you
want to mark for
deletion.
The shape is required
to "fit" entirely within a
dark blob to prevent its
removal.
Determine filter
size according to
the demension
required to "fit"
the filter element
entirely within a
dark blob to
prevent its
removal.
Use
reconstruction
for retaining
detail of the
remaining dark
blobs.
Smoothing dark, then
light objects using the
selected filter shape.
Determine which blobs,
(both bright and dark)
to mark for deletion.
Use sequential
filtering for best
visual results.
Use
reconstruction to
retain more
details of
remaining blobs.
Open-Close
Combines
Open and
Close
Smoothing light, then
dark objects using the
selected filter shape.
Determine which blobs,
(both bright and dark)
to mark for deletion.
Use sequential
filtering for best
visual results.
Use
reconstruction to
retain more
details of
remaining blobs.
Center Filter
MetaMorph
Smoothing light
and dark
Uses (at each pixel
location) the middle
406
Determine which blobs,
(both bright and dark)
Use sequential
filtering for best
Version 7.0
prior to eroding.
Erode moves the spati
location of object edge
so if edge localization i
important, use one of t
edge-preserving
morphological filters
(open, close, lomo,
center…).
Sensitivity to noise—
even a small number o
dark specs can cause t
loss of a bright blob.
You may need one of t
more advanced filters
(open-close, close-ope
center, lomo, etc.) if yo
haven’t already remove
the dark noise pixels.
Sensitivity to noise—
even a small number o
bright specs can cause
the loss of a dark blob.
You may need one of t
more advanced filters
(open-close, close-ope
center, lomo, etc.) if yo
haven’t already remove
the bright noise pixels.
Open-Close has a
slightly different intensi
bias than Open-Close
due to the filtering orde
Open-Close has a
slightly different intensi
bias than Close-Open
due to the filtering orde
Similar to the median
filter, this filter might no
User’s Guide
Lomo Filter
Reconstruct
from Above
Reconstruct
from Below
objects
simultaneously.
intensity value of three
choices: open-close,
close-open, and the
original image.
to mark for deletion.
Smoothing light
and dark
objects
simultaneously.
Uses (at each pixel
location) the mean
intensity value between
open and close filters,
and reapplies itself until
smoothness is
reached.
Determine which blobs,
(both bright and dark)
to mark for deletion.
Expanding
marker image
intensities from
above the
source image,
marking blobs
in one image
from blobs in
another.
Reconstructing cells
fluorescently stained in
one wavelength by
their nuclei stained in
another wavelength,
removing those cells or
other blobs without a
nuclear stain.
Use Reconstruct from
Above if you are
marking dark blobs to
retain, allowing
unmarked ones to
become bright.
Expanding
marker image
intensities from
below the
source image,
marking blobs
in one image
from blobs in
another.
Reconstructing cells
fluorescently stained in
one wavelength by
their nuclei stained in
another wavelength,
removing those cells or
other blobs without a
nuclear stain.
visual results.
Use
reconstruction to
retain more
details of
remaining blobs.
Use sequential
filtering for best
visual results.
smooth certain textures
or patterns. Use the
Lomo Filter for better
smoothness.
Slightly slower than
Close-Open, OpenClose, and Center filter
Use
reconstruction to
retain more
details of
remaining blobs.
N/A
After a filtering process,
use this filter to
regenerate the details
of the remaining
blobs/objects.
Use Reconstruct from
Below if you are
marking bright blobs to
retain, allowing
unmarked ones to
become dark.
The software
automatically cuts off
your marker image
intensities to be entirely
above or below (as you
selected) in case you
input a marker that is in
some places above an
other places below the
source image.
The actual marker imag
is not altered by this
intermediate calculatio
N/A
After a filtering process,
use this filter to
regenerate the details
of the remaining
blobs/objects.
The software
automatically cuts off
your marker image
intensities to be entirely
above or below (as you
selected) in case you
input a marker that is in
some places above an
other places below the
source image.
The actual marker imag
is not altered by this
intermediate calculatio
Morphology Extract Features Use Comparison Chart
Extract Feature
Best for…
Use for…
Best filter shape
Filter size
Cautions
Gradient
Showing local
contrast:
Detecting edges
between bright and
dark regions of an
image.
Usually you should use
circles, since biological
applications tend not to
have right angles or
any particular
orientation.
The typical width
of a bright to dark
or dark to bright
transition
between objects
(may not be a
perfectly sharp
transition due to
focus, and so
on.)
Sensitive to noise, so
you might want to use
another morphology fil
to smooth your image
before using gradient.
Reports
intensity of the
amount of local
contrast
computed by
dilate minus
erode
MetaMorph
Segmentation:
Creating a
"topographic
surface" image for
use in watershed
segmentation
407
A diamond or square
with a width of three
gives some precise
control over very small
neighborhoods
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Top Hat
Enhancing
small bright
objects by
computing the
original image
minus the result
of the open filter
Detecting bright
features of a known
spatial size or
shape.
Determine the shape
of the Open filter that
removes your objects
of interest. The top hat
result show what the
open filter removed.
Determine the
size of the Open
filter that
removes your
objects of
interest. The top
hat result show
what the Open
filter removed.
Sensitive to noise, so
you may want to use
another morphology fil
to smooth your image
prior to using top hat.
Bottom Hat
Reporting
brightness
where small
dark objects
were detected
by computing
the result of the
close filter
minus the
original image
Detecting dark
features of a known
spatial size or
shape.
Determine the shape
of the Close filter that
removes your objects
of interest. The bottom
hat result shows what
the close filter
removed.
Determine the
close filter’s size
that removes
your objects of
interest. The
bottom hat result
shows what the
close filter
removed.
Sensitive to noise, so
you may want to use
another morphology fil
to smooth your image
prior to using bottom h
Regional max
Reporting a
bright spot for
all local "peaks"
in intensity
(pixels of a
constant
intensity value
completely
surrounded by
lower intensity
pixels)
Detecting bright
spots.
n/a
n/a
Sensitive to slight
intensity fluctuations:
Returns a bright spot
even for the slightest
local "peak" in intensity
To set a threshold
amount of how high a
local peak of intensity
must be, use H-dome.
Regional min
Reporting a
bright spot for
all local
"valleys" of
intensity (pixels
of a constant
intensity value
completely
surrounded by
higher intensity
pixels)
Detecting dark
spots.
n/a
n/a
Sensitive to slight
intensity fluctuations:
Returns a bright spot
even for the slightest
local "valley" in intensit
To set a threshold
amount of how deep a
local valley of intensity
must be, use H-basin.
H-dome
Reporting bright
spots where
significant
intensity
"peaks" occur.
Similar to
regional max,
but allows
control over
how "significant"
the peaks.
Detecting local
bright spots whose
intensity "peaks" are
at least the threshold
amount brighter than
their surrounding
background.
None: Useful for when
you don’t have
knowledge of spatial
shape, but do know
the intensity above
local background of
the bright spots you’re
looking for.
None: Useful for
when you don’t
have knowledge
of spatial size,
but do know the
intensity above
local background
of the bright
spots you’re
looking for.
Requires intensity
knowledge of how brig
above background you
spots are—if instead y
have spatial knowledge
consider top hat as we
Reporting bright
spots where
significant
intensity
"valleys" occur.
Detecting local dark
spots whose
intensity "valleys"
are at least the
threshold amount
None: Useful for when
you don’t have
knowledge of spatial
shape, but do know
the intensity below
None: Useful for
when you don’t
have knowledge
of spatial size,
but do know the
Requires intensity
knowledge of how dark
below background you
spots are—if instead y
have spatial knowledge
H-basin
MetaMorph
Tip: Use Euclidean
distance in the
Binary dialog box to
find the furthest
points away from the
white pixels—maybe
the furthest points
inside your cells as
"markers" for
watershed
Finding bright spots
on a non-uniform
background.
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Watershed
Lines
Watershed
Basins
Similar to
regional min,
but allows
control over
how "significant"
the valleys.
darker than their
surrounding
background.
Partitioning an
"intensity
surface" image
where the
brighter
intensity
"ridges"
(watershed
lines) separate
the watershed
valleys or
basins.
Segmentation of an
image that can be
visualized as a
topographical
surface upon which
rain flows into
separate
watersheds.
Partitioning an
"intensity
surface" image
where the
brighter
intensity
"ridges"
(watershed
lines) separate
the watershed
valleys or
basins. Each
basin is given a
different color
for visualization.
Segmentation of an
image that can be
visualized as a
topographical
surface upon which
rain flows into
separate
watersheds.
local background of
the dark spots you’re
looking for.
intensity below
local background
of the dark spots
you’re looking
for.
consider bottom hat as
well.
n/a
n/a
Use the marker option
avoid over-segmentatio
(many little regions).
Each marker blob give
single watershed
region—no more, no
less.
n/a
n/a
Use the marker option
avoid over-segmentatio
(many little regions).
Each marker blob give
single watershed
region—no more, no
less.
Finding dark spots
on a non-uniform
bright background.
Tip: Use the Binary
dialog’s Euclidean
distance to make
surfaces from binary
images.
Select individual basin
of interest by using the
Binarize function in the
Binary dialog box, setti
both Low and High
values to the basin
number that you see a
the bottom of the scree
from placing your mou
pointer over the basin
the watershed image.
Tip: Use Euclidean
distance in the
Binary dialog box to
make surfaces from
binary images.
Segment Image (Process Menu)
Provides access to the Segment Image Modifications dialog box, and saves, stores, and
retrieves Image segmentation settings.
Drop-in: SEGIMAGE
Use this dialog box to access the Segment Image Modifications dialog box to define settings for image
segmentation. You can apply your image segmentation results to individual images directly from the
Segment Image Modifications dialog box, or you can use this Segment Image dialog box to store
satisfactory image segmentation settings for future use. Also use this dialog box to retrieve and load
previously stored image segmentation settings. The stored and loaded settings can be applied to
images directly from this dialog box. Use the output selections to specify whether you want to create a
binary mask from the processed image data or create a clipped version of the original image.
Segmenting Images
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To segment an image, complete the following procedure:
Step
Action
1
From the Apps Menu, click Segment Image.
The Segment Image dialog box opens.
2
From the File menu, open the image that
you want to segment. The image name is
displayed on the Source image button. If you
open more than one image, use the Source
image button to select the image you want to
segment.
3
If you have a prepared Setting file that you
want to use, click the Load button. The
Segment Image: Load Setting dialog box
opens.
4
In the Segment Image: Load Setting dialog
box, click the name of the setting (*.AST) file
that you want to load. Repeat this step to
load additional setting files into Segment
Image. You can then select the file that you
want to use in the Active Setting box.
5
Use the Result image selector to assign the
image name and characteristics to the
segmentation output image file. You can
specify New to create a new image file each
time you save, Overwrite, to overwrite an
image file with the same name, or Add To in
order to create an image stack from
sequentially saved images.
6
In the Active Setting list box, select the
active setting that you want to apply to your
image. The description associated with the
setting will appear in the Setting Description
box.
7
In the Setting Description box, view the
details of the setting assigned to the
selected active setting. Based on the setting
information, determine if you can apply the
setting in its present state, or if you need to
modify the setting.
8
If you do not have a Setting file or you need
to modify your loaded setting file, click
Modify. The Segment image modifications
dialog box opens.
