softWoRx User`s Manual Revision C

softWoRx User`s Manual Revision C
®
Imaging Workstation
User's Manual
Revision C
built with precisionware
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AppliedPrecision
®
softWoRx Imaging Workstation User’s Manual
Legal Notices
Revision C of the User’s Manual for the softWoRx Imaging Workstation. Part number 04-720103-000
Rev C.
© 1999-2008 Applied Precision, Inc. All rights reserved. No part of this manual may be reproduced,
transmitted, stored in a retrieval system, or translated into any language in any form by any means
without the written permission of Applied Precision, Inc.
Information in this document is subject to change without notice.
DeltaVision, Applied Precision, and softWoRx are registered trademarks of Applied Precision, Inc. All
other registered names and trademarks referred to in this manual are the property of their respective
companies.
Applied Precision, Inc
1040 12th Ave. NW
Issaquah, WA 98027
(425) 557-1000 FAX: (425) 557-1055
Other Manuals and Guides
The following documents are provided for softWoRx.
Document
Purpose
Available for…
Online Help
Provides reference information for
softWoRx and procedures that show
how to use softWoRx tools
All softWoRx workstations
Product Notes
Provide examples and tips for using
softWoRx
All softWorRx users (online at
The DeltaVision RT
Restoration
Microscopy System
User's Manual
Shows how to acquire data and how
to maintain the data acquisition
system
Acquisition workstations
Getting Started with
QLM
Shows how to acquire photokinetic
data with the QLM module
Acquisition workstations that have the
optional QLM module
RedHat Linux Bible
Shows how to use Linux
All softWoRx Users. (This is a third party
manual.)
www.appliedprecision.com)
Applied Precision
Contents
Preface ...............................................................................................i
About This Manual..................................................................................................................i
Document Conventions .........................................................................................................ii
Lists ....................................................................................................................................ii
Notes, Warnings and Cautions ......................................................................................ii
User Interface Description Conventions.......................................................................ii
Contacting Applied Precision, Inc.......................................................................................iii
Customer Service Hotline ..............................................................................................iii
Corporate Office ..............................................................................................................iii
Acknowledgements ........................................................................................................iii
Introduction .......................................................................................1
What is softWoRx?................................................................................................................... 1
What Can You Use softWoRx for?......................................................................................... 2
Acquiring Data ................................................................................................................. 2
Processing Data ................................................................................................................ 3
Visualizing and Presenting Data ................................................................................... 3
Analyzing Results ............................................................................................................ 4
Optional Components............................................................................................................ 5
The Quantitative Laser Module ..................................................................................... 5
softWoRx Explorer ............................................................................................................ 5
Part One: Processing Data...........................................7
1. Deconvolving Image Data .........................................................9
About Deconvolution Processing....................................................................................... 10
Deconvolution Tools ............................................................................................................ 10
Deconvolving an Image ....................................................................................................... 11
Deconvolving Several Images............................................................................................. 13
Common Deconvolution Options ...................................................................................... 14
More about the softWoRx Deconvolution Tools ............................................................... 16
2. Correcting Images.....................................................................17
About Correcting Images .................................................................................................... 17
Correcting Z Section Image Data ....................................................................................... 18
Equalizing Intensities in Time-lapse Image Data............................................................. 19
Calibrating Image Data........................................................................................................ 20
Aligning Adjacent Images ................................................................................................... 21
Correcting Chromatic Aberration ...................................................................................... 23
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3. Stitching ......................................................................................25
Stitching Images That Have a Single Z Section ................................................................26
Stitching Images That Have Multiple Z Sections .............................................................27
4. Importing Data ...........................................................................31
Converting TIFF Images ......................................................................................................32
Converting ISee Files............................................................................................................33
ISee Series Conversion...................................................................................................34
Converting Pic Files..............................................................................................................35
Converting STK Files............................................................................................................36
5. Data and Task Manipulation ....................................................39
Selecting Data ........................................................................................................................40
Selecting Rectangular Data Regions ............................................................................40
Selecting Irregular Data Regions..................................................................................42
Cropping and Trimming Data ............................................................................................45
Cropping a Rectangular Region ...................................................................................45
Cropping an Irregular Data Region ...................................................................................47
Trimming Time Data .....................................................................................................50
Combining Data of Two Images.........................................................................................52
Setting Up Process Chains with Task Builder ..................................................................54
Using Ratio Imaging.............................................................................................................57
Using the Multiplexed Wavelength Option......................................................................62
Setting Up the Multiplexed Wavelength Option .......................................................63
Designing a Multiplexed Wavelength Experiment ...................................................68
6. DMS Integration..........................................................................73
What is DMS? ........................................................................................................................73
Connecting to a DMS Database ..........................................................................................74
Uploading Images to a DMS Database ..............................................................................74
Uploading Images from the Image Window..............................................................74
Uploading Images Using Task Builder .......................................................................77
Auto-uploading Images after Acquisition ..................................................................80
Downloading Files from DMS ............................................................................................81
Browsing and Locating Images in a DMS Database ........................................................82
Browsing Image Files using P/D/I Hierarchy.............................................................82
Browsing Image Files using CG/C/I Hierarchy..........................................................83
Searching for Image Files Based on Annotation ........................................................84
Applied Precision
Contents
Part Two: Visualizing & Presenting Data ......................85
7. Viewing Image Data..................................................................87
Opening an Image ................................................................................................................ 88
The Image Window .............................................................................................................. 89
Viewing 5D Images .............................................................................................................. 90
Viewing Z Sections and Time Points........................................................................... 90
Viewing Areas ................................................................................................................ 92
Displaying or Hiding Channels ................................................................................... 94
Adjusting Brightness and Contrast .................................................................................... 95
Assigning Colors or Grayscale to Channels ..................................................................... 99
Grayscale Mode............................................................................................................ 100
Color Mode ................................................................................................................... 101
Blended Color Mode.................................................................................................... 101
Controlling the Image Window Display ......................................................................... 102
Hiding or Displaying Image Window Border Tools............................................... 103
The Image Window Scale Bar..................................................................................... 103
Resizing or Reorienting an Image .................................................................................... 105
Resizing an Image ........................................................................................................ 105
Rotating an Image ........................................................................................................ 107
Viewing Cross Sections...................................................................................................... 111
8. Viewing Movies ....................................................................... 113
Viewing Volumetric or Time-Lapse Movies................................................................... 113
Tracking Particle Movement with Trails Movies........................................................... 114
9. Viewing Projections and Volumes ........................................ 119
Creating 2D Projections ..................................................................................................... 119
Creating Volume Projections ............................................................................................ 122
Volume Rendering ....................................................................................................... 122
Axes of Rotation ........................................................................................................... 123
Methods for Projecting Volumes ............................................................................... 125
10. Filtering Image Data ............................................................. 137
About softWoRx Filters ....................................................................................................... 138
Using Convolution Filters ................................................................................................. 138
Enhancing Object Boundaries........................................................................................... 139
Using 2D Statistical Filters................................................................................................. 141
Using Image Arithmetic .................................................................................................... 142
Scaling Pixel Intensity to Enhance Local Contrast......................................................... 143
Setting an Intensity Threshold.......................................................................................... 144
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11. Saving, Exporting, and Printing.............................................147
Image Data Files and Image Graphic Files......................................................................148
Saving DeltaVision Files......................................................................................................148
Saving to the Data Management System (DMS) ............................................................150
Exporting DeltaVision Files...............................................................................................151
Exporting to TIFF Files ................................................................................................151
Exporting to PhotoShop Files .....................................................................................153
Exporting to Movie Files .............................................................................................153
Exporting to JPEG Files ...............................................................................................155
Capturing Screen Shots ......................................................................................................156
Archiving Files to CD/DVD...............................................................................................157
Printing Images ...................................................................................................................159
Part Three: Analyzing Results................................... 161
12. Examining Intensity Data ......................................................162
Examining Point Values.....................................................................................................162
Examining Intensity Data with Data Inspector ..............................................................163
Selecting a Region of Interest......................................................................................165
Viewing Intensity Line Profiles.........................................................................................166
Viewing the Line Intensity of a Row or Column .....................................................167
Viewing the Line intensity in Any Direction ...........................................................168
Calculating Statistics ..........................................................................................................169
Calculating Statistics for Selected Areas ...................................................................169
Calculating Statistics for Irregular Areas..................................................................171
13. Measuring Distance and Velocity .......................................175
Measuring Distances ..........................................................................................................175
Measuring Velocity.............................................................................................................177
14. Volume Modeling ..................................................................181
About Volume Modeling...................................................................................................182
Edit Polygon Dialog Box....................................................................................................182
2D Polygon Finder..............................................................................................................185
3D Object Builder................................................................................................................187
Creating and Viewing the 3-D Object........................................................................188
Volume Modeling Example...............................................................................................190
15. Detecting and Analyzing Colocalization............................193
Examining the Entire Image..............................................................................................194
Identifying Potential Colocalized Areas..........................................................................197
Detecting Colocalization with ROIs .................................................................................201
Applied Precision
Contents
16. Other Applications................................................................ 203
About Photokinetics ........................................................................................................... 203
Analyzing Fluorescence Recovery After Photo-bleaching ........................................... 204
About FRAP Experiments........................................................................................... 205
Analyzing Fluorescence Resonance Energy Transfer.................................................... 208
Using the FRET Analysis Tool ................................................................................... 208
Appendix A: Image Quality....................................................... 215
Using Deconvolution Residuals ....................................................................................... 216
Visually Evaluating Images .............................................................................................. 217
Index
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......................................................................................... 221
Preface
This manual shows how to use softWoRx to process, visualize, and analyze image
data.
„
About This Manual describes the information in the manual.
„
Document Conventions explains the typography, notes, and other conventions
used in this manual.
„
Contacting Applied Precision, Inc. provides information about how to contact
customer support.
About This Manual
This manual is divided into three parts that contain the following information:
„
Part One includes instructions for processing and importing data.
„
Part Two shows how to visualize data and prepare it for presentations. It also
shows how to save or export data in a variety of formats.
„
Part Three shows how to use softWoRx tools to perform quantitative analysis.
The manual also includes an appendix that shows how to analyze image quality.
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Document Conventions
In order to make the information in this manual as easy as possible for you to
locate and use, the following conventions are observed.
Lists
•
Round bullets indicate options in procedures.
1. Numbered items are sequential steps for completing a procedure.
„
Square bullets indicate items in a list.
X Arrows indicate single step procedures.
Notes, Warnings and Cautions
Note Indicates information about the previous paragraph or step in a procedure.
!
Important Indicates important or critical information about the previous
paragraph or step in a procedure.
Tip Indicates helpful advice.
 WARNING: Indicates important information regarding potential injury.
 WARNING: Indicates risk of explosion.
Ꮨ WARNING: Indicates risk of shock.
Indicates important information regarding potential damage to
 CAUTION:
equipment or software.
User Interface Description Conventions
Boldface indicates the names of buttons, menus, dialog box options, and fields.
Applied Precision
Preface
iii
Initial Capitals indicate the names of windows, dialog boxes, and tabs.
ALL CAPITALS SAN SERIF indicates the name of a key on your keyboard, such as
ENTER or DELETE.
Uniform width font indicates text to enter on a command line or in the GUI.
Contacting Applied Precision, Inc
If you have questions about DeltaVision, first refer to this manual or consult the
online Help system. If you don’t find the information you need, contact us at one
of the following addresses.
Customer Service Hotline
Phone: 800-862-5166
email: [email protected]
Hours: 8:00 AM – 5:00 PM, Pacific Time, Monday – Friday
Corporate Office
Applied Precision, Inc
1040 12th Avenue NW
Issaquah, WA 98027
USA
Phone: (425) 557-1000
Fax: (425) 557-1055
Internet address: www.appliedprecision.com
Acknowledgements
Applied Precision would like to thank Adrian Quintanilla and Dave McDonald of
the Fred Hutchinson Cancer Research Center for providing a data acquisition
workflow and other content that is used in this manual.
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Introduction
This introduction provides an overview of how you can use softWoRx to process,
visualize, and analyze multidimensional microscopy data. It also introduces
optional softWoRx components.
In the Introduction
What is softWoRx? ............................................................................................................... 1
What Can You Use softWoRx for? ..................................................................................... 2
Optional Components ........................................................................................................ 5
What is softWoRx?
softWoRx is a comprehensive software package designed for the analysis of multidimensional microscopy data. Although originally developed for use as a
component of Applied Precision’s DeltaVision Restoration Microscope System,
softWoRx is now also available on a stand-alone analysis workstation, giving you a
powerful yet friendly environment for exploring and refining your understanding
of specimen structure. The flexibility of the software makes it ideal for the study of
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images from fluorescence, brightfield, Differential Interference Contrast (DIC), and
electron microscopy.
softWoRx is available on two types of Linux workstations:
•
The Acquisition workstation is part of the DeltaVision data acquisition system
and is used to control the system. You can also use it to process, analyze, and
visualize data. (Refer to the DeltaVision Core and personalDV Restoration
Microscopy System User’s Manual for details on image acquisition.)
•
The Analysis workstation is a stand-alone workstation. You can use it only to
process, visualize, and analyze data.
The DeltaVision Core data acquisition system
What Can You Use softWoRx for?
You can use softWoRx to acquire, process, visualize, and analyze multidimensional renderings of a fluorescent specimen. You can also use it to save data
in a variety of formats.
Acquiring Data
If you are using an acquisition workstation, you can acquire images with the
DeltaVision Restoration Microscope System. The softWoRx Resolve3D module
provides various options for acquiring time-lapse data, data with multiple Z
sections, and data from multiple channels. (Refer to the DeltaVision Core and
Applied Precision
Introduction
3
personalDV Restoration Microscopy System User’s Manual for details on image
acquisition.)
If your system has the QLM module, you can use it to acquire photokinetic data
for a variety of experiments. (softWoRx photokinetics data includes photobleaching or photo-activation that results from a laser pulse. See the QLM Getting
Started Guide.)
Processing Data
Process image data to prepare it for visual examination and analysis. softWoRx
provides several types of modules for processing image data.
Deconvolving Image Data
Deconvolve image data acquired with the DeltaVision system. Deconvolving
improves contrast by relocating signal scatter and out-of-focus data.
Correcting Images
Correct image data for chromatic aberration (color shift) that results from oil
matching and other environmental conditions. You can also correct data collection
errors and equalize intensity values across Z sections.
Stitching
Stitch "panel" images collected with DeltaVision to generate a larger overall field of
view. Stitched images are organized as either a series of time points or Z sections.
Importing
Import data from the TIFF format (16-bit grayscale), BioRad’s Pic format,
InoVision’s ISee format, or MetaMorph’s STK format.
Selecting, Cropping, and Combining data
Select data to crop it or to combine it with other data.
Visualizing and Presenting Data
After processing data, you can view and present it in a variety of ways. softWoRx
provides several tools that you can use to visualize data and prepare it for
presentations.
Viewing Image Data
Open data files in softWoRx and adjust the way that the image is displayed (e.g.,
display a scale bar, set grayscale or color modes, or adjust brightness and
contrast). You can also rotate or resize image data or view data cross-sections.
Viewing Projections and Volumes
Render volumes and create 2D projections to visualize and explore threedimensional data. Several methods for rendering volumes that you can
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interactively rotate are available. The 2D projections quickly combine information
from multiple Z Sections into a single section.
Viewing Movies
Create movies of volume rendered data or time-lapse data. You can also create
movies to trace particle movement.
Filtering
Choose from several filters to improve the visual presentation of data, prepare
data for modeling, or for other types of analysis. You can use statistical filters that
are useful for removing noise from the image, threshold filters, and convolution
filters.
Saving, Exporting, and Printing
Save and present data in a variety of formats. Export images to PhotoShop or JPEG
formats or save image data in a DeltaVision file, a TIFF file, or a tabular format that
can be opened in a spreadsheet. You can also save time-lapse or volume-rendered
data as MPEG movies. All files can be archived to CD or DVD. If your system is
configured with a printer, you can print DeltaVision files from softWoRx. If you
have softWoRx Explorer, you can print DeltaVision files from a Mac or PC computer.
Analyzing Results
You can use measuring and modeling tools to perform quantitative analysis.
Examining Intensity Data
Study area and line profiles, calculate statistics, and display single point values.
Measuring Distance and Velocity
Measure features on an XY plane or across Z sections. You can also measure the
velocity of particle movement.
Modeling
Use tools to create line models or volume models.
Detecting Colocalization
Use Colocalization modules to create a scatter plots and measure the Pearson
Coefficient of Correlation to help determine whether colocalization is occurring.
Analyzing Fluorescence Resonance Energy Transfer Data
Use the Fluorescence Resonance Energy Transfer (FRET) module to analyze FRET
data.
Analyzing Fluorescence Recovery After Photo-bleaching Data
Use the Analyze Fluorescence Recovery After Photo-bleaching (FRAP) module to
analyze FRAP data.
Applied Precision
Introduction
5
Optional Components
You can purchase two optional components for softWoRx:
The Quantitative Laser Module
The Quantitative Laser Module (QLM ) is a DeltaVision component that adds a
laser beam into the back aperture of the microscope objective to provide a focused
illumination spot in the center of the optical field. This optional component
mounts to the Fiber Optic Module of a DeltaVision Core microscope, a DeltaVision
RT microscope, or a microscope that is upgraded to the DeltaVision 3.9 level.
If your system has the QLM hardware module, you can use softWoRx to analyze
Photokinetic (photo-bleaching and photo-activation) experiments. The softWoRx
FRAP analysis module discussed in Chapter 16 is only available for systems that
have QLM hardware.
softWoRx Explorer
®
softWoRx Explorer is a cross-platform image viewer available for many commonly
used operating systems.
softWoRx Explorer allows you to view and explore DeltaVision images and images
from other sources that contain spatial, temporal, and spectral ranges. In addition
to displaying data in the X and Y plane, you can scroll through Z sections and
time-lapse data. Individual spectrum (i.e., channels or fluorescent wavelengths)
can be hidden or displayed in a variety of colors.
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Applied Precision
Part One
PART ONE:
PROCESSING DATA
You will typically need to process data before you view or analyze it. Part one
includes instructions for processing and importing data.
In Part One
Chapter 1. Deconvolving Image Data ............................................................... 9
Chapter 2. Correcting Images .......................................................................... 17
Chapter 3. Stitching ............................................................................................ 25
Chapter 4. Importing Data................................................................................. 31
Chapter 5. Data and Task Manipulation .......................................................... 39
Chapter 6. DMS Integration ............................................................................... 39
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Applied Precision
1. Deconvolving Image Data
This chapter shows how to use the softWoRx Deconvolve tool to remove blur in
fluorescence optical sections.
You can deconvolve and view a single image or you can set up a queue to
deconvolve several images. You can also set options to deconvolve a region of an
image, select which wavelengths to include, or select the deconvolution method.
In This Chapter
Converting TIFF Images................................................................................................... 32
Converting ISee Files ........................................................................................................ 33
Converting Pic Files .......................................................................................................... 35
Converting STK Files ........................................................................................................ 36
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About Deconvolution Processing
After you acquire images, you'll need to process them to remove blurred data. The
process of relocating signal scatter and out-of-focus information present in digital
images is known as Deconvolution. Applied Precision’s proprietary deconvolution
algorithms preserve the amount of light throughout the entire Z-stack. Blurred
data is not dropped out. It is reassigned to its original location. This process
increases contrast and comparative intensities within each Z-stack image, making
for extremely sharp 3D reconstructions.
Unprocessed data on the left is deconvolved to create the image on the right
Deconvolution Tools
softWoRx provides two tools for deconvolving images: Deconvolve and Nearest
Neighbor. The Deconvolve tool (described in this chapter) provides the best
results for most applications. This method uses the iterative-constrained algorithm
described by Agard1. This tool should be used for experiments where the
quantification of intensities is required.
The Nearest Neighbor tool (described only in the online Help) uses an
approximate deconvolution approach, commonly referred to as deblurring, for
removing blur from optical sections. The Nearest Neighbor tool should not be
used in experiments where quantification of intensities is required.
See: Agard, D.A. (1984) Optical Sectioning Microscopy: Cellular Architecture in Three
Dimensions. Ann. Rev. Biophys. Bioeng. 13:191-219.
1
Applied Precision
Chapter 1: Deconvolving Image Data
Deconvolving an Image
To deconvolve an image:
1. On the softWoRx main menu, choose Process | Deconvolve to open the
Deconvolve dialog box.
2. Enter the original _R3D.dv image file (for example,
/usr/local/softWoRx/data/samples/oocyte_R3D.dv ) in the Input field.
You can do this in the following ways:
•
•
•
From the Linux file Manager, drag and drop the file in the Input field.
Click Input and browse to the file.
Type the path and file name into the Input field.
softWoRx creates an output file name by appending the _D3D extension to the
input file name. The new name (for example, oocyte_R3D_D3D.dv) is
displayed in the Output field.
3. Enter the .otf file (for example, the oocyte.otf file in the
usr/local/softWoRx/data/samples directory) into the OTF File field. You
can use the same methods (drag and drop, browse, or type) to enter this file as
you used in Step 2. For DeltaVision files, this is done automatically.
Note The .otf file is an Optical Transfer Function (OTF) file. In many
microscopy systems, there is only one OTF per objective lens and the correct
OTF is simply the one that corresponds to the lens used for measuring the optical
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sections. Refer to the lens identification number in the measured data and the
OTF to verify that the correct OTF is being used.
4. Click Do It.
The deconvolution status is displayed in the Deconvolve Output window and
on the bottom of the Deconvolve dialog box. When the deconvolution is
finished, messages appear in each of these windows and the deconvolved
image is displayed in the Image window.
The deconvolved image
Applied Precision
Chapter 1: Deconvolving Image Data
5. If you did not have the Show image when finished checkbox selected
(default) on the Deconvolve dialog box, choose File | Open to open the View
Image dialog box.
6. Enter the file name (for example, usr/local/softWoRx/data/samples/
oocyte_R3D_D3D.dv)into the Input field.
