important notes on chapter 5

important notes on chapter 5
IMPORTANT NOTES ON CHAPTER 5
LSM-FCS
Carl Zeiss
IMPORTANT NOTES ON CHAPTER 5
These operation instructions describe standard and optional hardware and software configurations.
Depending on your order and therefore on the configuration, the content of the screens may differ.
Software
Eight different configuration packages of the LSM Software Release 3.2 are available:
− Software "LSM control“ and an additional license
− Software "Physiology evaluation“
− Software “Topography evaluation”
− Software “Macro Recorder and Editor”
− Software "3D for LSM“
− Software “Multiple Time Series”
− Software “Image VisArt”
− Software “Deconvolution”
− Software “StitchArt”
If your configuration does not include the "Physiology evaluation“ software package, the following
functions are inactive:
− Mean of ROI scan button in Time Series control
− Mean of ROI button in the image display after frame scan
If your configuration does not include the “Topography evaluation” software package, the following
functions are inactive:
− Topo button in the image display after acquisition of image stacks
If your configuration does not include the “Macro Recorder and Editor” software package, the following
functions are inactive:
− New, Save and Save as buttons in the Macro Control
− Edit, Step, Delete, Editor buttons in the Macro Control
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5-I
IMPORTANT NOTES ON CHAPTER 5
Carl Zeiss
LSM-FCS
If your configuration does not include the “3D for LSM” software package, the following functions are
inactive:
Separate software “3D for LSM”
If your configuration does not include the “Multiple Time Series” software package, the following
functions are not available:
− Macro: “Advanced Time Series”
If your configuration does not include the “Image VisArt” software package, the following functions are
inactive:
− 3D button in the image window
If your configuration does not include the “Deconvolution” software package, the following functions
are inactive:
− DCV button in the image window and in the process main menu
If your configuration does not include the "StitchArt" software package, the following function is not
available:
− Macro: "StitchArt"
Hardware
Depending on whether the following hardware components are available or not, the content of the
screens may differ:
− HRZ 200 fine focusing stage
− Piezo objective focusing device
− X-Y scanning stage DC 4 × 4 or DC 100 × 90, each with MCU 28
− Depending on the configuration the Scan head equipment may differ in filters, beamsplitters and the
number of photomultiplier
− Transmitted-light PMT
− Stands:
Axiovert 100 M, Axiovert 200 mot
− Monitordiode
− Programmable AOTF
− Non Descanned Detectors
− META detection module
If your configuration does not include an AxioCam, the following functions are not available:
−
5-II
Camera in the Config Control window, Scan Control window
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IMPORTANT NOTES ON CHAPTER 5
LSM-FCS
Carl Zeiss
Limitations notes for the Release 3.2 (date of issue: 11/2002)
No.
Function
Hardware
1.
Hardware
Description
Fix
Starting the system new after a longer time, some errors Starting the system once
messages are possible in the error log
again
2.
Hardware
For precise measurements over long time ranges
constant environmental conditions are necessary
(temperature, humidity)
3.
Computer
The computer (generation Pentium 3) has problems to
initialize the operating system sometimes (blue screen)
Restart of the computer is
necessary
Software
Main menu
4.
Scan & system
information
File
5.
Import
6.
7.
Export/Import
Export/Import
The display of state of Ar-laser is off in the Scan &
system information if laser is in standby mode
For the import of more than one FCS image the view of
chambers is not correct
Export of 4D series as LSM 4 Tif is handled as z stack
*.eps – format will exported not correctly for more than
one channel
Acquire – Laser Control
Acquire – Microscope Control
8.
Microscope
You select another objective for an Axioplan 2
Control
Imaging if the LSM software is not active:
If you start the LSM software now – the last registered
objective in the LSM software moves back in the beam
path
Acquire – Configuration Control
9.
Configuration
The movement of the NDD switching unit (to HBO/HAL
mirror position) during changes from LSM to VIS
position will not updated in the configuration control
10.
Configuration
During visual adjustment with help of HBO/HAL lamp
the detector gain of NDD detectors must be minimized
to avoid decrease of sensitivity of photomultipler
11.
ConfigurationThere is a bug in the demo database: the slider of
META-Offline
lambda stacks shows a wrong spectral range and a
higher number of pmt elements
12.
NDDThe switching between NDD and descanned mode does
Configuration
not work properly, if the laserline attenuation is not
adjusted
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Make sure, that you use not 2
objectives for different
immersion media
Only in offline mode!
(ignore it)
Use the AOTFfit-macro to
adjust the laserlines
5-III
IMPORTANT NOTES ON CHAPTER 5
Carl Zeiss
Acquire – Scan Control
13.
Scan Control
14.
15.
16.
Scan Control
Scan Control
Scan Control
17.
Z Settings
18.
Z Settings
LSM-FCS
Auto Correction for bidirectional scanning does not
work for Lambda stacks
Spline scan with more than 4 channels does not work
The Gain Slider for monitordiode works upside down
Offset and Amplifier Gain Adjustment does not work for
Spot Scan
The movement of focus for Axioskop 2 FS is to slow for
Move First / Mid / Last – therefore it´s possible, that
the focus movement is not finished during the image
acquisition
Z Values in the LSM software and at the display of
Axiovert 200 are different
Acquire – Edit ROI
19.
Edit ROI
Creation of ROIs with 1 Pixel width is not possible
20.
Edit ROI
ROIs for xz planes are possible to define but not to scan
Acquire – Time Series Control
21.
Time series
xz-t-series: Roi/mean in the image menu shows Roivalues in xy-coordinates and calculated the area in xyunits and not in xz-units
Acquire – Bleach Control
Macros
22.
Multi Time Series
– macro
23.
AOTF Fit
24.
AOTF Fit
Analyse FCS
25.
Carrier
26.
Carrier
27.
Measure
Options
28.
FCM Settings
5-IV
There is no information about bleaching during the
measurement
Sometimes it occurs that after the linearisation the
maximum transmission value is only 99 %
Sometimes the macro creates wrong calibration files –
in this case you get messages in the error log about the
laserlines
You have to adjust the
scanner with a single track
before you start your Lambda
stack
In this case inform your service
Adjust the detector before
you start the spot series
Generate a second image if
the focus is finished
Ignore it
Minimum is a 2 pixel width
(rounding error) Ignore it
Adjust it again
The carrier window needs a bit time to open because
the little camera in the ConfoCor head has to initialize
The laser beam is on every time if the Carrier window is
open (also for the LSM-FCS combination system when
the slider are in the LSM position)
The coordinates for LSM and FCS are not the same for
the combination system
The coordinates for X and Y are exchanged for the case
that the LSM scan head is situated on the sideport and
the FCS head is situated on the baseport (for LSM-FCMCombi)
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IMPORTANT NOTES ON CHAPTER 5
LSM-FCS
Carl Zeiss
Maintain
29.
Objective
In the LSM software you get another information about Ignore it
the number of focus speed for the objectives than in the
external program AxioSet (for microscope settings)
Image
30.
Image
31.
At an image with 2k × 2k the µm-scale is to small in
Print Preview and in printed Picture.
The FCS pinholes are displayed in the LSM Pinhole
Control, but it is not possible to adjust FCS pinholes in
this dialog (for LSM-FCS-Combi)
Pinhole
Tools
32.
CLM
33.
Change Filters
Help
34.
Help
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CLM for Axioskop 2 FS don´t exist
Change Filter for Axioskop 2 FS does not exist
The heading of help text for Combi-system still contains
Release 2.8
5-V
IMPORTANT NOTES ON CHAPTER 5
Carl Zeiss
5-VI
LSM-FCS
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OPERATION IN EXPERT MODE
Contents
LSM-FCS
Carl Zeiss
CHAPTER 5
OPERATION IN EXPERT MODE
CONTENTS
Page
5
OPERATION IN EXPERT MODE ................................................................................. 5-11
5.1
5.1.1
5.1.2
5.1.3
5.1.4
5.1.5
Purpose of this Section and other Operating Manuals................................................... 5-11
Software ..................................................................................................................... 5-11
Windows and Window Elements ................................................................................. 5-12
Convention for the Text in this Manual ........................................................................ 5-13
Backup........................................................................................................................ 5-14
Software Operation ..................................................................................................... 5-14
5.2
5.2.1
5.2.2
5.2.3
Switching on the System ............................................................................................. 5-15
Log on to WINDOWS NT ............................................................................................. 5-16
Switching on the Enterprise UV Laser ........................................................................... 5-18
Starting the LSM-FCS Program..................................................................................... 5-18
5.3
Main Menu ................................................................................................................. 5-21
5.4
5.4.1
5.4.2
5.4.3
5.4.3.1
5.4.3.2
5.4.3.3
5.4.3.4
5.4.3.5
5.4.3.6
5.4.3.7
5.4.3.8
5.4.3.9
5.4.3.10
5.4.4
5.4.5
5.4.6
5.4.7
5.4.8
5.4.9
File Menu .................................................................................................................... 5-23
Create New Image Database........................................................................................ 5-23
Open Existing Image Database..................................................................................... 5-25
Image Database window ............................................................................................. 5-26
Form display mode ...................................................................................................... 5-27
Gallery display mode ................................................................................................... 5-29
Table display mode...................................................................................................... 5-30
Load function.............................................................................................................. 5-30
Subset function ........................................................................................................... 5-31
Refresh function.......................................................................................................... 5-31
Copy / Paste function .................................................................................................. 5-31
Filter function.............................................................................................................. 5-32
On Filter function ........................................................................................................ 5-35
Delete function ........................................................................................................... 5-35
Save an Image to the Image Database.......................................................................... 5-36
Import of Images......................................................................................................... 5-40
Export of Images ......................................................................................................... 5-41
Multi Print ................................................................................................................... 5-42
Open FCS - Load a FCS data file................................................................................... 5-45
Exit the Expert Mode ................................................................................................... 5-46
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5-1
OPERATION IN EXPERT MODE
Contents
Carl Zeiss
5.5
5.5.1
5.5.1.1
5.5.1.2
5.5.1.3
5.5.2
5.5.2.1
5.5.2.2
5.5.2.3
5.5.3
5.5.3.1
5.5.3.2
5.5.3.3
5.5.3.4
5.5.3.5
5.5.3.6
5.5.3.7
5.5.3.8
5.5.3.9
5.5.3.10
5.5.3.11
5.5.4
5.5.4.1
5.5.4.2
5.5.4.3
5.5.4.4
5.5.4.5
5.5.5
5.5.5.1
5.5.5.2
5.5.6
5.5.6.1
5.5.6.2
5.5.6.3
5.5.6.4
5.5.6.5
5.5.6.6
5.5.7
5.5.7.1
5.5.7.2
5.5.8
5.5.8.1
5.5.8.2
5.5.9
5-2
LSM-FCS
Acquire Menu............................................................................................................. 5-46
Laser Control .............................................................................................................. 5-47
Opening / Closing the Laser Control window ............................................................... 5-47
Function description.................................................................................................... 5-48
Settings ...................................................................................................................... 5-48
Microscope Control..................................................................................................... 5-50
Open the Microscope Control window ........................................................................ 5-50
Microscope Control window for Axiovert 200 M.......................................................... 5-51
Select the LSM mode .................................................................................................. 5-54
Configuration Control ................................................................................................. 5-55
Open / Close the Configuration Control window ......................................................... 5-56
Spectra button............................................................................................................ 5-56
Laserline button .......................................................................................................... 5-57
Config button............................................................................................................. 5-58
Settings for Single Track in the Channel Mode............................................................. 5-60
Settings for Multi Track in the Channel Mode .............................................................. 5-65
Ratio Settings panel .................................................................................................... 5-70
Settings in the Lambda Mode...................................................................................... 5-72
Settings in the Online Fingerprinting Mode ................................................................. 5-75
Non Descanned panel ................................................................................................. 5-76
Camera Detection panel.............................................................................................. 5-77
Scan Control............................................................................................................... 5-78
Open / Close the Scan Control window ....................................................................... 5-79
Frame ......................................................................................................................... 5-81
Line .......................................................................................................................... 5-101
Spot ......................................................................................................................... 5-107
Camera control......................................................................................................... 5-108
Edit ROI (Region Of Interest) ...................................................................................... 5-110
Open / Close the Edit ROI window............................................................................. 5-110
Function description.................................................................................................. 5-111
Time Series ............................................................................................................... 5-114
Open / Close the Time Series Control window ........................................................... 5-114
Function description.................................................................................................. 5-115
Time Series of a Frame .............................................................................................. 5-123
Time Series of a frame over Z Stack ........................................................................... 5-125
Time Series of a frame over a Z Stack over Lambda .................................................... 5-126
Time Series with Mean ROI........................................................................................ 5-127
Edit Bleach................................................................................................................ 5-129
Open / Close the Edit Bleach window ........................................................................ 5-129
Function description.................................................................................................. 5-129
Stage........................................................................................................................ 5-134
Open / Close the Stage and Focus Control window.................................................... 5-134
Function description.................................................................................................. 5-134
VIS, FCS and LSM Buttons ......................................................................................... 5-141
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OPERATION IN EXPERT MODE
Contents
LSM-FCS
5.6
5.6.1
5.6.1.1
5.6.1.2
5.6.1.3
5.6.1.4
5.6.1.5
5.6.2
5.6.2.1
5.6.2.2
5.6.3
5.6.3.1
5.6.3.2
5.6.4
5.6.4.1
5.6.4.2
5.6.5
5.6.5.1
5.6.5.2
5.6.6
5.6.7
5.6.7.1
5.6.7.2
5.6.7.3
5.6.7.4
5.6.8
5.6.9
5.6.9.1
5.6.9.2
5.6.9.3
5.6.9.4
5.6.10
5.6.10.1
5.6.10.2
5.6.10.3
5.6.10.4
5.6.11
5.6.11.1
5.6.11.2
5.6.11.3
5.6.12
5.6.13
5.6.13.1
5.6.13.2
5.6.13.3
5.6.13.4
B 45-0006 e
Carl Zeiss
Process Menu............................................................................................................ 5-142
Add .......................................................................................................................... 5-142
Open / Close the Add window................................................................................... 5-142
Source panel ............................................................................................................. 5-143
Destination panel ...................................................................................................... 5-144
Add panel ................................................................................................................. 5-144
Preview panel............................................................................................................ 5-145
Subtract .................................................................................................................... 5-145
Open / Close the Subtract window ............................................................................ 5-145
Performance of the Subtract function ........................................................................ 5-145
Multiply .................................................................................................................... 5-146
Open / Close the Multiply window ............................................................................. 5-146
Performance of the Multiply function......................................................................... 5-146
Ratio ......................................................................................................................... 5-147
Open / Close the Ratio window ................................................................................. 5-147
Performance of the Ratio function ............................................................................. 5-147
Copy (Channel) ......................................................................................................... 5-148
Open / Close the Copy window ................................................................................. 5-148
Performance of the Copy function ............................................................................. 5-148
Duplication (Image) ................................................................................................... 5-148
Filter ......................................................................................................................... 5-149
Open / Close the Filter window.................................................................................. 5-149
Image panel .............................................................................................................. 5-149
Filter List and Edit panel............................................................................................. 5-150
Preview panel............................................................................................................ 5-153
Contrast.................................................................................................................... 5-153
Interpolate ................................................................................................................ 5-154
Open / Close the Interpolate Brightness and Contrast window.................................... 5-154
Image panel .............................................................................................................. 5-154
Interpolation panel .................................................................................................... 5-155
Preview panel............................................................................................................ 5-156
Channel Shift ............................................................................................................ 5-156
Open / Close the Channel Shift window..................................................................... 5-156
Image panel .............................................................................................................. 5-157
Shift panel................................................................................................................. 5-157
Preview panel............................................................................................................ 5-157
Unmix ....................................................................................................................... 5-159
Open / Close the Unmix window ............................................................................... 5-159
Source panel ............................................................................................................. 5-159
Definition of Channels panel...................................................................................... 5-160
Ion Concentration ..................................................................................................... 5-162
3D DeConVolution (DCV) .......................................................................................... 5-168
Background............................................................................................................... 5-168
Open / Close the 3D Deconvolution window .............................................................. 5-169
Source panel ............................................................................................................. 5-170
Deconvolution panel.................................................................................................. 5-170
12/02
5-3
OPERATION IN EXPERT MODE
Contents
Carl Zeiss
LSM-FCS
5.7
5.7.1
5.7.1.1
5.7.1.2
5.7.1.3
5.7.1.4
5.7.2
5.7.2.1
5.7.2.2
5.7.2.3
5.7.2.4
5.7.3
5.7.3.1
5.7.3.2
5.7.3.3
5.7.3.4
3D View Menu.......................................................................................................... 5-172
3D DepthCod (Color Coded Depth Map) ................................................................... 5-172
Open / Close the Depth Coding window.................................................................... 5-172
Source panel............................................................................................................. 5-173
Depth Coding panel.................................................................................................. 5-173
Preview panel ........................................................................................................... 5-174
Projection ................................................................................................................. 5-175
Open / Close the Projection window.......................................................................... 5-175
Source panel............................................................................................................. 5-175
Projection panel ........................................................................................................ 5-175
Preview panel ........................................................................................................... 5-177
Stereo....................................................................................................................... 5-179
Open / Close the Stereo Images window ................................................................... 5-179
Source panel............................................................................................................. 5-179
Stereo Images panel.................................................................................................. 5-179
Preview panel ........................................................................................................... 5-181