9
After you have made modifications in the
Segment image modifications dialog box and
accepted the modifications, the Segment
Image dialog box reopens. Click Save to
save your modifications to either a new or
existing Setting (*.AST) file.
10
Determine the appearance that you want in
the final segmented image output : If you
want to output the image as a Binary Mask
(Binarize), click Create Binary Mask.
OR,
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If you want the segmented image data to
retain its gray-level value, while setting the
value of all non-segmented image data to 0,
click Clip to gray level.
11
Click Apply to Apply the setting values as
described in the Setting Description box.
OR,
Click Close to close this dialog box and
disregard any settings.
Segment Image - Dialog Box Options
Source image
Specifies the name of the source image file.
Result image
Specifies the name of the destination image file and enables you to select creating a new image file,
overwrite an existing image file, or create an image stack from saved images.
Setting
Defines the name and description assigned to the currently active setting.
Active Setting
Defines the name of the currently active setting.
Setting Description
Provides a summary of the segmentation settings in the currently active set.
Load
Loads an existing segmentation settings file.
Save
Saves your current segmentation settings to a segmentation settings file.
Modify
Opens the Segment Image Modifications dialog box to enable you to modify the currently active settings and
preview results of the new settings.
Output
Specifies that one of the following treatments is applied to your image output.
Create binary mask – Outputs your image segmentation result as a binary mask. With this
setting, details detected as image features are set to maximum level (white), and details not
detected as image features and also the background information are set to a zero level (black),
similar to the binarize operation.
Clip to gray level – Outputs your image as a clipped version of the original image. With this
setting, details detected as image features remain at their original gray levels, while details not
detected as image features are set to a zero level (black).
Apply
Applies the segmentation settings stored in the currently active segmentation settings file to the currently
open image.
Close
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Closes the Segment Image dialog box.
Segment Image Modifications
Provides a set of options for making image segmentation settings based on a standard set
of questions designed to determine the types of objects present in your image and their
general characteristics.
Drop-in: SEGIMAGE
Use this dialog box to evaluate the general and specific characteristics of an image and to determine the
image segmentation settings that it must apply to achieve successful image segmentation. Compare the
conditions described on each tab of this dialog box with the actual conditions present in your image.
Make radio button selections according to the conditions that exist in the image. After selections have
been made on the Image Features and More Settings tab, you can test and modify your settings on the
Adjustments tab before making your segmentation settings permanent.
To effectively segment an image type for which you have not already created a setting file, complete the
appropriate settings on the Image Features tab and the More Settings tab. Settings made on the
Adjustments tab and the Diagnostics tab are optional. The Adjustments tab enables you to interactively
view and modify the resulting segmented image using two controls. The Sensitivity Control enables you
determine how much of or how many of the objects in your original image will appear in your segmented
output image. The Segment Length control enables you to change the value for the maximum length of
a detectable object. By reducing the length of isolated segments, you can enable the program to better
distinguish and separate objects that are touching or overlapping. The Diagnostics tab enables you to
make additional modifications to the results of the settings you achieved on the other tabs.
Setting Segment Image Modifications
Use the following procedure to set options in the Segment image modifications dialog box to
specify your segmentation criteria. As you make settings, click Preview to update the output
image to enable you to see the results of your selections.
Step
Action
1
From the Segment Image dialog box, click
Modify. The Segment image modifications
dialog box opens.
2
Click the Source button to select a different
source image from the list (optional).
3
Click the appropriate image selector button
for the output image file destination to make
any changes. You can designate a New
output image file name or Overwrite or Add
To an existing image. (optional).
4
On the Image Features tab, click the
appropriate Illumination setting. Choose
Fluorescent for images created using a
fluorescence light source or for light objects
on a dark background. Choose Transmitted
for images created using a transmitted light
source or for dark objects on a light
background.
5
On the Image Features tab, click the best
method for Distinguishing objects from
background and other objects. Refer to the
individual descriptions on the Dialog Box
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Options page.
6
On the More Settings tab, in the Halos box,
click Correct halos around objects to reduce
or remove the halo effect that can be created
by light passing though transparent
mediums. If no halos are present or if it is
not necessary to remove them, click Objects
do not exhibit halos.
7
On the More Settings tab, in the Shading
Correction box, click Uneven illumination
requires correction, if there is a varying level
of illumination across the image. If the
illumination in the image appears even, click
No illumination shading artifacts.
8
On the More Settings tab, in the
Segmentation Goals box, click one of the
following buttons: Detect discrete object ,
Detect linear objects and structures, or
Segment into several homogeneous layers
or areas. Refer to the individual descriptions
on the Dialog Box Options page.
9
On the More Settings tab, in the Separate
Objects box, click Try to break apart joined
objects to separate objects that are touching
or overlapping. Click Do not adjust objects if
no objects are touching, or if it is not
necessary to separate touching objects.
10
Click Preview to review the your
segmentation results so far. A Preview
image window opens. The image in this
window will have the current segmentation
settings applied. Compare the results in the
output window with the image in the source
image window. Make the necessary
changes to create the segmented image that
you want. If the results are satisfactory, click
Accept to return to the Segment Image
dialog box where you can save your settings
ans create your final result.
11
If further segmentation settings are needed,
click the Adjustments tab. The Adjustments
tab moves to the front.
12
Click Display preview as an overlay on the
image to enable you to view the preliminary
output image superimposed on a copy of the
original image.
13
Click Overlay On/Off to toggle the image
overlay. This enables you to compare the
original image with the segmented image.
14
In the Feature Adjustments box, adjust the
Sensitivity control according to the results
that you want. Move the slider, click the
buttons on the spin box, or type a value in
the spin box. The greater the sensitivity
(100%) the more features are detected.
15
In the Feature Adjustments box, adjust the
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Length control according to the results that
you want. Move the slider, click the buttons
on the spin box, or type a value in the spin
box. The lesser the length the greater the
separation will be between objects that are
touching.
16
In the Feature Adjustments box, click Try a
range of settings. The Try a range of
settings dialog box opens along with an
image window showing a grid of nine
different thumbnail images that show nine
different segmentation possibilities.
17
In the Try a range of settings dialog box,
click the button that corresponds to the best
possible image. The selected image is
shown by itself in the image window. Click
Accept to accept this image,
OR,
Click Cancel to close the dialog box and the
image window without choosing an image.
18
Click the Diagnostics tab to try a different
approach for obtaining the best segmented
image.
19
On the Diagnostics tab, click one or more
symptom descriptions that describe the
visual symptom or problem that you are
observing in your image. Highlight the
symptom description to view an explanation
of the expected improvement to the image
when the selected symptom is checked.
20
Click Preview to view the changes that have
resulted from applying fixes for the selected
symptoms.
21
When the Preview image is satisfactory,
click Accept. The Segment image
modifications dialog box closes, and the
Segment Image dialog box opens.
Segment Image Modifications - Dialog Box Options
Image Features Tab
Selects and shows the Image Features tab. Use this tab to make settings based on the type of image
illumination and object characteristics that can be used to more effectively separate the objects from the
background.
More Settings Tab
Selects and shows the More Settings tab. Use this tab to apply algorithms to the image that can correct for
the appearance of halos, uneven illumination, detect objects that are completely separate from other
objects, and attempt to separate touching objects.
Adjustments Tab
Selects and shows the Adjustments tab. Use this tab to place an overlay of the proposed new image on top
of the original image. The Feature adjustments on this tab enables you adjust the grayscale threshold and
to set a value for the maximum acceptable line segment length.
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Diagnostics Tab
Selects and shows the Diagnostics tab. Use this tab to choose from a list of symptoms that best describe
the symptoms that you are observing in your preview image. View the corrections interactively, then click
Accept to accept the recommend changes based on the symptoms you selected.
Source
Specifies the name of the source image file.
Dest
Specifies the name of the destination image file and enables you to select creating a new image file,
overwrite an existing image file, or create an image stack from saved images.
Preview
Opens an image preview window to view the results of the changes you selected for your image before the
changes are permanently applied to the image.
Cancel
Cancels applying your selected changes and closes the dialog box.
Accept
Accepts and applies your selected changes and closes the dialog box.
Image Features Tab - Dialog Box Options
Illumination
Defines the type of light source used to create the image, or defines the general appearance of the lighting
in the image. This selection is mutually exclusive. Click one choice.
Fluorescent – Indicates that the image was produced using a fluorescence light source or has the
appearance of a light colored object on a dark background.
Transmitted – Indicates that the image was produced using a transmitted light source or has the
appearance of a dark colored object on a light background.
Distinguishing objects from background and other objects
Defines the algorithm that will be applied to improve the visual separation of objects from the background
and other objects. This selection is mutually exclusive. Click one choice.
Detect objects by intensity – Isolates objects that have a difference in intensity from the
background. The difference in object texture is not considered.
Detect objects by texture – Isolates objects that have a different texture than the background.
The difference in object intensity is not considered.
Detect objects by intensity and texture – Uses the difference in values in both intensity and
texture to isolate objects from the background.
Detect objects by color – Isolates objects from the background that have a different color than
the background.
More Settings Tab - Dialog Box Options
Halos
Removes or reduces the appearance of halos around objects in the image that have resulted from excessive
light diffusion through glass or other light transmissive media.
Correct halos around objects – Applies remove halo to Image features. Click when halos are
observed.
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Objects do not exhibit halos – Deactivates halo removal. Click when no halos are observed.
Shading Correction
Enables or disables automatic background correction. If the image background is uneven, this option
attempts to adjust the gray level of the background to the same level of intensity across the entire image.
Uneven illumination requires correction – Enables automatic background correction. Click if
the image background is uneven.
No illumination shading artifacts – Deactivates automatic background correction. Click if the
image background appears to be even.
Segmentation goals
Selects one of three criteria for segmenting objects in an image.
Detect discrete objects (blobs) – Detects large, unstructured binary objects that are completely
discrete (not touching or attached to other objects).
Detect linear objects and structures – Detects lines and coarse structures.
Segment into several homogeneous layers or areas – Separates individual objects or structures
into separate layers or areas.
Overlay colors on output – Assigns a different color overlay for each segment layer or
area.
Output multiple layers as stack planes – Organizes multiple layers of segmentation as
separate planes in a stack.
Separate objects
Enables or disables the option for separating objects that are touching or connected.
Try to break apart joined objects – Enables the option for separating joined or touching objects
into separate objects.
Do not adjust objects – Ignores objects that are joined or touching.
Adjustments Tab - Dialog Box Options
Display preview as an overlay on the image data
Overlays the Preview image on a copy of the original image. Use this option to help you determine how well
your segmentation results match their data.
Overlay On/Off
Activates or deactivates the Preview image overlay feature. Use this button to toggle the overlaid preview
image on and off to get a comparison of the two images.. This button is inactive if Display preview as an
overlay on the image data is unchecked.
Feature Adjustments
Use these options to interactively view your segmentation results. You can either accept your results or
adjust the segmentation settings on this tab to produce updated segmentation results.
Sensitivity (%) – Controls the level of grayscale threshold (either local or global) applied
to the image.
Length (units) – Specifies the estimated length of the largest object to be segmented in the image.
Try a range of settings on the image – Applies a range of settings for sensitivity and length to the
image and displays a panel of images from which you can select the best result.