Note You can use the same methods (drag and drop, browse, or type) to enter
this file as you used to enter the _R3D.dv file in Step 2 on page 11.
7. Click Do It to open the file in the Image window.
Deconvolving Several Images
You can create a queue to deconvolve several images and specify a time to start
the deconvolution.
To deconvolve several images:
1. On the softWoRx main menu bar, click Process | Deconvolve to open the
Deconvolve dialog box.
2. In the Input field, enter the _R3D.dv file (for example,
/usr/local/softWoRx/data/samples/oocyte_R3D.dv). You can use the
same methods (drag and drop, browse, or type) to enter this file as those used
in Step 2 of the previous procedure.
3. In the OTF File field, enter the .otf file (for example,
/usr/local/softWoRx/data/samples/Olympus_60X_142_10612.otf).
4. In the Deconvolve dialog box, click Run Options to open the Deconvolution
Run Options dialog box.
5. In the Run Options pull-down list, select Add to Queue. Then click Close.
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6. In the Deconvolve dialog box, click Do It. The file is added to the queue and
displayed in the Queue Manager.
7. Repeat Steps 2 and 3 and click Do It in the Deconvolve dialog box for each of
the remaining files.
Tip If your file names are all the same except for the last digit (for example
040600aq01, 040600aq02, etc.), you can simply overwrite the last digit and press
Do It for each file.
8. In the Queue Manager dialog box, choose one of the following options:
To perform the deconvolutions immediately, click Start Now.
or
To perform the deconvolutions later, click Start Later and select a time on the
clock that appears in the dialog box.
As the files are deconvolved, the deconvolution status is displayed in the
Queue Manager.
9. Click Quit to close the Queue Manager dialog box.
Common Deconvolution Options
You can set options to deconvolve a region of an image, deconvolve only data in
specified wavelengths, or change the deconvolution method.
Applied Precision
Chapter 1: Deconvolving Image Data
15
Use the Deconvolve dialog box to specify options for deconvolving
To
Do This
Deconvolve only part
of the image
Click Select Region and use your mouse to define a specific
region of the image to deconvolve. (This option is only
available when the input data comes from a window.)
Select which
wavelengths to
include
Select the Wavelengths options.
Select a
deconvolution
method
Choose one of the following deconvolution methods from
the Method list:
Ratio (conservative) method uses a more conservative
algorithm that generally finds an accurate solution. Images
with punctate fluorescence may deconvolve better using
this method.
Enhanced Ratio (the default method) is quicker because the
residuals stabilize in fewer iterations, typically 10 or less.
Additive uses a more conservative algorithm that generally
finds an accurate solution.
Enhanced Additive is faster than Additive because it requires
fewer iterations (typically ten or fewer).
The Additive and Enhanced Additive options are the
preferred deconvolution methods for data acquired with the
EMCCD electron multiplication camera. These methods are
more tolerant of noisy data (images with higher noise levels).
Display deconvolved
images immediately
after processing
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Select the Show image when finished checkbox.
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softWoRx Imaging Workstation User's Manual
You can typically use the default settings for the rest of the options in the
Deconvolve dialog box, including the options displayed when you click More
Options.
More about the softWoRx Deconvolution Tools
softWoRx provides two tools for deconvolving images: the standard Deconvolve
tool described in this section and the Nearest Neighbor Deconvolution tool. In
most instances, the standard Deconvolve tool provides the best results. The
Nearest Neighbor tool provides an approximate deconvolution approach for
removing blur from optical sections. If you wish to learn more about Nearest
Neighbor Deconvolution, refer to the online Help.
The standard Deconvolve tool uses the Constrained Iterative Deconvolution
algorithm to remove the out-of-focus blur in fluorescence optical sections. This
algorithm calculates a result using the following four steps:
1. The algorithm estimates what the object looks like.
2. The estimate is mathematically blurred to simulate the effects of the
microscope's limited aperture.
3. The blurred estimate is compared to the actual image. The difference between
the images is then used to modify the estimate.
4. The modified estimate is constrained to be non-negative, by setting pixels with
negative intensity to 0.
The algorithm repeats this sequence of steps until the estimate, convolved with the
point spread, closely approximates the actual image (see Agard2).
See: Agard, D.A. (1984) Optical Sectioning Microscopy: Cellular Architecture in Three
Dimensions. Ann. Rev. Biophys. Bioeng. 13:191-219.
2
Applied Precision
2. Correcting Images
softWoRx provides several tools for correcting image data. You may need to correct
data to prepare it for visualization and analysis.
In This Chapter
About Correcting Images................................................................................................. 17
Correcting Z Section Image Data .................................................................................... 18
Equalizing Intensities in Time-lapse Image Data ......................................................... 19
Calibrating Image Data .................................................................................................... 20
Aligning Adjacent Images ............................................................................................... 21
Correcting Chromatic Aberration................................................................................... 23
About Correcting Images
Correct Image is used to correct errors that are caused by photo-bleaching,
inconsistent illumination intensity, or CCD defects. The Correct Image tool
corrects the intensity values of sections within a Z series. With the exception of
photo-bleaching, the tool can also correct intensity values between time points of a
time-lapse experiment. By default, softWoRx automatically applies this tool during
the deconvolution process. (See Correcting Z Section Image Data on Page 18.)
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Equalize Time Points equalizes intensities of all time points to a reference time
point that you select. The tool uses the mean image intensity to help generate a
more uniform intensity display. You can use this tool to normalize time-lapse data
for display purposes. In general, the data generated by this tool should be used for
display only and should not be used for quantitative purposes. (See Equalizing
Intensities in Time-lapse Image Data on Page 19.)
Calibrate calibrates a raw image when you have a calibration file and a bad pixel
file that applies to the camera and conditions (array size, pixel size, and
wavelength) that were used to collect the image. (See Calibrating Image Data on
Page 20.)
Align Image corrects single wavelength images that have motion artifacts,
problems with Z sectioning, or problems with time series. It allows you to align
adjacent images by applying an XY shift with an optional rotation. (See Aligning
Adjacent Images on Page 21.)
Chromatic Aberration Corrector allows you to adjust channels relative to each
other to correct for shifts in color that result from oil matching, objective
anomalies, and other environmental conditions that use X-Z and Y-Z image
profiles. (See Correcting Chromatic Aberration on Page 23.)
Correcting Z Section Image Data
Correct Image options are used to correct systematic errors that occur during data
collection. The three basic systematic errors are caused by photo-bleaching,
inconsistent illumination intensity, and CCD defects.
By default, softWoRx automatically applies these options to images during the
deconvolution process (These options are specified in the More Deconvolution
Options dialog box).
If you are analyzing unprocessed images (images that are not deconvolved), use
the Correct options tool to apply these options to the images before you process
them. Applying correction options is especially important when you are
performing quantitative analysis on images that contain multiple Z sections or
Time series.
1. Open the Correct Image dialog box by choosing Process | Correct from the
softWoRx main menu.
Applied Precision
Chapter 2: Correcting Images
2. Enter the original _R3D.dv image file in the Input field.
3. Use the Correction Option toggles to select the desired image correction. See
the softWoRx online help for a description of each of the Correction and Run
options available from this dialog box.
4. When you are satisfied with your selections, select Do It to perform the
correction process. Details of the process are displayed in a Correct Image
Output window.
Equalizing Intensities in Time-lapse Image Data
Use Equalize Time Points to choose a reference time point and normalize the
intensities of all other time points to it. The tool uses the mean image intensity to
help generate a more uniform intensity display.
Note In general, the data generated by the Equalize Time Points tool should be
used for display only and not for quantitative purposes.
The Correct tool and the Correction options of the Deconvolve tool make
corrections to the intensity values of sections within a Z series and, with the
exception of photo-bleaching inconsistencies, can also make corrections to
intensity values between time points of a time-lapse experiment.
Note This tool is not intended to correct for photo-bleaching over time.
To equalize intensities to a time point:
1. Choose Process | Equalize Time Points from the main softWoRx menu to open
the Equalize Time Points dialog box.
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2. Enter the original _R3D.dv image file in the Input field.
3. Enter the time point to use as the standard for adjusting intensity values of
other time points in the Reference Time Point field.
4. To perform equalization based on background and minimize the influence of
the variation of higher intensities (signal), select Use Threshold. (This specifies
to use only values less than the Threshold value for each wavelength when
collecting Min/Max/Mean statistics for equalization.)
5. If you are using a threshold, specify the wavelengths in the Wave fields and
specify the threshold for each wavelength.
Note The number of Wave fields in the Equalize Time Points dialog box adjusts
automatically to the number of wavelengths in the selected image.
6. Click Do It to equalize the time points and then click Done.
Calibrating Image Data
Use the following instructions to calibrate an unprocessed image when you have a
calibration file and a bad pixel file that applies to the camera and conditions (array
size, pixel size, and wavelength) used to collect this image. If you do not have a
calibration file, you must create one before you calibrate the image.
To calibrate an image:
1. Choose Process | Calibrate from the main softWoRx menu to open the
Calibrate dialog box.
Applied Precision
Chapter 2: Correcting Images
2. Click Input and browse to the file that you want to calibrate.
3. To select a region of the file, click Details and use the Region Details dialog
box to select the region.
4. Click the Cal button for each channel and browse to the calibration file to use
for this image.
5. Click the Pix button for each channel and browse to the bad pixel file to use for
this image.
6. Select the types of calibration to perform (Calibrate Gain, Calibrate Offset, or
Replace Bad Pixels).
7. Click Do It to calibrate the image.
Aligning Adjacent Images
Use Align Image to correct motion artifacts, problems with Z sectioning, or
problems with time series. This tool allows you to align adjacent images by
applying an XY shift with an optional rotation. Use Align Image only for images
that have a single wavelength.
To align images:
1. Choose Process | Align Image on the softWoRx main menu to open the Align
Image dialog box.
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2. Click Input and browse to the appropriate file. The range fields and
wavelengths are filled in automatically when a file is selected.
Note This dialog box requires an image name for processing. You cannot provide
a window number for this field.
3. In the Align Adjacent list, select whether to align adjacent Z Sections,
Wavelenghs, or Time Points.
4. Click Do It to align the image.
Applied Precision
Chapter 2: Correcting Images
Correcting Chromatic Aberration
Use the Chromatic Aberration Corrector to adjust channels relative to each other.
This tool allows you to correct for shifts in color that result from oil matching and
other environmental and optical conditions.
To correct Chromatic aberration:
1. Choose Measure | Chromatic Correction from the softWoRx main menu to
open the Chromatic Aberration Corrector.
2. Select a multi-channel Image window to reference.
3. In the Image Profile: field, choose X-Z or Y-Z as the vertical profile to inspect.
4. Drag the yellow line in the Image window to adjust the X or Y position of the
profile.
5. In the Chromatic Aberration Corrector, use the colored toggle buttons on the
left to specify which channel you wish to adjust, relative to the others.
6. Click the up and down arrow buttons on the Chromatic Aberration Corrector
to move the selected channel up or down relative to the others.
7. Select File | Save Image with Corrections.
Note For correcting chromatic aberration introduced by the optics, it is
recommended that you make the measurement using a multi-colored bead so as
not to bias the data. Measure the offset using the bead, then apply the same
corrections to actual sample files.
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Applied Precision
3. Stitching
You can stitch images together to display images that are larger than a single field
of view. This is especially useful when you want to collect data at a high
magnification over a large area. You can also use it to display a sequence of time
points in a time-lapse image.
If you are using a DeltaVision Acquisition workstation, you can create stitched
images that are organized as either a series of time points or Z sections. Each time
point, or Z section, is treated as a panel of the stitched image.
Note Image stitching is only possible with certain types of DeltaVision image files.
In particular, the image file must contain a series of images, along with a
corresponding set of XY coordinates. To obtain images suitable for stitching, use
the Panel Collection feature of Resolve3D.
In this Chapter
Stitching Images That Have a Single Z Section............................................................. 26
Stitching Images That Have Multiple Z Sections ......................................................... 27
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Stitching Images That Have a Single Z Section
You can use stitching for simple 2D images or for time-lapse images.
Individual panels were stitched to create the final image on the right
Before you start
Collect Panel data with the DeltaVision Acquisition workstation.
To stitch an image:
1. Click View | Stitch in the softWoRx main menu. The Stitch Image dialog box is
displayed.
2. Click Input and browse to the file that you want to stitch. softWoRx reads the
wavelengths from the file header and automatically selects the appropriate
wavelengths. Change these settings only if your application does not require a
stitched file for a particular wavelength, in which case you can de-select the
appropriate wavelengths.
3. Click Output and browse to the same file previously selected. The file will
appear in the Output field with “_STC” added to the file name. (With this
Applied Precision
Chapter 3: Stitching
naming convention, the input source file is always associated with the output
stitch file.)
4. If desired, click More Options to display additional stitch options. For most
applications, the default settings should work quite well. When finished with
this dialog box, click Done to return to the main Stitch Image dialog box.
5. When all of the options have been specified, click Do It.
Stitching Images That Have Multiple Z Sections
For images that contain multiple Z sections, you will need to collect the images
with the Panel tool and crop them before you stitch them.
Before you start
Collect Panel data with the DeltaVision Acquisition workstation.
Determine the width of the border rolloff (in voxels) for the images. (To minimize
edge effects, the border rolloff is automatically set to about 1.5% of the image
dimensions.)
To crop a multilayered Image:
1. Collect 3D panel images.
2. Deconvolve the _R3D.dv file that you collected.
3. Open the deconvolved file in an Image window (this file has a _R3D_D3D.dv
file extension).
4. Choose Edit | Copy Region on the main softWorRx menu. The Copy Region
dialog box is displayed.
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5. Drag the deconvolved file into the Input field and click Details. The Region
Details dialog box is displayed.
6. Enter values in the X/Y/Width/Height, Z (Start, End, Inc), and Time
(Start/End/Inc) fields under the Output Options section. (The Dimensions
(XYZT) field displays the dimensions of the panel.) Use the following
equation to determine the Width and Height values under Output Options:
Width = x-2n
Height = y-2n
Where x and y are dimensions X and Y respectively and n is the number of
border rolloff voxels.
7. Click Close to close the Region Details dialog box. Then click Do It in the Copy
Region dialog box. (The cropped panel stack is displayed in a new Image
window.)
8. Choose File | Save on the Image window menu to save the new cropped panel
stack.
Applied Precision
Chapter 3: Stitching
9. Stitch the cropped deconvolved file as shown in Stitching Images That Have a
Single Z Section on Page 27.
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Applied Precision
4. Importing Data
You can convert the following image file formats to the DeltaVision file format.
„
TIFF
„
Inovision ISee™
„
BioRad MRC-600 Pic
„
UIC MetaMorph STK
Note Since Applied Precision does not own the STK, PIC, ISEE, or TIFF formats,
changes to those formats may occur that could make them incompatible with
softWoRx. If this occurs, try saving the file as an older version of the format.
In This Chapter
Converting TIFF Images................................................................................................... 32
Converting ISee Files ........................................................................................................ 33
Converting Pic Files .......................................................................................................... 35
Converting STK Files ........................................................................................................ 36
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Converting TIFF Images
You can convert a TIFF image file or series of TIFF files to a DeltaVision file format.
When converting a series of TIFF files, the files are converted to a Z section stack.
DeltaVision expects each TIFF file to be 16 bits of grayscale data representing a
single wavelength. If you have a multiple wavelength data set, you will need to
create single-wavelength DeltaVision files and merge them using the Copy Region
or Image Fusion tools.
When converting TIFF files to the DV format, you will need to provide pixel
dimensions and a wavelength value for the output file. Other information may be
added or modified using the Edit Image Header utility. You can also use this
utility to reorganize the description of the data as a series of time points instead of
Z sections.
To convert a TIFF image file to a DV image file:
1. Click Conversions | Import from TIFF in the softWoRx main menu. The
Convert TIFF to DeltaVision dialog box is displayed.
2. Select the file that you want to convert using the TIFF Input File Selection
options.
3. Type the name of the directory in which to place the TIFF file in the Output
Directory field.
4. Type a filename for the converted file into the Output File field.
5. Enter the X, Y, and Z spacing of the pixels in the data set (in microns) into the
Pixel Spacing (X/Y/Z) fields.
Note The deconvolution program and other softWoRx software rely upon the
presence of accurate wavelength and pixel spacing. Not all TIFF files contain
Applied Precision
Chapter 4: Importing Data
accurate pixel size and wavelength information, so it may be necessary to
manually enter some of the fields in TIFF conversion.
6. Enter the wavelength of the data to be converted (in nanometers) into the
Wavelength field.
7. Use the Stack of Images list and buttons to position the images in the desired
order.
8. Click Do It.
Converting ISee Files
The ISee conversion tool is used to convert Inovision ISee™ images to the
DeltaVision format.
The deconvolution program and other softWoRx software rely upon the presence
of accurate wavelength and pixel spacing. Not all ISee files contain accurate pixel
size and wavelength information. It may be necessary to manually enter values in
the ISee Conversion fields.
Note The image wavelength for a DeltaVision file indicates the wavelength of the
light imaged by the camera, rather than the illumination wavelength.
X/Y pixel spacing can be obtained in two ways: it can be measured with a test
target or it can be approximated from the CCD detector element size and the total
image magnification. For example, if the CCD detector has 6.7 µm pixels and the
image was acquired with a 100X lens and a 1.5X optivar, then the pixel size is
approximately 6.7 µm/(100 × 1.5) = 0.045 µm. The Z pixel spacing is the distance
between adjacent optical sections.
Figure 1. ISee Converter
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Options in this dialog box are described briefly in the following paragraphs. For
additional information regarding ISee file conversion, refer to your online Help
system.
ISee Series Conversion
The Convert Series option combines all files with a similar name into one
DeltaVision file. The conversion program looks for files that have the same prefix as
the input file. The program assumes that a series of Inovision files will be the
same, except for the sequence numbers in the last 3 characters.
For example, the following files would be automatically combined into a single
DeltaVision file:
my_file_name.001
my_file_name.002
my_file_name.003
my_file_name.004
Any of the above files could be entered into the ISee File option as the file to
convert. The use of wildcards, such as “*”, is not supported.
To convert an ISee image file to a DV image file:
1. Click Conversions | Import from ISee in the softWoRx main menu. The
convert Inovision ISee to DeltaVision dialog box is displayed, as shown in
Figure 1.
2. Select the file that you want to convert using the ISee File button and dataentry field.
3. Type a filename for the converted file into the DV File field.
4. Enter the wavelength of the data to be converted (usually in nanometers) into
the Wavelength field.
5. Enter the X, Y, and Z spacing of the pixels in the data set (usually in microns)
into the appropriate fields.
6. Enter the correct lens number in the Lens ID field.
7. Click Do It.
Applied Precision
Chapter 4: Importing Data
Converting Pic Files
The Pic conversion tool is used to convert BioRad MRC-600 Pic™ images to
DeltaVision format.
The deconvolution program and other softWoRx software rely upon the presence
of accurate wavelength and pixel spacing. Not all Pic files contain accurate pixel
size and wavelength information, so it may be necessary to manually enter values
in the Pic Conversion fields.
Note The image wavelength for a DeltaVision file indicates the wavelength of the
light imaged by the camera, rather than the illumination wavelength.
X/Y pixel spacing can be obtained in two ways: it can be measured with a test
target or it can be approximated from the CCD detector element size and the total
image magnification. For example, if the CCD detector has 6.7 µm pixels and the
image was acquired with a 100X lens and a 1.5X optivar, then the pixel size is
approximately 6.7 µm/(100 × 1.5) = 0.045 µm. The Z pixel spacing is the distance
between adjacent optical sections.
Figure 2
Options in this dialog box are described briefly in the following paragraphs. For
additional information regarding Pic file conversion, refer to your online Help
system.
To convert a Pic image file to a DV image file:
1. Click Conversions | Import from Pic in the softWoRx main menu. The Convert
Pic to DeltaVision dialog box is displayed, as shown in Figure 2.
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2. Select the file that you want to convert using the Pic File button and data-entry
field.
3. Type a filename for the converted file into the DV File field.
4. If the image file consists of two wavelengths that are arranged in two adjacent
panels, enable the Split Adjacent Panels option.
5. Enter the wavelength of the first image (or panel) into the Wavelength 1 (nm)
field.
6. If necessary, enter the wavelength of the second image (or panel) into the
Wavelength 2 (nm) field.
7. Enter the X, Y, and Z spacing of the pixels in the data set (usually in microns)
into the appropriate fields.
8. Enter the correct lens number in the Lens ID field.
9. Click Do It.
Converting STK Files
The STK conversion tool is used to convert MetaMorph STK images to DeltaVision
format. Unlike the ISee and Pic converters, the STK converter attempts to read the
wavelength and pixel size values from the STK file’s header immediately after you
specify the name of the input file. After reading these values from the header, the
converter enters this data into the fields in STK Conversion. If necessary, you may
change this information manually before you click Do It. (The ISee and Pic
converters do not read the input file until you click Do It.)
The deconvolution program and other softWoRx software rely upon the presence
of accurate wavelength and pixel spacing. As with ISee and Pic files, not all STK
files contain accurate pixel size and wavelength information, and it may be
necessary to manually enter some of the fields in STK Conversion.
Note The image wavelength for a DeltaVision file indicates the wavelength of the
light imaged by the camera, rather than the illumination wavelength.
X/Y pixel spacing can be obtained in two ways: it can be measured with a test
target or it can be approximated from the CCD detector element size and the total
image magnification. For example, if the CCD detector has 6.7 µm pixels and the
image was acquired with a 100X lens and a 1.5X optivar, then the pixel size is
approximately 6.7 µm/(100 × 1.5) = 0.045 µm. The Z pixel spacing is the distance
between adjacent optical sections.
Applied Precision
Chapter 4: Importing Data
Figure 3
Options in this dialog box are described briefly in the following paragraphs. For
additional information regarding STK file conversion, refer to your online Help
system.