5.8
5.8.1
5.8.2
5.8.2.1
5.8.2.2
5.8.3
5.8.3.1
5.8.3.2
5.8.3.3
5.8.4
5.8.4.1
5.8.4.2
5.8.4.3
5.8.4.4
5.8.4.5
5.8.4.6
5.8.5
Analyse FCS Menu .................................................................................................... 5-183
Select Method .......................................................................................................... 5-184
Select Carrier ............................................................................................................ 5-186
Opening / Closing the Carrier Position window .......................................................... 5-186
Function description.................................................................................................. 5-187
Measure Method ...................................................................................................... 5-191
Opening / Closing the Method Measurement ... window ........................................... 5-191
Function description.................................................................................................. 5-191
Measurement procedure ........................................................................................... 5-195
Optimize Method...................................................................................................... 5-196
Opening / Closing the Method Optimization ... window............................................. 5-196
Function description.................................................................................................. 5-196
Instrumentation ........................................................................................................ 5-197
Analysis Procedure .................................................................................................... 5-200
Result Handling......................................................................................................... 5-203
X,Y,Z-Scan for positioning the focus within structured samples .................................. 5-205
VIS, FCS and LSM Buttons ......................................................................................... 5-207
5.9
5.9.1
5.9.1.1
5.9.1.2
5.9.1.3
5.9.2
5.9.2.1
5.9.2.2
5.9.3
5.9.3.1
5.9.3.2
5.9.3.3
Config FCS Menu...................................................................................................... 5-208
Laser Control ............................................................................................................ 5-208
Opening / Closing the Laser Control window ............................................................. 5-208
Function description.................................................................................................. 5-209
Settings .................................................................................................................... 5-209
Micro........................................................................................................................ 5-210
Open the Axiovert Control window ........................................................................... 5-210
Function description.................................................................................................. 5-210
Adjust Pinhole........................................................................................................... 5-212
Open / Close the Pinhole Adjustment window ........................................................... 5-212
Function description.................................................................................................. 5-212
To adjust the pinhole proceed as follows ................................................................... 5-215
5-4
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OPERATION IN EXPERT MODE
Contents
LSM-FCS
Carl Zeiss
5.9.4
5.9.4.1
5.9.4.2
5.9.4.3
5.9.4.4
5.9.4.5
5.9.5
5.9.5.1
5.9.5.2
5.9.5.3
5.9.5.4
5.9.5.5
Define Beam Path...................................................................................................... 5-218
Open / Close the Beam Path Definition window ......................................................... 5-218
Function description .................................................................................................. 5-219
Define a new beam path configuration ...................................................................... 5-219
Save a new beam path configuration ......................................................................... 5-220
Delete a beam path configuration.............................................................................. 5-220
Define Carrier............................................................................................................ 5-221
Open / Close the Carrier Definition window ............................................................... 5-221
Function description .................................................................................................. 5-221
Define a new sample carrier ...................................................................................... 5-222
Save a new sample carrier ......................................................................................... 5-222
Delete a beam path configuration.............................................................................. 5-222
5.10
5.10.1
5.10.2
5.10.2.1
5.10.2.2
5.10.2.3
5.10.2.4
5.10.3
5.10.4
5.10.4.1
5.10.4.2
5.10.4.3
5.10.4.4
5.10.4.5
Macro Menu ............................................................................................................. 5-223
Macro Language ....................................................................................................... 5-223
Macro Control........................................................................................................... 5-224
Open / Close the Macro Control window ................................................................... 5-224
Edit Macro function................................................................................................... 5-224
Assign Macro to Button function ............................................................................... 5-227
Editing and debugging of macros .............................................................................. 5-228
Overview of available Macros (all LSM releases) .......................................................... 5-229
Sample Macros.......................................................................................................... 5-232
Distance macro ......................................................................................................... 5-232
Profile macro............................................................................................................. 5-232
Bleach macro ............................................................................................................ 5-233
Multiple Time Series macro, Rev. 3............................................................................. 5-234
StitchArt macro ......................................................................................................... 5-235
5.11
5.11.1
5.11.2
5.11.3
5.11.4
5.11.5
5.11.5.1
5.11.5.2
5.11.5.3
5.11.5.4
5.11.5.5
5.11.5.6
5.11.5.7
5.11.5.8
5.11.5.9
5.11.5.10
5.11.5.11
5.11.5.12
5.11.5.13
Options Menu ........................................................................................................... 5-237
Export RM Function ................................................................................................... 5-237
Load RM Function ..................................................................................................... 5-238
Dye DB Function........................................................................................................ 5-239
Spectra DB Function .................................................................................................. 5-240
LSM Settings Function ............................................................................................... 5-241
Open / Close the Settings for user : ... window .......................................................... 5-241
Autosave................................................................................................................... 5-242
Database General ...................................................................................................... 5-243
Database Table Viewer .............................................................................................. 5-244
Database Gallery Viewer ............................................................................................ 5-244
Import / Export .......................................................................................................... 5-245
Scan Information....................................................................................................... 5-245
Image Status Display.................................................................................................. 5-246
Print Status Display .................................................................................................... 5-246
Recording / Reuse...................................................................................................... 5-246
Time series ................................................................................................................ 5-247
Scan Mean of ROIs .................................................................................................... 5-247
Temporary Files ......................................................................................................... 5-248
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5-5
OPERATION IN EXPERT MODE
Contents
Carl Zeiss
LSM-FCS
5.11.5.14
5.11.5.15
5.11.5.16
5.11.5.17
5.11.6
5.11.6.1
5.11.6.2
5.11.6.3
5.11.6.4
5.11.6.5
5.11.7
Program Start ........................................................................................................... 5-249
Shutdown................................................................................................................. 5-249
Image Display Toolbars.............................................................................................. 5-249
Save ......................................................................................................................... 5-250
FCS Settings Function................................................................................................ 5-251
Open / Close the Settings for user : ... window .......................................................... 5-251
Open / Save tab ........................................................................................................ 5-251
Raw Data tab............................................................................................................ 5-252
Measure tab ............................................................................................................. 5-252
LSM+ConfoCor tab.................................................................................................. 5-253
FCS Collimator Settings Function ............................................................................... 5-253
5.12
5.12.1
5.12.1.1
5.12.1.2
5.12.2
5.12.2.1
5.12.2.2
5.12.2.3
5.12.3
5.12.3.1
5.12.3.2
5.12.3.3
5.12.4
5.12.5
5.12.6
5.12.7
5.12.8
5.12.9
5.12.10
5.12.11
Maintain Menu ......................................................................................................... 5-254
Scanner .................................................................................................................... 5-254
Calibration with Speed 1-10 (electr., unidirectional / bidirectional) and Speed 11-13 (electr.,
only unidirectional).................................................................................................... 5-254
Calibration with Speed 11-13 (optical, bidirectional) .................................................. 5-256
Objective .................................................................................................................. 5-261
Objective change ...................................................................................................... 5-261
Focus speed change .................................................................................................. 5-262
Parfocality Correction................................................................................................ 5-263
Pinhole Adjustment................................................................................................... 5-264
Open / Close the Pinhole & Collimator Control window ............................................. 5-264
Function description.................................................................................................. 5-265
Pinhole and collimator adjustment............................................................................. 5-267
DSP (Digital Signal Processor)..................................................................................... 5-269
Set Find .................................................................................................................... 5-270
Spline ....................................................................................................................... 5-270
DSP Trace ................................................................................................................. 5-271
Parameter ................................................................................................................. 5-271
Reboot ..................................................................................................................... 5-271
HW/Admin................................................................................................................ 5-271
Test Grid................................................................................................................... 5-271
5.13
5.13.1
5.13.2
5.13.3
5.13.4
Window Menu.......................................................................................................... 5-272
Full Screen ................................................................................................................ 5-272
Close All Image Display Windows .............................................................................. 5-272
Toolbar ..................................................................................................................... 5-273
Scan and System Information .................................................................................... 5-273
5.14
5.14.1
5.14.2
Help Menu................................................................................................................ 5-274
Help ......................................................................................................................... 5-274
About ....................................................................................................................... 5-274
5.15
5.15.1
5.15.2
5.15.3
Display and Analysis of Images .................................................................................. 5-275
Structure of the Image Display Window..................................................................... 5-275
Select - Chan ............................................................................................................ 5-278
Select - Zoom............................................................................................................ 5-281
5-6
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OPERATION IN EXPERT MODE
Contents
LSM-FCS
Carl Zeiss
5.15.4
Select - Slice .............................................................................................................. 5-283
5.15.5
Select - Overlay ......................................................................................................... 5-284
5.15.6
Select - Contr ............................................................................................................ 5-288
5.15.7
Select - Palette .......................................................................................................... 5-292
5.15.8
Select - Anim............................................................................................................. 5-295
5.15.9
Select - Reuse............................................................................................................ 5-297
5.15.10
Select - Crop ............................................................................................................. 5-298
5.15.11
Select - Copy............................................................................................................. 5-299
5.15.12
Select - Save.............................................................................................................. 5-300
5.15.13
Select - Save As ......................................................................................................... 5-300
5.15.14
Display - xy................................................................................................................ 5-301
5.15.15
Display - Split xy ........................................................................................................ 5-302
5.15.16
Display - Ortho .......................................................................................................... 5-303
5.15.16.1 Ortho - Select function .............................................................................................. 5-304
5.15.16.2 Ortho - Distance function .......................................................................................... 5-305
5.15.16.3 Ortho - 2D DeConVolution function........................................................................... 5-306
5.15.17
Display - Cut ............................................................................................................. 5-308
5.15.18
Display - Gallery ........................................................................................................ 5-309
5.15.19
Display - Histo ........................................................................................................... 5-311
5.15.20
Display - Profile ......................................................................................................... 5-323
5.15.21
Display - 2.5 D........................................................................................................... 5-326
5.15.22
Display - 3D (Image VisArt) ........................................................................................ 5-328
5.15.22.1 Shadow Projection..................................................................................................... 5-330
5.15.22.2 Transparency Projection............................................................................................. 5-332
5.15.22.3 Surface Rendering ..................................................................................................... 5-333
5.15.22.4 Appearance (Settings)................................................................................................ 5-334
5.15.22.5 Series ........................................................................................................................ 5-335
5.15.23
Display - Topo for LSM .............................................................................................. 5-337
5.15.23.1 Channel Selection...................................................................................................... 5-338
5.15.23.2 Generate Topography................................................................................................ 5-338
5.15.23.3 Topography Thresholds ............................................................................................. 5-339
5.15.23.4 Processing by Filtering ............................................................................................... 5-340
5.15.23.5 Display Modes........................................................................................................... 5-343
5.15.23.6 Context Menu of the 3D Display Mode ...................................................................... 5-346
5.15.23.7 Measurement Functions ............................................................................................ 5-353
5.15.23.8 3D Measurement Functions ....................................................................................... 5-365
5.15.23.9 Export data ............................................................................................................... 5-369
5.15.23.10 Topo ReUse............................................................................................................... 5-369
5.15.24
Display - Prev............................................................................................................. 5-370
5.15.25
Display - Info ............................................................................................................. 5-372
5.15.26
Additional Display Mode in Time Series ...................................................................... 5-373
5.15.26.1 Display - Mean .......................................................................................................... 5-373
5.15.27
Additional Display Modes in Lambda Mode................................................................ 5-377
5.15.27.1 Display - Coded......................................................................................................... 5-378
5.15.27.2 Display - Max ............................................................................................................ 5-378
5.15.27.3 Display - Mean .......................................................................................................... 5-379
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5-7
OPERATION IN EXPERT MODE
Contents
Carl Zeiss
LSM-FCS
5.15.27.4 Functionality for Emission Fingerprinting .................................................................... 5-383
5.15.27.5 Automatic Component Extraction.............................................................................. 5-385
5.16
5.16.1
5.16.1.1
5.16.1.2
5.16.1.3
5.16.2
5.16.2.1
5.16.2.2
Image Optimization................................................................................................... 5-387
Single Channel.......................................................................................................... 5-387
Requirements............................................................................................................ 5-387
Pinhole / Detector Gain / Ampl. Offset / Ampl. Gain ................................................... 5-392
Scan Speed, Scan Average and Pixel Depth ................................................................ 5-395
Multiple-channel ....................................................................................................... 5-396
Requirements............................................................................................................ 5-396
Image Optimization................................................................................................... 5-397
5.17
5.17.1
5.17.2
5.17.2.1
5.17.3
5.17.4
5.17.5
5.17.6
5.17.7
5.17.8
5.17.9
5.17.10
5.17.11
5.17.12
5.17.13
Data Evaluation and Result Presentation for FCS Measurements ................................. 5-399
Structure of the Data Evaluation Window .................................................................. 5-399
Open / Close the Data Evaluation Window................................................................. 5-400
Description of the Raw Data Format .......................................................................... 5-404
Display - Correlation.................................................................................................. 5-406
Display - Fit ............................................................................................................... 5-411
Display - Coincidence ................................................................................................ 5-419
Display - Preview ....................................................................................................... 5-420
Display - Save Data.................................................................................................... 5-422
Display - Copy Table.................................................................................................. 5-422
Display - Save Table................................................................................................... 5-422
Display - Copy Graphs ............................................................................................... 5-422
Display - Export ......................................................................................................... 5-422
Display - Columns ..................................................................................................... 5-423
Display - Info............................................................................................................. 5-424
5.18
5.18.1
5.18.1.1
5.18.1.2
5.18.2
5.18.2.1
5.18.2.2
5.18.3
5.18.3.1
5.18.3.3
5.18.4
5.18.4.1
5.18.4.2
Combined Application of LSM Scanning and FCS Measurement Procedure................. 5-425
Taking FCS Measurements within a Cell .................................................................... 5-425
Using the Method Measurement ... Window ............................................................. 5-426
Using the Method Optimization ... Window............................................................... 5-427
Performing FCS Measurements on the Cell Membrane............................................... 5-430
Manual Focusing on the Membrane .......................................................................... 5-430
Focusing on the Membrane by a Z Scan..................................................................... 5-430
Compensating Offsets between FCS and LSM............................................................ 5-432
Alignment in X, Y for the Method Measurement ... and the Method Optimization in the
Positions Mode ......................................................................................................... 5-432
Determination of the Fixed X,Y Position of the Laser Beam in the Method Optimization ...