Diagnostics Tab - Dialog Box Options
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Select symptoms that describe the segmentation result
Enables you to select and apply a number of symptoms from this list that best describe your anticipated
segmentation result.
Objects missing from the result
Defines the objects that were excluded from your segmentation result by your segmentation settings
selection.
Correlation Plot (Apps Menu)
Measures and displays the correlation between the intensities of corresponding pixels in
two images. Provides a correlation coefficient (r) of the pixel intensity data.
Availability: Included in MetaMorph Basic and MetaMorph Premier
Drop-in: CORRPLOT
Use this command when you want to display a graphical representation of the correlation between
corresponding pixel intensities in two images and log the measured correlation coefficient. The
correlation plot that is created represents a large amount of data in a resizable scatterplot. For every
image pixel being analyzed, MetaMorph examines the intensity of the corresponding pixels in the two
images, and uses the two intensity values as the X and Y coordinates in the scatterplot.
∑ xy
The Correlation Plot dialog box displays the correlation coefficient (r) of the data. This is defined as
r=
NS x S y
where
r = correlation coefficient,
xy = product of deviation scores,
N = sample size,
Sx = standard deviation of X (intensities in first image), and
Sy = standard deviation of Y (intensities in second image).
The range of values of the correlation coefficient is -1.0 to +1.0. A value of 1.0 shows that the data are
perfectly correlated with one another. This will only happen if the two images are identical. A correlation
coefficient of -1.0 is observed when there is an inverse relationship between intensities in the two
images.
Before you make your measurements, you may wish to use a Region Tool to define and select a specific
region of interest for measurement. MetaMorph allows you to move, resize, or switch between regions
while you follow the resulting measurements.
You also have the option of using thresholding in either or both of the images. Only pixels that have
intensities that are outside of the threshold range of both images will be excluded from the
measurement. If thresholding is turned off in either image, all pixels in the active region will be
measured. Thresholding will affect measurement of the correlation coefficient.
The correlation scatterplot that is displayed can be adjusted in two ways. First, the size of the plot
window can be adjusted up or down by using the Plot Size slider. Second, the range of gray values that
are displayed in the plot can be adjusted. The range of values selected in the first image will affect the Xaxis, and the range selected from the second image will affect the Y-axis. You can select the range
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manually by using the Min and Max spin boxes, or you can have MetaMorph select the ranges for you
automatically by selecting the Auto Scale check box. The range that you select will affect only the display
of the plot; it will not affect the measurement of the correlation coefficient.
QUICK TIP: To hide the Image Window Tools on the Plot window, right-click in the window and choose
Hide Image Window Toolbar from the pop-up context menu that appears.
Plotting Intensity Correlations
To display or print a correlation scatterplot of corresponding pixel intensities in two images, use
the following procedure:
Step
Action
1
If desired, define at least one region for
measurement. If regions have been defined,
skip to Step 2.
2
From the Apps menu, choose Correlation
Plot. The Correlation Plot dialog box and a
Plot window opens.
The currently selected region and the
correlation coefficient will be displayed in the
Correlation Plot dialog box, and the Plot
window will display a scatterplot of the
intensity correlation data.
QUICK TIP: To hide the Image Window
Tools on the Plot window, right-click in the
window and choose Hide Image Window
Toolbar from the pop-up context menu that
appears.
3
If necessary, use the X and Y image
selectors to select the appropriate images.
4
If you have applied thresholding and want to
restrict the data analysis to pixels that have
not been excluded from both images by
thresholding, select the Use Thresholds
check box. (Pixels that are included in the
threshold range in either image will be
measured.) The scatterplot and displayed
correlation coefficient will be updated
automatically.
5
To change the size of the Plot window, use
the Plot Size slider to specify a size. A
smaller size will be specified by sliding it to
the left.
6
If you want the X and Y axis of the Plot to be
scaled automatically, based on the intensities
in the two images, select Auto Scale.
OR
Use the Min and Max spin boxes in the X
and Y option groups to select the lower and
upper grayscale intensities to be plotted from
each image.
7
If you want to store the correlation
measurement in a log file and you do not
have a data log open, choose Open Log to
select a text-based log file or to open a DDE
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link to an open spreadsheet..
8
To configure data logging, choose Configure
Log. The Configure Log dialog box will
appear.
AND
From the Configuration table, select the
parameters you want to log, so that each is
marked by a check mark next to its entry
(you can choose Enable All or Disable All if
you want to select or deselect all of the
parameters listed). Then Choose OK to
return to the Log Pixels in Region dialog box.
9
When you are ready to log the correlation
measurement, choose F9: Log Data.
10
You can select another region to be plotted
by clicking it directly in the image window, or
you can resize the currently active region by
dragging its outline. The Correlation Plot
dialog box and the Plot window will update
automatically to reflect the new
measurement. Choose F9: Log Data
whenever you want to save the
measurements.
11
When you have finished, choose Close from
the Correlation Plot dialog box. Both the
dialog box and the Plot window will close.
Correlation Plot - Dialog Box Options
X (image selector)
Selects the first image, which will have its pixel intensities plotted as the X-coordinates of the points in the
correlation plot.
Y (image selector)
Selects the second image, which will have its pixel intensities plotted as the Y-coordinates of the points in
the correlation plot.
Min
Manually selects a minimum pixel intensity for the corresponding image (X or Y) to be plotted in the
correlation plot.
Max
Manually selects a maximum pixel intensity for the corresponding image (X or Y) to be plotted in the
correlation plot.
Image Whose Region Should Be Used
This option appears only in Journal Edit mode. Selects which image's region, if any, to use in the correlation
plot (Image X, Image Y, or None).
Auto Scale
Scales the X and Y axes of the correlation plot to include the entire range of pixel intensities available in the
two images. If there is an active region and/or thresholding has been applied, only pixels in the region and/or
with intensities within the threshold range will be plotted.
Use Thresholds
Restricts the data analysis to pixels with intensities in the threshold range of either of the images. Only pixels
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Drop-in Commands
with grayscale values outside of both threshold ranges will be excluded from measurement.
Plot Size
Changes the size of the Plot window.
Correlation Coefficient
Displays the correlation coefficient value (r) for the current correlation measurement data set.
Open Log
Opens a data log and/or a DDE link to an open spreadsheet application for logging data. This command
changes to F9: Log Data when a log file is open.
F9: Log Data
Logs the pixel data from the active region to an open data log or to an open spreadsheet application via a
DDE link. To assist you in logging the proper data when several measurement dialog boxes are open, "F9"
is added to the name of this option in the active dialog box to indicate which data will be logged when you
press [F9].
Configure Log
Opens the Configure Log dialog box so that you can select the parameters to be logged to the data log.
Parameters marked with a check mark will be logged for subsequent measurements.
If you select Log Column Titles, a line listing the measurement titles will be logged (1) the first
time you use the configured measurement, (2) whenever you enable/disable measurement
parameters, or (3) whenever the logged measurement is different from the previous measurement
in the log file.
If you select Place Log Data on Current Line, subsequently logged data will be appended to the
current line in the log file, rather than to a new line. Log Column Titles will be unavailable when you
select this option.
Close
Closes the Correlation Plot dialog box and the Plot window.
Log Pixels in Region (Log Menu)
Logs pixel grayscale data to a data log from the active region of interest of an image or
current plane of a stack. Copies pixel grayscale data from the active region to the Kernel
Editor if the region is smaller than 15 x 15 pixels.
Drop-in: LOGPIX
Use this command to log pixel grayscale data to a data log. You can also use this command to copy the
pixel grayscale data into the Kernel Editor. If you want to copy the data into the Kernel Editor, the region
must be smaller than 16 x 16 pixels. Before applying the kernel, you will need to change the Result
option in the Edit Kernel dialog box to the number of pixels in the kernel.
The Log Pixels in Region dialog box provides options for specifying the location and size of the region.
This dialog box also provides some region control options that, when enabled, provide full control over
the specified region characteristic, but will restrict it when disabled.
Note: Regions drawn with a two-dimensional Region Tool (Rectangular, Ellipse, Trace,
or Auto-Trace) must be at least 2x2 in size to be valid in MetaMorph.
Use the Open Data Log command to open a data log before using this command. You can use the Log
Data command or its keyboard shortcut, the [F9] function key, to log the data.
Logging Pixel Gray Scale Values from a Region
To log pixel intensity values from a region, use the following procedure:
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Step
Action
1
From the Log menu, choose Log Pixels in
Region. The Log Pixels in Region dialog box
opens.
2
Select the desired image for logging pixels
using the Image selector.
3
Choose Open Log to open a data log, if one
is not already open.
4
To configure data logging, choose Configure
Log. The Configure Log dialog box will
appear.
AND
From the Configuration list, select the
parameters you want to log, so that each is
marked by a check mark next to its entry
(you can choose Enable All or Disable All if
you want to select or deselect all of the
parameters listed).
Choose OK to return to the Log Pixels in
Region dialog box.
5
Draw a region of interest on the active image
using a Region Tool. Select it so that it is the
active region of interest.
Note: Regions drawn with a two-dimensional
Region Tool (Rectangular, Ellipse, Trace, or
Auto-Trace) must be at least 2x2 in size to
be valid in MetaMorph.
6
Left, Top, Width, and Height display the
location and size of the region. You can use
these options to reposition or resize the
region if desired.
The Region Control options provide greater
control over the region. You can enable or
disable these as desired.
7
If you want to copy the pixel data from the
active region to the Kernel Editor, choose
Copy to Kernel Editor.
8
Choose F9: Log Data to log the pixel data
from the region.
9
To measure another region, create and
select another region using the process
described in Steps 5 - 8.
10
Choose Close when you have finished .
Log Pixels in Region - Dialog Box Options
Image
Specifies the image from which regional pixel gray values will be logged.
Left
Specifies the X-coordinate for the upper left corner of the region to be used for logging the pixels.
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Top
Specifies the Y-coordinate for the upper left corner of the region to be used for logging the pixels.
Width
Specifies the width of the region to be used for logging the pixels.
Height
Specifies the height of the region to be used for logging the pixels.
Horiz. Movement
Allows horizontal movement of the region when enabled.
Vert. Movement
Allows vertical movement of the region when enabled.
Keep in Image
Forces the entire region to stay inside image when enabled.
Horiz. Resizing
Allows horizontal resizing of the region when enabled.
Vert. Resizing
Allows vertical resizing of the region when enabled.
Deletable
Region can be deleted when enabled.
Copy to Kernel Editor
Copies the pixel data into the Kernel Editor. If you want to copy the data into the Kernel Editor, the region
must be smaller than 16 x 16 pixels. Before applying the kernel, you will need to change the Result option
manually in the Edit Kernel dialog to the number of pixels in the kernel.
Open Log
Opens a data log and/or a DDE link to an open spreadsheet application for logging data. This command
changes to F9: Log Data when a log file is open.
F9: Log Data
Logs the pixel data from the active region to an open data log or to an open spreadsheet application via a
DDE link. To assist you in logging the proper data when several measurement dialog boxes are open, "F9"
is added to the name of this option in the active dialog box to indicate which data will be logged when you
press [F9].
Configure Log
Opens the Configure Log dialog box so that you can select the parameters to be logged to the data log.
Parameters marked with a check mark will be logged for subsequent measurements.
If you select Log Column Titles, a line listing the measurement titles will be logged (1) the first
time you use the configured measurement, (2) whenever you enable/disable measurement
parameters, or (3) whenever the logged measurement is different from the previous measurement
in the log file.