To convert an STK image file to a DV image file:
1. Click Conversions | Import from MetaMorph STK in the softWoRx main
menu. The Convert MetaMorph STK to DV dialog box is displayed, as shown
in Figure 3.
2. Select the file that you want to convert using the STK File button and dataentry field.
3. Type a filename for the converted file into the DV File field.
4. Enter the wavelengths (in nm) of the light collected by the camera for each
channel into the Wavelengths fields.
5. If necessary, modify the X, Y, and Z spacing of the pixels in the data.
6. Enter the correct lens number in the Lens ID field.
7. Click Do It.
8. Proceed to enter the appropriate values into the rest of the dialog box and
complete the desired manipulation.
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Applied Precision
5. Data and Task Manipulation
This chapter describes how to select data, crop data from images, and combine
images.
In this Chapter
Selecting Data........................................................................................................................ 40
Cropping and Trimming Data ............................................................................................ 45
Cropping an Irregular Data Region ................................................................................... 47
Combining Data of Two Images......................................................................................... 52
Setting Up Process Chains with Task Builder .................................................................. 54
Using Ratio Imaging............................................................................................................. 57
Using the Multiplexed Wavelength Option...................................................................... 62
Connecting to a DMS Database .......................................................................................... 74
Uploading Images to a DMS Database.............................................................................. 74
Downloading Files from DMS ............................................................................................ 81
Browsing and Locating Images in a DMS Database........................................................ 82
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Selecting Data
You can select either rectangular or irregular data regions:
„
Use the Select Region and Details buttons to select rectangular data regions.
These buttons are included at the top of softWoRx dialog boxes that allow you
to save, export, or select data.
„
Use the Edit Polygon tool to select irregular data regions.
Selecting Rectangular Data Regions
Use Select Region to select data for volume rendering, Rotate3D tool applications,
modeling, and other applications that require intensive processing. You can also
use this tool to crop data and save it in a new file (see Cropping a Rectangular Region
on Page 45).
To select a rectangular data region:
1. On any softWoRx process window that includes the Select Region button, click
Select Region and drag the mouse across the image to select a rectangular
area. The selected region is indicated by a dotted line.
2. Click Details to open the Region Details dialog box.
Applied Precision
Chapter 5: Data and Task Manipulation
3. In the Z (Start/End/Inc) field, select a Z section range to include. Start and End
are the beginning and end points. Inc (incremental) allows you to skip points
(e.g., entering an Inc value of 2 skips every other point).
4. In the Time(Start/End/Inc) fields, select a time data range to include. Then
click Close.
5. Select Do It on the process window you’re using. The selected region is
displayed in the chosen output window.
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Selecting Irregular Data Regions
Use the Edit Polygon tool to select irregular data regions. This tool is applied
differently than the tools for selecting rectangular regions. While selecting
rectangular regions is usually used within other tools, the Polygon Editor stands
alone. You must use the Polygon Editor tool with Cut Mask if you want to then
apply another tool only to the selected region.
To select an irregular data region:
1. Open the image in the Image window.
2. Choose Model | Edit Polygon to open the Edit Polygon menu.
Applied Precision
Chapter 5: Data and Task Manipulation
3. Choose a selection tool (e.g., ) from the Edit Polygon menu. Then press and
hold the left mouse button to draw a polygon around the area of interest
within the Image window.
4. To copy this region across wavelengths, time points, or through Z sections,
choose Edit | Propagate Polygons from the Edit Polygon menu and enter the
appropriate ranges.
Tip you can also select time points. You can select ranges or you can enter
selected points (e.g., 1, 3-5, 20-25).
5. Click Set All if you want to copy the polygons through all of the Z sections,
time points, and wavelengths. Click Do It to copy the polygons. Then view the
range of Z sections or time points to make sure that all of the data is included
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in the polygon for each section. Use the channel selectors to view the polygons
for each of the selected wavelengths.
In this example, all of the data for each of the selected wavelengths and between the
selected cross sections is within the polygons for each Z section.
Tip You can change selected points on a polygon using the
move a selected polygon by selecting it with the
new location.
button. You can
tool and dragging it to a
Applied Precision
Chapter 5: Data and Task Manipulation
Cropping and Trimming Data
You can selectively crop areas, Z sections, and channels from data files. You can
also trim time points from time-lapse data. Cropping and trimming are useful for
presenting data. It also helps prepare data for volume rendering, 3D rotation,
modeling, and other types of visualization.
Cropping a Rectangular Region
To crop a rectangular region to a new file:
1. Open an Image window. From the Image window menu, choose File | Save.
2. In the Input field, enter either a window number or an image file name. In the
Output field, specify an image file or window as output.
3. If your input is a window, you can select a region. To do this, click Select
Region and drag the mouse across the area to select it. Adjust the rectangle
that you've created until it contains the desired area. Then click outside the
Image window with the mouse.
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4. Click Details to open the Region Details dialog box.
5. In the Z (Start/End/Inc) field, select a Z section range to include. Start and End
are the beginning and end points. Inc allows you to skip points (e.g., entering
an Inc value of 2 skips every other point).
6. In the Time(Start/End/Inc) fields, select a time data range to include. Then
click Close.
7. In the Save File dialog box Wavelengths field, choose which wavelengths of
the input data to process and include in the output data set. If you don't have
the option of which wavelength to include, the toggle buttons are dimmed.
Applied Precision
Chapter 5: Data and Task Manipulation
47
8. Click Do It to save the cropped image file. Then open the saved file in another
Image window and view the results of the selections.
Cropping an Irregular Data Region
You can crop irregular data regions from image files.
To crop an irregular data region:
1. Open the image in the Image window.
2. Choose Model | Edit Polygon to open the Edit Polygon window.
3. Choose a selection tool (e.g.
interest.
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4. To copy this region across wavelengths, time points, or through Z sections,
choose Edit | Propagate Polygons from the Edit Polygon menu and enter the
appropriate ranges.
Tip You can also select time points. You can select ranges or you can enter
selected points (e.g., 1, 3-5, 20-25).
5. Click Do It to copy the polygons. Then view the range of Z sections to make
sure that all of the data is included in the polygon on each section.
Tip You can change selected points on a polygon using the
move a selected polygon by selecting it with the
new location.
button. You can
tool and dragging it to a
6. From the main softWoRx menu, choose Edit | Cut Mask.
Applied Precision
Chapter 5: Data and Task Manipulation
7. In the Input field, enter the Image window number. Then click Details to open
the Region Details dialog box and enter the Z or T sections to include. Click
Close.
8. In the Cut Mask window, choose which wavelengths to include. In Create
Mode, choose one of the following modes and specify whether to act on the
inside or the outside of the polygons.
•
Choosing Data cuts all of the data inside or outside of each polygon and
copies it to the output destination.
•
Choosing Mask creates an output file with only 1's and 0's representing
either the inside or the outside of the polygons.
9. In the Threshold field, set a background intensity to remove from the
selection. (For example, setting a threshold of 200 selects only data with an
intensity value greater than 200.)
Note With Trim Output selected, the smallest area containing all the polygons is
the area written to the output window. With Trim Output unselected, the size of the
file has the same x-y dimensions as the original file, but only the part defined by
polygons has intensity.
10. Click Do It to crop the image.
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Trimming Time Data
You can trim time points from time-lapse images.
Starting image with 61 time points
To trim time points:
1. Open the image in the Image window.
2. From the main softWoRx menu, choose Edit | Copy Region.
Applied Precision
Chapter 5: Data and Task Manipulation
3. In the Input field, enter the Image window number.
4. Click Details to open the Region Details dialog box.
5. In the Time (Start/End/Inc) field, enter the first and last time points to include
and the increment between points. For example, entering 15, 45, 1 includes all
of the points between 15 and 45. You could skip every other point in this
interval by entering 15, 45, 2. (In the example above, every third point is
included.)
6. Click Close to quit Region Details.
7. In the Copy Region dialog box, click Do It to create the new image.
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Ending image with 11 time points
Combining Data of Two Images
Use the Image Fusion dialog box to combine time points, Z sections, or
wavelengths from two DeltaVision images into one output file. The input images
may come from windows or files. After selecting the input images, you can specify
exactly which wavelengths, time points, and Z sections you want to combine.
You can either append selected wavelengths to a single file or fuse time points or
Z sections of the same wavelength, creating a single data set for each output
wavelength.
To combine data of two image files:
1. Choose Edit | Image Fusion from the main softWoRx menu to open the Image
Fusion dialog box.
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2. Enter image file names or window numbers for the two files that you want to
combine in the Image 1 and Image 2 boxes.
3. Select which wavelengths, time points, and Z sections to combine from the first
image in Image 1 Selections.
4. Select which wavelengths, time points, and Z sections to combine from the
second image in Image 2 Selections.
5. Specify how to combine the data under Fusion Options as follows:
•
To append all selected wavelengths to the output data set, choose Append
Wavelengths. (With this option, if Image 1 had wave 490 selected and
Image 2 had wave 490 selected, the output data set would have two
separate 490 wavelengths.)
•
To combine timepoints from all selected matching wavelengths into a
single final wavelength data set, choose Combine timepoints for like
wavelengths.
•
To combine Z sections from all selected matching wavelengths into a single
final wavelength data set, choose Combine z-sections for like
wavelengths.
6. Click Do It.
Notes
#1 If error messages are displayed referring to differences in file types that include
image size, pixel size, lens info, data type, etc., use Copy Region and Edit Header
to manipulate these items.
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#2 If the number of sections varies from wavelength to wavelength during an
operation, Blank Z sections have been added to the image is displayed.
These blank sections are added to balance the number of sections between each
wavelength of the output image. Each one is a zero intensity image added to the
end of the appropriate wavelength.
Setting Up Process Chains with Task Builder
A process chain is a series of tasks that are predefined for a given collection of data.
Task Builder is a unique feature of softWoRx that allows you to set up process
chains for one or several tasks to be performed on a single set, or multiple sets of
data. You can use the Task Builder dialog box to select files and define multiple
operations to be performed. When you’ve finished providing the data information
and the processes you want to accomplish, you can choose to either start the jobs
immediately or start the jobs at a specified time.
To set up process chains with Task Builder:
1. Select Process | Task Builder from the main softWoRx menu to open the Task
Builder dialog box.
2. On the dialog box, select File | Add Files. You are presented with a list of files
from which you can choose the specific files you want to add to your process
chains.
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3. Select the file(s) you want to add to the Task Builder dialog box and click OK.
The files are displayed in the Input Files section of the dialog box.
Note You can select files from this dialog box using the SHIFT key to select multiple
contiguous files or the CTRL key to select multiple files from various parts of the list.
4. Next, use the Task options in the Processing Tasks section of the dialog box to
select the tasks and the order in which you want these processes performed on
the selected file(s). The Task options to choose from are Deconvolution,
Correction, Crop Image, Quick Projection, Volume Rendering, and Export
As. Use the Add button to include additional tasks and the X buttons to
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remove tasks from the chain. Use the Options buttons next to each task to
view a dialog box of available options for the specific task.
The task options you specify will be performed on each file in the exact order
they appear in the Task Builder dialog box. Each of the selected files is run
through the entire list of tasks before the Queue Manager moves on to the next
file in the list.
Tip You can use the left mouse button to drag image file icon, a group of file
icons, or folder icons to the Task Builder or the Queue Manager.
5. When you are satisfied with your selections and the order in which the tasks
will occur, click Submit Tasks to the Queue. The softWoRx Queue Manager
dialog box is displayed.
6. To start the process chain,
•
Immediately, click Start Now.
•
At a later time, click Start Later. When you choose this option, a set of time
option buttons is displayed as shown in the example below. Set the time at
which you want the process chain to start. You can use the Change It
button if you decide you want to change the time to begin the process
chain.
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7. Select Quit to exit the Queue Manager dialog box.
Using Ratio Imaging
softWoRx provides a ratio imaging acquisition function that allows you to view a
graphic representation of the ratio of two channels as the images are being
collected. In addition, a ratio graph displays the mean value (of an area in the
middle of the image) vs. time. Both the ratio image and the ratio graph are for
monitoring purposes only. The ratio imaging experiment results in a two-channel
time-lapse image.
Sample image for ratio imaging experiment
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To set up a ratio imaging experiment:
1. From the softWoRx main menu, select File | Acquire (Resolve3D) to open the
Resolve3D window.
2. On the Resolve3D window, click the Experiment button to open the
Design/Run Experiment dialog box.
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3. Select the Sectioning tab and unselect the Z Sectioning toggle.
4. Select the Channels tab and specify the two channels you want to use for this
experiment.
5. Select the Time-lapse tab and specify the time-lapse and total time for this
experiment.
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6. Select the Actions tab and select Ratio Image as the action for this experiment.
The Time Points specification will default to all and the When control will be
After Imaging.
7. Select the Run Experiment tab and enter the image file name and a title for the
ratio image. You can also enter text into the Add note to log field to include
the text in your image log file.
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8. Click the Start Scan button to begin the imaging process. The ratio imaging
process will occur in a separate Image viewer similar to the following:
The square outline in the center of the image
represents the mean value of the image data.
When the ratio imaging experiment is complete, a two-channel time-lapse
image is displayed along with a ratio graph showing the mean value (the
outlined area in the middle of the image) vs. time. The square outline
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represents the portion of the image used to calculate the mean for a ratio
graph.
Using the Multiplexed Wavelength Option
The optional Multiplexed Wavelength module for the DeltaVision system allows
you to perform nearly simultaneous two-channel imaging without the drawbacks
associated with true simultaneous two-channel imaging. This option uses two
shuttered illumination sources and a dual-band emission filter to eliminate filter
wheel movement between channels, and therefore greatly reduces the time
required for the DeltaVision system to acquire a set of two-channel images. The
combined light path ensures no registration artifacts are introduced and
independent excitation of probes helps to ensure minimal crosstalk.
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Conceptual view of Multiplexed Wavelength functionality
Before you use the Multiplexed Wavelength option, you must first have it installed
and configured correctly. Your Applied Precision representative will assist you in
setting up this option and help you ensure that all hardware and software to
support Multiplexed Wavelength functionality is installed properly.
After the option has been installed and configured, the menus, tools, and other
infrastructure necessary to use the feature will be available on your workstation.
Setting Up the Multiplexed Wavelength Option
Before you begin designing your Multiplexed Wavelength experiment, you’ll need
to perform the steps described in the following procedures to activate a
Multiplexed Wavelength filter set and prepare the DeltaVision system for
Multiplexed Wavelength operation.
To activate the Multiplexed Wavelength filter set:
1. To change the active filter set to a filter set that is Multiplex capable, select
Settings in the Resolve3D main menu.
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Settings
The Resolve3D Settings window is displayed.
2. In the Resolve3D Settings window, click on the Misc tab.
Misc tab
3. In the Resolve3D Settings window, select the EX and EM filter sets you want to
use. When these fields are changed, the <<<Pending Activation message is
displayed in the window (as shown).
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Select the EX and
EM filter sets
Message
displayed when
filter wheel
settings are
changed
4. Click Activate Filter Sets. The following confirmation window is displayed.
Note If you select filter sets for the Excitation filter wheel and Emission filter wheel
fields and then click Done in this window, your selections are retained until you
either activate the filter sets or exit Resolve3D.
Note For the filter sets to be activated, the selected multiplexed filter set filters
must exist in the currently installed excitation and emission filter wheels.
5. If the selected filter wheels are installed on your DeltaVision, click Next to
continue.
Note If you click Skip from this window, the selected filter wheels are activated
immediately and the remainder of the activation wizard is skipped.
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6. Insert the selected secondary filter insert (mCherry is used in this example)
into Filter Slot 1 of the secondary light path and click Next to continue.
The system gathers the information for this window (in this case, “position 4 (500
LP)” from the MXWSetup.ini file, not from the Instrument Controller.
7. Move the beam combiner to the appropriate position and click Next to
continue.
Again, softWoRx gets the information for this window (in this case, “position 2
(GFP/mCherry)”) from the MXWSetup.ini file, not from the Instrument Controller.
8. Click Finish to complete the Multiplexed Wavelength filter activation process.
After the selected filter set is activated, the Design/Run Experiment window
will look similar to the following.
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At this point, you have completed activating the Multiplex Wavelength filter
set. You should now continue with the steps in the next procedure for viewing
a sample with the Multiplexed Wavelength operation.
To view a sample using the Multiplexed Wavelength option:
1. Rotate the eyepiece filter wheel to the POL or BLANK position.
2. From the Resolve3D main menu, select the Excitation filter currently in the
primary light path (CFP or GFP).
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Select the EX filter
currently in the
primary light path.
3. Use the EX button on the keypad to open the primary EX shutter and view the
primary light path.
4. Use the EX2 (formerly CAMERA SHUTTER) button on the keypad to open the
EX2 shutter and view the secondary light path.
Note With the Multiplexed Wavelength option, you can view both selected
wavelengths simultaneously by opening both EX shutters at the same time.
Designing a Multiplexed Wavelength Experiment
Use the following procedure to begin the design process for Multiplexed
Wavelength experiments.
To design a Multiplexed Wavelength experiment:
1. From the Resolve3D main menu, click the Experiment button to open the
Design/Run Experiment window.
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69
Experiment
Button
2. If the Multiplexed Wavelength option is enabled on your DeltaVision system,
you’ll see the Multiplexed tab in the Design/Run Experiment window. Click on
the Multiplexed tab to view the options for Multiplexed Wavelength
experiments.
Multiplexed
Tab
The Multiplexed tab of the Design/Run Experiment window is displayed as
shown.
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Select
Checkbox
3. From the Multiplexed tab, select the Do Multiplexed Channel Imaging
checkbox.
If the currently active filter set is not Multiplex capable, the following window
is displayed.
You will need to change the active filter set to continue. Press OK to return to
the Design/Run Experiment window. To change the active filter set for
Multiplexed Wavelength experiments, see the procedure for activating a
multiplexed wavelength filter set in “Setting Up the Multiplexed Wavelength
Option.”
If the currently active filter set is Multiplex capable, the Design/Run
Experiment window is displayed and will look similar to the following.
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At this point, you have completed the initial portion of the Multiplexed
Wavelength experiment design setup. You should now continue with the
standard steps for the remainder of the experiment design, such as Sectioning,
Timelapse, and so on.
Note As soon as you activate the Do Multiplexed Channel Imaging checkbox, all
conventional imaging settings are disabled. This is also true of all multiplexed
settings when you reactivate conventional imaging.
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Applied Precision
6. DMS Integration
This chapter describes how to use the DMS (Data Management System) with
softWoRx to manage image files for your particular application.
What is DMS?
The DMS (Data Management Solution) product provides a functional
infrastructure for the storage of biological images and their associated metadata.
The DMS Server contains a data management system that centralizes all image
data management. Once configured, the DMS Server becomes a repository for all
data generated by a laboratory’s image acquisition system(s). All visualization and
analysis processes are performed on client workstations connected to the DMS
Server.
In This Chapter
Connecting to a DMS Database .......................................................................................... 74
Uploading Images to a DMS Database.............................................................................. 74
Downloading Files from DMS ............................................................................................ 81
Browsing and Locating Images in a DMS Database........................................................ 82
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Connecting to a DMS Database
Before you attempt to connect to a DMS database, you’ll need to have a user name
and password for the database you want to use. Check with your system
administrator for this information.
To connect to a DMS database:
1. From the softWoRx main menu, select File | Connect to DMS Database. The
Connect to DMS Database login box is displayed.
2. Enter the name of host computer in the Database Host Name field.
3. Enter your user name and password in the appropriate fields and click Connect. A
pop-up message confirms that you are connected to the database.
If the connection fails, make sure you are using the correct user name and
password combination for the specific database.
Uploading Images to a DMS Database
You can upload image data from your local file system to the DMS database in a
number of ways. You can upload image data:
•
•
•
Directly from an Image Window.
As part of a Task Builder processing chain.
As part of an experiment, in which the data is auto-uploaded at the conclusion
of the experiment.
Each of these methods is discussed in the following sections.
Uploading Images from the Image Window
The simplest method for uploading image data to the DMS database from your
local file system is to upload the data directly from the Image Window.
To upload image data from the Image Window:
1. From the softWoRx main menu, select File | Open. When the Open Image
window is displayed, click the Files tab and select the appropriate directory.
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2. Select the file you want to upload and click OK. The Image Window is
displayed with the file you selected.
3. From the Image Window, select File | Save to DMS. The Save To Data
Management System menu is displayed as shown.
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From this menu, select the Project and Dataset to which you want to save the
data on the DMS database. You can optionally select a Category Group and
Category for the saved image data and add initial annotation to the file for
future reference. When you are satisfied with the image data to be uploaded,
click Do It.
When the upload is complete, “Finished” is displayed on the status line as
shown.
4. Click Done to exit the Save To Data Management System menu.
5. To confirm that the selected image file has been uploaded to the DMS
database, select File | Open from the softWoRx main menu. When the Open
Image window is displayed, click the DMS Database tab.
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Note To activate the DMS Database tab, you must be currently connected to a
DMS database. Refer to the previous section, “Connecting to a DMS Database”
for details.
The file you uploaded should appear in the displayed list of files.
Uploading Images Using Task Builder
To upload image data from your local file system to the DMS database from
within Task Builder, use the following procedure.
To upload image data to the DMS database using Task Builder:
1. From the softWoRx main menu, select Process | Task Builder. The Processing
Task Builder menu is displayed.
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2. In the Processing Task Builder menu, select the file(s) you want to process and
upload to the DMS database. You can accomplish this by simply dragging and
dropping the file or folder icon(s) into the Image Files to Process area of the
menu. Alternatively, you can use File | Add Files from this menu to select the
file(s) to include.
3. Next, select the processes to perform on the image file(s). For the final task,
select Save to DMS as shown.
4. Click the Options button next to the Save to DMS selection. The Save to DMS
Options menu is displayed.
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5. From this menu, select the Project and Dataset to which you want to save the
data on the DMS database. You can optionally select the Category Groups and
Category for the saved image data and add initial annotation to the file for
future reference. When you are satisfied with the image data to be uploaded,
click OK. You are returned to the Processing Task Builder menu.