Window in the Crosshair Mode ................................................................................. 5-439
Alignment in Z .......................................................................................................... 5-441
Piezo Stage ............................................................................................................... 5-442
Activating the Piezo Stage ......................................................................................... 5-442
Working with the Piezo stage.................................................................................... 5-443
5.19
5.19.1
5.19.2
Shut-Down Procedure ............................................................................................... 5-444
Exiting the LSM-FCS Program .................................................................................... 5-444
Shut Down the WINDOWS Operating System ............................................................ 5-445
5.18.3.2
5-8
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OPERATION IN EXPERT MODE
Contents
LSM-FCS
Carl Zeiss
5.19.3
Turning Power Off..................................................................................................... 5-446
5.20
5.20.1
5.20.2
Software and Hardware Options................................................................................ 5-447
Software ................................................................................................................... 5-447
Hardware.................................................................................................................. 5-449
5.21
System Configuration Tool......................................................................................... 5-450
5.22
Courses on How to Operate the System in an Optimized Way .................................... 5-451
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5-9
OPERATION IN EXPERT MODE
Contents
Carl Zeiss
5-10
LSM-FCS
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OPERATION IN EXPERT MODE
Purpose of this Section and other Operating Manuals
Software
LSM-FCS
5
OPERATION IN EXPERT MODE
5.1
Purpose of this Section and other Operating Manuals
Carl Zeiss
This section describes the operation of the LSM-FCS Combination System exemplified by typical
applications in conjunction with the LSM-FCS software and its graphic user environment.
When starting up and operating the microscope system, mind the operating instruction manual for the
Axiovert 200 M microscope:
–
B 40-080 e
5.1.1
Axiovert 200 M, Operating Manual
Software
The LSM-FCS software, Version 3.2, controls the microscope, the scanning and laser modules, tools
(filters, stand, CLM) and the image acquisition process, and displays and analyzes the images. It is based
on the network-capable graphic 32-bit Microsoft ® WINDOWS NT 4.0 operating system and WINDOWS
2000, respectively.
Portions © Copyright 1996, Microsoft Corporation. All rights reserved.
The installation of the software for the LSM-FCS and the basic settings of the equipment
components are carried out by Carl Zeiss service staff. This job includes the creation of a
customized software configuration in line with the specific hardware components of the
customer's microscope system.
The LSM-FCS software is menu-controlled and normally uses its own windows for the activation of the
various functions; within these windows, further submenus (panels) can be displayed and removed.
Images of the specimens to be examined, created by scanning, are displayed in separate Image Display
windows.
Theoretically, the number of simultaneously opened windows for software operation or image display is
unlimited, but should not be too excessive so that an overview is still possible.
Identical functions, e.g. Laser Control, can be performed in several software windows. Changes made
by the software are recorded immediately and are automatically transferred to all the other windows
concerned.
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5-11
OPERATION IN EXPERT MODE
Purpose of this Section and other Operating Manuals
Windows and Window Elements
Carl Zeiss
5.1.2
LSM-FCS
Windows and Window Elements
Window element
Description / Explanation
Window (e.g.: Laser Control window)
– Window displayed after activation of a function
button (e.g.: Laser button in the toolbar of the
Expert Mode).
Panel (e.g.: Argon panel)
– Limited function range within a window
List box or selection box
– Selection of one of the displayed options at a click of
the mouse.
–
Open the box by clicking on the arrow button.
Input box
– Input of text or numeric values via the keyboard.
Scrollbar with slider
– Setting of numbers in the relevant input box by
moving the slider or clicking on the arrow buttons or
clicking on the slider and moving via the arrow keys
of the keyboard.
Press the Shift or Ctrl key while clicking on the
arrow button to change the numeric values in coarse
or fine steps.
5-12
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OPERATION IN EXPERT MODE
Purpose of this Section and other Operating Manuals
Convention for the Text in this Manual
LSM-FCS
Window element
Carl Zeiss
Description / Explanation
Check box
– Activates / deactivates setting options.
Button
– Selection / performance of a function via mouse
click.
5.1.3
Convention for the Text in this Manual
All the originally used terms of the software interface, e.g.
–
names of windows,
–
panels,
–
input boxes,
–
list / selection boxes,
–
check boxes,
–
menu items,
–
names of buttons and
–
keyboard keys,
are displayed in bold letters to allow easier identification.
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5-13
OPERATION IN EXPERT MODE
Purpose of this Section and other Operating Manuals
Backup
Carl Zeiss
5.1.4
LSM-FCS
Backup
System backup
–
A complete backup is contained on the enclosed optical disk.
User files backup
The following user-generated files need to be included in a backup procedure (keep directory structure):
–
Image database files: *.mdb (but not system_configuration_*.mdb
–
LSM Image files: *.lsm
–
Exported images: *.* (*.Tiff, *.LSM-Tiff, *.BMP, ...)
–
Palette files: AIM \ Palette \ *.lut
–
Filter files: AIM \ Filter \ *.krn
–
Pinhole setting files: AIM \ PH*.pos
–
Log files: AIM \ *.log
The following files generated during the system integration should also be included in a backup
procedure:
–
Parameter file for pinhole setting: AIM \ *.set
–
Parameter file after pinhole adjustment: AIM \ *.adj
–
Scanner files: AIM \ bin \ *.bin
–
Microscope stand database: AIM \ database \ system_configuration_*.mdb
5.1.5
Software Operation
The LSM-FCS software can be operated using the mouse, the PC keyboard, or both.
The operation of the mouse and the keyboard is identical to that of the Microsoft ® WINDOWS
operating system and is therefore not dealt with in detail in this manual.
If required, see the Microsoft manual or online help for relevant information.
5-14
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OPERATION IN EXPERT MODE
Switching on the System
LSM-FCS
5.2
Carl Zeiss
Switching on the System
The LSM system is turned on with the REMOTE CONTROL switch. This switches all the system
components on except for the "Enterprise" UV laser.
If the UV laser shall be used, it can be switched on after the start of the WINDOWS ® NT operating
system - but must always be switched on before the LSM-FCS software is started.
If REMOTE CONTROL switch is not used, turn the system on with the "I" button on the laser module;
in addition, the jumper plug supplied must be connected at the POWER REMOTE CONTROL terminal.
• Turn the REMOTE CONTROL switch to "ON"
position (see Fig. 5-1).
–
This switches the entire system on.
–
Microscope and laser will be ready for
operation after a short time.
–
Computer boots up.
–
Computer hardware system test runs.
Drive "A" of the computer must not
contain a diskette.
The monitor shows a dialog box for selection of
the operating system version.
Fig. 5-2
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Fig. 5-1
REMOTE CONTROL switch
Selecting the operating system version
5-15
OPERATION IN EXPERT MODE
Switching on the System
Log on to WINDOWS NT
Carl Zeiss
LSM-FCS
• Confirm the default setting of the "Windows
NT Workstation Version 4.00" by pressing the
Enter key.
Fig. 5-3
–
WINDOWS NT operating system is being
loaded.
–
The Begin Logon window appears on the
screen.
Begin Logon window
5.2.1
Log on to WINDOWS NT
• Press the three keys Ctrl, Alt and Del at the
same time.
–
The Logon Information window appears
on the screen, permitting you to log on to
the WINDOWS NT 4.0 operating system.
• Enter the valid user name into the User name
text box.
Fig. 5-4
5-16
Logon Information window
• Enter your password into the Password text
box.
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OPERATION IN EXPERT MODE
Switching on the System
Log on to WINDOWS NT
LSM-FCS
Carl Zeiss
• After entries, confirm by clicking the OK button or Enter.
–
The WINDOWS NT operating system desktop appears on the screen, showing a number of icons.
Change Filters icon
Stand Select icon
LSM-FCS icon
LSM-5 Image Examiner icon
LSM-5 Image Browser icon
Fig. 5-5
WINDOWS NT operating system desktop
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5-17
Carl Zeiss
OPERATION IN EXPERT MODE
Switching on the System
Switching on the Enterprise UV Laser
5.2.2
LSM-FCS
Switching on the Enterprise UV
Laser
• If the UV laser is required, switch it on via the
toggle switch (5-6/1) of the power supply.
–
It will be ready for operation after a few
seconds.
Only for LSM.
Fig. 5-6
Power supply of UV-Ar laser
5.2.3
Fig. 5-7
Starting the LSM-FCS program
Starting the LSM-FCS Program
The LSM-FCS software program can be operated
in two different modes (with or without connected
instrument system). In the on-line mode, the entire
program package (image recording and analysis) is
available, while only a part of the software
functions (image analysis only of already stored
images) and no hardware functions are available in
the off-line mode. Of course, the off-line mode
can also be started when the instrument system is
connected. In that case, it is not necessary that the
lasers and the microscope are switched on.
• Double-click on the LSM-FCS icon on the desktop of WINDOWS to start the LSM-FCS software
program (see Fig. 5-5).
–
5-18
The LSM-FCS Switchboard menu appears on the screen (see Fig. 5-8).
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OPERATION IN EXPERT MODE
Switching on the System
Starting the LSM-FCS Program
LSM-FCS
Fig. 5-8
Carl Zeiss
LSM-FCS Switchboard menu
The LSM-FCS Switchboard menu presents the following items for selection:
–
Scan New Images
Clicking on this button activates the complete LSM hardware (on-line mode).
–
Use Existing Images
This item allows you to process and analyze previously acquired images with the LSM-FCS software. In
this mode, control of the hardware (laser module ...) is not possible (off-line mode).
Please note that the Scan New Images button must be activated before setting up the Routine
Mode or the Expert Mode. Otherwise, the hardware can not be controlled by the LSM-FCS
software.
–
Start Routine Mode
Click on this button if you want to work with pre-configured system settings (typical applications).
In the Routine mode, the FCS measurement functions are not available.
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5-19
OPERATION IN EXPERT MODE
Switching on the System
Starting the LSM-FCS Program
Carl Zeiss
–
LSM-FCS
Start Expert Mode
Use of this mode requires to be thoroughly familiar with the exact microscope procedures and
interrelations.
You need to set all parameters and functions upon your own decision; this mode therefore provides
you with the greatest flexibility of operation.
It is also possible, however, to call up stored configurations and to modify the parameters / settings if
necessary.
After the start of the Expert Mode or the
Routine Mode, instrument initialization is
performed and can be monitored in the
Initialization window and interrupted with a click
on the Cancel button, if required.
Fig. 5-9
OFF LINE Initialization window
Depending on the selected option (Scan New
Images or Use existing Images), initialization is
performed in the offline or online mode.
Existing images can only be loaded and processed in the Expert Mode.