If you select Place Log Data on Current Line, subsequently logged data will be appended to the
current line in the log file, rather than to a new line. Log Column Titles will be unavailable when you
select this option.
Close
Closes the dialog box.
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Measure Grid (Apps Menu)
Creates a user-configurable measurement grid on an image and performs measurements,
or runs a journal to process and/or measure the image regions, within each element in the
grid.
Availability: Included in MetaMorph Basic and MetaMorph Premier
Drop-in: MEASGRID
Use this command to divide an image into evenly spaced regions and run a journal to measure each
region. This function is particularly useful for images with regularly spaced areas of interest, such as
those from a gene chip. Measurement parameters include the location (coordinates of the upper left
corner), width, height, area, perimeter, thresholded area, average, minimum, maximum, and integrated
intensity value, standard deviation and signal-to-noise level of the intensity, and the percent thresholded
area.
When you first open the Measure Grid dialog box, the measurement grid and three regions will appear in
an overlay on the selected image: an Anchor region, an Angle line region, and a Reference region. The
following sample figure illustrates the placement of the Measure Grid regions and measurement grid:
In their default configuration, the Anchor and Reference regions appear in the upper left and lower right
corners of the measurement grid, respectively. Their placement defines the initial horizontal and vertical
extent of the measurement grid. These two regions can be resized and moved in the same manner as
any other region of interest. Alternatively, you can use the Horizontal Spacing and Vertical Spacing spin
boxes in the Measure Grid dialog box to define the size of each element in the grid, and then set the
number of rows and columns with the Columns and Rows spin boxes in the Grid option group. To apply
a change you have made from the dialog box, click another option in the dialog box, or press the [TAB]
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key.
The Angle line region is used to change the angle of the grid to correspond to the angle of the data
regions. This may be necessary for images in which such regions are not arranged in a perfectly vertical
and horizontal orientation. After you make the Angle line region the active region by clicking it, you can
move the region by dragging it. You can change its angle or length by double-clicking one end of the line
and dragging the rounded handle that appears. Alternatively, you can use the Grid Angle spin box to
select an angle with respect to the X-axis. Again, to apply the configured change, click another option in
the dialog box, or press the [TAB] key.
Many of the settings in the Grid, Anchor Region, and Reference Region option groups have an
interactive effect on one another and the on the size and placement of the measurement grid. For
example, if you increase the Column setting in the Reference Region group, the setting in the Columns
spin box in the Grid option group will also increase. Simultaneously, the number of columns in the
measurement grid will increase, and the Horizontal Spacing setting will decrease, reflecting the
diminished width of each element in the grid. If you then decrease the Column setting in the Reference
Region group, the number of columns in the measurement grid will stay at its higher setting, and the
Reference region will stay in its current location on the image, but the size and width of the measurement
grid will increase. You will need to experiment to find your optimum settings for the various options in the
dialog box.
After you have configured the measurement grid to your specifications, you will need to configure the
size and shape of the measurement regions within each grid element using the Measure Each option
button group and the Region Width and Region Height spin boxes.
Regions by their nature must lie on discrete pixels. In practice, however, samples in the image rarely
align with discrete pixels. For this reason, the grid spacing can be set to non-integer values. When
regions are placed on the image, the vertices will fall on discrete pixels. Thus, because the size of the
regions will not change, the Unit Square sizes may differ slightly from the displayed grid.
A data log can be opened and configured directly from the Measure Grid dialog box. Choosing F9: Log
Data will measure the region within each grid element. You can use the Configure Log button to select
which measurement values to record. If you need measurements other than those provided in the
Configure Log dialog box, or if you wish to perform some additional processing, you can use the Run
Journal for Each Region option. (But you will not be able to run Measure Grid in a journal from within
itself: doing so will generate an error message.) When you use Run Journal for Each Region, a region
will be placed over the first grid element and made active. The journal will then be run for that region.
The region is then moved to the next element, and the process will be repeated for each grid element in
turn. Nothing will be logged, however, unless the journal being run explicitly performs the logging
function.
Measuring Grids
Overview of Grid Measurement
Configuring the Grid
Overview of Grid Measurement
To use the Measure Grid command to measure an image, use the following procedure as a
general guideline. If you intend to run a journal that makes measurements, be sure to threshold
the image before proceeding.
Step
1
Action
From the Apps menu, choose Measure Grid.
The Measure Grid dialog box opens, and a
measurement grid (green), an Anchor region
(cyan), an Angle line region (yellow), and a
Reference region (magenta) appears in an
overlay on the active image.
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2
If necessary, use the Image selector to select
the image you want to measure.
3
Follow the directions for configuring the
measurement grid.
4
If you want to measure the regions and
simultaneously log your measurements,
choose Open Log and select a data log. The
Open Log button label will change to "F9:
Log Data." Continue to Step 5.
OR
If you want to run a journal to process or
measure each measurement region, skip to
Step 6.
5
To configure the data log for logging, choose
Configure Log. The Configure Log dialog box
will appear.
AND
From the Configuration list, select the
parameters you want to enable for logging,
so that each is marked by a check mark next
to its name (you can choose Enable All or
Disable All if you want to select or deselect
all of the parameters listed). Then choose
OK to return to the Measure Grid dialog box.
Now skip to Step 7.
6
To run a journal to process or measure each
measurement region, choose Select a
Journal. The Select Journal dialog box will
appear.
AND
Select the icon for the desired journal. (You
will not be able to run Measure Grid in a
journal from within itself: doing so will
generate an error message.) If necessary,
use the Look In list or Up One Level icon
button to select the pertinent drive and folder.
Choose Open. Then continue to Step 7.
7
If you are running a journal, choose Run
Journal for Each Region. The selected
journal will be run for each region in the grid.
OR
If you are logging data, choose F9: Log Data,
or press the [F9] function key. The selected
measurements will be made and logged to
the data log.
8
When you have finished, choose Close.
Configuring the Grid
To configure the measurement grid, use the following procedure:
Step
1
Action
Click the Anchor region (a cyan-colored
elliptical region in the upper left corner of the
grid) to make it the active region. Then use
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the pointer to drag the Anchor region to the
uppermost, leftmost sample that you want to
measure in the image.
Alternatively, you can use the X Position and
Y Position spin boxes in the Anchor Region
option group to specify the placement of the
Anchor region. Be sure the Column and Row
spin boxes in the Anchor Region option
group correspond to the column and row in
the measurement grid that you want to use to
define the Anchor position (typically Column
1, Row 1).
2
Click the Angle region (a horizontal yellow
line region across the first row in the grid) to
make it the active region. Drag the Angle
region up, down, to the left, or to the right to
move it to the desired position.
To change the line's length and angle,
double-click one end of the line and drag the
round handle that appears. It is best to place
the left handle over the center of the leftmost
sample and the right handle over the
rightmost sample.
Alternatively, you can change the Angle
region's angle setting by selecting a value
with the Grid Angle spin box in the Measure
Grid dialog box's Grid option group.
3
Click the Reference region (a magentacolored elliptical region in the lower right
corner of the grid) to make it the active
region. Then use the pointer to drag the
Anchor region to another clearly identifiable
sample. This typically will be the lowermost,
rightmost sample that you want to measure
in the image (but does not need to be, as
long as it is not in the same row or column as
the Anchor region).
4
Adjust the Reference Region option group's
Column and Row settings to reflect the
configuration of the image samples. If
necessary, readjust the Grid option group's
Column and Row settings. These must have
a width and height of at least two pixels.
5
For added visual feedback, select the Show
Complete Grid check box to see the grid
lines for all of the measurement grid's
columns and rows. Clear the check box if
you want to see just the bounding rectangle
of the grid and the Anchor and Reference
regions' rows and columns.
6
From the Measure Each option button group,
select the type of region that you want to
measure for each grid element. Select
Unit Square if you want to measure the entire
rectangular region that is defined by the
column and row grid lines. If you select this
option, the Region Width and Region Height
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spin boxes will become unavailable, and will
appear dimmed.
OR
Circle Region if you want to measure an
elliptical region that corresponds in size to
the magenta-colored Reference region. You
can adjust the dimensions of the Reference
region by dragging its outline. Alternatively,
you can use the Region Width and Region
Height spin boxes to specify the respective
horizontal and vertical sizes, in pixels, of the
measurement regions. These must have a
width and height of at least two pixels.
7
If your image has been thresholded and you
want to measure just the thresholded
regions, select the Use Threshold for
Measurement check box.
OR
If you want to measure all the entire area in
each grid element, leave the Use Threshold
for Measurement check box cleared.
8
If necessary, adjust your settings for
optimum size and placement of the
measurement grid and its elements. Then
continue with Step 4 of the procedure
described in the Overview of Grid
Measurement.
Measure Grid - Dialog Box Options
Image
Selects the image to be measured.
GRID
This option group specifies the size, angle, and number of rows and columns of the measurement grid.
Grid Angle
Determines the angle of the measurement grid, in degrees, with respect to the X-axis (horizontal). The grid
will be rotated around the Anchor region, which serves as the pivot point. Changing the grid angle will
change the position of the Reference region.
Columns
Selects the number of columns of samples in the horizontal (X-axis) direction that will be measured.
Rows
Selects the number of rows of samples in the vertical (Y-axis) direction that will be measured.
Vertical Spacing
Specifies the vertical size, in pixels, of each element in the measurement grid. This measurement is based
on the distance between the centers of two successive elements in a column. Changing this distance will
change the position of the Reference region, and vice-versa. The spacing must be at least two pixels high.
Horizontal Spacing
Specifies the horizontal size, in pixels, of each element in the measurement grid. This measurement is
based on the distance between the centers of two successive elements in a row. Changing this distance will
change the position of the Reference region, and vice-versa. The spacing must be at least two pixels wide.
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Show Complete Grid
Determines the display of the measurement grid. If you select this check box, all of the elements of the
measurement grid will be displayed. This is useful for checking the configuration of the grid before you
perform the measurement. If you clear this check box, only the outermost rows and columns of the grid will
be displayed, along with the lines marking the row and column of the Anchor and Reference regions.
ANCHOR REGION
This option group specifies the location of the Anchor region on the image and its position within the
measurement grid.
Column (Anchor Region)
Selects the horizontal position of the Anchor region within the measurement grid. Increasing this setting will
move the grid and the Reference region to the left, one column at a time. Decreasing the setting will move
the grid and the Reference region to the right.
Row (Anchor Region)
Selects the vertical position of the Anchor region within the measurement grid. Increasing this setting will
move the grid and the Reference region upwards one row at a time. Decreasing the setting will move the
grid and the Reference region downwards.
X Position
Specifies the vertical location of the center of the Anchor region, in pixels, from the top border of the image.
Y Position
Specifies the horizontal location of the center of the Anchor region, in pixels, from the left border of the
image.
REFERENCE REGION
This option group specifies the location of the Reference region on the image and its position within the
measurement grid.
Column (Reference Region)
Selects the horizontal position of the Reference region within the measurement grid. Increasing this setting
will increase the number of columns in the grid and decrease the width of the columns without moving the
Anchor or Reference regions on the image. Decreasing this setting without altering the Grid option group's
Columns setting will increase the width of the columns, and the Reference region will be positioned one
column to the left in the measurement grid, although the Reference region will not itself be moved from its
location on the image.