6. From the Processing Task Builder menu, click Submit to Queue to open the
softWoRx Queue Manager dialog box.
7. To start the process chain,
•
Immediately, click Start Now.
•
At a later time, click Start Later. When you choose this option, a set of time
option buttons is displayed. Set the time at which you want the process
chain to start. You can use the Change It button if you decide you want to
change the time to begin the process chain.
8. Select Quit to exit the Queue Manager dialog box.
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Auto-uploading Images after Acquisition
To auto-upload image data to the DMS database after an experiment is
completed, use the following procedure.
To auto-upload image data immediately following an acquisition:
1. Set up your experiment as normal by clicking Experiment from the Resolve3D
window to open the Design/Run Experiment window.
2. On the Design Experiment tab, enter the experiment name and configure the
experiment. (For experiment setup details, see the “Setting Up and Running
Experiments” Chapter in your DeltaVision System User’s Manual.)
3. Select the Run Experiment tab and enter the image file name (or drag and drop
from another location), the image title, and any annotation you want to add.
4. Click the DMS Setup button. The DMS Destination Setup window is
displayed.
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5. Be sure that the Upload files to Database after collection checkbox (at the top
of this screen) is activated.
6. Enter the project and dataset where you want the acquired image data to
upload.
7. Optionally, you can also enter the category group and the category for the
uploaded data and include any annotation necessary.
8. When you are satisfied with the information entered in the DMS Destination
Setup window, click Done.
9. Run the experiment macro as normal. The resulting image acquisition will be
added to the DMS database as specified.
Downloading Files from DMS
You can use softWoRx to download image data from the DMS database to your
local file system.
To download a file from the DMS database:
1. From the softWoRx main menu, select File | Open. When the Open Image
window is displayed, click the DMS Database tab and select the file you want
to download.
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2. Click the Download button. The Choose Destination Folder dialog box is
displayed.
3. Select the directory on your local file system where you want the selected
image file downloaded and click OK. The image file is downloaded from the
DMS database to the specified location in your local file system.
Browsing and Locating Images in a DMS Database
The softWoRx software provides the ability to browse images within a DMS
database by either the Project/Dataset/Image or the Category
Group/Category/Image hierarchies. You can also search for specific images
based on the image file’s annotation.
Browsing Image Files using P/D/I Hierarchy
To browse through image files by Project/Dataset/Image:
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1. From the softWoRx main menu, select File | Open. When the Open Image
window is displayed, click the DMS Database tab.
2. Use the drop-down menu in the Database View field to select
Project/Dataset/Image.
3. Select the specific projects and datasets to browse using the dropdown menus
in the Project and Dataset fields.
Browsing Image Files using CG/C/I Hierarchy
To browse through image files by Category-Group/Category/Image:
1. From the softWoRx main menu, select File | Open. When the Open Image
window is displayed, click the DMS Database tab.
2. Use the drop-down menu in the Database View field to select
CategoryGroup/Category/Image.
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3. Select the specific category groups and categories to browse using the dropdown menus in the Category Group and Category fields.
Searching for Image Files Based on Annotation
You can locate images in a DMS database by performing a search based on the
image file’s annotation.
To search for an image file based on its annotation:
1. From the softWoRx main menu, select File | Open. When the Open Image
window is displayed, click the DMS Database tab.
2. Use the drop-down menu in the Database View field to select Search
Annotations.
3. In the Search Annotations For field, enter the annotation for the desired image
file and click the Search button.
Note Holding your mouse cursor over the Search Annotations For field displays a
tooltip window containing specific wildcard use information.
Applied Precision
Part Two
PART TWO: VISUALIZING & PRESENTING DATA
softWoRx provides several tools that you can use to visualize data and prepare it
for presentations. You can also save or export data in a variety of formats.
In Part Two
Chapter 7. Viewing Image Data ....................................................................... 87
Chapter 8. Viewing Movies.............................................................................. 113
Chapter 9. Viewing Projections and Volumes ............................................... 119
Chapter
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10. Filtering Image Data....................................................................................137
Chapter
11. Saving, Exporting, and Printing ..................................................................147
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7. Viewing Image Data
softWoRx provides several options for viewing data. You can open DeltaVision or
TIFF files in the Image window and use slide bars and controls to view different Z
sections and time points. You can also adjust the brightness and contrast of each
channel in an image and assign a color or grayscale to each channel. To prepare
your data for presentations, you can display a scale bar on the image and hide the
Image window controls. You can also resample image data to change the size or
orientation of the display and view cross sections of the data.
In This Chapter
Opening an Image.................................................................................................................88
The Image Window ..............................................................................................................89
Viewing 5D Images...............................................................................................................90
Adjusting Brightness and Contrast ....................................................................................95
Assigning Colors or Grayscale to Channels......................................................................99
Controlling the Image Window Display .........................................................................102
Resizing or Reorienting an Image ....................................................................................105
Viewing Cross Sections ......................................................................................................111
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89
Opening an Image
You can open image data files in DeltaVision or TIFF image file formats.
To open an image data file:
1. Choose Applied Precision | Start softWoRx from the CentOS
softWoRx main menu is displayed.
menu. The
2. Click the
icon on the softWoRx toolbar, or File | Open from the menu. The
Open Image dialog box is displayed. Click the Files tab.
Select and preview DeltaVision images in the Open Image dialog box.
Tip For DeltaVision images, you can adjust the thumbnail image in the Preview
area. Change the brightness by dragging the left mouse across the preview image.
View different Z sections or time points by pressing the right and middle mouse
buttons.
3. Select an image file (for example, Nuclear_PoreD3D.dv) and click OK to
open the image in the Image window.
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The image opens in the Image window
Tip You can also open an image by clicking Data Folder and then double-clicking
on the .dv file in the data1 directory.
The Image Window
DeltaVision images are displayed in the Image window.
Menu Bar
Tool Bar
Quick Scale Tools
Tool Buttons
Wavelength
Selectors
Status
Bar
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Chapter 8: Viewing Image Data
The Image Window
The Image window provides controls and tools that you can use to view and
analyze image data:
•
The Menu Bar allows you to open or save images, control the display of
the data, and open tools to analyze intensity data.
•
The Toolbar provides buttons as alternatives to the menu bar above.
The buttons can be used to open or save image data files, play movies,
view intensity line profiles, or inspect intensity data.
•
The Tool Buttons are used to change the image view. These controls allow
you to scroll through Z sections and time points, zoom, pan the image
vertically or horizontally, select which channels to view, and scale intensity
to adjust brightness and contrast.
•
The Wavelength Selectors show or hide the wavelengths (channels) in
the Image window. When the Image is displayed in color, each button has
the same color as the wavelength that it controls. The number on each
control button indicates its wavelength. When the data is displayed as
grayscale, use these controls to choose which wavelength is displayed. The
buttons are white with black numbers when on and black with white
numbers when off.
•
The Status Bar shows which Z section or time interval is displayed. It also
displays the intensity of the pixel currently under the mouse pointer.
•
The Quick Scale Tools provide a convenient method by which you can
adjust the intensity scale of each wavelength in the image.
Viewing 5D Images
After opening an image file, use the softWoRx Image window controls for:
ƒ
Viewing Z sections and time points in the data.
ƒ
Viewing different areas of a section.
ƒ
Zooming in on a selected area.
ƒ
Displaying or hiding channels.
Viewing Z Sections and Time Points
Use the Z and T sliders on the left side of the Image window to display different Z
sections and time points. If images contain both Z and T data, you can use the Z
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slider to show all of the sections at a time point or use the T slider to show how a
section changes with time.
To navigate through Z sections and time points:
X Move the Z slider down to show deeper Z sections (the top section is displayed
when the slider is at the top). Move the T slider down to show data acquired at
later time points (delta time increases as the slider is moved down).
The Z Slider
The T Slider
Move the Z and T sliders to display Z sections and time points
Tip You can also use the right and middle mouse buttons to scroll through Z
sections. To scroll through time points, hold down the CTRL key and press the right or
middle mouse buttons.
Viewing Z Sections in Several Image Windows
The Section slider allows you to scroll through Z sections of all open windows
simultaneously. (Only the windows that are open at the time that you activate the
Section slider are affected.)
To scroll Z sections simultaneously:
1. Choose View | Slider from the softWoRx main menu to open the Section Slider
tool.
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The Section Slider
2. Move the slider to the left to decrease the current Z Section or to the right to
increase it.
Viewing Areas
You can view different areas of a section by sliding the vertical and horizontal
scroll bars and the tool buttons to reposition the image.
Tool Buttons
Zoom
Use tool buttons, scroll bars, and zoom to view different image areas.
You can also use the following tool buttons to change display characteristics or
reposition the image.
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Use this tool
To…
Adjust the intensity scale of the image.
Pan across the image.
Center the image on a point. Select the tool and click the point that you want to
center.
Position the image on its original center.
Zoom in or out. Move the Zoom wheel up to zoom out or down to zoom in.
Return to a 1:1 zoom level.
Zoom to fit image to window.
Tip From the Image window, you can also use Alt + Mouse Wheel to zoom the view
in and out.
Zooming In or Out on Specific Points
One common way to use the tool buttons is to position the image and zoom in on
a specific point.
To zoom in on a specific point:
1. On the Image window, click the Choose New Window Center
button and
then click the point in the image on which you want to zoom. The image is
centered on the selected point.
Center
Image
Use the Center Image
control to re-center the
image
Zoom
Wheel
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2. Use the Zoom wheel to zoom in on the point that you selected. The new zoom
level is displayed in the status bar.
Use the Zoom wheel to zoom on the point that is selected as the center point
3. To return the zoom to a 1:1 display, click the Reset Zoom to 1:1
button.
Tip You can specify to interpolate (smooth) images when the zoom level is greater
than 1. Interpolated images provide better quality results but take longer to display.
To interpolate, choose Options | Display and select the Interpolate zoom option.
Displaying or Hiding Channels
You can display or hide channels with the Wavelength Selectors on the Image
window. When the channel is displayed, the channel displays the color that is
assigned to it. When the channel is not displayed, the selector is black.
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Channels not
displayed
Hiding channels in the Image window
To hide a channel:
X Double-click the Wavelength Selector of a displayed channel. The Wavelength
Selector turns black to indicate that the channel is not displayed.
To display a channel:
X Click the Wavelength Selector of an undisplayed channel. The Wavelength
Selector displays the color of the channel and the channel is displayed.
Adjusting Brightness and Contrast
You can improve the contrast of selected data in a channel by changing the
channel's intensity scale.
softWoRx uses shades of the color selected for a channel to display an intensity
scale. The darkest shade is mapped to the lowest (dimmest) intensity value in the
wavelength and the lightest shade is mapped to the highest (brightest) intensity
value. The remaining shades are mapped to values between the lowest and
highest values.
Color shades can be mapped to create linear or nonlinear intensity scales. In linear
scales, the color shades are mapped to values that are distributed evenly from the
minimum to the maximum intensity values. In nonlinear scales, the shades are
mapped to values that are distributed unevenly throughout the range.
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You can adjust the intensity scale for an individual image window or for all open
image windows simultaneously.
Note Changing intensity scale values only adjusts the display of the data. It does
not alter the image data.
To change the intensity scale for the current image window:
button to open the
1. On the Image window, click the Scale Image Intensities
Image Scaling dialog box. This dialog box shows the image intensity scale.
The Scale Image dialog box is used to adjust the intensity scale. The histogram on this
dialog box is a frequency plot that shows the distribution of pixel intensities in the image
file. The Y-axis shows the number of pixels for a given intensity.
2. In the Wave field, select which channel to scale.
3. To change the minimum or maximum scale value, click and drag on the Left or
Right handles.
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As you move the handles, the
image displayed in the Image
window changes interactively.
Data values higher than the
maximum value are assigned
the lightest shade. Values lower
than the minimum value are
assigned the darkest shade.
Right Handle
Left Handle
The Left and Right handles change the scale range.
4. To slide the range to the left or right, click and drag on the Center handle.
Use the Center handle to
slide the scale back and
forth across the histogram.
Center Handle
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5. To change the intensity scale distribution, click anywhere in the histogram
(except on the handles) and drag the mouse up or down.
Dragging the mouse up or down in the histogram changes the scale distribution.
Tips
#1 You can improve contrast at the low end of the intensity range by reducing the
gamma value. To improve contrast at the high end of the range, increase the
gamma value.
#2 Another way to scale the image is to enter values into the Min/Max/Exp fields.
#3 You can restore all of the default values by clicking Restore Default Scale.
To change the intensity scale for all open image windows:
1. Select View | Scale All Windows from the softWoRx main menu. The Scale
All Windows dialog box is displayed.
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2. Similar to the Image Scaling dialog box, the right side of the Scale All
Windows dialog box includes a line with three nodes (or handles) that
graphically represent the intensity scale. Use the mouse to click and drag the
white handles on the line as follows:
•
•
To change the minimum or maximum scale value, click and drag on the
left or right handle.
To slide the scale range to the left or right, click and drag on the center
handle.
As you move the handles, the images displayed in all of the currently open
Image windows change accordingly. Changing the minimum and maximum
intensity values changes brightness and contrast by mapping all 256 color
shade values to a larger or smaller range of data. Data values higher than the
maximum value are assigned the brightest shade. Data values lower than the
minimum value are assigned the dimmest shade.
3. To change the intensity scale distribution, change the shape of the curve by
clicking anywhere on the histogram (except on one of the handles) and
dragging the mouse up or down.
•
•
To improve contrast at the low end of the intensity range, increase the
slope of the curve at the left side of the graph.
To improve contrast at the high end of the range, increase the slope of the
curve at the right side of the graph.
Changing the scale distribution increases the contrast at one end of the data
range and decreases it at the other end.
4. Use the Restore Channel button to restore the selected channel to its original
intensity scale.
5. Use the Restore All button to restore all channels to their original intensity
scales.
6. Press Done when finished with the Scale All Windows dialog box.
Assigning Colors or Grayscale to Channels
You can view image data in grayscale or in two different color modes:
Grayscale Mode is useful for studying detail in a single wavelength. Because of
the way the eye reacts to colors, you may be able to see more detail in
Grayscale than in a Color mode. You can only view one channel at a time in
Grayscale mode.
Color Mode can be used to visually compare intensities of two or three
wavelengths. It also allows you to use the Volume Viewer with RGB opacity,
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improve speed of volume rendered images, or save multi-channel DeltaVision
images as a series of TIFF images.
Blended Color Mode allows you to overlay nonfluorescent data such as
Differential Interference Contrast (DIC) data sets onto fluorescent data sets or
to visually compare intensities of more than three channels simultaneously.
Grayscale Mode
You can switch between Color and Grayscale modes. Because of the way the eye
reacts to colors, you may be able to see more detail in Grayscale than in Color. In
general:
ƒ
Use Grayscale when you want to see more detail in a single wavelength of
an image.
ƒ
Use Color mode when you want to visually compare intensities of two or
more wavelengths.
To switch between Grayscale and Color mode:
X On the Image window, choose View | Color to toggle between Color and
Grayscale.
Grayscale shows more detail in a single wavelength
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Color Mode
If you choose a basic color for each channel (red, green, or blue), you can display
up to three channels in this mode. If you select any other colors (e.g., cyan,
magenta, or yellow), the two colors used to create these mixtures are disabled for
the other channels and they are turned off (Black). Colors such as Cyan, Magenta,
or Yellow can be viewed in combination with only one other color.
To assign a basic color to a channel:
1. Choose View | Select Image Colors on the Image window menu to open the
Select Image Colors dialog box.
Set channel color options in Select Image Colors
2. Select the wavelength for each channel in the Display Color option lists. The
new colors are displayed in the Image window as they are selected.
3. Click Done to set the colors that you selected.
Note You can assign basic red, blue and green colors to as many as three channels.
You can assign other colors to two channels.
Blended Color Mode
In Blended Color Mode, you can assign any color to each channel. You can view
up to five channels as separate colors. You can also assign grayscale as a color (this
is useful for DIC data).
You can select an arbitrary color for each wavelength or you can specify to use the
true color that is normally associated with each wavelength in the color spectrum.
To set Colors in Blended Color Mode:
1. On the Image window menu, choose View | Blended Color to set Blended
Color mode.
2. Choose View | Select Blended Colors to open the Blended Colors dialog box.
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3. To specify the colors normally associated with a wavelength, click True
Colors.
4. To specify a custom color for a channel, click the color under the channel to
open the Choose Color for… dialog box and use the Red, Green, and Blue
sliders to choose the color.
Choose Color for the selected channel
To assign grayscale to a channel in Blended Color Mode:
1. On the Image window menu, choose View | Blended Color to set Blended
Color mode.
2. Choose View | Select Blended Colors to open the Blended Colors dialog box.
3. Click the color under the channel you want to modify and move all three color
sliders all the way to the right to assign White as the Current Color, then press
OK.
Controlling the Image Window Display
You can hide the Image window Display controls, toolbar, and scroll bars. You can
also display and set a scale on your images. This is useful for preparing images for
presentations.
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Hiding or Displaying Image Window Border Tools
The border tools are the icons and controls on the left of the Image window. The
toolbar is the set of icons above the Image window. These tools are displayed by
default. You can hide them to capture a JPEG of the image.
Hiding border tools and the toolbar can focus the screenshot on your data.
The Image window with the border tools and toolbar hidden
To switch border tools on and off:
X Choose Options on the Image window menu and display or hide the border
tools as follows:
To display the tools, select the Show Border Tools toggle on the Options
menu.
To hide the tools, clear the Show Border Tools toggle on the Options menu.
To switch the toolbar on and off:
X Choose Options on the Image window menu and display or hide the toolbar
as follows:
To display the toolbar icons, select the Show Toolbar toggle on the Options
menu.
To hide the tools, clear the Show Toolbar toggle on the Options menu.
The Image Window Scale Bar
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You can display or hide a scale bar to show the scale of the image. You can also set
options to move the scale bar (to show the scale of a point of interest in the image)
or to control how the scale bar is displayed. The scale bar is displayed by default.
To change the image scale bar:
1. From the Image window menu, choose Options | Display to open the Display
Attributes dialog box.
Set Scale Bar Attributes
2. Choose whether to display or hide the scale bar.
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To display the scale bar, select the Displayed option.
To hide the scale bar, clear the Displayed option.
3. To display the scale as a vertical bar, select Vertical (the default is horizontal).
4. Use the Show Value option to select whether or not you want the scale bar
value displayed.
5. Adjust the Position, Length, Color, and Thickness of the scale bar. The scale
bar changes interactively as you set these properties.
6. Select the Move button and use the mouse to drag the scale bar to any position
within the Image window.
Resizing or Reorienting an Image
You can use the Resample2D tool to resize an image or to reorient an image in X,
Y, and Z directions.
Resizing an Image
When Resample2D magnifies an image, it interpolates values to add pixels to the
image. When it reduces an image, it combines or eliminates pixels to create a
subset of the original pixel data.
To resize an image:
1. Choose View | Resample2D from the softWoRx main menu to open the
Resample2D dialog box.
2. Enter the window number in the Input field.
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3. To resize part of the image, click Select Region and then drag the mouse
across the area in the Image window.
Select a region in the Image window
4. To magnify the image, enter a magnification factor in the Magnification in XY
field.
5. To reduce an image, enter a reduction factor in the XY Reduction Factor field.
6. Select Keep Cell Dimensions to keep the X and Y pixel size constant. (If
unselected, a new size is calculated, based on the current settings for
magnification.)
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7. Click Do It.
Rotating an Image
To rotate an image:
Image before rotation
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1. Choose View | Resample2D from the softWoRx main menu to open the
Resample2D dialog box.
2. Enter the window number in the Input box.
3. If you want to reorient part of the image, click Select Region and then drag the
mouse across the area in the Image window.
4. Select the wavelengths that you are interested in.
5. Enter the angle to rotate the image in the Z direction in the Z Rotation field.
6. Enter the distance to shift the data in the Shift in XY field.
7. Enter the angle to rotate the data on the XY plane in the Angle Between Axes
field.
8. If you want to maintain the same relative pixel size in the rotated image,
activate the Keep Cell Dimensions checkbox.
Enter the angle to rotate the axes
9. Click Do It.
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Image after rotation
To reorient images:
Image before reorientation
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Chapter 8: Viewing Image Data
1. Open the Rotate3D dialog box by choosing View | Rotate3D on the main
softWoRx menu.
2. Enter the Image window number in the Input field.
3. Specify the X, Y, and Z rotation angles to apply to the data in the Angle field.
When viewing an image in a window, a positive X rotation rotates the top of
the image towards you, and the bottom away from you. A positive Y rotation
rotates the right side of the image towards you and the left away from you. A
positive Z rotation indicates counter-clockwise rotation.
4. To modify the output size that softWoRx creates from the rotation angle and
the dimensions of the input image, select Options from the Rotate 3D dialog
box. The Rotate Options dialog box is displayed. Enter the new dimensions in
the Output Dimensions field.
5. Specify an X,Y,Z vector for translating the image (pixels) in the Translation
field. The size of each pixel in real-world coordinates (usually microns) is
displayed in the Pixel Size field.
6. Specify a center point about which the rotation occurs in the Rotation Center
field. The Rotation Center is by default the center of the image. You can
specify a different center point (in pixel coordinates).
7. Click Do It to generate the image.
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Image after reorientation
Viewing Cross Sections
You can view cross sections of the data by creating orthogonal projections. The
Orthogonal Viewer allows you to interactively view YZ and XZ plane cross
sections.
To view YZ or XZ cross sections:
1. Open the image in the desired Image window.
2. Open the Orthogonal Viewer by choosing Tools | Orthogonal Viewer from
the Image window. The orthogonal projection is displayed. The new Image
window displays the original image. It also displays an XZ projection (at the
bottom of the window) and a YZ projection (on the right side of the window).
Projection lines show the areas of the image that are displayed on the cross
sections.