If you want to change from the Expert Mode to the Routine Mode and vice versa, close all
the windows first.
Some printers (for example KODAK Thermo Printer) will produce an error message "hard key
not found" in case the printer is not switched on.
Remedy: turn on the printer before starting the LSM-FCS software.
Don’t switch off the KODAK printer during the scanning process.
5-20
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OPERATION IN EXPERT MODE
Main Menu
Starting the LSM-FCS Program
LSM-FCS
5.3
Carl Zeiss
Main Menu
• Click on the Start Expert Mode button.
–
The LSM-FCS - Expert Mode Main menu
appears on the screen.
The File button is active automatically, and the
submenus selectable in it are shown in the second
(bottom) toolbar.
Fig. 5-10
LSM-FCS Switchboard menu
Main menu (pull-down)
Main menu toolbar
Subordinate toolbar
(File button activated)
Fig. 5-11
LSM-FCS - Expert Mode Main menu
The major functions can be selected in the Main menu of the Expert Mode either via the pull-down
menus in the menu bar or via a toolbar which can be displayed or removed as required.
Further subordinate toolbars are available below this toolbar, depending on which button has just been
pressed (File, Acquire, etc.).
In the standard setting of the LSM-FCS software, the toolbars are automatically displayed after the start
of the Expert Mode. However, display of the toolbars can be deactivated via the Window pull-down
menu (see Toolbar, page 5-273).
However, since the LSM-FCS software is operated more conveniently with the help of the toolbars, only
this method of function activation will be described in the following.
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5-21
Carl Zeiss
OPERATION IN EXPERT MODE
Main Menu
Starting the LSM-FCS Program
LSM-FCS
The buttons of the Main menu (upper toolbar) have the following meanings:
File button
Open, save, import and export of image data or FCS measurement results.
Printing individual or several images on one page. Ending (Exit) the Expert Mode.
Acquire button
Calling up and setting the necessary operating parameters. During the preparation
for and execution of laser scan image acquisition, this menu item is used as the
working dialog between the computer and the microscope.
Process button
Used for processing of acquired images.
3D View button
Used for three-dimensional reconstruction.
Analyse FCS button Selecting measurement method and carrier for FCS measurements. Executing
measurement and data analysis. Optimizing measurement method.
Config FCS button
Calling up and setting the necessary operating parameters for FCS measurements.
During the preparation for and execution of measurements via laser excitation.
This menu item is used as the working dialog between the computer and the
microscope.
Macro button
Makes it possible for the user to store frequently used processes (Macro recorder)
and to run them automatically (Macro play). It is possible to write new macros or
to edit existing ones.
Options button
For custom-configuration of software and hardware options, and for exporting
system operating sequences to the Routine Mode. This menu item enables access
to the coloring table. In the Settings for User window you can specify essential
operating modes and informative help, organized by tabs, which have an effect
on the user interface.
Maintain button
Service mode for adjustment and setting of other parameters (e.g. objectives).
5-22
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OPERATION IN EXPERT MODE
File Menu
Create New Image Database
LSM-FCS
5.4
Carl Zeiss
File Menu
The functions of the File menu permit images and the relevant information to be managed and handled
completely in a database system. You can also create your own databases. The databases allow images
to be stored, loaded and deleted. The additional functions Import and Export permit images from other
systems to be made available to the LSM 5 software, or the export of images to other software
packages. The Print function allows individual or several images to be arranged on a print page for
printout. The Expert Mode can be ended via the Exit function.
• In the Main menu toolbar, click on File.
–
This opens another, subordinate toolbar in the Main menu.
File subordinate
toolbar
Fig. 5-12
File menu
5.4.1
Create New Image Database
The New function permits the creation of a new
image database.
• Click on the New button in the
subordinate toolbar of the Main menu.
–
File
This opens the Create New Database
window for the selection of drives,
directories and subdirectories.
• Enter the name of the image database you
want to create in the File name text box, e. g.
Convallaria.
Fig. 5-13
Create New Database window
• If you want to create the image database in a certain folder (drive / directory), click on the arrow
button next to the Create in box.
–
This opens a drop-down list box showing all folders available for selection.
To move up one layer of folders, click on the
• If you want to create a new folder, click on the
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button. Cancel allows you to stop the process.
button.
5-23
OPERATION IN EXPERT MODE
File Menu
Create New Image Database
Carl Zeiss
LSM-FCS
• Click on the Create button.
–
This creates the new image database in the selected drive and directory.
–
The database files of the LSM-FCS software have the filename extension *.mdb.
The Convallaria.mdb window appears, presenting the opened image database with 0 of 0 image
entries.
Fig. 5-14
New Database window
The new image database can be used to store an acquired or processed image (see Saving an Image,
page 5-36).
The start settings and the extent of the parameters displayed in the image database window
can be changed as required via the Settings function in the Options subordinate toolbar (see
Settings Function, page 5-241).
5-24
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OPERATION IN EXPERT MODE
File Menu
Open Existing Image Database
LSM-FCS
5.4.2
Carl Zeiss
Open Existing Image Database
The Open function allows one or several
databases to be opened. The images stored in the
database(s) are displayed in thumbnail form; they
can be selected and loaded into the Image
Display window (see page 5-275).
• Click on the Open button in the File
subordinate toolbar of the Main menu.
–
This opens the Open Database window for
selection of image databases.
Fig. 5-15
Open Database window
• If you want to load an image database from another folder (drive / directory), click on the arrow
button to the right of the Look in box.
–
This opens a drop-down list box in which you can select from all available folders.
The window displays the various image databases with the file extension *.mdb.
• Open the image database by a double click on the respective key icon (e.g. Test-Rel-3_0.mdb), or
click on the name of the image database for selection and open it by clicking on the Open button.
–
This opens the image database window, e.g. Test-Rel-3_0.mdb - AIM, in which you can select
from a variety of options.
• Click on the
B 45-0006 e
button in the title bar of the Database window (see Fig. 5-16) to close this window.
12/02
5-25
OPERATION IN EXPERT MODE
File Menu
Image Database window
Carl Zeiss
5.4.3
LSM-FCS
Image Database window
The Image Database window allows you to choose one of three different display modes:
–
Form
–
Gallery
–
Table
To choose the required mode, activate the relevant button on the right-hand side of the Image
Database window. Loading of images into the Image Display window is possible in every display
mode.
The buttons on the right have the following functions:
Form button
Show image database in form display mode.
Gallery button
Show image database in gallery display mode.
Table button
Show image database in table display mode.
Load button
Load image and parameter from image database to Image Display window.
Subset button
Load image and parameter with size reduction from image database to Image
Display window.
Reuse button
Reuse scan parameters of the selected image without loading the image.
Refresh button
Refresh Image Database window.
Copy button
Copy selected images to clipboard.
Paste button
Paste images from clipboard into image database.
Filter button
Select or edit search filter from image in the image database.
On Filter button
Switch search filter on or off.
Delete button
Delete selected images from the image database.
The status line, which displays the following current parameters, is at the bottom of the Image
Database window:
Total: ...
Display of storage volume of the entire image database
Selected: ...
Display of storage volume of the selected image(s)
Current: ...
Display of storage volume of the current image
Clipboard: ...
Display of storage volume of the image(s) contained in the clipboard
5-26
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OPERATION IN EXPERT MODE
File Menu
Image Database window
LSM-FCS
5.4.3.1
Carl Zeiss
Form display mode
When a image database is opened, the Form display mode is used, if no other settings were made under
Settings in the Options subordinate toolbar.
Fig. 5-16
Image Database window (Form display mode)
In the Options menu in the function Settings it is possible to define
– the start mode of the image database (Form, Gallery, Table)
– the Recordset displayed (first, last, middle) and
– the parameters shown.
The number of the image currently displayed from a set of images is indicated in the Recordset text box.
• From the image database you can display images using the recording slider, or in one of the following
ways:
show the next image
show the previous image
show the last image of the image database
show the first image of the image database
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5-27
OPERATION IN EXPERT MODE
File Menu
Image Database window
Carl Zeiss
LSM-FCS
The currently selected image is displayed as thumbnail in the Image Display window on the right. In the
case of Z Stacks or time series, the Slice slider appears on the screen beside the Image Display window.
• You can browse through the series by dragging the Slice slider using the mouse.
• Click on the Load button to open the selected
image.
A double-click on the Image Display
window of the database will also load the
selected image.
For a description of the toolbars Select
and Display see Display and Analysis
of Images, page 5-275).
Fig. 5-17
Opened image
The name of the image is displayed in the Name input box of the Image Database window.
• If you want to change the name, click on the input box and enter the new name directly via the
keyboard.
• The Description and Notes input boxes allow you to subsequently add descriptions or special notes
on the recorded image via the keyboard.
The acquisition parameter settings of the image are displayed in the Acquisition panel.
Changes to an original scan image are automatically recorded in the Processing panel under History. If,
for example, the image was added to the database via the clipboard, the entry Imported file will be
shown under History.
Under Image Size, the size of the image in pixels for XY(Z) and the number of used channels are
displayed.
5-28
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OPERATION IN EXPERT MODE
File Menu
Image Database window
LSM-FCS
5.4.3.2
Carl Zeiss
Gallery display mode
• Click on the Gallery button. All images of the image database, e.g. Test-Rel3_0.mdb, (image series)
are shown in a tiled arrangement of thumbnails on the screen.
Fig. 5-18
Database window (Gallery display mode)
In the Options menu in the function Settings it is possible to define
– the start mode of the image database (Form, Gallery, Table)
– the recordset displayed (first, last, middle) and
– the parameters shown.
• To select one of the images of the database for normal-size presentation, double-click on the desired
image. The same can be achieved by clicking on the desired image in the gallery and then clicking on
the Load button.
• To select several single images press & hold down the Ctrl-key and select each desired image by a
click of the mouse. If several images have been selected, they will all be opened and displayed one
after the other.
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OPERATION IN EXPERT MODE
File Menu
Image Database window
Carl Zeiss
LSM-FCS
• To select a number of consecutive images press & hold down the Shift-key, click on the first and the
last image to be selected. All the images between these two will also be included in the selection. If
several images have been selected, they will all be opened and displayed one after the other.
Selected images are highlighted in blue. Furthermore, the current image selected last receives a
patterned frame.
5.4.3.3
Table display mode
• Click on the Table button.
–
All images of the image database, e.g. Test-Rel3_0.mdb, (image series) are shown in Table
display mode on the screen.
Fig. 5-19
Database window (Table display mode)
In the Options menu in the function Settings it is possible to define
– the start mode of the image database (Form, Gallery, Table)
– the recordset displayed (first, last, middle) and
– the parameters shown.
• To select one of the images of the database for normal-size presentation, double-click on the desired
line. The same can be achieved by clicking on the desired image in the table and then clicking on the
Load button. If several images have been selected, they will all be opened and displayed one after the
other.
The selection of several images is performed in the same way as in the Gallery mode, i.e. by pressing
the Shift and Ctrl keys.
5.4.3.4
Load function
• Click on the Load button to load the selected images into the Image Display window.
5-30
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OPERATION IN EXPERT MODE
File Menu
Image Database window
LSM-FCS
5.4.3.5
Carl Zeiss
Subset function
The Subset function allows images to be loaded
with reduced resolution. For this purpose, the
image pixels in XY(Z) are reduced. It is also possible
to reduce the number of slices (in stacks and time
series).
• Click on the Subset button to open the Load
with reduction in size window.
• Enter one value each for n under Pixel (x and
y), Pixel (z) and Stack (Time) in the Load
every nth panel.
• If required, turn on the
Fig. 5-20
Load with reduction in size window
Load 12 bit as 8 bit check box.
• Click on the Load button to load the selected images with reduction in size, time slices and stack
slices.
• Use Cancel to exit the window without any selection.
5.4.3.6
Refresh function
• Click on the Refresh button to update the Image Database window.
5.4.3.7
Copy / Paste function
• Select the image(s) to be copied. You can use the Shift and Ctrl keys for multiple selection.
• Click on the Copy button.
–
The image(s) is(are) copied to the clipboard and can be inserted in either the same or another
database or in other software packages.
• Click on the Paste button to insert the image in the current database.
Identical to the WINDOWS function "Drag & Drop", one or several images can be copied or moved from
one database to another.
The Form mode allows only one image to be copied or moved. The Gallery and Table modes permit
several images to be copied or moved simultaneously by multiple selection (keeping the Shift key pressed
on clicking).
• Open the relevant databases and position both windows side to side.
• Select the required images (multiple selection by keeping the Shift key pressed) from one database.
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OPERATION IN EXPERT MODE
File Menu
Image Database window
Carl Zeiss
LSM-FCS
• Click on a selected image and keep the mouse button pressed, move the mouse button to the
window of the other database (a small rectangular appears near the mouse button) and release the
mouse button again (Drag & Drop).
The images are now being moved to the other image database, i.e. they are deleted from the first image
database and are then only available in the second image database.
• If the images shall only be copied, also press the Ctrl key during the Drag & Drop procedure (in
addition to the rectangular, a "+" sign will also appear near the mouse button).
The images will then be available in both image databases.
5.4.3.8
Filter function
The Filter function permits the display of the
database to be modified in such a way that only
images with certain features are displayed. This
requires the definition and following activation of
a relevant filter. Defined filters can be stored,
reloaded and also deleted.
Fig. 5-21
5-32
Filter window
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OPERATION IN EXPERT MODE
File Menu
Image Database window
LSM-FCS
Carl Zeiss
Edit panel
The following features can be used as filter functions under Field:
Name
Words or row of letters from the name of the image
Description
Words or row of letters from the description of the image
Scan Mode
Scan Mode in which the image was created: Stack or Plane
Date / Time
Date / Time of image acquisition
# Planes (z)
Pixel size of the image in the Z-direction (e.g.: 10)
# Lines (y)
Pixel size of the image in the Y-direction (e.g.: 512)
Samples (x)
Pixel size of the image in the X-direction (e.g.: 512)
Z-Step
Distance of Z Slices in a Z Stack in µm
User
Name of the user as entered in the WINDOWS NT login
Time series
Selection of time series
• Open the Field list box and select the filter feature (e.g.: Scan Mode).