Row (Reference Region)
Selects the vertical position of the Reference region within the measurement grid. Increasing this setting will
increase the number of rows in the grid and decrease the height of the rows without moving the Anchor or
Reference regions on the image. Decreasing this setting without altering the Grid option group's Rows
setting will increase the height of the rows, and the Reference region will be positioned one row higher in the
measurement grid, although the Reference region will not itself be moved from its location on the image.
Measure Each
Determines the type of region that will be measured at each element in the measurement grid. Unit Square
will measure the entire rectangular region that is defined by the column and row grid lines. If you select this
option, the Region Width and Region Height spin boxes will become unavailable, and will appear dimmed.
Circle Region will measure an elliptical region, centered within each grid element, that corresponds in size to
the dimensions of the Reference region. Use the Region Width and Region Height spin boxes to specify the
respective horizontal and vertical sizes, in pixels, of the measurement regions.
Use Threshold for Measurement
When selected, this check box specifies that only the thresholded regions in each grid element are to be
measured. When this check box is cleared, the entire area within each cell will be measured.
Region Width
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Specifies the horizontal size of each elliptical measurement region. This option is available only when you
select Circle Region from the Measure Each group. The width must be at least two pixels across.
Region Height
Specifies the vertical size of each elliptical measurement region. This option is available only when you
select Circle Region from the Measure Each group. The height must be at least two pixels across.
Open Log
Opens a data log and/or a DDE link to an open spreadsheet program for logging data. This command
changes to F9: Log Data when a log file is open.
F9: Log Data
Measures the samples and logs the measured data to an open data log or to an open spreadsheet by way
of a DDE link. The values that can be measured are similar to those for the Show Region Statistics
command. Measurements themselves will not be displayed. To assist you in logging the pertinent data when
several measurement dialog boxes are open, "F9" is added to the name of this option in the active dialog
box to indicate which data will be logged when you press the [F9] function key.
Configure Log
Displays the Configure Log dialog box, from which you can select the parameters to be logged to the data
log. Parameters marked with a check mark will be logged for subsequent measurements.
If you select Log Column Titles, a line listing the measurement titles will be logged (1) the first time you use
the configured measurement, (2) whenever you enable/disable measurement parameters, or (3) whenever
the logged measurement is different from the previous measurement in the log file.
If you select Place Log Data on Current Line, subsequently logged data will be appended to the current line
in the log file, rather than on a new line. Log Column Titles will be unavailable when you select this option.
Select a Journal
Displays the Select Journal dialog box, from which you can select the processing and/or measurement
journal that you want to run for each measurement region in the grid.
Run Journal for Each Region
Starts the grid measurement process, placing an active measurement region over each element in the
measurement grid in turn, and running the selected journal at each position. This function will not log any
data unless the journal being run does so explicitly.
Close
Clears the grid and region overlays from the image and closes the dialog box.
Measure XYZ Distance (Apps Menu)
Measures the spatial distance between pairs of points in different image planes in a stack.
Plays back three-dimensional wireframe rotations of the track lines. Distance
measurements are displayed in the dialog box, and can be saved in a data log.
Drop-in: MEASXYZD
Use this command to measure the straight-line distance between pairs of points or between endpoints of
a multi-plane track. The points can be in the same image or image plane, or they can be in different
planes in an image stack.
Distances can be expressed either in pixels or in distance units that have been calibrated with the
Calibrate Distances command (Measure menu). Similarly, the Z-distance between successive planes
can be set to use either calibrated units or a user-specified distance.
Measurement lines and their distance values are drawn in a measurement overlay. You can show or
hide the overlay as desired. You can also select whether to show all lines and values, or just those for
the last distance measured, and you can enable or disable display of the measurement values on the
image. All measurements and their associated overlay elements will be retained even when their display
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is disabled.
An additional feature of the Measure XYZ Distance command is its ability to play back the threedimensional tracks between points in a wireframe rotation. The wireframe can be rotated along either the
horizontal or vertical axis. This feature is similar to those seen with the 3D Reconstruction drop-in. This
command can also used simultaneously with the View Orthogonal Planes drop-in command, which is
also a component of the 3D Module. The tracking line can be observed as an overlay on both the original
image stack and on the XZ and YZ view orthogonal plane stacks.
Note: The measurement overlay will be retained with the image stack if you choose to
save it. If you do not want the overlay to be saved with the stack, be sure to choose No
from the Image Has Been Modified dialog box when you close the stack.
Measuring XYZ Distance
Measuring XYZ Distance - Overview
Configuring Display
Performing a Wireframe Rotation
Measuring XYZ Distance - Overview
To measure multi-plane distances between pairs of points, use the following procedure.
Note: If you want to use calibrated distance values, you will need to apply the Calibrate
Distances command (Measure menu) before using this procedure.
Step
Action
1
From the Apps menu, choose Measure XYZ
Distance. The Measure XYZ Distance dialog
box opens.
2
Use the Stack image selector to select the
image you want to measure. (Note: The
image does not need to be an image stack;
this command works equally well on singleplane images.)
3
From the Display option group, configure
the display for the points and the tracks
between them.
4
Use the Use Z Distance radio button group to
select the method for calculating distance.
(The X and Y values will always be the
calibrated values.) Select
Calibrated if you want to use the currently
active distance calibration (this will be
measured in pixels if you have not applied
the Calibrate Distances command, or in
calibrated units if you have), or
User Specified if you want to specify the
between-plane distance. Then specify the Zdistance with the Z Step spin control that
appears in the dialog box.
5
If you want to log the distance
measurements, choose Open Log and select
the icon for the desired data log you want to
overwrite or append, or type a name for a
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new data log in the File Name text box. Then
choose Save. The label on the Open Log
command button will change to "Log Data."
AND
If necessary, choose Configure Log and
select the data parameters that you want to
log from the Configuration list. Then choose
OK.
6
Open the Change Plane dialog box for the
image to be measured by choosing its
Change Plane Tool.
7
Select the plane for the starting point of the
distance you want to measure and click the
first point with the pointer using the left
mouse button.
8
Select the plane for the next point in the track
whose distance you are measuring and click
the point. If this is the last point in the track,
use the left mouse button. If you make a
mistake or otherwise want to remove a point
you have just added, choose Undo Click.
OR
If this is not the last point in the track, use the
right mouse button to click the point. Then
when you finally come to the last point in the
track, use the left mouse button to click the
point.
9
Depending on the display selections you
made, measurement lines and values will
appear in the image overlay, and the
distance will be displayed in the Measure
XYZ Distance dialog box's Distance status
line.
10
Repeat Steps 7 - 9 for all distance
measurements you want to make.
11
If you opened a data log, choose Log Data to
store all your measurement data.
All the points displayed will be logged. If you
select Accumulated, all distances measured
for the image since the dialog box was
opened, or since you last chose the Clear
Measurements command button, will be
logged. If you leave Accumulated cleared,
only the last distance measured will be
stored.
12
If you want to perform a three-dimensional
wireframe rotation of the images and the
measurement track overlays, follow the
procedure for performing a wireframe
rotation.
13
When you have finished, choose Close.
Configuring Display for Measure XYZ Distance
To configure the point and measurement track display for XYZ distance measurements, use the
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following procedure:
Step
Action
1
If you have not already done so, follow the
first two steps in the Measuring XYZ
Distance Overview.
2
To enable display of the measurement
overlay, select the Show Overlay check box
so that a check mark appears in it.
3
If you want to display measurement lines
(and values) for all pairs of points, select the
Accumulated check box.
OR
If you only want to display the measurement
lines (and values) for the most recently
measured pair of points, leave the
Accumulated check box cleared.
4
If you want to display the individual points
along a multi-point track, select the Show
Nodes check box.
5
If you want to display measurement values
directly in the image overlay, select the
Stamp Distances check box.
OR
If you want to display only the measurement
lines in the image overlay, but not the
distance values, leave the Stamp Distances
check box cleared. (The distance
measurement will still be displayed in the
Line Length status line in the Measure XYZ
Distance dialog box.)
6
Make your color selections for the
measurement overlay. Select a color for the
distance line from the Line Color drop-down
list box. Then select a color for the starting
point of the distance being measured from
the Pt 1 Color list.
Performing a Wireframe Rotation with Measure XYZ Distance
To perform a three-dimensional wireframe rotation of the images and the measurement track
overlays, use the following procedure:
Step
Action
1
If you have not already done so, follow the
first 11 steps in the Measuring XYZ
Distance Overview.
2
To adjust the angle settings for the wireframe
view, use the Angle of First View, Angle of
Last View, and Angle Between Views options
to select the desired settings.
3
From the Rotation option button group, select
a wireframe rotation direction: Horizontal or
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Vertical.
4
If you want to see a preview of the wireframe
before building the actual three-dimensional
display, select the Show Preview check box.
A preview of the wireframe will be displayed
in a Preview window.
AND
Then select an angle of view from the
Preview Angle spin box. This angle will also
be affected by the direction of rotation you
selected in Step 3.
5
When you are ready to build the wireframe
display, choose Build Wireframe. The threedimensional wireframe representation will be
created and displayed in a Wireframe
window.
6
You can "play" the wireframe display back
and forth by choosing the Change Planes
Tool from among its Image Window Tools
and manipulating the slider in the Change
Plane dialog box that appears.
Measure XYZ Distance - Dialog Box Options
Stack
Selects the image stack to be used for measuring distance.
Show Overlay
Enables or disables display of the measurement overlay.
Accumulated
Determines whether all distance measurements will be displayed in the image overlay (checked) or only the
last measurement (unchecked). Clearing this check box may aid in viewing image details without
interference from the overlay.
Note: All measurements and their graphical elements will be retained even if the
Accumulated check box is left cleared. If you then select the Accumulated check box, all
measurements and their graphical elements will be displayed.
Show Nodes
Determines whether or not the individual points in a measurement track are to be indicated by a dot in the
measurement track overlay.
Stamp Distances
Determines whether or not measurement values will be displayed in the image overlay. Note: The distance
will be still be displayed in the Measure XYZ Distance dialog box's Line Length status line.
Line Color
Selects a color for the distance measurement lines and their values displayed in the image overlay.
Pt 1 Color
Selects a color for the starting point of the distance measurement lines. This color will also be used to
display the current point while you are selecting a series of points.
Use Z Distance
Determines how the Z-distance is to be calculated. Calibrated will use the currently active distance
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calibration (measured in pixels if you have not applied the Calibrate Distances command, or in calibrated
units if you have). User Specified allows you to specify the between-plane distance with the Z Step spin box.
X:Y:Z Units
Indicates the ratio between X, Y, and Z distances, as well as the units used. If the Calibrate Distances
command has not been applied, distances will be expressed in pixels.
Z Step
Specifies a step size for the between-plane Z-distance. This option will only appear if you select User
Specified from the Use Z Distance group.
Line Length
Indicates the distance between the last measured pair of endpoints.
Preview Angle
Selects an angle of view for the wireframe Preview window. This view will also be affected by your selection
from the Rotation option button group.
Show Preview
Displays a wireframe preview of the measurement track in a separate Preview window.
Angle of First View
Sets the angle by which the first reconstructed view will be offset from zero degrees.
Angle of Last View
Sets the angle by which the last reconstructed view will be offset from zero degrees.