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113
YZ Projection
Chapter 8: Viewing Image Data
XZ Projection
3. To orient the image in "real world" coordinates, select Options on the
Orthogonal Viewer, and then select the Cover Slip at Bottom/Right toggle.
This orients the image so that the display in the window represents the
orientation of the sample when the data was collected (e.g., on the XZ
projection, the cover slip is down).
4. To change the cross sections that are displayed on the projections, use the
mouse to drag the projection lines across the Image window.
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8. Viewing Movies
Movies greatly enhance the analysis of certain types of image data. When used on
a volume rendering, a movie shows the relationships between objects in 3-D space.
When used with a time-lapse data file, a movie allows you to visualize the course
of events captured in the study. You can also use movies to trace particles in timelapse data.
In This Chapter
Viewing Volumetric or Time-Lapse Movies ............................................................... 113
Tracking Particle Movement with Trails Movies........................................................ 114
Viewing Volumetric or Time-Lapse Movies
At least one Image window must be open in order to apply the movie function.
There are two ways to access the movie function: from the softWoRx main toolbar
and from the Image window. Accessing the movie function from the softWoRx
toolbar applies the movie function to all open windows. Opening the movie
function from an Image window applies it only to that Image window.
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Chapter 8: Viewing Movies
To view one image data set as a movie:
1. Open the file that you want to view as a movie in an Image window.
2. Click View.
3. Click Movie.
4. Unselect the Paused check box.
To view two or more image data sets as movies:
1. Open the files that you want to view as movies in Image windows.
2. Click View on the softWoRx main toolbar.
3. Click Movie.
4. Unselect the Paused check box.
To view time-lapse Z series data:
1. Open the file that you want to view as a movie in an Image window.
2. Click View.
3. Click Movie.
4. Unselect the Paused check box.
5. Select the Time Lapse check box.
6. Adjust the Manual Z Control for Time Data slider.
Tracking Particle Movement with Trails Movies
You can trace the movement of particles in time-lapse data with the Trails Movie
tool.
To trace particle movement:
1. Open an image that contains time-lapse data in the Image window.
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2. From the main softWoRx window, choose View | Trails Movie. The Trails
Movie dialog box is displayed.
3. Drag the Image window number into the Input field.
4. Click Select Region to select a region of interest. Then select which
wavelengths to include in the movie.
5. Enter the length of the trace history in the Window field (e.g., for a value of 5,
the trace includes the previous 4 time frames and the current time frame).
6. To emphasize the display of the current point, enter a value greater than 1.0 in
the Enhancement Factor field (typical values for this field are 1.0 - 2.0).
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Chapter 8: Viewing Movies
Note The displayed intensity value of each point is a weighted average of the
corresponding points in the previous time frames. The previous points all have a
weight of 1.0. The Enhancement factor is assigned as the weight for the intensity of
the current value.
7. Click Do It to create the trails display.
8. To create a Trails movie, choose File | Save As Movie on the Image window
and save the movie. The Save As Movie dialog box is displayed.
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9. From this dialog box, you can use the appropriate fields to select the movie
format (Quicktime, AVI, or MPEG), the animation style (Forward, Backward,
or Forward and Back), the compression quality, the frame rate, and the time
increment to use. You can also select whether to animate the movie through Z
sections or time.
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9. Viewing Projections and Volumes
You can create two types of data projections of multiple Z sections:
Two-dimensional projections can help you to visualize how the data are oriented
in the XY direction. These projections allow you to view the paths of individual
fibers, chromosomes, or other types of linear data.
Volume projections can help you to understand the three dimensional nature of
the data.
In This Chapter
Creating 2D Projections .................................................................................................. 119
Creating Volume Projections ......................................................................................... 122
Creating 2D Projections
Use the Quick Projection tool to quickly combine information from multiple Z
Sections. Averaging all of the sections into one provides an approximation of a
volume rendering of the image looking directly down the Z axis.
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A multiple Z section image before projection
1. Choose View | Quick Projection from the main softWoRx menu to open the
Quick Projection dialog box.
2. Enter an image file name or window number in the Input field.
3. If you want to include only selected data, click Details to open the Region
Details dialog box. Then specify the ranges of data that you want to include in
the Output Options fields.
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4. In the Quick Projection dialog box, select which wavelengths to include.
5. In the Quick Projection dialog box, choose how to group sections as follows:
To group sequential sets of sections into output sections, specify how many
input sections to average for each output section in the Number of
Sections to Average field.
To group all of the sections into one section, select All.
6. Choose one of the following ways to combine the sections in the Method list:
•
To add the intensity of each pixel to create the output values, choose Sum.
(Be careful when using this option. If the output intensity values are too
large for the output data type specified, the output image will appear to be
saturated.)
•
To average the input data values to create the output image, choose
Average.
•
To use the largest intensity value of all the input intensities to create an
output value, choose Max Intensity. (This method may give you the most
realistic representation of a volume rendered image, especially when
combining all of the images in the input data set.)
7. Click Do It. The projected image is displayed in a new Image window.
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Chapter 9: Viewing Projections and Volumes
A single Z section image after projection
Creating Volume Projections
The Volume Viewer provides you with the ability to view the image data in 3-D.
This tool allows greater visual understanding of the image data and comparison of
features within the image data. It also allows quantitative assessment of structures
throughout the entire data set on a single image.
Volume Rendering
A brief understanding of how softWoRx creates a volume rendering will help you
utilize this tool for various image data sets. Theoretically, a set of parallel rays is
sent through the data set at various angles to analyze the data in those paths and
collect new data from that perspective. Each time a set of rays is passed through
the data, a projection is created from the resulting data. These projections
constitute the volume rendering.
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Figure 4. Volume Rendering Projections
3-D Projection Image
Image Data
Parallel Rays
Axes of Rotation
Volume Viewer enables you to create a movie of the data rotating around an axis.
This axis of rotation can be any of the three common axes, X, Y, or Z.
Given a Z series of data, the coordinates are defined as shown in the following
figure.
Figure 5. Axes of Rotation
Y
Rotations about the axes are as shown in the following figures.
Figure 6. Rotation About the X Axis
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Chapter 9: Viewing Projections and Volumes
Figure 7. Rotation About the Y Axis
Figure 8. Rotation About the Z Axis
According to the parameter settings, softWoRx enables you to see the desired
portion of the Z section, in the desired wavelength, using a variety of methods.
Options in Volume Viewer help you to perform a volume rendering.
The most important options are Select Region, Details, and Method. By limiting
the size of the data set using Select Region and Details, the time needed to create
projection images can be drastically reduced.
For detailed information about each of Volume Viewer options, refer to the online
Help.
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Methods for Projecting Volumes
The following table summarizes the six methods of data collection.
Method
Functionality
Application
Maximum Intensity
Each ray collects the data
from the voxel with maximum
intensity.
Best choice for showing internal
detail of a translucent image.
Additive
Each ray collects and sums
data from all the voxels in its
path and scales it down to an
appropriate intensity between
0-255.
Generates quantitative
projections. This data can be
used for comparison of intensity
in various structures within the
image data.
Progressive
Each ray collects the data in
the voxel that is closest to the
front of the image.
Clearly displays opaque features
within an image. Works well for
objects whose internal details are
not needed, such as metaphase
chromosomes.
RGB/Opacity
If the image data has been
processed using Blend Colors,
with the Opacity option
toggled on, then this data can
be used to form a volume
rendering in multiple
wavelengths. The resulting
image captures the positional
information and relationship
among all the wavelength
data sets.
Realistically renders a volume in a
multi-wavelength image. Clearly
relates the data points of the
various wavelengths.
Mixed
Different methods (maximum
intensity, additive, and
progressive) can be assigned
to different wavelengths.
One wavelength contains
diffuse, cloudy features and is set
to the maximum intensity method
and another wavelength has
opaque features and the
method is set to progressive.
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127
Method
Functionality
Application
VolPack
Generates images that use
lighting techniques to highlight
surfaces in the 3D rendered
image.
Substantially faster than the other
methods supported, although
the method may not be optimal
for all image types.
Note VolPack uses libraries obtained from the Stanford Computer Graphics
Laboratory. It is an implementation of the shear-warp volume rendering algorithm
as described in Lacroute, P. and Levoy, M., Fast Volume Rendering Using a ShearWarp Factorization of the Viewing Transformation, Proc. SIGGRAPH 1994, ACM.
To render a volume:
1. Open an image file. Click to open the intensity scaling dialog box and
subtract background from the image by setting the left control point at the
middle of the first intensity peak.
2. Choose View | Volume Viewer in the softWoRx main menu to open the
Volume Viewer dialog box.
Use Volume Viewer to render volumes
3. Drag the Image window number into the Input field.
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4. Click Select Region and drag the mouse across the region of interest to select a
rectangular region. Scroll through the Z sections to insure the region of interest
includes all the image data that you want to include in the data set.
5. Specify the Viewing Parameters and Movie Options.
6. Click Do It to process the image data.
Volume created with the Max Intensity method
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7. Move the Z slider up and down to view the projections or use the Movie tool.
To rotate the image using interactive image rotation:
1. Open the image file and choose View | Volume Viewer in the SoftWoRx main
menu to open the Volume Viewer dialog box.
2. Drag the Image window number into the Input field.
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Image before interactive rotation
3. Click Select Region and drag the mouse across the region of interest.
4. Click Interactive to open the Interactive Volume Viewer Parameters dialog
box.
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5. Choose which wavelengths to display in the window in the Wavelengths
options.
Tip VolPack is the preferred method for interactive viewing. With VolPack, you can
view up to three wavelengths at a time. To use VolPack, select this option under
Viewing Parameters in the Volume Viewer dialog box.
6. Select Low in the Resolution During Move option.
7. Drag the cursor on the image to rotate the image to the desired orientation.
8. Click Done in the Interactive Volume Viewer Parameters dialog box.
9. Click Do It in the Volume Viewer to render the volume with the new
orientation.
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Image after interactive rotation
To render a volume using the RGB Opacity Method:
1. Open the desired image file.
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2. Choose View | Blend Colors in the softWoRx main menu. The Blend Colors
dialog box is displayed.
3. Drag the Image window number to the Input field.
4. Select Max Opacity in the Method option and select the Opacity option.
5. Click Do It to create a blended color image.
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6. In the softWoRx main menu, select View | Volume Viewer to open the Volume
Viewer dialog box.
7. Drag the Image window number of the blended image to the Input field in the
Volume Viewer dialog box.
8. To select which data to view, click Select Region and drag the mouse across
the Image window. (The area inside of the rectangle that is displayed as you
drag the mouse is the region of interest.) Scroll through the Z sections to insure
the region of interest includes all the image information you want to include in
the data set.
AppliedPrecision
Chapter 9: Viewing Projections and Volumes
9. In the Viewing Parameters section of the Volume Viewer dialog box, select
RGB/Opacity as the Method option and adjust the Movie Options.
10. Click Do It to create the RGB opacity volume.
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11. To view the volume, move the vertical Z scroll bar up and down or use the
Movie tool.
To save the volume rendered images:
1. Click File | Save in the Image window menu to open the Save to File window.
2. Examine the Output field to ensure that the desired name is used. The
softWoRx software automatically adds a _VOL tag near the end of the filename.
3. Click Do It to save the image.
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10. Filtering Image Data
Use the softWoRx filters to prepare data for modeling and other types of analysis.
In This Chapter
About softWoRx Filters.................................................................................................... 138
Using Convolution Filters .............................................................................................. 138
Enhancing Object Boundaries........................................................................................ 139
Using 2D Statistical Filters ............................................................................................. 141
Using Image Arithmetic ................................................................................................. 142
Scaling Pixel Intensity to Enhance Local Contrast...................................................... 143
Setting an Intensity Threshold....................................................................................... 144
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About softWoRx Filters
softWoRx provides the following types of filters:
„
Convolution filters perform high pass, low pass, and other digital filtering.
„
The Edge Enhancement filter enhances object boundaries.
„
2D Filter limits noise-like intensity.
„
The Image Arithmetic filter scales images, combines information from images,
or subtracts images to isolate features.
„
The Local Contrast Enhancement filter enhances local contrast around pixels.
„
The Threshold filter removes data below an intensity threshold.
Using Convolution Filters
Use the Convolution3 tool to perform basic digital filtering, such as high-pass and
low-pass filtering. When you select a filter, the fields in this dialog box are
updated to indicate the kernel values.
Tip You may add or change convolution kernels to the list by modifying the
CONVOLUTION_FILTERS file in the softWoRx configuration directory. Use the Revise
Convolution Kernels menu item within the softWoRx Utilities menu to access this file.
To use Convolution filters:
1. Choose Filter | Convolution from the softWoRx main menu to open the
Convolution dialog box.
For more about Convolution filters, see Digital Image Processing, by Kenneth R. Castleman,
Prentice Hall, 1995.
3
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2. Enter an image file name or Window number in the Input field.
3. If you want to include only selected data, use the Select Region button and
drag the mouse across the portion of the image you want to include.
Alternatively, you can click Details to open the Region Details dialog box.
Then specify the ranges of data that you want to include in the Output
Options fields.
4. Select which wavelengths to filter.
5. Select which filter to use in the Filter option list.
6. To customize the filter, change the values in the numbered grid.
7. Click Do It to run the filter.
Enhancing Object Boundaries
Use Edge Enhance to enhance object boundaries. This tool uses the image intensity
gradient to calculate boundaries. The result is calculated from the following
expression:
Result = Input * {Fraction + (1 - Fraction) * gradient[F (Input)] }
where Fraction is a number between 0 and 1, and F is either a linear or an arc
tangent function.
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To enhance object boundaries:
1. Choose Filter | Edge Enhance from the softWoRx main menu to open the Edge
Enhancement dialog box.
2. Enter an image file name or window number in the Input field.
3. If you want to include only selected data, use the Select Region button and
drag the mouse across the portion of the image you want to include.
Alternatively, you can click Details to open the Region Details dialog box.
Then specify the ranges of data that you want to include in the Output
Options fields.
4. Select which wavelengths to filter.
5. If you want to use the arc tangent method to calculate the gradient, select
Atan. To use a linear method, unselect this option.
6. Specify the relative contribution of the original image and the gradient image
by entering a value between 0 and 1 in the Fraction field. Increasing the
Fraction value increases the relative contribution of the original image.
7. Click Do It to apply the filter.
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Using 2D Statistical Filters
Use 2D filters to remove pixels with noise-like intensity or to achieve other effects.
To use statistical filters:
1. Choose Filter | Filter2D from the softWoRx main menu to open the 2D Filter
dialog box.
2. Enter an image file name or window number in the Input field.
3. If you want to include only selected data, use the Select Region button and
drag the mouse across the portion of the image you want to include.
Alternatively, you can click Details to open the Region Details dialog box.
Then specify the ranges of data that you want to include in the Output
Options fields.
4. Select which wavelengths to filter.
5. Select one of the following filters in the Method option list:
•
•
•
•
Median uses the image intensities within a box-shaped region around each
pixel and selects the median value for the resulting image. (This is useful
for removing noise.)
Mean is similar to the median filter except that the mean value is used
instead of the median.
Variance calculates the statistical variance of the image about the mean
intensity within the local region.
Weighted Mean is similar to the mean filter, except that mean is weighted
by the variance.
6. Specify the size (pixels) of the square box used to calculate the filtered images
in the Kernel Size field. (Kernel sizes of 3 and 5 are most useful.)
7. Specify the number of times to run the filter in the Iterations field.
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8. Click Do It. The filtered image is displayed in a new Image window.
Using Image Arithmetic
Use Image Arithmetic to scale images, combine information from multiple images,
and subtract images in order to isolate features.
Image Arithmetic calculates an output image from one or two input images by
applying one of several operations. The input images may come from one or two
windows or files. To create the image, specify the input images (files or windows),
the wavelength number of Image 1, the wavelength number of Image 2, the
arithmetic operation, and any necessary coefficients.
Note In order for the calculation to be processed, the input images must have the
same X, Y, and Z dimensions.
To perform an image arithmetic calculation:
1. Click Filter | Image Arithmetic in the softWoRx main menu to open the Image
Arithmetic dialog box.
2. Drag a window number from an Image window into the Image 1 field, or click
Image 1 and browse to an image file.
3. If desired, repeat Step 2 for the Image 2 field.
4. Define the destination file or window for the calculated result. (The default is
Window 1.)
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5. Enter the appropriate input wavelength from Image 1 into the Image 1 Wave
field.
6. If you are using a second image, enter the appropriate wavelength from Image
2 to use as input into the Image 2 Wave field.
7. Select the appropriate operation from the Operation "o" list.
8. Set the Process Image 1 and Process Image 2 options to enable or disable the
processing of the appropriate images.
9. Enable or disable the other options in the dialog box as necessary for your
calculation.
10. Click Do It. The results of the calculation are displayed in the file or window
defined in the Results field.
Scaling Pixel Intensity to Enhance Local Contrast
Use Local Contrast Enhancement to scale the intensity of each pixel in the image
based on the Local Mean and Local Contrast of the pixel. (The term “local” refers
to the fact that the mean and contrast are calculated from the pixel elements that
form a box around the pixel of interest.)
To use nonlinear local contrast enhancement:
1. Choose Filter | Enhance Contrast from the softWoRx menu to open the Local
Contrast Enhancement dialog box.
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2. Enter an image file name or window number in the Input field.
3. If you want to include only selected data, use the Select Region button and
drag the mouse across the portion of the image you want to include.
Alternatively, you can click Details to open the Region Details dialog box.
Then specify the ranges of data that you want to include in the Output
Options fields.
4. Select which wavelengths to filter.
5. Specify the box size (in pixels) that determines the size of the region used for
the local mean and contrast calculations. Only odd values are valid.
6. To reposition the intensity scaling curves based on the minimum/maximum
values of the local mean and contrast, select AutoRange.
7. Specify the minimum and maximum values of the intensity scaling curve in
the Min/Max fields.
8. To modify the intensity scaling curves displayed on the LM weight function
and LC weight function histograms, select one of the three points on the
curves with the mouse and move the point to the left or right.
9. Choose one of the following options for applying the contrast enhancement in
the Apply To option list:
•
All Sections creates a regular output image file or window.
•
Current Section stores the result in a temporary ("scratch") window.
10. Click Do It to create the filtered output.
Setting an Intensity Threshold
Use the Threshold dialog box to remove all data in an image that is below a certain
intensity cutoff value. You can choose to output either the image values above the
threshold or a binary mask, where all pixels below the cutoff are 0 and all above
are set to 1.
To remove data below an intensity threshold:
1. Choose Filter | Threshold from the softWoRx main menu to open the
Threshold dialog box.
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2. Enter an image file name or window number in the Input field.
3. If you want to include only selected data, use the Select Region button and
drag the mouse across the portion of the image you want to include.
Alternatively, you can click Details to open the Region Details dialog box.
Then specify the ranges of data that you want to include in the Output
Options fields.
4. Select which wavelengths to filter.
5. Select one of the following options in the Create Mode list:
•
Choose Data to create an output image of all intensities equal to or above
the threshold (the cutoff intensity value). If a pixel has an intensity value
below the threshold, the pixel in the output image is set to this value
(usually 0).
•
Choose Mask to put zeros in all pixels where the intensity is below the
threshold and ones for all pixels equal to or above the threshold.
6. Specify a Threshold and Background value for each wavelength.
7. Click Do It
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11. Saving, Exporting, and Printing
softWoRx provides several options for saving, exporting, and printing image data.
You can save complete DeltaVision files or save only selected image data (e.g.,
selected wavelengths or an XYZ region). You can also export DeltaVision files to
TIFF, Movie, or PhotoShop formats and capture screenshots of images.
After you save images or data, you can archive them to a CD. You can also print
TIFF or DeltaVision files as images directly from the Image window.
In This Chapter
Image Data Files and Image Graphic Files .................................................................. 148
Saving DeltaVision Files .................................................................................................. 148
Exporting DeltaVision Files ........................................................................................... 151
Capturing Screen Shots .................................................................................................. 156
Archiving Files to CD/DVD ........................................................................................... 157
Printing Images................................................................................................................ 159
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Image Data Files and Image Graphic Files
softWoRx distinguishes between image data files and image graphic files. Image
data files contain more information than image graphic files; image data files can
be used for analysis while image graphic files are used as graphics.
•
5D Image data files include spatial and possibly temporal and spectral data.
•
Image graphic files that are created when you save or copy Image windows
include only the 2D pixel data that is displayed inside the window.
Saving DeltaVision Files
You can save complete DeltaVision files or you can choose to save only specific
data. softWoRx allows you to select an area and to specify which Z sections and
time points to include. You can also choose which channels to save.
To save a .dv file:
1. From the Image window, choose File | Save to open the Save File dialog box.
2. Enter an input file or window number in the Input field.
Tip You can also specify an existing window by dragging an Image window button
from the main softWoRx menu to the Input field, or by dragging the window number
indicator in the upper left hand corner of the Image window to the Input field.
3. Enter an output file or window in the Output field. If the window or file exists,
you will need to choose whether to overwrite the data or to append the new
data as new channels.
4. To save a region of a window, choose Select Region and select an area in the
Image window by dragging the mouse across the area. Adjust the rectangle
you've created until it contains the desired area. Then click outside the Image
window with the mouse.
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The Region of Interest
Note Selecting a region is optional and you can do this only when your input is
a window.
5. Click Details to open the Region Details dialog box. Specify the ranges of the
X, Y, Z, and time data to save in the selected region. Then set any other output
details.
6. On the Save File dialog box, choose which wavelengths of the input data to
process and include in the output data set.
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Chapter 11: Saving, Exporting, and Printing
7. If you want to scale the output data according to the current minimum and
maximum intensity scale factors, click Scale to Display Min/Max. (Scaling
only works when the output data type is 16-Bit integer or 8-Bit).
8. Click Do It to save the file.
Saving to the Data Management System (DMS)
You can save DeltaVision image files from your local file system to the DMS
database if you have this feature set up. See Chapter 6, “DMS Integration” for
details.
4. From the Image Window, select File | Save to DMS. The Save To Data
Management System menu is displayed as shown.