The following operator signs can be activated under Operator:
=
>
<
>=
<=
<>
equals
larger
smaller
larger, equals
smaller, equals
smaller, larger
• Select the relevant operator sign (e.g.: =) from the Operator list box.
The relevant value or a combination of characters for the filter function (Field) is entered under Value
via the keyboard:
• Enter the relevant text or value (e.g.: Stack).
• Click on Add. The defined filter feature is displayed in the work box of the Edit panel and is therefore
activated (e.g.: Scan Mode = stack).
If a further filter feature is to be linked with the already defined one, proceed as follows:
• Activate the relevant entries under Field and Operator and enter a value or text (e.g.: Name =
Convallaria) under Value.
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OPERATION IN EXPERT MODE
File Menu
Image Database window
Carl Zeiss
LSM-FCS
If groups of letters shall be searched, the * sign can be entered for undefined letters (e.g.: if you
search for the letter row Conv, enter Conv*).
• Activate the required link type And, Or or Not with a click of the mouse (e.g.: And).
• Click on the Add button. The created filter feature is added to the work box of the Edit panel (e.g.:
AND Name = Convallaria).
The Modify button enables you to edit an already defined filter feature:
• Activate the required feature on the work box.
• Make the necessary changes under Field, Operator and Value. Select the link type And, Or or Not.
• Click on Modify. The filter feature will be changed accordingly.
The Delete button enables you to delete a defined filter feature:
• Activate the required feature in the work box.
• Click on Delete. The filter feature will be deleted from the work box.
• Clicking on OK will activate the filter (the entire set of filter features) displayed in the work box and
close the Filter window. On Filter is activated right on in the Database window and the filter
function will be performed. Only those images which fulfill with the defined filter features will then be
displayed. The procedure is interrupted via Cancel.
If required, the filter features displayed in the work
box can be stored.
• Click on the Add to List button. The Add to
List window will be opened.
• Enter a name for the filter and click on OK. The
filter will be included in the Filter List panel.
Fig. 5-22
5-34
Add Filter to List window
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OPERATION IN EXPERT MODE
File Menu
Image Database window
LSM-FCS
Carl Zeiss
Filter List panel
All the stored filters are displayed in the Filter List
panel and can be activated any time at a click of
the mouse.
• Click on the name of the required filter. The
linked filter features will be displayed in the
work box.
Fig. 5-23
Filter List panel
Filters which are no longer needed can be deleted.
• Click on the filter to be deleted in the Filter List panel.
• Click on Remove from List. The filter will be removed.
5.4.3.9
On Filter function
The On Filter function is a toggle switch to activate or deactivate selected filter settings.
5.4.3.10
Delete function
• Select the images to be deleted from the image database.
• Click on the Delete button. Confirm the safety inquiry then displayed by pressing OK.
–
The images and the acquisition parameters will be removed from the image database.
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OPERATION IN EXPERT MODE
File Menu
Save an Image to the Image Database
Carl Zeiss
5.4.4
LSM-FCS
Save an Image to the Image Database
The Save function allows to store an image together with the acquisition parameters (and processing
information) to be stored in an image database.
In the Options menu in the function Settings it is possible to define an Autosave function. When
Autosave is off, the Save dialogue is the Save As dialogue.
Proceed as follows to save an acquired or an edited / processed image:
• Click on the Save or Save As button in the File subordinate toolbar of the Main menu.
–
The Save Image and Parameter As window appears on the screen.
Save
Stores a newly created or changed image. Newly created images must be given a name and
assigned to an existing or new database.
Save As
Stores a previously stored and called up image under a different name. If images are called up
and stored again, the original data and time display will be retained.
Clicking on either of these buttons opens the Save As window to create and open an image
database.
When the Compress Files
form.
check box is activated, the images are stored in a compressed
• If necessary, enter a description of the image or comments on it in the appropriate text boxes.
• The default display in the User text box is the name of the logged-on user. If you want, you can enter
a different user name for the current image.
• Click on the Open MDB button if you want to open an existing image database in which you want to
save the current image. Click on the New MDB button if you want to create a new database to save
the current image.
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OPERATION IN EXPERT MODE
File Menu
Save an Image to the Image Database
LSM-FCS
Fig. 5-24
Carl Zeiss
Save Image and Parameter As window
• Enter the name of the image in the Name text box, e.g. Conv-7.
• Click on the New MDB button.
–
This opens the Create New Database window in which you can create a new image database.
• Enter the name of the database you want to create in the File name text box, e.g. Projections.
• If you want to create the image database in a certain folder (drive / directory), click on the arrow
button next to the Create in box.
–
This opens a drop-down list box showing all folders available for selection.
• After selection, click on the Create button.
–
This creates the image database in the selected drive and directory.
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OPERATION IN EXPERT MODE
File Menu
Save an Image to the Image Database
Carl Zeiss
Fig. 5-25
LSM-FCS
Save Image and Parameter As window and
Create New Database window
• The Projections.mdb - AIM window appears.
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OPERATION IN EXPERT MODE
File Menu
Save an Image to the Image Database
LSM-FCS
Fig. 5-26
Carl Zeiss
Database window
• Click on the OK button in the Save Image and Parameter As window.
–
The Projections.mdb - AIM window now shows the saved image.
–
The Recordset box indicates the current number of the image in the image series contained in this
database.
• In the Description text box you can enter, for example, the configuration of the image.
• In the Notes text box you can enter further information about the image content.
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OPERATION IN EXPERT MODE
File Menu
Import of Images
Carl Zeiss
5.4.5
LSM-FCS
Import of Images
The Import function enables the import of externally created images into the Image Display window
and the image database of the LSM-FCS software.
• Click on the Import button in the File subordinate toolbar of the Main menu.
–
This opens the Import Images window.
Fig. 5-27
Import Images window
• Select the data medium and the directory where the relevant image is contained in the Look in
selection box.
• Select the image file by clicking on it.
–
The selected image will be shown for checking in the Image Display window (on the left)
together with the relevant details (size, channels, storage volume).
• Select the image type (Single Image or Image Series) in the Image type selection box.
• Click on Open.
–
The image is displayed in a new Image Display window.
All the usual image and movie formats (e.g. .tif, .jpg, .bmp, .pcx, .avi, .mov etc.) are supported.
When importing series, please make sure to select the first image for the representation of the
entire series and to select the Image Series option under Image type.
• Finally, save the image in the desired image database via the Save As function.
• In Processing History this file is marked as imported file.
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OPERATION IN EXPERT MODE
File Menu
Export of Images
LSM-FCS
5.4.6
Carl Zeiss
Export of Images
The Export function allows the export of acquired images and images loaded from the image database.
• Select the image to be exported.
• Click on the Export button in the File subordinate toolbar of the Main menu.
–
This opens the Export Images and Data window.
• Under Save in, select the data medium and the
directory to which the image is to be exported.
• Enter a name for the image under File name.
• Select the image format into which the image is
to be exported under Image type (Single
Image with raw data, Contents of the
Image Display window, Full resolution).
• Click on the Save button.
–
The image is stored on the relevant data
medium / directory.
All the usual image and movie formats (e.g. .tif,
.jpg, .bmp, .pcx, .avi, .mov etc.) are supported.
Fig. 5-28
Export Images and Data window
When stacks or time series are exported, each frame is stored as an individual image.
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OPERATION IN EXPERT MODE
File Menu
Multi Print
Carl Zeiss
5.4.7
LSM-FCS
Multi Print
This function permits you to arrange several
images on one print page and to print them out
together.
• Click on the Multi Print button in the File
subordinate toolbar of the Main menu.
–
This opens the Print ... window.
The main area of the Print ... window is used for
the display of the print page in the selected paper
orientation and for the arrangement of the images
to be printed.
The Print toolbar with the following buttons is
displayed on the right-hand side of the window:
Fig. 5-29
Print - AIM window
Paste button
Paste from
sheet.
Delete button
Delete marked image.
Print button
Start printing.
Setup button
Printer setup.
Landsc. button
Landscape paper orientation.
Portrait button
Portrait paper orientation.
Zoom slider
Zoom function for page preview.
clipboard
to
The following functions can be performed on activation of the buttons in the Overlay toolbar (left-hand
side):
Arrow (selection) button: Activation of the mouse button for selection, resizing or
movement of an overlay element in the Image Display window.
Resizing: Click on the handle and hold down the mouse button, drag the handle, release the
mouse button.
Movement: Click on the line and hold down the mouse button, move the entire element,
release the mouse button.
Line button: Creation of a straight line in the Image Display window.
Click and hold down the mouse button, draw a line in any required direction, release the
mouse button to end the procedure.
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File Menu
Multi Print
LSM-FCS
Carl Zeiss
Rectangle button: Creation of a rectangle in the Image Display window.
Click and hold down the mouse button, draw a rectangle in any required direction, release
the mouse button to end the procedure.
Closed polyline button: Creation of a closed polyline figure in the Image Display window.
The first click sets the starting point, each additional click adds a further line, a click with the
right mouse button closes the figure and ends the procedure.
Open polyline button: Creation of an open polyline figure in the Image Display window.
The first click sets the starting point, each additional click adds a further line, a click with the
right mouse button ends the procedure.
Ellipse button: Creation of an ellipse in the Image Display window.
The first click sets the center point, the displayed line permits the determination of the first
dimension, the second click sets the first dimension, the second dimension and rotation
direction can then be determined, the third click sets the second dimension and direction
and ends the procedure.
Closed free-shape curve button: Creation of a closed Bezier figure in the Image Display
window.
The first click sets the starting point, each additional click adds a further line, a click with the
right mouse button closes the figure and ends the procedure.
Open free-shape curve button: Creation of an open Bezier figure in the Image Display
window.
The first click sets the starting point, each additional click adds a further line, a click with the
right mouse button closes the figure and ends the procedure.
Circle button: Creation of a circle in the Image Display window.
Clicking and holding down the mouse button sets the center point, drag the diameter and
release the mouse button to end the procedure.
Line with arrow button: Creation of a line with arrow in the Image Display window.
Click and hold down the mouse button, drag the line in any required direction, release the
mouse button to end the procedure.
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Carl Zeiss
OPERATION IN EXPERT MODE
File Menu
Multi Print
LSM-FCS
A (Text) button: Creation of a text box in the Image Display window.
After clicking on A, the Text window will be displayed, and text can be entered via the
keyboard. The Font ... button enables you to select the font style and size in the Font
window. The entered text will be displayed in the left upper corner of the Image Display
window after clicking on OK and can be moved to the required position using the mouse.
The Text window can also be activated with a double-click on a created text box, and the
entered text can be edited subsequently.
Recycle bin button: All the overlay elements and dimensions dragged to the scanned image
are deleted. If one overlay element was marked before, this element is now deleted from the
scanned image.
Line button:
This button allows you to determine the line thickness of the area outline.
Color button: After clicking the Color button, the Color selection box will be opened. The
colors displayed in the Color selection box can be assigned to the overlay elements with a
click of the mouse. The currently selected color is displayed in the Color button. A selected
color is automatically assigned to the currently selected overlay element and then to all the
elements created afterwards.
To print out several images on one page, proceed as follows:
• Use the Overlay functions to additionally illustrate the graphics and images to be printed.
• Select the paper orientation by clicking on the Landsc. or Portrait button.
• Open the image to be printed or select it from the relevant image database.
• Click on the Copy button. The image is copied to the clipboard.
• In the Print - AIM window, click on the Paste button.
The copied image appears in the work area of the Print - AIM window. You can click on it with the
mouse and move it to any position on the print page or you can adapt the image size.
• Proceed in the same way with all other images you want to print out.
• Finally, arrange all images on the print page as required.
• Click on the Print button to start the printout.
• Close the Print - AIM window by clicking on the
5-44
button.
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OPERATION IN EXPERT MODE
File Menu
Open FCS - Load a FCS data file
LSM-FCS
5.4.8
Carl Zeiss
Open FCS - Load a FCS data file
This function is intended for use of already
measured data in the FCS mode. It also allows you
to import data stored with the ConfoCor or the
ConfoCor 2.
• To open previously stored data, click on the
Open FCS button in the File subordinate
toolbar of the Main menu.
–
This opens the Evaluate window for the
selection of drives, directories and
subdirectories in which data files have been
stored.
Fig. 5-30
Evaluate window
• If you want to load a data file in another folder (drive / directory), click on the arrow button to the
right of the Look in box.
–
This opens a drop-down list box in which you can select from all available folders.
• Select the appropriate data file(s) via mouse click and click on the Open button.
–
This opens the FCS data file in a separate window (see Fig. 5-31).
Fig. 5-31
FCS data file window
When the files have been opened, the data display window as described in the beginning of chapter
5.17, Data Evaluation and Result Presentation for FCS Measurements, will be shown. It offers the
same possibilities as described in chapter 5.17, page 5-399.
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OPERATION IN EXPERT MODE
Acquire Menu
Exit the Expert Mode
Carl Zeiss
5.4.9
LSM-FCS
Exit the Expert Mode
• Make sure to save all required images in the image database or export them.
• Close all open windows of the LSM-FCS program by clicking on the closing icon
corner of each window.
in the top right
• Click on the Exit button in the File subordinate toolbar of the Main menu.
–
The LSM-FCS - Expert Mode Main menu will be closed and the LSM-FCS Switchboard menu
appears on the screen.
5.5
Acquire Menu
• In the Main menu toolbar, click on Acquire.
–
This opens another, subordinate toolbar in the Main menu.
Acquire subordinate toolbar
Fig. 5-32
Acquire menu
For preparing and acquiring a scanning image, it is recommended to call up and use the tools of the
subordinate toolbar in the following order:
Conventional microscope setting.
– Laser setting.
– Configuring the optical system for the Scanning Mode.
– Setting of scan parameters.
– EditROI permits up to 99 regions within a frame to be defined and scanned.
– TimeSeries permits user-specific time series to be selected for the scan procedure.
– The EditBleach function is used to bleach a defined, freely selectable area within the scanning field.
– Upon selecting Stage you can set the focus (Z coordinate) and the Z step size between successive
slices. If the optional, motorized X/Y-stage is connected, the X and Y-positions of the sample can also
be selected.