Angle Between Views
Sets the interval between adjacent views in the stack.
Rotation
Selects a direction (Horizontal or Vertical) for the wireframe rotation.
Build Wireframe
Creates a three-dimensional wireframe representation of the measurement track and displays it in a
separate Wireframe window.
Undo Click
Removes the last point added. You can use this command button repeatedly to remove several points you
have just added.
Clear Data
Clears all measurements and removes the image overlay.
Log Total Line Only
When this option is selected, only the data for the entire measurement track will be logged. If you leave this
check box cleared, the distance data for each pair of points in the track will also be logged.
Open Log/Log Data
Opens a data log and/or a DDE link to an open spreadsheet application for logging data. This command will
change to F9: Log Data when a log file is open. Subsequently choosing the button or pressing the [F9] key
will save the distance measurement data for all currently displayed distance lines (distances, starting and
ending XYZ coordinates, elapsed time, etc.) in the data log. If you selected the Accumulated check box, all
distances measured for the image since the dialog box was opened, or since the you last chose Clear
Measurements, will be logged. If did not select Accumulated, only the last distance measured will be stored.
Configure Log
Opens the Configure Log dialog box so that you can select the parameters to be logged to the data log.
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Parameters marked with a check mark in the Configuration list will be logged during subsequent
measurements.
If you select Log Column Titles, a line listing the measurement titles will be logged (1) the first time you use
the configured measurement, (2) whenever you enable/disable measurement parameters, or (3) whenever
the logged measurement is different from the previous measurement in the log file.
If you select Place Log Data on Current Line, subsequently logged data will be appended to the current line
in the log file, rather than to a new line. Log Column Titles will be unavailable when you select this option.
Close
Closes the dialog box.
Measure Volume (Apps Menu)
Measures the volume of a thresholded object through all of the planes in a stack.
Availability: Available for MetaMorph Basic; included in MetaMorph Premier
Drop-in: MVOLUME
Use this command to measure the volume of an object in a Z-series stack of through-focus images. This
measurement requires that the images first be thresholded to separate the object to be measured from
its background. You must also draw a region of interest around the object, to delineate the extent of the
thresholded region.
The thresholded area in each image plane will be used to determine the volume of the object between
that plane and the next. MetaMorph does not interpolate the outline of the object as it passes between
planes, but, rather, simply extends the outline of the object from one plane to the next, as illustrated in
the following figure. Thus, the measured volume is only an approximation of the actual volume of the
object.
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Note: The algorithm used for measuring object volume instructs MetaMorph to assume
that small, poorly defined objects may not show up in every image plane. Thus, if an
object in one plane is "missing" in the very next plane but reappears in the subsequent
plane, MetaMorph will interpolate the volume of the object from its positions in the first
and third planes.
If you have not used the Calibrate Distances command, volumes will be expressed in terms of pixel
volumes (voxels). If you want to express volume in another unit, such as cubic microns, you must first
apply Calibrate Distances.
If necessary, you should also specify a between-planes distance in the Change Plane dialog box's Z
Distance text box. Alternatively, you can specify a user-defined relationship between the Z-distance unit
on the one hand, and the X and Y distance unit on the other.
Measuring Volume
To measure the volume of an object through a stack, use the following procedure.
Note: If you want the volumes to be expressed in calibrated distance units, rather than
pixels, you must first use the Calibrate Distances command.)
Step
Action
1
Threshold the image stack using either the
Threshold Tool and Slider or the Threshold
Image command (Process menu).
2
Define a region of interest around the object
using the Rectangular Region, Ellipse
Region, or Trace Region Tool.
3
If necessary, specify a between-plane
distance using the Set Plane Z Distance
command.
4
From the Apps menu, choose Measure
Volume. The Measure Volume dialog box
opens. The Volume status line will inform you
if any steps have been overlooked, and will
automatically indicate the measured volume.
5
Use the Image selector to select the image
stack that contains the object to be
measured.
6
To specify how the Z-distance units relate
to the X and Y units, select Calibration from
the Relationship of Z to X-Y group if you
want the relationship to be defined by the
Calibrate Distances command and the
Z Distance setting in the Change Planes
Tool's dialog box.
OR
Select Custom if you want to use a userdefined Z-distance. Then specify the
arithmetic relationship of the Z-distance unit
to the X and Y distance unit in the 1 XY Unit
= text box.
The X:Y:Z status line will indicate the
selected relationship between distances in
the three dimensions.
7
If you want to save the volume measurement
in a log file, choose Open Log and open
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either a text-based data log file or a Dynamic
Data Exchange (DDE) link to an open
spreadsheet. The Open Log button text will
change to "F9: Log Data."
OR
If you do not want to log the measurement
data, skip to Step 10.
8
If desired, choose Configure Log to select
the measurement parameters to be logged.
The Configure Log dialog box will appear.
Double-click the entries in the Configuration
list to select the parameters you want to log.
Then choose OK to return to the Measure
Volume dialog box.
9
When you are ready to log the measurement
data, choose F9: Log Data, or press the [F9]
function key to use the keyboard shortcut.
10
When you have finished, choose Close.
Measure Volume - Dialog Box Options
Image
Selects the image stack that contains the object you want to measure.
Volume
Displays the measured volume. If the image has not been thresholded, or if a region of interest has not been
defined, this status line will indicate this to you.
Relationship of Z to X-Y
Specifies the method by which the Z-distance is to be related to the X and Y distances. Select Calibration if
the Z-distance is to be derived from the calibration applied with the Calibrate Distances command and the
Change Planes Tool's Z Distance option. Select Custom if you want to specify a relationship in the 1 XY Unit
= text box.
1 XY Unit =
If you selected Custom from the Relationship of Z to X-Y radio button group, this text box specifies the
arithmetic relationship between the Z-distance units and the X and Y distance units. If you type 1, the Zdistance units will be equal to the X and Y distance units.
X:Y:Z
Indicates the relationship between the X, Y, and Z distance units.
Open Log/Log Data
Opens a data log and/or a DDE link to an open spreadsheet application for logging volume measurement
data. This command will change to F9: Log Data when a log file is open. Subsequently choosing the button
or pressing the [F9] key will save the volume measurement data in the data log.
Configure Log
Opens the Configure Log dialog box so that you can select the parameters to be logged to the data log.
Parameters marked with a check mark in the Configuration list will be logged when you choose F9: Log
Data.
Close
Closes the dialog box.
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Measure Object Distance (Measure Menu)
Displays the distance of that part of a drawn line which is within the boundaries of a binary
or thresholded object.
Drop-in: OBJDIST
Use this command in conjunction with a region drawn with a Line Region Tool to determine the distance
of a thresholded or binary object. The line region should pass through the object completely. Assuming
the line is straight, this command reports both the distance of the entire line and the distance of the line
over the object.
The distance measurement is displayed in calibrated units, which can be selected using the Calibrate
Distances command. If the units have not been calibrated, MetaMorph will display the distance in pixels.
You can log each measurement to an open data log if desired. First, use the Open Data Log command
to open a data log. You can use the Log Data command or its keyboard shortcut, the [F9] function key,
to log the data.
Measure Object Distance allows you to measure the length of a thresholded or binary object for all
planes in a stack, all images in a directory, or from live video at near video rate (for example, a
contracting cell whose edges are thresholded). To measure objects in a stack, use this command with
Loop for All Planes (Journal menu). To measure objects in all images in a directory, use this command
with Loop for All Images in Directory (Journal menu). To measure objects in Live Video, use the Loop a
Journal command (Journal menu) with an extended line region drawn over the thresholded Live Video
image window.
Measuring Object Distance
To measure the distance of an object, use the following procedure:
Step
Action
1
From the Measure menu, choose Measure
Object Distance. The Measure Object
Distance dialog box will appear.
2
Select the desired image using the Image
selector.
AND
Threshold the image using the Threshold
Tool.
3
If you want to log measurement data, open a
data log using the Open Data Log
command.
4
To configure the data log for logging, choose
Configure Log. The Configure Log dialog box
will appear.
AND
From the Configuration list, select the
parameters you want to enable for logging,
so that each is marked by a check mark next
to its entry (you can choose Enable All or
Disable All if you want to select or deselect
all of the parameters listed).
Choose OK to return to the Measure Object
Distance dialog box.
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5
Draw a line across the object using the
Single Line Tool, Multi-Line Tool, or Traced
Line Tool. The line must cross the object
completely.
6
Select the desired line so that it is the active
region. MetaMorph will measure the distance
of the line and display it in the dialog box.
7
If you want to log the measured distance,
choose F9: Log Data.
8
To measure another distance, create and
select another line using the process
described in Steps 5 - 7.
Note: You can edit the distance of the line by
double-clicking the mouse pointer on the line
with the left mouse button and moving the
vertices.
9
Choose Close when you have finished.
Measure Object Distance - Dialog Box Options
Image
Selects the image for measuring object distances.
Distance
Displays the data from the distance measurement for the entire line region.
Object Distance
Displays the data from the current object distance measurement, based on the thresholding. This assumes
that the original line is straight.
Open Log
Opens a data log and/or a DDE link to an open spreadsheet application for logging data. This command
changes to F9: Log Data when a log file is open.
F9: Log Data
Logs the currently displayed data from the dialog box to an open data log or to an open spreadsheet
application by way of a DDE link. To assist you in logging the proper data when several measurement dialog
boxes are open, "F9" is added to the name of this option in the active dialog box to indicate which data will
be logged when you press [F9].
Configure Log
Opens the Configure Log dialog box so that you can select the parameters to be logged to the data log.
Parameters marked with a check mark will be logged for subsequent measurements.
If you select Log Column Titles, a line listing the measurement titles will be logged (1) the first
time the you use the configured measurement, (2) whenever you enable/disable measurement
parameters, or (3) whenever the logged measurement is different from the previous measurement
in the log file.
If you select Place Log Data on Current Line, subsequently logged data will be appended to the
current line in the log file, rather than to a new line. Log Column Titles will be unavailable when you
select this option.
Close
Closes the dialog box.
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Track Objects (Apps Menu)
Tracks one or more selected objects through each image in an image stack or a sequential
series of single images. You can derive measurements of the paths, positions, and
velocities of the points.
Availability: Available for MetaMorph Basic; included in MetaMorph Premier
Drop-in: TRACKOBJ
Use this command when you want to measure the movement of one or more objects between
successive images in a stack or a sequential series of images. You can track the movements over time
of individual tagged particles, such as fluorescently labeled cell surface molecules, microtubules, nucleic
acids, or lipids. Typically, this procedure will be performed to determine whether or not the molecules
being tracked are stationary, move in a straight line, or move in a "random walk."
This procedure works particularly well when used with differential interference contrast (DIC) images, as
the tracking region can be made to encompass both the white and black "spots" produced by this
method. This gives you twice as many pixels to track, leading to a proportionate increase in the tracking
precision.
After you have configured the tracking parameters and defined the objects to be tracked, MetaMorph will
determine the intensity centroids of the defined target regions, and track their displacements
automatically through the planes in the source image stack. Each particle is imaged as an Airy disk
covering many pixels. The Airy disk is imaged with high contrast, and its position is determined with subpixel accuracy. The image of the particle is then tracked using a cross-correlation centroid-finding
algorithm to determine the best match of the particle position in successive images. A search based on
image thresholding is also available.