5. From this menu, select the Project and Dataset to which you want to save the
data on the DMS database. You can optionally select a Category Group and
Category for the saved image data and add initial annotation to the file for
future reference. When you are satisfied with the image data to be uploaded,
click Do It.
When the upload is complete, “Finished” is displayed on the status line as
shown.
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6. Click Done to exit the Save To Data Management System menu.
Exporting DeltaVision Files
®
You can export DeltaVision files to TIFF, PhotoShop or MPEG Movie files.
Exporting to PhotoShop and Movie files saves only the pixel image data (the
intensity data is not preserved).
Exporting to TIFF Files
Exporting to TIFF files allows you to share data sets in a nonproprietary format.
To export a DeltaVision file to a TIFF file:
1. Open the image that you want to export in a softWoRx Image window.
2. From the Image window menu, select File | Save As TIFF to open the Save as
TIFF dialog box.
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Chapter 11: Saving, Exporting, and Printing
Use Save As TIFF to specify export options
3. If you want to specify a particular time point or a single Z section, click Details
and set the applicable options in the Region Details dialog box.
4. Enter the directory and file name in the Output directory. The default is the
directory in which the input file is located.
5. Enter the prefix of the file name in the File Prefix field.
6. Select the TIFF options from the lists on the dialog box. (The default settings
should work well for most applications.)
7. Under Output Size, select one of the three possible TIFF output formats:
•
8-bit Grey scale – generates compressed data, with each channel separate.
•
16-bit Grey scale – generates uncompressed data. Use this option for
quantitation.
•
24-bit RGB – generates compressed data; a 3-channel color TIFF with 8-bits
per channel.
8. Click Do It to export the data to a TIFF file.
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Exporting to PhotoShop Files
You can save the visible contents of an Image window as an Adobe PhotoShop
24-bit, RGB color, image file. You can choose to:
„
Save the image with the overlay graphics (scale markers, etc) merged into a
single image.
„
Save the image without the overlay graphics.
„
Save only the overlay graphics.
Exporting to PhotoShop saves only the pixel image data (the original intensity
data is not preserved).
To export to a PhotoShop file:
1. Open the folder containing the image file that you want to export. Then
double-click the image file name to automatically open that image in a
softWoRx Image window.
2. From the Image window File menu, choose Save As Photoshop to open the
Save Photoshop dialog box.
3. Enter the directory and file name in the Output directory. The default is the
directory in which the input file is located.
4. Select the one or both of the following options for saving the file:
•
To save the image, select Save Image.
•
To save the overlay graphics, select Save Overlay.
5. Click Do It to complete the export.
Exporting to Movie Files
You can save the contents of an Image window as an MPEG movie in 24-bit, RGB
color. You can choose to:
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„
Save the image with the overlay graphics (e.g, scale markers) merged into a
single image, save it without overlay graphics, or save only the overlay
graphics.
„
Save movies that include Z sections, time-lapse data, or both types of data.
„
Specify a range of data to include in the movie file.
You can play the MPEG movie format on the QuickTime viewer, the Windows
Media Player, or a variety of Linux or Macintosh movie players. You can also
import movies into PowerPoint. Double clicking on an MEPG file in a file browser
opens the
PlayMPEG viewer, which you can use to vary the speed of the movie.
To save a movie:
1. From the Image window, choose File | Save As Movie to open the Save Movie
dialog box.
2. Drag the Image window number into the Movie File field, or click Movie File
and select a name and path for the movie.
3. At the top of the window, select one or both of the following options:
•
To save the image, select Save Image.
•
To save the overlay graphics, select Save Overlay.
4. In the Movie Format list, select from Quicktime, AVI, or MPEG movie
formats.
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5. In the Animation Style list, set the movie looping mode by selecting one of the
following options:
•
Forward & Back records a movie that starts on the first frame selected,
plays to the last frame, and then back again to the last frame.
•
Forward records a movie that starts on the last frame, plays to the first
frame, and stops.
•
Backwards records a movie that starts on the last frame selected, plays to
the first frame, and stops.
6. Adjust the Compression Quality slider to specify image quality and file size.
Moving the slider to the left produces better quality and bigger file sizes.
Moving the slider to the right produces lower quality and smaller file sizes.
7. Adjust the Frame Rate slider to specify the playback speed.
8. Select whether to animate through Z sections. Then select the range of data to
include in the movie and the increment between frames.
9. Select whether to animate through Time. Then select the range of data to
include in the movie and the increment between frames.
10. Click Do It to save the movie.
Tip Select the Preview button to preview the speed and other settings before
saving.
Note softWoRx Task Builder provides the capability of exporting Image window
contents to AVI and QuickTime movies. For details on using the Task Builder, see
Page 54.
Exporting to JPEG Files
You can save the contents of any window that includes an image as a JPEG file.
This includes:
„
The Image window
„
The 3DModel window
„
The Statistics window
To save a JPEG file:
1. In a window that contains graphical data, choose File | Save JPEG Snapshot
to open the JPEG Snapshot dialog box.
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2. Enter a directory and file name and click Do It.
Capturing Screen Shots
You can capture a screen shot of any window and save it as a JPEG image. This is
a useful way to save images for presentations.
To capture a screen shot:
1. From the main window, choose Utilities | Image Snapshot to open the JPEG
Snapshot window.
2. Select the directory and file name for the file.
3. To use the same prefix for a series of screen shots, click Auto sequence names.
(A sequential number is added to the prefix to create a unique name for each
file.)
4. To set the delay between when the image is selected and captured, move the
Capture Delay slider.
5. Set the JPEG slider to the right to increase the quality of the screen shot.
(Higher quality results in larger file sizes.)
6. Choose whether to save a window or the entire screen.
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7. Click the window (or screen) to capture the image.
8. To view the screen shot, click Display Last.
Tip You can also open Snapshot from the Image window, the 3DModel window,
the Chromatic Correction dialog box, and the Orthogonal Viewer window.
Archiving Files to CD/DVD
You can open the Linux K3b “CD Kreator” tool directly from the softWoRx main
menu to archive your softWoRx files to CDROM or DVD discs. This tool supports
650 or 700 MB capacity CD-RW discs and 4.3 GB DVD discs.
To copy files to a CD using K3b:
1. Place a blank CD into the CD drive.
2. On the softWoRx main menu, choose Utilities | Archive Data to CD to open
the K3b CD/DVD creation window.
3. Click on the New Data CD Project icon. The Current Projects window opens.
Note The process for archiving files to a DVD is the same, except you choose
New Data DVD Project in this step.
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4. Navigate to the files you want to archive and double-click on each file you
want to copy to the CD, or drag and drop the files into the Current Projects
window.
Tip You can select multiple files in the K3b window by holding down the CTRL
key while selecting files.
5. When you are satisfied with your selections, click the Burn… button in the
bottom right corner of the window. The Data Project – K3b dialog box is
displayed.
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6. Click the Burn button at the top right corner of the window to begin the CD
creation process. For more complete information on using the K3b “CD
Kreator” tool, select Help | K3b Handbook on the K3b main window to view
the entire user’s manual for K3b.
Printing Images
softWoRx allows you to print DeltaVision images from an Image window.
To print a DeltaVision image:
1. Open the .dv image that you want to print in the Image window.
2. From the Image window menu, choose File | Print.
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3. In the Image Print dialog box, enter the window number of the image in the
Window field.
4. Click Do It to print the image.
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Part Three
PART THREE:
ANALYZING RESULTS
Part Three shows how to use softWoRx tools to perform quantitative analysis.
In Part Three
Chapter 12. Examining Intensity Data .............................................................162
Chapter
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163
13. Measuring Distance and Velocity............................................................. 175
Chapter
14. Volume Modeling ....................................................................................... 181
Chapter
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15. Detecting and Analyzing Colocalization..................................................193
Chapter
16. Other Applications ......................................................................................203
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165
12. Examining Intensity Data
This chapter introduces the tools used to examine intensity data.
In This Chapter:
Examining Point Values ................................................................................................. 162
Examining Intensity Data With Data Inspector.......................................................... 163
Viewing Intensity Line Profiles..................................................................................... 166
Calculating Statistics....................................................................................................... 169
Examining Point Values
You can examine intensity values for individual pixels in the Image window. The
wavelength and intensity value of the point under the mouse are displayed at the
bottom of the Image window.
To view point values:
X From the main softWoRx toolbar, choose Measure | Point Values. Select a
channel (Wave) and move the mouse across the image to view individual
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point values for that channel. The point coordinates and intensity of the point
under the mouse are displayed in the Point Values dialog box.
Tip You can also view the intensities of individual points for the selected channel on the
status bar (e.g., 457:1216 in this example).
Examining Intensity Data with Data Inspector
The Data Inspector includes several tools for examining the intensity data in the
image file. You can simultaneously view a graphical image, a table of intensity
values, a 3-D graph, and a histogram of intensity values. With these views open,
you can select various regions of interest (ROIs) to explore the data. As you select
an ROI, the data in each view is updated for that ROI.
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Chapter 12: Examining Intensity Data
To open the Data Inspector tools:
1. Open an image file in the Image window.
2. Choose Tools | Data Inspector on the Image window to open the Data
Inspector window.
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3. In Data Inspector, click Show 3D Graph.
4. Click Show Histogram.
5. Arrange the windows on the screen.
Selecting a Region of Interest
The region of interest, or ROI, is a region of the image that you can resize and
move to visually examine image details. You can select a region of interest (ROI) in
the Image window to display its intensity values in the 3D graph, the Histogram,
and the table in Data Inspector. The display in each window changes
automatically as you change the ROI in the Image window.
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The ROI is the area within the box
To define an ROI:
1. Choose Tools | Data Inspector on the Image window to open the Data
Inspector window.
2. On the Data Inspector window, click Select Box.
3. In the Image window, click the top left corner of the ROI and drag the
mouse to enclose a region of interest with the ROI box.
4. To make the ROI a circle instead of a square, select Constrain to Circle.
5. To change the position of the ROI, click on any point in the Image window.
(The ROI is centered on the point that you click.)
Viewing Intensity Line Profiles
A line profile is a plot of intensity values for pixels along a straight line. Two types
of line profile tools are available:
„
Line Profile displays a plot of intensity values for pixels in a row or column of
the Image window. This profile is overlain on the image. You can view a
profile of a line of pixels or a band of pixels. This data can be saved as a text
file.
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„
Arbitrary Profile displays a plot of intensity values along a line segment that is
oriented at any angle in the Image window. This profile is displayed in a
separate window. You can interactively change the orientation of the line. This
data can be saved in an .slk spreadsheet compatible file.
Viewing the Line Intensity of a Row or Column
1. Open an image in the Image window and choose Tools | Line Profile on the
Image window to view the Line Profile dialog box.
2. Drag the window number from the Image window to the Window field in the
Line Profile dialog box.
3. Click on the image to display a horizontal line profile.
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4. To get the average line profile of several rows and columns of pixels, enter the
width in the Band field.
5. To display a vertical line profile, choose Vertical in the Direction list.
6. To change the position of the profile, click another point on the image or use
and
the
buttons next to the XY field.
7. Create a group of profiles to save in a file as follows:
•
To save only selected profiles, unselect AutoSave. Then click Save after
each profile that you want to save is displayed.
•
To save all profiles, select AutoSave before you create the profiles.
8. To save a group of profiles, click Write to File, enter the file name and other
options in the Profile Output Options dialog box, and click Do It.
Tip You can change the Line Profile colors that are associated with different
wavelengths. From the Image window, choose View > Select Graphics
Colors.
Viewing the Line intensity in Any Direction
To display an arbitrary line profile:
1. Open an image in the Image window and choose Tools | Arbitrary Profile to
open the Arbitrary Line Profile dialog box.
2. Click and drag on the image to display a line profile.
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3. Choose how to scale the profile in the Intensity Scaling list.
•
Constant Min/Max sets the scale range to the minimum and maximum
intensity values in the image.
•
Autoscale sets the range to the minimum and maximum intensity values in
the profile.
4. To save a group of profiles in a spreadsheet-compatible text file, click Save
after each profile that you want to save is displayed. When you are finished
collecting profiles, click Write to File, enter the file name and other options in
the Save as SYLK spreadsheet dialog box, and click OK.
Calculating Statistics
Calculating Statistics for Selected Areas
Use Data Inspector to calculate data for selected areas.
To open Data Inspector:
X Select Tools | Data Inspector on the Image window to open the Data Inspector
window.
AppliedPrecision
Chapter 12: Examining Intensity Data
To gather statistics:
1. Click on a point of interest in the Image window and press the space bar. (The
statistics for a rectangular area around that point are displayed in the Data
Inspector Statistics field.)
2. From the Data Inspector window, choose File | Save Statistics Record to open
the Statistics Record File dialog box.
3. To save the statistics file, enter a file name in the Statistics Record File dialog
box and click OK. The file is saved as a text file similar to the following.
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File Generated By softWoRx DataInspector
/data1/statisticsRecord_sample2
Tue Feb 17 16:55:01 2004
spot
wave
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box
diameter width
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total
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min
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max
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46.83
Calculating Statistics for Irregular Areas
The Edit Polygon tool can be used to calculate statistical values for the area of data
that is inside of a polygon. You can draw multiple polygons on different Z sections
and view the statistics for each. You can also save statistical reports to text files or
to SYLK spreadsheet compatible files.
To select polygon areas:
1. Choose Model | Edit Polygon on the main softWoRx menu.
2. Click a polygon option (e.g.,
freehand) on the Edit Polygon window.
AppliedPrecision
Chapter 12: Examining Intensity Data
3. To automatically find the borders around objects (based on changes in
intensity values) select the Guided Mode option.
4. Drag the mouse to draw the polygons on the Image window. You can draw
sets of polygons on the same Z section and on different Z sections.
5. To copy a polygon to other sections or wavelengths, use the arrow tool in the
Edit Ploygon window to select the polygon. Then choose Edit | Propagate
Polygons.
6. Choose Statistics | Table to display a table that shows statistical values for
each polygon.
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7. Choose Statistics | Graph to open the Plot Polygon Graph dialog box.
8. In the Y axis field, choose which type of statistical parameter (e.g., SD) to plot.
9. Choose other options in the Plot Polygon Graph dialog box and click Do It to
display a graph of the statistics values.
AppliedPrecision
Chapter 12: Examining Intensity Data
10. From the Polygons window File menu, choose Save As SYLK to save the file
as a spreadsheet compatible file.
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13. Measuring Distance and Velocity
This chapter shows how to measure distance and velocity.
In This Chapter
Measuring Distances....................................................................................................... 175
Measuring Velocity ......................................................................................................... 177
Measuring Distances
The Distance tool allows you to measure distances between points in one Z plane
or between points in many different Z planes. The measurement data can be saved
to a file for off-line analysis.
The Measure Distances dialog box contains the options for measuring distances.
This section briefly describes these options and contains step-by-step instructions
for completing the most common procedures.
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Chapter 13: Measuring Distance and Velocity
Options in Measure Distances are summarized below and are described in further
detail in the softWoRx online Help.
Select
To…
Standard Two
Point
Measure the distance between two points.
Single Reference
Measure the distance from a single reference point to other points.
Leap Frog
Measure the distance between two consecutive selected points.
Multiple
Segment
Measure the sum of the distance between consecutive selected
points.
The following procedures describe the most common measurement tasks
performed using softWoRx.
To measure distance using the Standard Two Point method:
1. Open an image in the Image window.
2. On the Image window menu, click Tools | Measure Distances to open the
Measure Distances dialog box.
3. Enter the desired window number in the Window field.
4. In the Measure Method list, select Standard Two Point.
5. Select the appropriate unit of measurement from the Units option.
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6. Select two points using the mouse. The distance will be displayed in the gray
information window.
To measure distance using the Multiple Segment method:
1. Open an Image in the Image window.
2. On the Image window menu, click Tools | Measure Distances to open the
Measure Distances dialog box.
3. Enter the desired window number in the Window field.
4. In the Measure Method list, select Multiple Segment.
5. Select the appropriate unit of measurement from the Units option.
6. Drag the mouse across the image to select consecutive points.
7. Click Get Distance to display the coordinates of all the selected points and the
sum of the distance in the gray information window.
Note If an error occurs, click Delete Last.
Measuring Velocity
You can use the distance tool to measure the velocity of objects in time-lapse data.
Use the Measure Distance tool to measure the velocity of particle movement.
1. Open an image in the Image window.
2. On the Image window menu, click Tools | Measure Distances to open the
Measure Distances dialog box.
AppliedPrecision
Chapter 13: Measuring Distance and Velocity
181
3. In the Measure Method list, select the Standard Two Point method.
4. In the Units list, select the appropriate unit of measurement.
5. In the Image window, click on the particle that you want to measure.
Original Time
Point
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6. Move the T (time) slider on the Image window to display the particle at a
different time point.
7. Click the same particle that you selected at the original time point.
New Time Point
The distance and velocity of the particle movement are displayed at the bottom
of the Measure Distances dialog box.
AppliedPrecision
Chapter 13: Measuring Distance and Velocity
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14. Volume Modeling
Modeling features of your image data can help you to understand the threedimensional nature of your data. Volume models have been used to study objects
such as nuclei and cell boundaries.
Note softWoRx also provides Line modeling tools that have proven to be useful for
studying chromosomes, neurons, and other complex three-dimensional structures.
For information about Line modeling, see the online Help.
In this Chapter:
About Volume Modeling ............................................................................................... 182
Edit Polygon Dialog Box ................................................................................................ 182
2D Polygon Finder .......................................................................................................... 185
3D Object Builder ............................................................................................................ 187
Volume Modeling Example ........................................................................................... 190
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About Volume Modeling
The 2D Polygon Finder and the 3D Object Builder are used to create threedimensional models of features within the image data and obtain quantitative
information. Initially, the 2D Polygon Finder is used to specify the object of
interest. The 3D Object Builder is then used to create a three-dimensional model
from the two-dimensional polygons in each Z section. Finally, the real space
coordinates are saved to an ASCII file (which can be viewed from a table of
measurements) and the 3D Model Display can be viewed.
In summary, this is the process:
Image Data
(Z Series)
Find/Edit Polygon
Polygons Defined in
each Z Section
Build 3-D object
3-D Object
3D Modeling Flow
Edit Polygon Dialog Box
When a satisfactory polygon is not obtained using the 2D Polygon Finder, you can
use the Polygon Editor to define a polygon.
Several controls enable the Polygon Editor to define the intended feature.
Descriptions of the most useful controls follow. For additional information, refer
to the online Help.
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Window
Sets the Image window number of the image data to be processed.
Polygon Data
Defines the Image window to be used by a file name.
Snap Selected
Determines the boundaries of the polygon after you have manually selected an
approximation of the polygon. Polygon Editor finds the closest pixel to your
manually selected polygon that matches the criteria of the Guide Options.
Options | Guide Mode Options
Defines the parameters for the Polygon Editor to evaluate when you use Snap
Selected. For additional information, refer to the online Help.
Done
Closes the Edit Polygon dialog box.
File | Save
Allows you to specify a name for the polygon file and save the new data.
The following table provides a brief description for each of the buttons on the Edit
Polygon dialog box.
Edit Polygon Tools
Button
Name
Functionality
Select polygon
Selects, moves, or modifies existing polygons or
points within a polygon.
Add polygon
Connects points which you select using the
mouse.
Add polygon
(freehand)
Allows you to draw the polygons freehand. It is
especially useful when used with Guided mode.
Automated
polygon creation
tools
Allows you to define parameters for the
automatic detection of polygons.
Insert point into
polygon
Adds a point to a line segment in a selected
polygon. Drag and drop this point to change the
shape of the polygon.
Close current
polygon
Connects the last point selected with the first
point in order to close the polygon.
Add circle
Allows you to define a circular area.
Add box
Allows you to define a rectangular area.
Delete selected
polygon point
Deletes the selected or most recently added
point.
Delete selected
Deletes the selected polygon.
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187
polygon
Delete all polygons
Deletes all polygons.
Copy polygon for
pasting
Allows you to select a polygon to copy.
Paste polygon
Pastes the polygon that is selected for copying.
Undo last action
Restores the last deleted object or moves the
object to its former position.
To create polygons using Snap Selected in Polygon Editor:
1. Open the desired image data file.
2. Click Model | Edit Polygon in the softWoRx main menu.
3. Drag the Image window number to the Window field.
4. Click the Add Polygon
button.
5. Click points to form the polygon. To connect the last point with the first, click
the Close Polygon
button.
6. Select Guided Mode.
7. Select Options | Guided Mode Options on the Edit Polygon dialog box. The
Guided Polygon Select Parameters dialog box is displayed.
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8. Adjust the values in the Guided Polygon Select Parameters dialog box. For
more information on this topic, consult the online Help.
9. Click Close.
10. In the Edit Polygon dialog box, click Snap Selected.
11. Repeat this process for each Z section that will be used in the 3-D model.
12. In the Edit Polygon dialog box, select File | Save.
13. Enter a name for the polygon file and click OK.
14. Use the polygon file to create a 3-D model using the 3D Object Builder.
2D Polygon Finder
The first task that must be performed is to isolate the object that you want to
study. This is accomplished by finding the 2-D representation in each Z section
and combining them into a 3-D object. Setting a threshold value in the wavelength
intensity and allowing 2D Polygon Finder to create a polygon in each Z section can
often isolate two-dimensional features in a Z series. A simple adjustment of the
threshold value allows you to modify the polygon created. The softWoRx Polygon
Editor includes additional tools for more complex adjustments.
The 2D Polygon Finder Dialog Box
AppliedPrecision
Chapter 14: Volume Modeling
The main options in the 2D Polygon Finder dialog box are summarized below. For
information regarding the other options and for additional details on these, refer
to the online Help.
Window
Sets the Image window number of the image data to be processed.
Select Region
Defines the region of interest.
Reset
Erases the selected region.
Minimum Perimeter
Sets a value for the minimum number of points that can define a polygon.
Polygons with less than this number of points will be discarded.