– The VIS, FCS and LSM buttons switch the beam path and indicate which beam path has been set in
the binocular tube of the microscope (VIS for viewing, FCS for FCS measurements via laser excitation
and monitor observation, LSM for laser scanning operation with monitor observation).
For the scanning process, the LSM button in the toolbar subordinate to the Acquire item must be
activated.
–
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OPERATION IN EXPERT MODE
Acquire Menu
Laser Control
LSM-FCS
5.5.1
Carl Zeiss
Laser Control
The Lasers panel shows the types, excitation
wavelengths and operating status of all lasers
available.
The subordinate laser settings panel shows the
relevant and currently set Maximum Power,
Wavelength, Status, Tube Current and
Output [%] values of the current laser. The
buttons On, Off and Standby permit the current
laser to be set in the required status, and the laser
intensity (Output) can be set using the slider or
the input box. The name of the selected laser
(Enterprise, Argon, HeNe1 or HeNe2) is displayed
in the headline of this setting panel for checking.
5.5.1.1
Fig. 5-33
Laser Control window
Opening / Closing the Laser Control window
• Click on the Laser button in the Acquire subordinate toolbar.
–
This opens the Laser Control window, which shows all lasers connected to the system.
When the setting of the required lasers has been finished, the Laser Control window can be closed
again.
• Click on the Close button to close the Laser Control window.
–
The Laser Control window will be closed.
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OPERATION IN EXPERT MODE
Acquire Menu
Laser Control
Carl Zeiss
5.5.1.2
LSM-FCS
Function description
Lasers panel (upper)
List of available lasers, including the display of relevant wavelengths and
switching status.
Selection of the laser to be switched on / off and setting of the laser
output is performed in the subordinate setting panel.
Laser settings panel (lower)
Switch on / off the required laser or set Standby operation.
Display Maximum Power, Wavelength, Status and Tube Current (only
Enterprise and Argon) of the relevant laser.
Set the laser output for Enterprise and Argon.
5.5.1.3
Settings
• Click on the desired laser on the (upper) Lasers panel.
–
This highlights the selected laser.
On the lower panel of the Laser Control window, activate the laser as follows:
This applies to the Coherent UV-laser 653 II (Enterprise) and the Ar-multiline laser:
• Click on the Standby button.
–
Wait for the laser to heat up, until the Status ready - Standby message appears (approx. two
minutes).
• Click on the On button.
–
Status ready - On appears.
• Use the Output [%] slider to set the laser power which is ideal for the measurement job.
Thus, the laser needed for image acquisition is available.
Argon:
Set output between 25 and 100 % of the maximum tube current.
Optimum operation is at 8 A (lowest laser noise). However, the laser life is reduced
if the laser is constantly operated at 8 A. Therefore, 8 A should be used only if this
is absolutely necessary.
Enterprise:
Set output between 50 and 100 % of the maximum tube current.
Optimum operation is at 20 A (Tube Current; lowest noise). However, the laser life
is reduced if the laser is constantly operated at 20 A. Therefore, 20 A should be
used only if this is absolutely necessary.
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Acquire Menu
Laser Control
LSM-FCS
Carl Zeiss
To switch on the Enterprise laser, proceed as follows:
• The internal water cooling LP 5 is running.
• Start the PC, wait until NT system is booted.
• Switch on the power supply of the Enterprise laser, power potentiometer turned to maximum.
• Start the LSM-FCS software.
Please bear in mind that a cooling phase of at least 5 minutes is required between switching off
of the laser via the software and switching off of the entire system via the REMOTE CONTROL
main switch or the Power Supply switch of the Enterprise UV laser.
If the LSM-FCS software is already running and you want to use the UV laser, do the following:
• Close the LSM-FCS software.
• Switch on the power supply of the Enterprise, power potentiometer turned to maximum.
• Start the LSM-FCS software again.
This applies to HeNe Diode lasers:
• After selecting the laser, click on the On button.
–
The required laser for image acquisition is now available.
The Argon, HeNe1 and HeNe2 lasers are available for FCS measurements.
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OPERATION IN EXPERT MODE
Acquire Menu
Microscope Control
Carl Zeiss
5.5.2
LSM-FCS
Microscope Control
The Microscope Control (Micro button) window permits motorized functions (objective and reflector
change and filter settings) and the illumination mode (transmitted or reflected light) of the connected
microscope to be controlled via the software.
Without any difference to software control, these microscope functions can also be operated directly on
the stand via the relevant controls. In that case, any changes are recorded by the software and displayed
in the relevant windows / panels.
5.5.2.1
Open the Microscope Control window
• Click on the Micro button.
–
This opens the Axiovert Control window on the screen (Fig. 5-34).
After conclusion of the conventional setting of the connected microscope, the Axiovert Control
window can be closed again.
• Click on the Close button in the Microscope Control window.
–
5-50
The Axiovert Control window will be closed.
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Microscope Control
LSM-FCS
5.5.2.2
Carl Zeiss
Microscope Control window for Axiovert 200 M
Transmitted Light button
Transmitted light is switched on / off via ON button in the Transmitted
Light frame, setting of light intensity can be varied via input box or
slider. 3200 K color temperature for photo documentation can be
switched on via 3200 K button in the Transmitted Light frame. The
transmission light control potentiometer on the stand is disabled via the
Remote button. By clicking on the Close button the Transmitted Light
frame is closed.
Condensor button
Numerical aperture of the condensor is set via input box or slider. Turret
position selected from graphical pop-up menu (only for motorized
condensors). By clicking on the Close button the Condensor frame is
closed.
Objective button
Objective can be selected via graphical pop-up menu.
Reflector button
Push and click, reflector cube can be selected via graphical pop-up menu.
Tube Lens button
Push and click, tube lens can be selected via graphical pop-up menu.
Reflected Light button
The shutter is switched on and off.
Recording of microscope settings
The upper part of the Axiovert Control window
shows the recording functionality of microscope
configurations.
Complete microscope
created and applied.
configurations
can
be
The Store button permits existing microscope
configurations to be stored under any name.
The Apply button permits existing
microscope configurations to be loaded.
stored
The Delete button permits existing microscope
configurations to be deleted.
The Assign button permits the assignment of a
microscope configuration to a button.
Load a microscope configuration
An existing microscope configuration can be
loaded as follows:
• Click on the arrow button.
–
This opens a list box of all stored microscope
configurations.
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Fig. 5-34
Axiovert Control window
5-51
Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Microscope Control
LSM-FCS
• Browse through the microscope configurations by clicking, or use the scroll bar at the side of the list
box.
• Click on the desired microscope configuration.
–
The selected microscope configuration is shown in the first line of the Microscope
Configurations list box.
• Click on the Apply button.
• Click on the Close button to close the microscope window.
Only those microscope settings which are encoded and motorized can be reloaded.
Store a microscope configuration
A newly created or changed microscope configuration can be stored under a new name as follows:
• Enter the desired name in the first line of the microscope setting list box.
• Click on the Store button.
• The actual configuration with the chosen name is added to the microscope settings list.
• Click on the Close button to close the microscope window.
Delete a microscope configuration
A no longer required microscope configuration can be deleted as follows:
• Select the microscope configuration to be deleted from the microscope configuration list box.
• Click on the Delete button.
• Click on the Close button to close the microscope window.
Assignment of a microscope configuration to a button
A microscope configuration can be assigned to a button as follows:
• Click on the Assign button.
• This opens the Assign-Microscope-Settings-To-Button window.
• Click on the arrow in the Button list box and select a button out of the list.
With increasing numbers the buttons are arranged from the upper to the lower row from lefthand side to right-hand side.
• Click on the arrow in the Settings list box and select a microscope configuration.
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Microscope Control
LSM-FCS
Carl Zeiss
• Click on the Apply button. A new button with the name of the selected microscope configuration
has been created.
• Click on the Close button to close the Assign-Microscope-Settings-To-Button window.
• Click on the Close button to close the microscope window.
For the conventional setting of the Axiovert 200 M, proceed as follows:
• Click on the VIS button in the Acquire subordinate toolbar.
• Place specimen on microscope stage.
–
The cover slip must be facing down.
• In the Objective list box, select the required objective.
• Use the focusing drive (5-35/4) to focus the required specimen plane.
• Select specimen detail by moving the stage in X and Y via the XY stage fine motion control (5-35/3
and 2).
(1)
Transmitted-light observation
• Click on the Reflected Light button and set the shutter to the Closed position.
• Click on the Transmitted light button. Click on the On button in the Transmitted Light panel and
set the transmitted light intensity via the slider or click on 3200 K. Click on Close to close the
Transmitted Light panel.
• Click on the Condensor button and set the aperture via the slider in the Condensor panel. Set the
filter in the Filter selection box. Click on Close.
• Click on the Objective button and select the objective by clicking on it.
• Click on the Reflector button and select the None.
(2)
Reflected-light observation (Epi-fluorescence)
• Turn on the HBO 50 power supply switch (5-35/1).
• Click on the Reflected Light button and set the shutter in the Open position.
• Click on the Reflector button and select the desired filter set by clicking on it.
–
The filter is automatically moved into the beam path to enable observation in epi-fluorescence.
• Click on the Tube Lens button and select the tube lens.
• Click on the Objective button and select the objective.
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OPERATION IN EXPERT MODE
Acquire Menu
Microscope Control
Carl Zeiss
LSM-FCS
HA
L
10
0
SN
T
12
V
4
OI
10
0W
5
St
6
7
8
an
32
10
12
Ex
db
00
te
y
K
rn
1:1
ZE
RO
FO
CU
S
Fig. 5-35
LSM-FCS with Axiovert 200 M BP
5.5.2.3
Select the LSM mode
Switchover to the scanning mode is then required.
• Click on the LSM button in the Acquire subordinate toolbar.
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Configuration Control
LSM-FCS
5.5.3
Carl Zeiss
Configuration Control
The setting of the beam path for the scanning procedure, i.e. the definition of channels (single detector,
META detector) and tracks and the setting of the Acousto-Optical Tunable Filters (AOTF) of the various
laser lines is performed in the Configuration Control window.
A track is:
- a set of parameters for the detection channels and for illumination (wavelength and intensity)
- scanned simultaneously and identified and handled by the system with one name
The Configuration Control window has a different appearance, depending on which selection button
has been activated (Channel mode or Lambda Mode). The Lambda Mode is only available if the
system contains the META detector. Only in the Channel Mode a subordinate toolbar exist which
contains a Single Track, Multi Track or Ratio button. In the Channel mode use the Single Track and
Multi Track buttons to toggle between the two image acquisition modes single tracking and
multitracking. In addition, you can activate the Ratio button for the activation of up to two Ratio
channels.
Performed settings can be stored as Track Configurations for single tracking. The number of
traditional channels to be defined in one track is limited to 8 (incl. monitor diode, transmission and ratio
channels), depending on the configuration. The number of channels to be defined in one track for LSM
510 META systems is 8 (including monitor diode and transmission detector). Furthermore, 2 ratio
channels can be used.
In case the number of available channels is not sufficient for the scanning procedure, further tracks can
be added and configured. The combination of these tracks can also be stored as Recording
Configurations for multitracking. A recording configuration may contain the maximum of 4 tracks.
Regardless of the number of included tracks, the maximum of 8 channels (incl. monitor diode,
transmission and ratio channels) can be used in a recording configuration in multitracking.
If several tracks have been activated, they are processed one after the other during the scan
procedure.
If the maximum number of channels to be used in a Single Track or a Multi Track has already been
achieved, it is no longer possible to add further channels or tracks.
If a second track or further tracks are used, the scan parameters can be changed as required. This avoids
"cross-talk" from one channel to another when different tracks are used.
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Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
5.5.3.1
LSM-FCS
Open / Close the
Control window
Configuration
• Click on the Config button in the Acquire
subordinate toolbar.
–
The Configuration Control window is
opened with the display last selected.
The
Beam
Path
and
Channel
Assignment panel differs according to
the hardware configuration supplied.
• Click on the Close button to quit the
Configuration Control window.
5.5.3.2
Spectra button
The Spectra button opens the Detection Spectra
& Laser Lines window. This window displays the
laser wavelengths activated for excitation (as
colored vertical lines) and the activated channels
(as colored horizontal bars).
Fig. 5-36
Configuration Control window,
Single Track activated
The color of the bar corresponds to the one
assigned to the relevant channel. Non-active
channels receive a gray bar over the entire spectral
range.
The length and position of the bar corresponds to
the emitted spectral range which is overlaid with
the filter and beam splitters selected in the
Configuration Control window or number of
selected channels of the META detector.
• Click on the Spectra button to open the
Detection Spectra & Laser Lines window and
to check the settings you made. The window is
closed via Close.
All amendments made in the Configuration
Control or Laser Control window are updated
on-line in the Detection Spectra & Laser Lines
window.
Fig. 5-37
5-56
Detection Spectra & Laser Lines
window
• A click on the Laser button enables you to
open the Laser Control window, switch lasers
on and off, if required, and control the laser
output.
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Configuration Control
LSM-FCS
5.5.3.3
Carl Zeiss
Laserline button
The Laserline button opens the Wavelength Switch Control window.
If more than 6 excitation laser lines (wavelengths) are available from the connected lasers, these can no
longer be completely displayed in the Line Active column of the Excitation window. In such a case, the
required laser lines (if not displayed) must be allocated.
• For this purpose, click on the Laser Line
button. The Wavelength Switch Control
window is opened.
• Select the required laser lines in the selection
boxes and confirm the selection with a click on
Store.
• Click on the Close button of the Wavelength
Switch Control window (Close is used to close
the window without accepting the changes).
The laser lines are now available in the Line active
column of the Excitation window.
If the laser lines 351 nm, 364 nm and / or 405 nm
have been switched on via the relevant lasers, they
are automatically entered into the Line Active
column as non switchable.
Fig. 5-38
Wavelength & Switch Control
window
• Click on the Close button to conclude the laser
settings procedure.
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Configuration Control
Carl Zeiss
5.5.3.4
Fig. 5-39
Track panel
LSM-FCS
Config button
The Config button permits existing track
configurations to be loaded, stored under any
name, or deleted.