WARNING:
You will not be able to switch to another stack or add a plane to an image stack after you have already
measured it. Doing so will generate an error message informing you the track data are no longer valid,
and the data will be cleared and all data displays will be closed.
As you select your options to configure the object tracking, you will also have the opportunity to select a
journal to run during the tracking. In this way, you can perform processing on the image prior to tracking
each point. Because you will need to configure your processing journal to overwrite the original images
(so as to retain the original images' file name), we suggest that you make duplicates of the original
images and track the objects in them if you do not want the original images to be altered by the journal.
For dealing with displacement data, you will need to define an "origin point" to which object positions can
be referred. One option (Corner of Image) is to select the upper left corner of the image. In this mode, all
positions for each object will be expressed as absolute positions in the image. A second option (First
Point in Track) uses the location of the object at its first position in the track. In this mode, the position of
each object will be expressed in terms of its starting point. The third option (Corresponding Point of First
Object) expresses the positions of all objects in terms of the location of the first defined object within the
same image plane. This method can be particularly useful for accounting for drift when the first "object" is
a fixed point. Alternatively, you can use this method to measure such phenomena as elongation of
microtubules or transport of labeled proteins.
QUICK TIP: You can erase all tracks with the Clear Measurement Stamps command
(Graphics menu) or use the keyboard shortcut, ALT + C.
When the tracking procedure is complete, you will have the opportunity to edit the data. You can change
the coordinates of a point or delete it altogether. You can then display and print the motion
measurements that have been derived, such as particle X and Y coordinates, velocity, mean
displacement, and mean vector length. Other measures include the mean angle (the angle of the mean
vector of the object) and the angular deviation (analogous to the standard deviation in linear statistics).
The selected variables can be displayed in a configurable and printable scatterplot graph, which, like the
data tables, can be sent to a printer or copied to the Clipboard for use in a graphics or word processing
program.
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Note: This command does not support 24-bit color or binary (1-bit) images.
Tracking Objects - Procedures
Overview of Object Tracking
Configuring Object Tracking:
Configuring Data Logging
Configuring the Search
Configuring the Time Intervals
Configuring the Track Overlays
Selecting and Tracking Objects
Viewing and Editing Object Tracking Data:
Viewing Track Data
Viewing Point-by-Point Data
Viewing Data Graphs
Editing Track Data
Overview of Object Tracking
To track objects and measure their movement through a series of images, use the following
procedure:
Step
Action
1
From the Apps menu, choose Track Objects.
The Track Objects dialog box opens.
2
From the Source for Images option button
group, select the type of images that you will
use for object tracking: Stack, or Sequential
Files.
3
If you selected Stack in Step 2, use the Stack
image selector to select the stack that you
want to use. Then use the Plane… to… spin
boxes to select which planes in the stack that
you want to use.
OR
If you selected Sequential Files in Step 2,
choose the Select Files button, and select
the first and last image in the series from the
Select First Image and Select Last Image
dialog boxes that appear.
4
If you want to run a journal to process each
source image before the tracking protocol is
applied, choose Select Journal, and choose
the journal from the Select a Journal to Run
dialog box that appears. Then choose Open.
5
If you wish, open and configure a data log
to store the object tracking data.
Note: You can also perform this step after
you have already carried out the tracking
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procedure.
6
Choose Search Options, and configure the
search algorithm and its behavior from the
Search Options dialog box that appears.
7
If you want to configure the time units in
the data tables and displays, choose Set
Interval and make your selections from the
Track Objects Interval Options dialog box
that appears.
If you want to configure the origin, click Set
Origin to open the Set Origin dialog box.
8
If necessary, use the Set Overlay command
to configure the track overlays that will be
displayed during object tracking.
9
To define an "origin point" to which object
positions can be referred, choose Set Origin.
Then make a selection from the Origin
Options dialog box that appears: Corner of
Image, First Point in Track, or Corresponding
Point of First Object.
When you have finished, choose OK to
return to the Track Object main dialog box.
10
When you are ready, choose Track, and
select the objects to be tracked. Object
tracking will then proceed automatically.
11
If you are saving the tracking data in a data
log (see Step 5), choose Log Data when
object tracking has completed.
12
You can choose any of three ways to display
the object position and path data.
Choose Display Statistics to view a table
that shows data for each object's entire path.
Choose Display Data to view a table that
shows the frame-by-frame data for each
object.
Choose Graph Data to view configurable
scatterplots that show point or path data for
each of the objects.
13
If you need to edit the data points after
tracking is complete, you will have the
opportunity to do so. Choose Edit Data, and
make your changes from the Track Objects:
Edit Data dialog box.
14
If, for clarity's sake, you want to view an
otherwise blank image that displays the track
overlays, click the Duplicate Overlay button
that has appeared in the dialog box.
15
When you have finished, choose Close.
Configuring Data Logging for Track Objects
To configure logging of object tracking data, use the following procedure:
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Step
Action
1
If you have not already done so, follow the
procedures in the first five steps of the
Overview of Object Tracking.
2
If you want to organize the tracking data in
the data log by frame, select the Log by
Frame check box.
OR
If you want to organize the data by object
number, leave the Log by Frame check box
cleared.
3
If you are processing a large number of
images (several hundreds) and need to
conserve program memory, select the Log
Only: No Data Display check box. This will
prevent the display of the tracking data, and
will accordingly reduce the risk of memory
resource depletion.
OR
If memory usage is not a consideration,
leave the Log Only: No Data Display check
box cleared.
4
Choose Open Log and select a log file for
data storage. When you have finished, the
title on the Open Log button will change to
"Log Data."
5
Choose Configure Log to select which data
are to be enabled for logging.
6
After you finish configuring the other tracking
options and carry out the tracking, you can
choose Log Data to save the tracking data in
the log file.
7
If you want to save the statistical summary
data for each object's entire path, you will be
able to open and configure a summary log
and log the data from the Track Objects
Statistics dialog box.
Configuring the Search Options for Track Objects
To configure the Track Objects search algorithm and its behavior, use the following procedure:
Step
Action
1
If you have not already done so, follow the
procedures in the first four steps of the
Overview of Object Tracking.
2
Choose Search Options. The Search Options
dialog box will appear.
3
From the Algorithm drop-down list, select the
method by which MetaMorph decides
whether an object it finds is the same as the
object it found in the preceding frame:
Template Match filters each new frame
using a convolution mask that is based
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on the object's intensity values in the
preceding frame. The centroid of the
object will be determined from the
convolved image’s intensity peak, using a
percentage of the original peak intensity
(specified by Minimum % for Match) as
the lowest acceptable value. (For more
on image convolution and masks, be
sure to read the chapter on Using Image
Filters in your MetaMorph Task Guide.)
Threshold Result simply detects the
center of the intensity peaks within the
thresholded target areas and determines
which object in the preceding frame is
closest.
4
To configure the search behavior for cases in
which MetaMorph is unable to find an object
in a given frame, make a selection from the If
Object Not Found option button group. Select
Click on Position if you want to use your
pointer to click on what you perceive to
be the object,
Quit Object if you want MetaMorph to
stop searching for the object in all
subsequent frames, or
Skip Frame if you want MetaMorph to
omit this frame as a data point for the
object and to continue the search for the
object in the next frame.
5
If you want MetaMorph to extrapolate where
the center of an object should be based on
the "velocity" of its movement calculated from
the previous two frames, select Use Velocity
for Center of Next Search.
6
If you want to slow down the tracking
process, for example to be able to catch
errors as they occur, use the Delay spin box
to specify the time, in seconds, that each
frame will be displayed before proceeding to
the next.
7
If you selected Template Match in Step 3,
several more options will be displayed.
Continue to Step 8.
OR
If you selected Threshold Result in Step 3,
skip to Step 10.
8
Ordinarily, the Template Match algorithm will
use the same convolution mask for every
frame. If you are tracking objects that change
in shape and intensity, you will need to have
the template recalculate the convolution
mask for each frame. If so, select the Update
Template Each Frame check box.
9
In performing the Template Match
convolution, MetaMorph looks for intensity
maxima in the convolved image. If an
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object’s shape or intensities change
considerably from their original state, the
intensity levels in the convolved image may
be quite low. Accordingly, you will need to
inform MetaMorph of a cutoff intensity level
below which it should discard the object
"match."
Use the Minimum % for Match spin box to
specify what the cutoff should be, expressed
as a percentage of the original peak object
intensity. Then skip to Step 11.
10
If you selected Threshold Result as the
search algorithm in Step 3, the Object Size
Match Requirement (as %) spin box will
appear. Because an object may appear to
change in size from frame to frame, you
should use this option to select a range of
object sizes within which an object detected
in a subsequent frame will be considered as
a "positive match."
11
When you have finished, choose OK to
return to the Track Objects dialog box.
Configuring the Time Interval Options for Track Objects
To configure the time units used for the object tracking data, use the following procedure:
Step
Action
1
If you have not already done so, follow the
procedures in the first six steps of the
Overview of Object Tracking.
2
Choose Set Interval. The Track Objects
Interval Options dialog box will appear.
3
From the Table Time Units drop-down list,
select the time units to be used: Milliseconds,
Seconds, Minutes, or Hours.
4
From the Time Interval Options button group,
select the method by which the image time is
to be determined. Select
Time Image was Created if you want to
take the image time from the image's
timestamp, which stores the time of
creation or last modification, or
Check User Defined if you want to
specify a time interval between frames. If
you select this option, the Time Interval
spin box and drop-down list will become
available.
5
If you selected User Defined in Step 4, select
a time interval between frames from the Time
Interval spin box and drop-down list.
Otherwise, continue to Step 6.
6
When you have finished, choose OK to
return to the Track Objects dialog box.
If you have already performed object
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tracking, the values in the displayed table
and graphs will be updated based on the
new time intervals, and subsequent logging
of data will use the updated values. Data
already logged will not be changed.
Configuring the Overlay Options for Track Objects
To configure the track overlays that will be displayed during object tracking, use the following
procedure:
Step
Action
1
If you have not already done so, follow the
procedures in the first seven steps of the
Overview of Object Tracking.
2
Choose Set Overlay. The Track Overlay
Options dialog box will appear.
3
To change the color of the Track Objects
marker and path, select the desired color
from the Track Points Color list. To change
the shape of the Track Points marker, select
the desired shape from the Point Marker
Type list.
If you choose a Circle marker and want to fill
in the circle, select the Fill Circle Markers
check box. You can change the size of the
Track Points marker with the Point Marker
Size spin box.
Note: The size of the Dot marker can not be
changed. For a larger dot marker, select a
Circle marker shape and select the Fill Circle
Markers check box.
4
Use the Point Marker Display Mode radio
button group to choose between an overlay
display that shows all points in a track
(Display All Points) and one that shows only
the point in the current frame or image plane
(Display Point on Current Plane).
5
The track path and number can be displayed
or hidden by selecting or clearing the Display
Track Path and Display Track Numbers
check boxes, respectively.
6
A track "pattern" can be added to the overlay,
showing lines that connect the points in an
individual plane. The patterns from each
plane will be displayed simultaneously. This
option can be enabled and disabled from the
Display Track Pattern check box.
AND
The color of the pattern in the plane being
viewed can be changed in the Track Pattern
Color list. The patterns from other planes will
continue to be displayed in red. This option
also controls the color of the track numbers
displayed in the image window overlay.