Maximum Perimeter
Sets a value for the maximum number of points that can define a polygon.
Polygons with more than this number of points will be discarded.
Polygon Smoothing
Sets the desired number of pixels between points on the polygon. The 2D Polygon
Finder will use greater detail when able.
Exclude Edge Objects
Specifies not to use those polygons that touch the edge of the image.
Outer Objects Only
When enabled, specifies that 2D Object Finder will only find the outermost
continuous polygons. When disabled, specifies that polygons that are fully
contained within others will be created if detected.
Wave # Threshold
Defines the minimum intensity to be included in the polygon. (Default Value:
Minimum Value for the Wavelength + 20% of the Dynamic Range).
Launch
Opens either the Polygon Editor, to enable more sophisticated selection of a
polygon, or 3D Object Builder, to continue creating the three-dimensional model.
Done
Closes the 2D Polygon Finder.
Do It
Begins the process of creating polygons.
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Save Polygon File
Allows you to specify a name for the polygon file and save the new data.
To create polygons using 2D Polygon Finder:
1. Open the desired image data file.
2. Select Model | 2D Polygon Finder in the softWoRx main menu. The 2D
Polygon Finder dialog box is displayed.
3. Drag the Image window number to the Window field.
4. Click Select Region.
5. Using your mouse, draw a box around the region of interest. It may be helpful
to scroll through the Z sections to ensure that all of the desired areas are
included.
6. Select the desired wavelengths in the Wavelengths check boxes.
7. Enter the desired values in the Threshold fields. In order to estimate an initial
threshold value, use Point Values (located in the Tools menu of the Image
window) to view the image intensity at various points.
8. Click Do It.
9. Click Save Polygon File. Then enter a name for the polygon file and click OK.
By default, softWoRx uses the previous file name and replaces the file extension
with POL.
10. Create a 3-D model using the 3D Model Builder or use Polygon Editor to create
new polygons or modify existing ones.
3D Object Builder
The 3D Object Builder joins the 2-D polygons together to create a threedimensional model. This is helpful for quantitative analysis as well as visual
understanding of the image data.
AppliedPrecision
Chapter 14: Volume Modeling
The 3D Object Builder Dialog box
Information loaded into 3D Object Builder must include the polygon data. The two
methods of accomplishing this are described in the following procedures.
To load data from an Image window:
X Select Model | 3D Object Builder from the softWoRx main menu and drag the
desired Image window number into the Input Image field.
To load data from a saved polygon file:
1. Load the image file which corresponds to the polygon file into the Input
Image field by dragging and dropping the Image window number.
2. Click Polygon Data and select the appropriate polygon file. (You may move
up a directory level by clicking on the path bar above the desired directory.)
3. Click OK.
Creating and Viewing the 3-D Object
Once the polygon file is loaded into the 3D Object Builder, you are ready to create
the 3-D object. Before a 3-D object can be viewed, it must be saved as a solid
model. Before the measurements can be viewed, it must be saved as a
measurement file. These measurement files are saved as tab delimited text files
that can easily be exported to spreadsheet programs such as Microsoft Excel.
To build and save a 3-D object:
1. Select Model | 3D Object Builder on the softWoRx main menu.
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2. Load the data into the 3D Object Builder as described in the previous section.
3. Choose the desired wavelengths to be modeled.
4. Click Build 3D Objects.
5. Click Model in the 3D Object menu.
6. Click Save Solid Model.
7. Enter the desired name in the field. By default, softWoRx will use the previous
file name and replace the file extension with SOL.
8. Click OK.
To view a 3-D object (not available without optional 3D Model):
1. Click Model in the 3D Object Builder menu.
2. Click View Model.
Note You must have the original Image window open in order to view the 3-D
model.
To measure the area of an object:
1. Click Measurements in the 3D Object Builder menu.
2. Click Table of 2D Measurements. The Save Measurements File dialog box is
displayed.
3. Type the desired name for the 2-D measurement file.
4. Click OK.
5. Open the folder containing the saved measurement file.
6. Double-click the icon of the desired measurement file to view a text file similar
to the following.
AppliedPrecision
Chapter 14: Volume Modeling
To measure the volume of an object:
1. Select Measurements in the 3D Object Builder menu.
2. Select Table of 3D Measurements. The Save Measurements File dialog box is
displayed.
3. Enter the desired name for the 3-D measurement file.
4. Click OK.
5. Open the folder containing the saved measurement file.
6. Double-click the icon of the desired measurement file to view a text file
containing the data.
Volume Modeling Example
The following steps show how to use the Polygon Finder and 3D Object Builder in
a specific set of image data. The image data file Nuclear_Pore_D3D (included
with your system) is used in this tutorial.
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1. Open Nuclear_Pore_D3D.
2. Select Model | 2D Polygon Finder in the softWoRx main menu.
3. Drag the Image window number to the Window field.
4. Click Select Region.
5. Using your mouse, draw a rectangle around the region of interest. It may be
helpful to scroll through the Z sections to ensure that all of the desired areas
are included.
6. Select the wavelength 457 in the Wavelengths check box.
7. Type 750 in the Wave 3: Threshold field. In order to estimate an initial
threshold value, use Point Values in the Tools menu in the Image window
menu to view the image intensity at various points.
8. Click Do It.
9. Click Save Polygon File.
10. Type a name for the polygon file and click OK.
11. In the Launch: field, click 3D Object Builder. The 3D Object Builder dialog box
is displayed. Notice that softWoRx automatically loads the polygon data from
the open Image window which was used to make the polygon.
12. Select the wavelength 457 in the Wavelengths check box.
13. Click Build 3D Objects.
14. Click Model | Save Solid Model in the 3D Object Builder menu.
15. Type the desired name in the File Name field and click OK.
16. Select Model | View Solid Model to view the model.
17. Use the center mouse button to rotate the model for viewing from other angles.
AppliedPrecision
Chapter 14: Volume Modeling
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15. Detecting and Analyzing
Colocalization
softWoRx provides two tools that you can use together to detect and analyze
colocalization.
First, use the Colocalization tool to examine the entire image and identify areas
that appear to have colocalized data. You can use the data generated by this tool to
create volume views and graphically examine them to find structures or specific
areas that appear to be colocalized.
Then use the ROI Colocalization tool to examine the specific structures or areas
that you have identified. The data selection features of this tool can be used to
select many types of areas.
In This Chapter
Examining the Entire Image .......................................................................................... 194
Identifying Potential Colocalized Areas ...................................................................... 197
Detecting Colocalization with ROIs.............................................................................. 201
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Chapter 15: Detecting and Analyzing Colocalization
Examining the Entire Image
Use the Colocalization tool to identify possible areas of colocalization throughout
the data set. This tool generates a product image of two channels after subtracting
a threshold value for each. Then a scatter plot of the results is created and the
Pearson Coefficient of Correction is measured.
To use Image Colocalization:
1. Open the image to analyze in the Image window.
The Nuclear Pore image displays two proteins: channel 528
is tagged to a protein that regulates the gateway to the cell.
Channel 617 is VOM, an HIV protein.
2. Choose Measure | Colocalization from the softWoRx main menu to open the
Colocalization dialog box and enter the number of the Image window that you
want to analyze in the Input field.
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3. To analyze a region of a window, click Select Region and select an area in the
Image window by dragging the mouse across the area. Adjust the rectangle
you've created until it contains the desired area. Then click outside the Image
window with the mouse.
4. Click Details to open the Region Details dialog box. Specify the ranges of the
X, Y, Z, and time data to analyze in the selected region.
For example, in this case we are interested in colocalization that is occurring below the
surface of the cell.
5. Select which channels to analyze in the Input Channels lists.
6. Select a background threshold for each channel by clicking Get and then
clicking on a background area of the image. The background is an averaged
value within a box of the size specified. Click Get again when you are finished
selecting the background.
AppliedPrecision
Chapter 15: Detecting and Analyzing Colocalization
Tip You can change the size of the box.
7. Click Do It to run the colocalization analysis for the selected data.
The Colocalization Graph and a new window
with the colocalization data are displayed
The Colocalization graph is a plot of the two intensities on a pixel-by-pixel basis
(each spot is a pixel). The Pearson Coefficient of Correlation indicates how closely
the two intensities are colocalized (full colocalization is 1.0) and calculates the
Pearson Coefficient of Correlation. This value is displayed in the Colocalization
dialog box.
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The new window contains a third channel (*) that is the product of the two
intensities at each data pixel. This channel indicates possible areas of
colocalization. (If the intensities of both channels are high for a given pixel, the
product of the intensities is high. If one of the intensities is low or zero, the
product is much lower.)
Identifying Potential Colocalized Areas
To visually identify areas that may be colocalized, you can select specific points or
groups of points to display them on the three channel image. You can also render
a volume projection of the three channel image that includes the selected product
channel in white.
To identify colocalized structures in Image Colocalization Tool data:
1. Select the points on the colocalization graph that have higher intensities by
dragging the mouse across the graph as shown below.
AppliedPrecision
Chapter 15: Detecting and Analyzing Colocalization
2. Select additional points by holding the CTRL button as you drag the mouse
across additional areas.
As you select points, they are highlighted in red on the image graph and
displayed in white on the three channel image.
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3. Choose View | Volume Viewer on the softWoRx main menu to open the
Volume Viewer window and drag the output window number (postcolocalization) into the Input field.
4. Select Volume Viewer parameters and click Do It to render the volume.
5. Click Interactive on the Volume Viewer and examine the image from several
angles to find intense white areas that indicate potential colocalization.
AppliedPrecision
Chapter 15: Detecting and Analyzing Colocalization
Interactive Viewer shows product channel and original data
6. To view only the colocalized channel, select the (*) channel.
Alternatively, you can view the colocalized channel by changing the grayscale
color map for the (*) channel to a rainbow or cold to hot color map so the bright
intensity is displayed in a different color.
To change the grayscale color map (from the previous example):
1. While viewing only the (*) channel, choose View | Select Image Colors on the
output window menu. The Select Image Colors dialog box is displayed.
2. In the Color Display Mode field, select Grayscale. The Modify “Grayscale”
Colormap button is activated.
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3. Click the Modify “Grayscale” Colormap button. The Select Image Colormap
dialog box is displayed.
4. In the Current Colormap field, change the color map from Grayscale Default
to one of the rainbow or temperature color maps (for example, Rainbow 1 or
Cold To Hot).
Detecting Colocalization with ROIs
After you use the Colocalization tool to identify possible areas of colocalization,
you can use Region-Of-Interest (ROI) Colocalization to selectively analyze those
areas.
ROI Colocalization allows you to create ROI polygons from which a scatter plot
and Pearson Coefficient of Correlation are derived.
To use ROI colocalization:
1. Choose Measure | Colocalization (ROI) from the softWoRx main menu to
open the Colocalization Analysis dialog box. Enter the number of the Image
window that you want to analyze in the Input Window field.
2. Select which channels to analyze in the Input Channels lists.
3. To select specific areas of the image, use the Create Freehand ROI Polygon
button to create polygons on each section that you want to analyze. If the
position and shape of the structure are consistent through time and Z intervals,
you can use the Copy Selected Polygon and the Paste Polygon buttons to
copy and paste the polygons through time points or Z sections.
AppliedPrecision
Chapter 15: Detecting and Analyzing Colocalization
4. Select a background threshold for each channel by clicking Get and then
clicking on a background area of the image. The background is an averaged
value within a box of the size specified. Click Get again when you are finished
selecting the background.
5. Click Do It to plot the Colocalization graph and calculate the Pearson
Coefficient of Correlation value for the selected region (this value is displayed
in the Colocalization Analysis dialog box).
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16. Other Applications
This chapter shows how to analyze experiments that are performed with the
optional Quantifiable Laser Module (QLM).
Contents in this Chapter
About Photokinetics........................................................................................................ 203
Analyzing Fluorescence Recovery After Photobleaching ......................................... 204
Analyzing Fluorescence Resonance Energy Transfer ................................................ 208
About Photokinetics
Photokinetics refers to the reactivity of fluorescent molecules while they are in the
excited state. Photokinetic reactions can be used to study the interactions of
molecules within living cells.
Photo-bleaching, FRET, and photo-activation are examples of photokinetic
reactions.
The following table shows photokinetic experiment methods and which biological
applications can be studied with those methods.
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207
Photokinetic Methods and Applications
Photokinetic Methods
Biological Applications
FRAP Single-Point and MultiPoint
Affinity, Biomolecular Cycling, Biomolecular
Environment, Structural Kinetics
Pattern Bleaching
Compartmental Analysis, Biomolecular Cycling,
Transport
FLIP
Compartmental Analysis, Biomolecular Cycling,
Transport, Structural Visualization
Background Reduction
Structural Visualization
Combinations
Compartmental Analysis, Biomolecular Cycling,
Transport
-FRAP/FRET
-Repeat during cell cycle
Affinity, Biomolecular Cycling, Biomolecular
Environment
-Rapid Repeat
FRET
Affinity, Biomolecular Environment
-Sensitized emission
-Donor Photo-bleaching
-Acceptor Depletion
Photo Activation
Compartmental Analysis, Affinity, Biomolecular Cycling,
Biomolecular Environment, Transport, Cell Fate,
Structural Kinetics, Structural Visualization
Analyzing Fluorescence Recovery After Photobleaching
The Fluorescence Recovery After Photo-bleaching (FRAP) experiment method
consists of photo-bleaching a point (or points) of interest and then observing the
recovery of fluorescence in the bleached area. For detailed instructions, see the
Fluorescence Recovery After Photo Bleaching Product Note at:
www.appliedprecision.com.
An example of a Single-Point FRAP experiment is shown below.
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A point of interest is photo bleached and monitored
About FRAP Experiments
There are two types of FRAP data:
•
Single-Point FRAP data is collected in experiments that monitor a single
location or monitor several locations in a sequential fashion.
•
Multi-Point FRAP data is collected in experiments that monitor several
locations in the sample at the same time.
Single and Multi-point FRAP experiments can be used for the following types of
studies:
„
Affinity Studies
„
Biomolecular Cycling
„
Biomolecular Studies
„
Environment Studies
„
Structural Kinetics
To analyze FRAP data:
1. Open an image in the Image window. From the softWoRx main menu, choose
Measure | PK Analysis. The Photokinetic Data Analysis dialog box is
displayed.
AppliedPrecision
Chapter 16: Other Applications
Tip You can use the Photokinetic Data Analysis dialog box to specify a recovery
model, type of ROI, beam profile shape, and number of sites. You can also use it
to remove background intensity, select which Z sections and wavelengths to
include in the analysis, and specify other options.
2. Drag the window number into the Input Image field.
3. Select the desired Response type, Recovery Model, and Data Recovery options.
4. If you are analyzing a Multi-point FRAP data set, enter the number of laser
sites in the Number of Laser Sites field.
5. If you are using background subtraction, enter a background value in the
Background Intensity field.
6. To determine a number to enter in the Spot Radius field, use the Measure
Distances tool to make an approximate measurement of the bleach spot.
7. Click Do It to run the analysis. The following files are generated:
The three channel output image file includes the original time lapse image, the
ratio of the current time point to average pre-bleach time points, and the ratio
data at the location used for analysis.
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The three channel image generated by the Photokinetic Data Analysis tool
A JPEG file contains a recovery graph that is a plot of the fluorescence intensity
before and after the event.
The Fluorescence Recovery Graph
AppliedPrecision
Chapter 16: Other Applications
A log file contains the analysis results.
The FRAP Analysis log file
Analyzing Fluorescence Resonance Energy Transfer
FRET (Fluorescence Resonance Energy Transfer) is a method for determining
whether two types of molecules are in close proximity. FRET occurs when there is
a quantum physical exchange of energy between dipoles. The presence of FRET
indicates that the molecules are within 60 Å (6 nm).
FRET is orientation specific. Negative FRET does not mean the molecules are not
interacting. Positive FRET means that they are close, but does not necessarily mean
that the molecules are interacting.
There are many ways to measure FRET. Experiments are simple, but controls are
essential.
Tip Before you conduct a FRET experiment, consider using the Colocalization tool
to determine whether the molecules that you are studying are close to each other.
(Colocalization experiments provide more approximate results than FRET, but they
are much simpler to prepare and analyze.)
Using the FRET Analysis Tool
The FRET analysis tool provided assists in analysis of sensitized emission FRET
experiments. Acceptor photo-bleaching can also be done with Deltavision. Use the
Ratio Analysis tool or Polygon Editor to analysis acceptor photo-bleaching
experiments.
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Before You Begin
Be sure to follow the conventions described in the online Help topic Acquiring and
Preparing FRET Data.
Before you begin, make sure that you have the following data:
You must have the following three image files:
„
A Donor Control data file that contains data from a slide prepared for the
Donor probe only.
„
An Acceptor Control data file that contains data from a slide prepared for the
Acceptor probe only.
„
The FRET Experiment file that contains data from a slide prepared for both
Donor and Acceptor probes.
Each image must have the following three channels:
Channel
Description
1
Donor (Donor Excitation, Donor Emission)
2
Acceptor (Acceptor Excitation, Acceptor Emission)
3
FRET (Donor Excitation, Acceptor Emission)
Analyzing FRET Data
Analysis of Direct FRET data consists of determination of Donor and Acceptor
crosstalk factors, calculation of Net FRET and FRET efficiency, and analyzing the
Net FRET and FRET Efficiency statistics. Use the FRET analysis tool and the
following process to analyze the FRET data:
Calculate CrossTalk
Calculate FRET
Analyze Results
Calculating Crosstalk
Calculation of Donor and Acceptor crosstalk factors involves creating Region of
Interest (ROI) polygons on Donor and Acceptor Control images and determining
representative background values to be subtracted from intensities in ROIs.
To calculate crosstalk:
1. From the softWoRx main menu, choose Measure | FRET Analysis. The FRET
Analysis dialog box is displayed. Make sure Calculate Crosstalk is the active
tab.
AppliedPrecision
Chapter 16: Other Applications
2. Open the Donor and Acceptor images in Image windows.
3. Drag the Image window number icon from each of the Donor and Acceptor
Image windows to the appropriate Donor and Acceptor Window fields in the
FRET Analysis dialog box to connect to these windows.
4. Validate that the Probe Channel for the Donor Control Crosstalk is set to the
correct (Donor) channel and the Acceptor Channel for the Acceptor Control
Crosstalk is set to the correct (Acceptor) channel.
5. Validate that the FRET Channel is set to the correct channel for each control
image.
6. Specify the background for the Probe and FRET channels of each image using
the Get buttons. The "Get" functions average intensity values in a box
(specified in the Box fields) while you drag the cursor around in the Image
window with the left mouse button held down. Click the Get button a second
time to disable the "Get" function.
7. For each image, define ROIs using the tools on the toolbar at the top of the
FRET Analysis dialog box to define representative areas where FRET would
occur if these were Experiment images.
8. Click Calculate Crosstalk to analyze the background and defined ROIs to
generate a crosstalk factor for both the Donor and Acceptor control images.
Typical Donor Crosstalk factors are 50-70%; typical Acceptor crosstalk factors
are 15-30%, if using CFP and YFP and the FRET pair.
The Donor and Acceptor crosstalk factors are loaded in to the appropriate
fields for FRET calculation.
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Calculating FRET
Calculation of Net FRET and FRET Efficiency uses a FRET Experiment image as
input along with Donor and Acceptor Crosstalk factors and background values for
each channel. It generates an image with 2 channels: Net FRET and FRET
Efficiency (%E).
To calculate FRET:
1. Choose the Calculate FRET tab on the FRET Analysis dialog box.
2. Open the FRET Experiment image you would like to analyze in an Image
window.
3. Drag the Image window number icon from the FRET Experiment Image
window to the FRET Input Image field in the FRET Analysis dialog box. An
output FRET Results image file name is automatically generated. (The FRET
Results image is saved to the disk and an Image window containing the saved
image is displayed after the calculation is completed).
4. Validate that the correct channels have been assigned to the Donor, Acceptor
and FRET channels of the input image.
5. Validate that the crosstalk factors are reasonable (These factors were calculated
when you calculated crosstalk).
6. Use the "Get" function to specify a background value for each of the 3 channels
(see Step 6 in To calculate crosstalk on page209).
7. Once you are satisfied that all parameters are set up correctly, click Generate
FRET Results Image. When this process is finished, the image opens.
AppliedPrecision
Chapter 16: Other Applications
Analyzing Results
Analyzing Net FRET and FRET Efficiency statistics involves specifying a FRET
Results Image and one or more ROI polygons to generate a table and graph of
statistics.
To analyze FRET results:
1. Choose the Analyze Results tab of the FRET Analysis dialog box.
2. If it isn't already being viewed, open the FRET Results image that you would
like to analyze in an Image window.
3. If it isn't already specified, drag the window number of FRET Results Image
window to the appropriate Input field in the FRET Results Statistics area.
4. Use the ROI creation tool icons at the top of the FRET Analysis dialog box to
specify one or more regions of interest.
5. If you have an image with multiple time points or multiple Z sections, you
may want to propagate ROI polygons through time or Z. To do this, create a
polygon and make sure it is selected. Then, choose Copy Through Time or
Copy Through Z to propagate the polygons. (As changes to ROI polygons are
made, the table of statistics is updated to reflect the statistics of the chosen ROI
set.)
6. To export the table of numbers in a form that can be used in a spreadsheet,
choose Save Results As SYLK (Symbolic Link format) or Save Results As
CSV (Comma-Separated Values) from the File menu on the FRET Analysis
dialog box.
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7. To view the results in a graph, choose Plot Results to generate an X/Y plot of
parameters that you choose. If the X axis is time-related, the software
associates ROIs from time point to time point and plots the values as
connected sets on the graph.
Tips
#1 You can modify the details of how the graph is displayed after it is created with
the Graph-Properties tool.
#2 You can optionally save the FRET Results graph by selecting Save As JPEG from
the FRET Results graph's File menu.
AppliedPrecision
Chapter 16: Other Applications
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Appendix A: Image Quality
Because many restoration problems can be attributed to problems with data
collection, you’ll find it worthwhile to become familiar with the common problems
and their solutions.