(1)
Load a track configuration
A configuration stored in the system, whether
factory-supplied or user-created, can be accepted
or selected for active operation as follows:
Fig. 5-40
Track Configurations window
• Click on the Config button, the Track
Configurations window appears on the
screen.
On the Store / Apply Configuration panel, click on the arrow button
–
.
This opens a list box of all stored track configurations.
• Browse through the configurations by clicking, or use the scroll bar at the side of the list box.
• Click on the desired configuration.
–
The selected configuration is shown in the first line of the Configurations list box (e.g.:
FITC/Rhod).
• Click on the Apply button.
–
This results in the stored instrument parameters being taken over for active use. The track
configuration selected before is overwritten.
The optical diagram of the configuration selected appears on the Beam Path and Channel
Assignment panel. The newly loaded track has been automatically activated for the scanning
procedure. The Track Configurations window is closed automatically.
In the Options menu in the function Settings it is possible to define the parameters to be used
when applying a track configuration.
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Configuration Control
LSM-FCS
(2)
Carl Zeiss
Store a track configuration
A newly created or changed track configuration can be stored under a new name as follows:
• Click on the Congig button, the Track Configurations window appears on the screen.
• Enter the desired name in the first line of the Configurations list box.
• Click on the Store button.
• Close the window by clicking on Close.
During storage via the Store/Apply function, all the data of the Beam Path and Channel Assignment
and the Detector Gain, Ampl. Offset, Ampl. Gain and Data Depth (8 / 12 Bit) scan parameters of the
current track (single tracking) will be stored.
(3)
Delete a track configuration
A no longer required track configuration can be deleted as follows:
• Click on the Config button, the Track Configurations window appears on the screen.
• Select the configuration to be deleted from the Configurations list box.
• Click on the Delete button.
• Close the window by clicking on Close.
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Carl Zeiss
5.5.3.5
OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
LSM-FCS
Settings for Single Track in the Channel Mode
The settings of the beam path for the scanning procedure with regard to the main dichroic beam splitter
(HFT), secondary dichroic beam splitter (NFT), emission filters (EM) to be used and the assignment of
channels, excitation wavelengths and laser intensities are performed in the Beam Path and Channel
Assignment panel.
The setting can be performed manually or by using existing track configurations.
• Click on the Single Track button, unless it has already been activated.
–
The Configuration Control window for single tracking is displayed.
• Click on the Descanned button, unless it has already been activated.
(1)
Beam Path and Channel Assignment panel
The Beam Path and Channel Assignment panel displays the selected track configuration which is used
for the scan procedure.
You can change the settings of this panel using the following function elements.
Activation / deactivation of the excitation wavelengths (check box) and setting of
excitation intensities (slider). Open the Laser Control window via the Laser
button.
Selection of the main dichroic beam splitter (HFT) or secondary dichroic beam
splitter (NFT) position through selection from the relevant list box.
Selection of an emission filter through selection from the relevant list box.
Activation / deactivation of the selected channel (Ch 1-4, monitor diode ChM,
META detectors ChS1-8, transmission ChD) for the scanning procedure by
assigning an existing color icon or defining a new one. Deactivation of the channel
via deactivation of the check box.
The Fiber Out port of the LSM 510 permits connection of a MCS or another
detector. The connected MCS is operated via a macro which can be started via the
Macro function (see page 5-223ff).
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Configuration Control
LSM-FCS
Carl Zeiss
For the configuration of the beam path, please refer to the application-specific configurations depending
on the used dyes and markers and the existing instrument configuration (e.g.: module LSM - config. 16)
listed in the annex.
The assignment of the numbered emission filters (1-4), NFT secondary dichroic beam splitters (1-3) and
HFT main dichroic beam splitters in the Beam Path and Channel Assignment panel is shown in the
Configuration Control window (Fig. 5-41). The numbers of the emission filters correspond to those of
the channels lying behind (PMT photomultipliers).
Emission filter
NFT 3
NFT 2
NFT 1
HFT
Fig. 5-41
(2)
Configuration Control window
Beam path - HFT main dichroic beam splitters and NFT secondary dichroic beam splitters
• On the Beam Path and Channel Assignment panel, click on the HFT main dichroic beam splitters
(see Fig. 5-41).
–
This opens a graphical pop-up window of all beam splitters available.
• To select a beam splitter, click on the respective line of the list.
–
The selected beam splitter moves into the beam path.
• Proceed accordingly to configure the NFT secondary dichroic beam splitters
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Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
(3)
LSM-FCS
Beam path - Emission filter
• On the Beam Path and Channel Assignment
emission filter symbol.
panel, click on the
–
This opens a graphical pop-up window
of all available emission filters (e.g. BP for
band pass, or LP for long pass) with their
wavelengths.
• To select an emission filter, click on the
respective filter in the pop-up window.
–
The emission filter selected moves into the
beam path in front of the PMT
photomultiplier.
• Depending on the application, it may be
necessary to insert additional mirrors, secondary
dichroic beam splitters or neutral glass filters
between the HFT main dichroic beam splitter
and the PMT photomultiplier. To select these
components, click on the respective
symbols.
Fig. 5-42
5-62
Configuration Control window
For channels 1 and 2, it is possible to
change the filters directly on the
LSM scan module (see Annex: Filter
change in the beam path of channels 1
and 2).
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Configuration Control
LSM-FCS
(4)
Carl Zeiss
Beam path - Activation / Deactivation of
Channels and Channel Color Assignment
• On the Beam Path and Channel Assignment
.
panel, click on the channel symbols, e.g.
–
This opens the Channel Color Selection
window on the Beam Path and Channel
Assignment panel.
• Click on the desired color bar.
Fig. 5-43
Channel Color Selection window
This changes the color of the channel symbol.
• To close the Channel Color Selection box,
click on the Close button.
Further colors for the corresponding channel can
be produced as follows:
• Clicking on the Define button will open a
further Channel Colors window.
All the available colors are shown as buttons in the
Current Set of Channel Colors panel.
Reticule
• Via a reticule in the Define Color panel, any
desired color can be produced.
• Clicking on the Add button allows the color to
be used for further channel coloring.
• Choose the desired color with the reticule (the
reticule is in the left corner at the bottom of the
color range).
• Define the brightness by use of the scroll bar.
• Use the Add button to add the color to the
color range.
• To delete a defined color, click on the relevant
color button and then on the Remove button.
Fig. 5-44
Channel Colors window
Standard colors (black for OFF, red, green, blue and white) cannot be removed.
• Click on the Close button to close the Channel Colors window.
–
Newly defined colors are accepted and displayed in the Channel Color Selection window. They
can then be used in the same way as standard colors.
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OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
LSM-FCS
The PMT1 photomultiplier is activated / deactivated by the check box.
• Proceed in the same way for the other PMT photomultipliers.
The
symbols for the transmitted-light PMT photomultiplier (ChD Transmission) and monitor
diode (ChM) can be activated in the same way as the photomultipliers of channels 1 to 4. The
use of the monitor diode function is described in detail in the annex of this manual.
When changing from the NFT 1secondary dichroic beam splitter, the appropriate setting of the
NFT 3 secondary dichroic beam splitter is performed automatically.
(5)
Beam path - Laser attenuation
• On the Beam Path and Channel Assignment
panel, click on the Excitation button.
–
This opens a dialog box of all available lasers
with their wavelengths and their usable
Acousto-Optical Tunable Filters (AOTF)
attenuation.
• To select the desired laser line, activate the
check box for Line Active.
Fig. 5-45
Configuration Control window
• Use the Transmission [%] slider to set the
utilizable laser intensity (recommendation: start
at 50 %).
–
The transmittance of the Acousto-Optical
Tunable Filter (AOTF) changes accordingly.
• This allows you to adapt the laser intensity very sensitively to the job. Activate the check box for Line
Active.
–
This activates the selected laser power for use. This is indicated by the Laser Power displaying
lamps (status display green / grey).
By clicking on the Excitation button you can check at any time which lasers are available for
active operation.
If you deactivate Line Active, the laser wavelengths for Enterprise and argon lasers are
deselected by means of the Acousto-Optical Tunable Filters (AOTF), i.e. these lasers change into
standby status.
If you interrupt your work with the LSM for a break, it is recommended not to switch the
Enterprise and argon lasers off by hardware action, but to put them into standby status as
described.
Excitation filters, emission filters, HFT main dichroic beam splitters and NFT secondary dichroic
beam splitters can be switched online, channels (PMT photomultipliers) only off-line.
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Configuration Control
LSM-FCS
5.5.3.6
Carl Zeiss
Settings for Multi Track in the
Channel Mode
The Multi Track function permit several tracks to
be defined as one configuration (Recording
Configuration) for the scan procedure, to be
stored under any name, reloaded or deleted.
The maximum of four tracks with up to 8 channels
can be defined simultaneously and then scanned
one after the other. Each track is a separate unit
and can be configured independently of the other
tracks with regard to channels, Acousto-Optical
Tunable Filters (AOTF), emission filters and dichroic
beam splitters.
• Click on the Multi Track button.
–
The Configuration Control window for
multitracking appears, which means that the
List of Tracks panel is additionally
displayed.
The tracks required for multitracking can either be
configured manually one after the other (identical
to single tracking) and then stored as recording
configuration, or already existing recording
configurations can be used and changed as
required.
It is also possible to load already stored track
configurations (single tracking) in a recording
configuration.
(1)
Fig. 5-46
Configuration Control window,
Multi Track activated
Beam Path and Channel Assignment panel
The Beam Path and Channel Assignment panel displays the track configuration of the track currently
selected in the List of Tracks panel (highlighted in blue or gray).
The settings for this panel are performed separately for each track, in the same way as for single
tracking. To do this, select the track to be configured from the List of Tracks panel (see the following
description of the List of Tracks panel).
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Configuration Control
Carl Zeiss
(2)
LSM-FCS
List of Tracks panel
In the List of Tracks panel, the available tracks are
displayed with names, activated channels and laser
lines.
Fig. 5-47
List of Tracks panel
The Line, Frame and Fast Switch buttons are
used to determine in which way switching
between tracks is made during the scan
procedure.
Furthermore, the sequence of tracks to be
processed can be changed for the scan procedure.
The Add Track, Store/Apply Single Track and Remove buttons permit individual tracks to be added,
saved or deleted.
In addition, this panel is used to activate / deactivate the tracks for the scan procedure.
• To activate or deactivate one or several tracks for the scan procedure, activate / deactivate the check
box of the relevant tracks.
The configuration of the selected track is displayed in the Beam Path and Channel Assignment ...
panel.
• To select a track for the display of the beam path configuration, click on its name.
–
The selected track is highlighted in gray or blue.
When you switch from multitracking to single tracking, the track selected in the multitracking
mode (highlighted in blue or gray) is always transferred and automatically activated for the scan
procedure. All other tracks are deactivated, and they remain deactivated when you switch back
to the multitracking mode afterwards.
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Carl Zeiss
The following functions are available in the List of Tracks panel:
Switch tracks after each
Line button
Switch tracks after each
Frame button
Frame Fast button
Tracks are switched during scanning line by line. The following settings
can be changed between tracks: Acousto-Optical Tunable Filters (AOTF)
and the Amplifier Offset.
Tracks are switched during scanning frame by frame. The following
settings can be changed between tracks: Acousto-Optical Tunable Filters
(AOTF), the emission filters, the dichroic beam splitters, the channels, the
settings of the pinhole position in XY(Z)-direction and pinhole diameter
and Gain and Amplifier Offset.
The scanning procedure can be made faster. Only the Acousto-Optical
Tunable Filters (AOTF) for the selected laser line and the Amplifier
Offset are switched, and no other hardware components. The tracks are
all matched to the current track with regard to emission filter, dichroic
beam splitter, setting of Detector Gain, pinhole position and diameter.
When Line button is selected, the same rules apply as for Frame Fast.
Settings
Add Track button
An additional track is added to the configuration list. The maximum of
four tracks can be added. One track each with basic configuration is
added, i.e.: one Ch 1 channel is activated, all laser lines are switched off,
emission filters and dichroic beam splitters are set in accordance with the
configuration last used.
Remove button
The single track previously marked in the List of Tracks panel in the
Name column is deleted.
Store/Apply button
Opens the Track Configurations window. A selected track defined in a
Recording Configuration can also be stored as a single track for single
tracking applications. Also, it's possible to load a single track in a
multitracking configuration.
A click on this arrow button will move the selected track (highlighted in
blue) one position upwards in the list box.
A click on this arrow button will move the selected track (highlighted in
blue) one position downwards in the list box.
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LSM-FCS
When adding new tracks, the following sequence should be followed:
• Add a track by clicking on the Add Track button.
• Determine the configuration of the track in the Beam Path and Channel Assignment panel or
select an existing one via the Store/Apply Single Track button of the List of Tracks panel.
• Store the name of a track configuration defined via the Store/Apply button of the List of Tracks
panel. The new track name will then be displayed in the List of Tracks panel.
If this way of storing is performed, the created track will also be available as a single track and can
therefore also be activated individually.
• Add the next track via the Add Track button and then configure and store it again.
The name of a track can also be changed directly in the List of Tracks panel. In that case, however, the
edited track is not available as a single track configuration, but only within the recording configuration.
To edit a track name within Recording Configurations, proceed as follows:
• To select the track, click on the relevant track name in the List of Tracks panel. Then click on the
name again to open the text editing field.
• Change the track name via the keyboard. Use Esc to undo the procedure.
• Click once in the area outside the text editing box to close this box.
The channels of the individual tracks with the relevant scan parameters can be displayed in the
Scan Control window after activation of the Channels button. The description of channel 1 in
Track 1, for example, is Ch1-T1.
(3)
Config button in Multi Track mode
The Config button in the Multi Track mode permits all tracks to be loaded, stored under any name, or
deleted.
Load a recording configuration
An existing recording configuration can be loaded as follows:
• Click on the Config button, the Recording
Configurations window appears on the
screen.
• On the Store / Apply Configuration panel,
click on the arrow button .