7
When you are satisfied with all of your
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selected graphics options, choose OK. Your
options will then take effect.
Selecting and Tracking Objects
To select the image objects to be tracked and to perform the tracking, use the following
procedure:
Step
Action
1
If you have not already done so, follow the
procedures in the first eight steps of the
Overview of Object Tracking.
2
When you are ready to select the objects to
be tracked, choose Track. The Select
Objects dialog box will appear.
3
Select the objects in the first image frame by
holding down the [CTRL] button and clicking
the objects using the left mouse button.
A rectangular object region will appear
around each object as you click it, and a
larger search region will appear around the
object region.
4
If you want to use the same size for all object
and search regions, select the Lock Region
Sizes check box.
OR
If you want to use different sized object and
search regions for the various objects, leave
the Lock Region Sizes check box cleared.
5
You should configure the search region so
that it is large enough that no part of the
object will extend beyond the edge of the
search region in the subsequent frame. You
can modify the size of the object and search
regions by dragging the outlines with your
pointer. Alternatively, you can use the four
spin boxes in the Select Object dialog box to
specify the object and search region heights
and widths, expressed in pixels.
AND
You can move object and search regions by
clicking inside the region and dragging it to
the desired location.
6
When you are satisfied with the size and
placement of the object and search regions,
choose OK.
Object tracking will proceed automatically.
Colored object tracks will be drawn in the
images, updating as successive frames are
processed.
WARNING:
You will not be able to switch to another
stack or add a plane to an image stack after
you have already measured it. Doing so will
generate an error message informing you the
track data are no longer valid, and the data
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will be cleared and all data displays will be
closed.
7
If an object is "lost" during the tracking,
MetaMorph will proceed according to your
selection in the If Object Not Found options
group in the Search Options dialog box.
You can also stop object tracking by pressing
the [ESC] key. Doing so will display the
Tracking Halted dialog box, from which you
can make a selection for what MetaMorph
should do next: End Tracking, Quit Object,
Skip This Point, Step Back, Update Position
and Continue, or Update Template and
Continue. If you need to resize the tracking
regions, make sure that the Lock Region
Sizes check box is cleared and use the
Locator Tool to drag the edges of the
regions. You also have the option of
switching the tracking overlay on and off with
the Overlay option buttons.
Note: The Update Template and Continue
option will be available only when Template
Match has been selected as the Algorithm in
the Search Options dialog box.
Viewing Track Data
To display the track statistics for individual points, use the following procedure:
Step
Action
1
From the Track Objects dialog box, choose
Display Statistics. The Track Objects
Statistics dialog box opens.
2
You can use the horizontal and vertical
sliders, at the lower edge and at the right of
the data table, respectively, to scroll from
side to side or up and down through the table
to see the data for each object.
If necessary, you can increase the size of the
Track Objects Statistics dialog box by
dragging its borders. You can also change
the width of the data columns by placing the
pointer between the columns at the top of the
table and dragging the column border.
3
If you want to log the track data, choose
Open Log. When you have finished selecting
a summary log file, the text on the Open Log
button will change to "Log Data."
AND
If you want to select which track parameters
to store in the summary log, choose Config
Log and make your selection from the
Configuration table of the Configure Log
dialog box that appears. Then choose OK to
return to the Track Objects Statistics dialog
box.
4
To save the track data in the summary log
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you opened in Step 3, choose Log Data.
5
If you want to print the data table, choose
Print Table. A message box will appear,
asking you to confirm the print request.
Choose Yes to proceed with the printing.
6
When you have finished, choose Close.
Viewing Point-by-Point Data
To display the point-by-point measurement data for individual objects, use the following
procedure:
Step
Action
1
From the Track Objects dialog box, choose
Display Data. The Track Objects Data dialog
box will appear.
2
You can use the horizontal and vertical
sliders, at the lower edge and at the right of
the data table, respectively, to scroll from
side to side or up and down through the table
to see the data for each object at each
position. Clicking on a table entry will switch
the image display to the corresponding
frame.
If necessary, you can increase the size of the
Track Objects Data dialog box by dragging
its borders. You can also change the width of
the data columns by placing the pointer
between the columns at the top of the table
and dragging the column border.
3
To switch between data views, make a
selection from the Data Type option button
group.
4
If you want to print the data table, choose
Print Table. A message box will appear,
asking you to confirm the print request.
Choose Yes to proceed with the printing.
5
When you have finished, choose Close.
Viewing Data Graphs
To display scatterplot graphs showing object data over time or plane number, use the following
procedure:
Step
Action
1
From the Track Objects dialog box, choose
Graph Data. The Track Objects Graph dialog
box will appear.
2
From the Data Type option button group,
select the data that you want to display.
Position: The X and Y coordinate at
each position.
Distance: The absolute distance
between each position and the one
immediately preceding.
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DeltaXY: The change in X and Y values
at each position, relative to the
immediately preceding position.
Velocity: The distance moved per time
unit or from one position to the next.
Angle: The angle of the path taken by
the object from its previous position.
Angles are measured from the "nine
o'clock" position and will range from 0 to
180 degrees. Downward angles are
expressed as positive numbers, and
upward movements are expressed as
negative numbers.
Dist. to Origin: The straight-line distance
between the object's current position and
the Origin.
3
If you want to display the data points for just
a single object, select the Graph Single
Object check box. Then use the associated
spin box to select the number of the desired
point.
OR
If you want to display the data points for all
objects, leave the Graph Single Object check
box cleared.
4
To display the data in terms of time, select
Time from the X-Axis radio button group.
OR
To display the data in terms of plane number,
select Plane from the X-Axis radio button
group.
5
To change the colors or labeling in the graph,
click the Down Arrow button and choose the
appropriate command from the configuration
pop-up menu that appears.
6
If you want to print the graph, click the Down
Arrow button and choose Print from the
configuration pop-up menu.
7
You can also copy the graph to the
Clipboard, so that you can paste it into
another program, such as a graphics or word
processing program. To do so, click the
Down Arrow button and choose Copy to
Clipboard from the configuration pop-up
menu. Then use the appropriate Paste
command to import the graph to the other
program (most programs support the
CTRL + V keyboard shortcut).
8
When you have finished, choose Close.
Editing Object Tracking Data
To edit the object tracking data, use the following procedure:
Step
Action
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1
From the Track Objects main dialog box,
choose Edit Data. The Track Objects: Edit
Data dialog box will appear.
2
Use the Frame # spin box to select the frame
containing the "bad" or missing point, and
use the Object # spin box to select the
number of the object being tracked.
Clicking on the data in the graph or either
data table will also update the source stack
and the Edit Data dialog box to the
appropriate plane. Changing the plane in the
source stack will also update the setting in
the Frame # spin box.
3
To change the location of a "bad" point, use
the Position on Image spin boxes to select
the position, relative to the Origin. Then
choose Done, or go to the next frame.
OR
Alternatively, you can hold down the [CTRL]
key and click on the location in the image
where a "bad" or missing point ought to be.
4
If you need to undo your change and you
have not yet moved to a new frame or object,
choose Reset Point.
5
If you need to remove the active point (that
is, the currently selected point), choose
Delete Point.
CAUTION: Be sure to verify that you have
selected the point to be removed. The Object
# spin box should be set to the desired object
before you remove the current point.
6
When you have finished, choose Done.
Track Objects - Dialog Box Options
Track Objects Main Dialog Box
Configuring Object Tracking:
Search Options Dialog Box
Track Objects Interval Options Dialog Box
Track Overlay Options Dialog Box
Origin Options Dialog Box
Selecting and Tracking:
Select Objects Dialog Box
Tracking Halted Dialog Box
Displaying and Editing Data:
Track Objects Statistics Table
Track Objects Data Table
Track Objects Graph
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Edit Data Dialog Box
Track Objects: Overview of Dialog Box Options
Source for Images
Selects the type of images that will be used for object tracking: Stack or Sequential Files. If you select Stack,
the Stack image selector will appear. If you select Sequential Files, the Select Files command button will
appear.
Plane… to…
Selects the range of planes in the source stack that you want to use in the object tracking. This set of spin
boxes becomes available when you select Stack from the Source for Images option button group.
Stack
Selects the image stack to be used for object tracking. This option appears only if you select Stack from the
Source for Images option button group.
Select Files
Opens the Select First Image dialog box, from which you can select the first image in the sequential series
to be used for object tracking. After you choose OK, the Select Last Image dialog box will appear, from
which you can select the last image to be included in the tracking procedure. The first and last image that
you select must be from the same sequential image series. The Select Files button appears only if you
select Sequential Files from the Source for Images option button group.
Select Journal
Opens the Select a Journal to Run dialog box, from which you can select a journal to process each source
image before the tracking protocol is applied to it. Because you will need to configure your processing
journal to overwrite the original images (so as to retain the original images' file name), we suggest that you
make duplicates of the original images and track the objects in them if you do not want the original images to
be altered by the journal.
Log by Frame
When you select this check box, data being sent to a data log will be organized by position (image frame)
number. If you leave this check box cleared, the data will be organized by object number.
Log Empty Lines
When selected, the log will show a line for every frame even when no object is found. This ensures that
every frame will be logged.
Log Only: No Data Display
When selected, this check box prevents the display of the tracking data, thereby conserving system memory
resources. Data will be logged automatically, but the data tables and graphs will be unavailable, as will their
corresponding Display Statistics, Display Data, and Graph Data command buttons. When you use Log Only,
be sure that the interval is set correctly before tracking, because changing an incorrect interval after tracking
will not correct any data that are already logged.
Open Log/Log Data
Opens a data log for storing the frame-by-frame object data. After you open the data log, the text on this
button will change to "Log Data." Choosing this button will then save the object tracking data set to the data
log.
Config Log
Allows the selection of tracking data that are to be included or excluded from data logging. Also allows a
choice of whether column titles are to be included and if data are to be listed on a single line.
Search Options
Opens the Search Options dialog box, from which you can configure the search algorithm and its behavior.
Set Interval
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Opens the Track Objects Interval Options dialog box, from which you can configure the time units used for
the object tracking data.
Set Overlay
Opens the Track Overlay Options dialog box, from which you can configure the track overlays that will be
displayed during object tracking.
Set Origin
Opens the Origin Options dialog box, from which you can configure the origin that the object will be
measured against.
Display Statistics
Opens the Track Objects Statistics dialog box, which displays the track statistics for each object's entire
path.
Display Data
Opens the Track Objects Data dialog box, which displays the frame-by-frame data for each object.
Graph Data
Opens the Track Objects Graph dialog box, which displays configurable scatterplot graphs showing point or
path data for each of the objects.
Edit Data
Opens the Track Objects: Edit Data dialog box, which you can use after tracking to change the position of a
point or to remove the point altogether.
Duplicate Overlay
Creates a blank image with a copy of the track overlays, as currently displayed on the tracking image.
Track
Opens the Select Objects dialog box, which you use to define regions that contain objects that you want to
track. After you create and configure the object and search regions and choose OK, the tracking procedure
and data analysis will proceed automatically.
Close
Closes the Track Objects dialog box.
Track Objects: Search - Dialog Box Options
Algorithm
Selects the method by which MetaMorph decides whether an object it finds is the same as the object it
found in the preceding image frame. Two methods are available:
Template Match filters each frame using a convolution mask that is based