All of these methods are simple to perform on a regular basis.
Note Although the softWoRx restoration algorithms contain many refinements that
improve their ability to handle experimentally obtained optical sections, there are
certain types of data that simply cannot be properly restored.
In This Appendix
Using Deconvolution Residuals .................................................................................... 216
Visually Evaluating Images ........................................................................................... 217
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219
Using Deconvolution Residuals
What is a Residual?
The deconvolution residual is a measure of the difference between the measured
image and the solution convolved with the point spread function (PSF). In
mathematical terms:
Residual = (Measured Image) - (Deconvolved Image * PSF)
where * represents convolution.
In principle, the two quantities on the right side of the equation are equal, so that
the residual should be zero. Not surprisingly, however, the use of experimental
data prevents perfectly precise results and the residuals are not zero.
For the purpose of digital deconvolution, the residual is calculated from the
average of the residuals measured at each point in the three-dimensional image.
As a general rule, a small residual is better than a large residual.
The most useful form of the residual is the "Average Counts Residual," which is
the average difference between the measured image and the result convolved with
the PSF.
The Standard Residual
The "standard residual" is the sum of all residuals divided by the sum total
intensity of the image. Images with a large total intensity may therefore yield an
unrealistically small residual. For this reason, the standard residual may not be as
useful as the average count residual. Use the standard residual to compare
deconvolution performance results with deconvolutions prior to version 2.10 of
the DeltaVision softWoRx software.
Refer to the following table when assessing the "standard residual."
Value
Quality
Suggested Action
> 0.1
Poor deconvolution
quality
Check experiment conditions and data quality.
0.1 -0.05
Marginal
Deconvolution
May not be appropriate for this data.
0.05-0.01
Reasonable
Typical for data with a low signal-to-noise ratio
or a large amount of spherical aberration.
< 0.01
Good
Normalized Residual
As a means of watching the deconvolution progress, the "Normalized Residual" is
also displayed. By definition, the normalized residual equals 1 after the first
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iteration. Subsequent residuals are then scaled in the same way as the first
iteration to yield numbers between 0 and 1. A residual larger than 1 indicates that
the deconvolution algorithm has encountered serious difficulties. The table below
gives a guideline for assessing normalized residuals.
Value
Quality
Suggested Action
> 1.0
Worse than before the first
iteration.
Review experimental conditions.
1.0
No improvement.
0.5
Reasonable improvement.
0.25
Substantial improvement.
0.10
Excellent improvement.
Most deconvolutions yield residuals somewhere between 0.1 and 0.3. Consistent
results between 0.3 and 1.0 should prompt a review of experimental conditions
and the deconvolution problems listed in Visually Evaluating Images. In particular,
you should check pixel sizes, wavelength, and PSF selection.
Visually Evaluating Images
The second method of assessing a deconvolution is to simply study the resulting
images. Comparison of the measured and deconvolved images is an excellent way
of verifying that structures present in the results are a valid representation of the
actual object. With the aid of the deconvolution image, it is usually possible to
understand the structures present in the measured image.
It is expected that certain deconvolutions will be less successful than others, due to
the dependence upon experimental data. It is not always possible to meet the
conditions required for deconvolution microscopy. Fortunately, there are only a
few characteristic problems.
Weak Convergence of the Residual
A common cause of poor convergence is that the optical sections were measured
in the presence of spherical aberration. As a consequence, the standard PSF is not
appropriate for deconvolution. Flip the image on its side (using the "Flip" or
"Rotate" program) and study the quality of the image along the optical axis ("Z").
Asymmetric blurring and greatly elongated points indicate spherical aberration.
Use of very low intensity images (with a corresponding low signal-to-noise ratio)
can also limit convergence.
Dark Halo Around Bright Structures
The halo problem is often caused by the presence of excessive spherical aberration.
As always, ensure that the image properties are correct before beginning an
exhaustive study of this sort of problem.
AppliedPrecision
Appendix A: Image Quality
Use the Line Profile tool to assess the relative magnitude of the dark intensity
region. Although very noticeable, the intensity of the halo is often quite small (e.g.,
about 5% below the adjacent background).
Another source of halos is refractive index changes.
Bright Spot Problem
In situations where the object contains concentrated areas of fluorescent probe, it is
natural for the deconvolution process to yield even brighter spots in the resulting
image. For example, a region with 2000 counts of fluorescence could deconvolve to
a brightness of 8000 counts. The intensity of such areas can be so great that low
intensity structures present in the 16-bit image are not visible on a standard 8-bit
computer screen. Although these low intensity data exist, these data are simply
not visible next to the bright spots. To view low intensity areas, adjust the image
contrast, brightness, and intensity scaling factor.
Pebble Grain Texture
Low intensity texture patterns are often visible in low intensity images where the
signal-to-noise ratio has dropped below about 10 to 1. Increasing the image
intensity is the obvious method of avoiding this problem.
This can also be caused by problems with the CCD camera elements.
Deconvolution Holes
A bright ringed hole in background areas of the image is probably a result of
subtracting too much background intensity during deconvolution. The
"deconvolution hole" is the edge of the region that has reached the minimum
possible intensity (typically 0). Deconvolve the image again using a background
subtraction of 0, rather than the default value.
Increasing the "Z Transform Size" can also help control deconvolution holes. In
some situations, the measured data may actually have a low intensity threshold
caused by the loss of detector response. In this case, it is not possible to simply add
intensity to the image. The edge of the low intensity threshold is indistinguishable
from the edges being sought by the deconvolution process.
Poor Z Resolution
Elongated blurring in the Z directions is characteristic of spherical aberration.
Also, note that the football shape along the Z-axis is normal for an optical
microscope. The conical extensions above and below an object represent the
uncertainty involved with measuring light through a lens with less than a 90
degree cone angle. At present, the best available lenses have a cone angle of
approximately 68 degrees. Information between 68 and 90 degrees is not measured
by the lens.
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Section Intensity Fluctuation
Since each optical section is measured separately, it is common for the image
intensity to be slightly different. To view this experimental situation, flip the
optical sections on their side and look at the sections in the XZ plane. Striations
perpendicular to the Z-axis indicate that the relative intensity of the sections
varies. The striations are typically caused by arc lamp fluctuations during data
acquisition.
Use the image correction program before deconvolution to compensate for arc
lamp fluctuation. In some cases, the striations are visible after the image correction
process. This is a serious problem that needs to be addressed! Such images will
yield disastrous deconvolutions since the striations will be enhanced by the image
processing.
Invalid Optical Transfer Function
There are at least two ways that an optical transfer function (OTF) can be invalid.
•
The corresponding PSF may not have been well measured.
•
Incorrect OTF may have been applied for deconvolution. Check that the lens
ID number of the measured image corresponds to the lens ID of the OTF.
AppliedPrecision
Appendix A: Image Quality
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Index
2D Polygon Finder, 182–85
Dialog options, 186
Excluding edge objects, 186
introduction, 182
Minimum perimeter, 186
Smoothing pixel distance, 186
Specifying outer object only, 186
3D graph, 163–65
3D Object Builder, 187–90
creating the object, 188
example, 190
introduction, 182
loading the input image, 188
measuring the area of an object, 188
measuring the volume of an object, 188
viewing the object, 188
3D, viewing data in. See Volume Viewer
5D image data files, 148
Acquisition
data, 2
workstation, 2
Additive method of data collection, 125
Adjacent images, 21–22
Adjusting thumbnail images, 88
Aligning adjacent images, 18
Analysis workstation, 2
Analyzing data, 4
Applied Precision, LLC, contacting, iii
Arbitrary profile, 166
Arbitrary profiles, 168–69
Archiving, 157–59
Area of object, measuring, 188
ASCII file, saving coordinates to, 182
Assigning colors, 101
Auto-Polygon Creation icon tool, 182
Axes of rotation, 123–24
Background value, 145
Bad pixel file, 18, 20–21
BioRad MRC-600 Pic, 35
Blended color mode, 100, 101–2
Border rolloff, 27
Bright spot problem, 218
Brightness, 95–99
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Burning discs, 157–59
Calibration, 20–21
Capturing screens, 156–57
CCD defects, 17
CD Kreator tool, 157–59
CDROM discs, 157–59
Channels, assigning colors to, 101
Chromatic aberration, 21–22
Chromatic Aberration Corrector, 18
Close Polygon icon tool, 182
Colocalization
graph, 196
indentifying potential areas of, 197–201
Pearson Coefficient of Correction, 194
ROIs, 201–2
tool, 194–97
Color
assigning to channels, 99–102
blended, 101–2
true, 101
Color mode, 100
Colormap, 95–99
changing, 100
Combining data, 119–22
with image arithmetic, 142–43
Combining data files, 52–54
Combining multiple tasks, 54–57
Connecting to a DMS database, 74
Constrained iterative algorithm, 16
Contact Applied Precision, iii
Contrast, 95–99
Conventions, document, ii
Converting file formats, 31
Convolution filters, 138–39
Correct Image tool, 17, 18–19
Correcting images, 17–25
chromatic aberration, 18, 21–22
inconsistent illumination intensity, 17
motion artifacts, 18
photo-bleaching, 17
Cover slip, 112
Create Polygon icon tool, 182
Cropping regions, 44–50
Appendix A: Image Quality
Cross sections, 111–12
Crosstalk, calculating, 209
Customer Service Hotline, iii
Cut Mask, 49
Dark halo, 218
Data
Presenting, 85
Visualizing, 85
Data collection, methods of, 125
additive, 125
maximum intensity, 125
mixed, 125
progressive, 125
RGB/opacity, 125
VolPack, 125
Data files, 148
combining, 52–54
defined, 148
filtering, 137–45
opening, 88–89
Data Inspector, 163–65
3D graphing with, 165
and statistics, 169–74
Database integration, 73–84
Deconvolution, 9–16
about, 10
constrained iterative algorithm, 16
Deconvolve dialog box, 12
holes, 218
nearest neighbor method, 10, 16
options, 14–16
residuals, 216–17
run options, 13
single image, 11–13
standard method, 10, 16
using a queue, 13–14
visually assessing, 217
Delete Selected Point icon tool, 182
Delete Selected Polygon icon tool, 182
DeltaVision
archiving files from, 157–59
exporting files from, 151–56
saving files for, 148–51
Display border tools, 103
Display channel, 95
Distance, measuring, 175–77
options, 176
Tutorial, 176
DMS
about, 73
connecting to database, 74
integration, 73–84
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uploading images, 74–81
Document conventions, ii
DVD discs, 157–59
Edge enhance, 139–40
Edge objects, excluding in 2D Polygon
Finder, 186
Edit polygon tools, 182–85
Edit Polygon tools
for gathering statistics, 41–44
Enhance contrast, 143–44
Equalize intensities. See Equalize time points
Equalize time points, 18, 19–20
Experiments
FRAP, 204–8
FRET, 208–13
Explorer, 5
Exporting
DeltaVision files, 151–56
to JPEG files, 155
to Movie files, 153–55
to PhotoShop, 153
to TIFF, 151
Files
archiving, 157–59
data, 148
exporting DeltaVision, 151–56
formats of, 31
graphic, 148
saving as JPEG, 155
saving as movie, 153–55
saving as TIFF, 151
saving DeltaVision, 148–51
Filter2D, 140–42
Filtering images, 137–45
convolution, 138–39
enhancing boundaries, 139–40
removing noise, 140–42
softWoRx filters, 138
Filters
activating multiplexed sets, 63–67
Fluctuation of intensity, 219
Fluorescence recovery after photo-bleaching.
See FRAP
Fluorescence resonance energy transfer. See
FRET
Fluorescence, punctate, 15
FRAP, 204–8
FRET, 203, 208–13
analysis tool, 208–13
analysis workflow, 209
analyzing results, 212
calculating, 211
calculating crosstalk, 209
efficiency statistics, 212
prerequisites, 209
Graph, ratio, 62
Grayscale, 100
about, 99
assigning from blended color mode, 102
colormap, 100
Hide border tools, 103
Hide channel, 95
Histogram, 163–65
Image arithmetic, 142–43
Image fusion, 52–54
Image graphic files, 148
Image viewer
cross sections, 111–12
Image window, 102–7
about, 89–90
border tools, 103
display controls, 92
menu, 90
scale bar, 103–5
status bar, 90
toolbar, 90
zoom wheel, 94
Images
adjacent, 21–22
analyzing quality, 215–19
and colocalization, 194–97
and colocalized structures, 197–201
and intensities, 162–74
and line intensity, 167–68
archiving, 157–59
arithmetic, 142–43
calibrate, 20–21
calibrating, 18
combining data, 52–54
contrast and brightness, 95–99
correction, 17–25
cropping, 27, 44–50
cross sections, 111–12
deconvolve single, 11–13
deconvolving, 9–16
enhancing boudaries, 139–40
filtering, 137–45
graphic files, 148
Inovision ISee, 33
intensity threshold, 144–45
interpolation, 93, 94
local contrast enhancement, 143–44
opening, 88–89
ratio, 57–62
reorienting, 105–11
repositioning, 92
resizing, 105–7
RGB opacity method, 131–35
rotating, 107–9
saving as JPEG, 155
saving as movie, 153–55
saving as PhotoShop files, 153
saving as TIFF files, 151
saving volume rendered, 135
scaling, 95–99
scaling pixel intensity, 143–44
selecting, 40–44
stitching, 25–29
time-lapse, 19–20
uploading to DMS, 74–81
viewing as movies, 107–12
viewing data, 87–112
visually evaluating, 217–19
window, 102–7
with bright spots, 218
with dark halo issues, 218
with deconvolution holes, 218
with invalid OTF, 219
with multiple Z sections, 27–29
with pebble grain texture, 218
with poor Z resolution, 218
Importing
BioRad MRC-600 Pic files, 35
file formats, 31
Inovision ISee files, 33
TIFF images, 32
Inconsistent illumination intensity, 17
Inovision ISee™, 33
Insert Point icon tool, 182
Intensity data, 162–74
and viewing rows or columns, 167–68
Intensity fluctuation, 219
Intensity scale, 95–99
restore defaults, 98
Intensity threshold, 144–45
Intensity values, plotting, 166
Interactive image rotation, 128–31
Internet Address, Applied Precision, iii
Interpolated images, 93, 94
Invalid OTF, 219
Irregular data regions
cropping, 47–50
selecting, 41–44
JPEG files, 155
K3b, 157–59
AppliedPrecision
Appendix A: Image Quality
Large images, 25–29
Line intensity, 167–69
viewing in any direction, 168–69
Line Profile tool, 167
Line profiles, 167–68
Linear intensity scales, 95–99
Live specimen tracking, 114–18
Local contrast enhancement, 143–44
dialog box, 143
Local mean, 143
Maximum intensity, 125
Maximum parimeter, 186
Measuring
area of an object, 188
chromatic correction, 21–22
colocalization, 194–97
colocalized ROI, 201–2
distance. See Distance, measuring
FRAP data, 204–8
FRET data, 208–13
residuals, 216–17
velocity of particle movement, 177–79
volume of an object, 188
Methods of data collection. See Data
collection, methods of
Minimum perimeter, 186
Mixed method of data collection, 125
Modeling, 181–92
2D Polygon Finder, 182–85
3D Object Builder, 187–90
Edit Polygon, 182–85
Edit Polygon tool, 182–85
example, 190
summary of process, 182
Modes
blended color, 101–2
color, 101
color and grayscale, 99–102
switching between, 100
Motion artifacts, 18, 21
Movie files, 153–55
Movie players, 154
Movies
time-lapse, 113–14
trails, 114–18
viewing, 107–12
volumetric, 113–14
Multi-layered images, 27–29
Multiple Segment method, 177
Multiplexed wavelength
experiment design, 68–71
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filter set activation, 63–67
option, 62–71
overview, 63
Multi-Point FRAP, 205
Navigating Z sections, 90–92
Nearest neighbor, 16
deconvolution, 10
Nonlinear intensity scales, 95–99
Nonlinear local contrast enhancement. See
Local contrast enhancement
Normalize time points, 18, 19–20
Normalized residual, 217
Opening files, 88–89
Optical File Transfer function, 11
Optional components, 5
Orthogonal viewer, 111–12
OTF, 11
invalid, 219
Panel collection feature, 25
Panels, 25–29
Particle movement, 114–18
measuring velocity of, 177–79
Pearson Coefficient of Correction, 194
Pebble grain texture, 218
Photo-activation, 203
Photo-bleaching, 203
correcting, 17
for FRAP, 204
Photokinetics, 3, 203–4
PhotoShop files, 153
Point values, 162
Polygon Editor, 182–85
maximum points, 186
minimum points, 186
Polygon smoothing, 186
Polygons. See 2D Polygon Finder, 3D Object
Builder, Modeling
Poor Z resolution, 218
Presenting data, 85
Primary light path, 63
Printing, 159
screens, 156–57
Process chains, 54–57
Processing data, i, 3, 7
Progressive method of data collection, 125
Projections, 119–35
quick, 119–22
volume, 122–35
Punctate fluorescence, 15
QLM. See Quantifiable Laser Module
Quality of images, 215–19
Quantifiable Laser Module, 3, 203
Queue
for deconvolution, 13–14
manager dialog box, 14
Queue manager, 56, 79
Quick projection, 119–22
Ratio graph, 62
Ratio imaging, 57–62
Rectangular data regions
cropping, 45–47
selecting, 40–41
Reducing time data, 50–52
Region of interest, 165
Removing noise, 140–42
Rendering with volume viewer, 122–24
Reorienting images, 105–11
Repositioning images, 92
Resample2D, 105–7
Residuals, 216–17
weak convergence of, 217
Resizing images, 105–7
Resolve3D, 2
RGB opacity method, 131–35
RGB/opacity method of data collection, 125
ROI. See Region of interest
ROI colocalization, 201–2
Rotating images, 107–9, 128–31
Rotation angles, 107–9
Rotation axes, 123–24
Rotation center, 110
Save as SYLK spreadsheet dialog box, 169
Saving
as JPEG, 155
as Movie, 153–55
as PhotoShop, 153
as TIFF, 151
DeltaVision files, 148–51
statistics records, 170
volume rendered images, 135
with overlay graphics, 153–55
Scale bar, 103–5
Scaling pixel intensity, 143–44
Screen shots, 156–57
Scrolling Z sections, 91
Secondary light path, 63
Section intensity fluctuation, 219
Select Polygon icon tool, 182
Selecting
irregular regions, 41–44
rectangular regions, 40–41
Selecting data, 40–44
Show channel, 95
Single-Point FRAP, 205
Smoothing pixel distance in 2D Polygon
Finder, 186
Snapshots, 156–57
Spherical aberration, 218
Standard residual, 216
Standard Two Point method, 176
Statistics
calculating for selected areas, 169–74
using Data Inspector for, 170
with Edit Polygon tools, 41–44
Status bar, 90
Stitching, 25–29
border rolloff, 27
example, 26
images with multiple Z sections, 27–29
Table of 3-D measurements, 190
Task Builder, 54–57
Task chains, 54–57
Technical support, iii
Threshold
default value for 2D Polygon Finder, 186
Thumbnails, adjusting, 88
TIFF output formats, 152
TIFF, importing, 32
Time points
reducing, 50–52
viewing, 90–92
Time-lapse image data, 19–20, 59
Time-lapse movies, 107–12, 113–14
Tools
Align Image, 18, 21–22
calibrate, 18
calibration, 20–21
chromatic aberration corrector, 18, 21–22
colocalization, 194–97
convolution, 138–39
copy region, 53
Correct Image, 17, 18–19
correction, 17–25
Cut Mask, 49
Data Inspector, 163–65
deconvolve, 9–16
edge enhance, 139–40
edit header, 53
Edit Polygon, 41–44
equalize time points, 18, 19–20
Filter2D, 140–42
AppliedPrecision
Appendix A: Image Quality
for calculating statistics, 169–74
for identifying colocalized structures, 197–
201
for measuring velocity, 177–79
image arithmetic, 142–43
image fusion, 52–54
image window, 90, 102–7
intensity threshold, 144–45
interactive image rotation, 128–31
Line Profile, 167
local contrast enhancemnt, 143–44
measuring distances, 175–77
nearest neighbor, 10
orthogonal viewer, 111–12
Point values, 162
Polygon Editor, 182–85
projections, 119–35
queue manager, 56, 79
quick projection, 119–22
removing data below a threshold, 144–45
reorienting, 105–11
Resample2D, 105–7
resizing, 105–7
ROI colocalization, 193, 201–2
rotating, 107–9
scale bar, 103–5
scale image, 95–99
switching border on/off, 103
time-lapse movies, 113–14
trails movie, 114–18
viewing movies, 107–12
volume rendering, 122–24
volume viewer, 122–35
volumes, 119–35
Tracking particle movement, 114–18
Trimming time data, 50–52
True color, 101
Tutorials
Measuring distances, 176
modeling, 190
04-720103-000 Rev C /1008
229
UIC MetaMorph STK, 36
Undo Last icon tool, 182
Uploading images to DMS, 74–81
URL, Applied Precision, iii
Velocity, 177–79
measuring, 175
Viewing data in 3D. See Volume Viewer
Viewing image data, 87–112
Viewing movies. See Movies
Viewing time points, 90–92
Viewing Z sections, 90–92
Visualizing data, 3
VolPack, 126
Volume models, 181–92
Volume of object, measuring, 188
Volume projections, 122–35
Volume rendering, 122–24
Volume viewer, 122–35
axes of rotation, 123–24
interactive image rotation, 128–31
limiting size of data set, 124
rendering, 122–24
RGB opacity method, 131–35
using with colocalization, 199
Volumes, 119–35
Volumetric movies, 113–14
Voxels, 27
Weak convergence, 217
Web site, Applied Precision, iii
Z sections
and blurring, 218
data projections, 119–35
measuring points in, 175–77
slider, 91
viewing, 90–92
viewing as movie, 107–12
Zooming, 93
Z-stack image, 10
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