Fig. 5-48
Recording Configurations window
–
5-68
This opens a list box of all stored recording
configurations.
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OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
LSM-FCS
Carl Zeiss
• Browse through the configurations by clicking, or use the scroll bar at the side of the list box.
• Click on the desired configuration.
–
The selected configuration is shown in the first line of the Configurations list box (e.g.: DAPI).
• Click on the Apply button.
–
The program loads those parameters of the selected Recording Configuration which have been
activated in the Options menu under Settings / Recording Configuration (see section 5.11.5,
page 5-241). The Recording Configurations window is automatically closed.
The optical diagram of the configuration selected appears on the Beam Path and Channel
Assignment panel. The entire recording configuration has been activated for the scanning
procedure.
Store a recording configuration
A newly created or changed recording configuration can be stored under a new name as follows:
• Click on the Config button, the Recording Configurations window appears on the screen.
• Enter the desired name in the first line of the Configurations list box.
• Click on the Store button.
• Close the window by clicking on Close.
During storage via the Config button, all the data of Beam Path and Channel Assignment and the
Detector Gain, Ampl. Offset, Ampl. Gain and Data Depth (8 / 12 Bit) scan parameters of all the defined
tracks (multitracking) are stored. Furthermore, the used objective, the Frame Size, Zoom, Rotation &
Offset and Scan Direction parameters and the bleach parameters are stored.
Delete a recording configuration
A no longer required recording configuration can be deleted as follows:
• Click on the Config button, the Recording Configurations window appears on the screen.
• Select the configuration to be deleted from the Configurations list box.
• Click on the Delete button.
• Close the window by clicking on Close.
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Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
5.5.3.7
LSM-FCS
Ratio Settings panel
The Ratio Settings panel permits you to activate
two additional Ratio channels.
• Click on the Ratio button.
–
The Ratio Settings panel is displayed at the
bottom of the Configuration Control
window. The settings of the selected
tracking mode (Single Track / Multi Track)
remain unchanged.
The Ratio Settings panel is only available in the
Single Track and Multi Track mode.
Source 1 in ratio settings
Selects source 1 data channel in Configuration
Control.
Source 2 in ratio settings
Selects source 2 data channel in Configuration
Control, including the option to select "1st
Image" for R1 and/or R2 (e.g. to calculate F/F0 for
single wavelength dyes).
R1/R2 in Scan Control
Fig. 5-49
Configuration Control window;
Ratio activated
R1/R2 can be selected as channels in the Scan
Control window. Five preset formulas can be
chosen for online display of radiometric or single
wavelength dyes.
Set by min/max (in Scan Control window Channels mode)
Allows the definition of the display scaling
according to the expected minimal and maximal
values.
5-70
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OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
LSM-FCS
Carl Zeiss
The following function elements are provided in the Ratio Settings panel:
Activation of the Ratio channel (R1, R2) through assignment of an existing color
or definition of a new one. Activation / deactivation of the Ratio channel via the
check box.
Selection of the channels of which the ratio is to be formed from the relevant list
box.
A suitable color can be assigned to each of the two Ratio Channels R1 and R2, in the same way as for
the photomultiplier channels.
The channels of which a ratio will be formed are selected via the Source 1 and Source 2 list boxes.
• Click on the
opened.
arrow button to select the required channel for Source 1 and 2 from the list box now
The ratio to be formed between the selected channels can be defined more precisely using
three formulas in the Scan Control window after activation of the Channels button and a click
on the relevant ratio button (e.g.: R1).
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Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
5.5.3.8
LSM-FCS
Settings in the Lambda Mode
The Lambda Mode settings are only available if
the LSM 510 contains the META detector. With
this spectral module the overall emission from the
sample is directed onto a wavelength-dispersive
element and is imaged on a 32 channel detector.
All 32 photomultipliers of the detector cover a
spectral width of approximately 340 nm, a single
PMT covers a spectral range of 10.7 nm. In the
Lambda Mode, images, image stacks or time
series can be recorded in a wavelength selective
way.
Fig. 5-50
Configuration Control window;
Lambda Mode activated
This new experiment is called Lambda Stack. For
the acquisition of a Lambda Stack, the
fluorescence signal of 8 PMTs out of the 32 can be
read out at once. The settings of the beam path
for the Lambda Mode scanning procedure with
regard to the main dichroic beamsplitter and the
META detector settings are performed in the
Beam Path and Channel Assignment panel.
• Click on the Config Button in the Acquire
Subordinate toolbar of the main menu.
–
The Configuration Control window opens.
• Click on the Lambda Mode button, unless it
has already been activated.
–
5-72
The Beam Path and Channel Assignment
panel for the Lambda Mode is opened.
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OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
LSM-FCS
(1)
Carl Zeiss
Beam Path and Channel Assignment panel
The Beam Path and Channel Assignment panel displays the configuration of laser lines, the main
(dichroic) beamsplitter and the spectral range of the META detector to be covered.
You can change the settings of this panel using the following function elements:
Beam Path configuration
Activation / deactivation of the excitation wavelengths (check box) and
setting of excitation intensities (slider). Open the Laser Control window
via the Laser button. The active laser lines are automatically displayed in
the wavelength color bar.
Selection of the main dichroic beam splitter (HFT) through selection from
the relevant list box.
META detector slider
Definition of number of PMTs out of the 32 PMT Detectors to fit the
required spectral range.
Control buttons
Close
Closes the Configuration Control window.
Spectra
The Spectra button opens the Detection Spectra & Laser Lines
window (see page 5-56).
Laserline
The Laserline button opens the Wavelength Switch Control window
(see page 5-57).
Config
The Config button permits existing track configurations to be loaded,
stored under any name, or deleted (see page 5-58).
(2)
Beam path - Excitation
• On the Beam Path and Channel Assignment panel, click on the Excitation button.
–
This opens a dialog box of all available lasers with their wavelengths and their usable Acousto-Optical
Tunable Filters (AOTF) attenuation.
• To select the desired laser line, activate the check box for Line Active.
• Use the Transmission [%] slider to set the utilizable laser intensity (recommendation: start at 50 %).
–
The transmittance of the Acousto-Optical Tunable Filter (AOTF) changes accordingly.
• This allows you to adapt the laser intensity very sensitively to the job. Activate the check box for Line
Active.
– This activates the selected laser power for use. This is indicated by the Laser Power displaying
lamps (status display green / gray).
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Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
LSM-FCS
By clicking on the Excitation button you can check at any time which lasers are available for
active operation.
If you deactivate Line Active, the laser wavelengths for Enterprise and argon lasers are
deselected by means of the Acousto-Optical Tunable Filters (AOTF), i.e. these lasers change into
standby status.
If you interrupt your work with the LSM for a break, it is recommended not to switch the
Enterprise and argon lasers off by hardware action, but to put them into standby status as
described.
Excitation filters, emission filters, HFT main dichroic beam splitters and NFT secondary dichroic
beam splitters can be switched online, channels (PMT photomultipliers) only off-line.
(3)
Beam path - HFT main (dichroic) beam splitter
• On the Beam Path and Channel Assignment panel, click on the HFT main dichroic beam splitters
.
–
This opens a graphical pop-up window of all beam splitters available.
• To select a beam splitter, click on the respective line of the list.
–
The selected beam splitter moves into the beam path.
• Proceed accordingly to configure the NFT secondary dichroic beam splitters
(4)
.
Beam path - META detector settings
On the Beam Path and Channel Assignment panel use the two sliders to define the spectral detection
range of the META module. The current position of the slider is displayed in a white box when keeping
the left mouse button pressed.
Both slider values are updated in the Start and End Input box.
The wavelength values can also be typed directly into the Start and End input boxes. The position of the
sliders are updated after the next mouse click. The Number of Passes displays the number of successive
scans to be performed in order to cover the required spectral range.
Step check box:
5-74
Step size can be increased to 21.4 nm by binning of a pair of adjacent channels.
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OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
LSM-FCS
5.5.3.9
Carl Zeiss
Settings in the Online Fingerprinting Mode
The use of this function permits the selection of
reference spectra together with the excitation
settings, to allow an immediate display of the
unmixing results during the scanning.
• Click on the Config button in the Acquire
subordinate toolbar of the main menu.
–
The Configuration Control window opens.
• Click on the Online Fingerprinting button.
–
The Beam Path and Channel Assignment
panel for the Online Fingerprinting Mode
is opened.
Beam Path configuration
Activation / deactivation of the
excitation wavelengths (check
box) and setting of excitation
intensities (slider). Open the
Laser Control window via the
Laser button. The active laser
lines are automatically displayed
in the wavelength color bar.
Fig. 5-51
Configuration Control window;
Online Fingerprinting Mode
activated
Selection of the main dichroic beam splitter (HFT) through selection from
Control buttons for reference spectra
RS1 ... 8
Menu for selecting a display color and a reference spectra (reference
spectra derive from earlier experiments via mean of ROI or ACE tools);
selected spectra and colors appear in the wavelength chart. Unmixed
results will be displayed during scanning, lambda stack will neither be
displayed nor stored.
META detector slider
Definition of number of PMTs out of the 32 PMT Detectors to fit the
required spectral range. To optimize acquisition speed, a range detected
in 1 or 2 passes is recommended.
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OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
Carl Zeiss
Start box
Start value of required spectral range.
End box
End value of required spectral range.
Step box
Step width in spectral range (10.70 recommended).
5.5.3.10
LSM-FCS
Non Descanned panel
The functions of the Non Descanned panel are described in chapter 9 of this manual (section 9.11,
page 9-35).
5-76
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OPERATION IN EXPERT MODE
Acquire Menu
Configuration Control
LSM-FCS
5.5.3.11
Carl Zeiss
Camera Detection panel
The use of this function permits the use of a Zeiss
AxioCam HR camera as an alternative external
detector.
• Click on the Config button in the Acquire
subordinate toolbar of the main menu.
–
The Configuration Control window opens.
• Activate one of the Single Track or Multi
Track buttons and click on the Camera button.
–
The Beam Path and Channel Assignment
panel for camera detection is opened.
Control buttons
TV
Menu for selecting a display
color for the camera image.
Reflector
Selects a beamsplitter for the
excitation/emission.
Add Track
Adds a second track to the
acquisition in Multi Track
mode,
e.g.
a
different
fluorescence filter cube or
transmitted light.
Fig. 5-52
If TV and LSM tracks are mixed, the active
detection port of the microscope has to be set
according to the first track.
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Configuration Control window;
camera detecting activated
5-77
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
Carl Zeiss
5.5.4
LSM-FCS
Scan Control
The scan parameters for image acquisition are set
in the Scan Control window.
The microscope must be in the LSM mode, i.e. the
relevant sliders on the relevant microscope stand
must be in the LSM position. The LSM button in
the Acquire subordinate toolbar is activated when
the LSM mode has been set.
The scanning actions are started via the buttons on
the right-hand side of the Scan Control window,
and the scan parameters are set in the main part
of the window.
An acquired image is displayed in a separate
Image Display window. If an Image Display
window is not yet available, a new Image Display
window is automatically opened during the
acquisition.
Fig. 5-53
Scan Control window
The following scanning modes can be performed:
Spot
–
scanning of a spot (Spot + Time Series)
Line
–
–
scanning of a line in the XY-plane (Line, Line + Time Series)
scanning of a line with different Z-values (Line + Z Stack, Line + Z Stack + Time Series)
Frame
–
–
–
–
5-78
scanning of an XY frame (Frame, Frame + Time Series)
scanning of XY frames with different Z-values (Frame + Z Stack, Frame + Z Stack + Time Series)
scanning of XY frames in defined ROIs (Frame + Use ROI + Time Series)
scanning of XY frames with different Z-values in defined ROIs (Frame + Z Stack + Use ROI + Time
Series)
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
5.5.4.1
Carl Zeiss
Open / Close the Scan Control window
• Click on the Scan button in the Acquire subordinate toolbar of the Main menu.
–
This opens the Scan Control window, which shows all lasers connected to the system.
• Click on the Close button to quit the Scan Control window.
The following main function buttons are available in the Scan Control window:
Generally available buttons
Mode button
When the button is activated, the following panels are available for the
setting of the scanning parameters for the line and frame modes:
Objective Lens, Image Size & Line Step Factor, Speed, Pixel Depth,
Scan Direction & Scan Average and Zoom, Rotation & Offset.
Channels button
When the button is activated, the Channel Settings and
Excitation of Track ... panels are available for the setting of the
channels and the laser excitation.
Spot button
Activate the Spot scan mode
Line button
Activates the Line scan mode.
Frame button
Activates the Frame scan mode.
Use ROI button
Activates the scanning procedure only within a ROI (region of interest) to
be defined first.
Z Stack button
Activates the Z Stack scan mode, display of additional buttons on the
right-hand side of the Scan Control window.
Z Settings button
When the button is activated, the Z Settings panel is available for the Zscan parameter definition. The Z Stack scan mode must be active.
Close button
Closes the Scan Control window.
New button
Opens a new Image Display window.
Find button
Automatic optimization of image brightness and contrast. The settings
for the Find function can be varied as required using the Maintain
menu, Set Find (see page 5-270).
Fast XY button
Continuous scan with high speed. This function should be used to a
limited extent and only for a short period of time. Fast XY switches
temporarily to 512 x 512 frame size.
Single button
Single scan (named Start in the Z Stack mode).
Stop button
Stops the current scan procedure, no matter in which window the button
is pressed (also see the Time Series Control and Bleach Control
windows).
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Carl Zeiss
Cont. button / Finish button
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Continuous scan (not available in the Z Stack mode). If you select the
option Frame for Mode and the option Continuous for Number in the
Pixel Depth, Scan Direction & Scan Average panel, the Finish button
is displayed instead of the Cont. button. In this case, continuous
averaging is performed when you have started the scan. If you click on
the Finish button, the scan/averaging process is stopped after the scan
of the current image has been completed.
Additional button in the Spot mode
Spot Sel button
Automatically defines spot on the Image Display window by positioning
of two perpendicular lines
Additional button in the Line mode
Line Sel button
Automatically defines a line in the center of the Image Display window
(Frame) for creation of the intensity profile; using the mouse, the line for
the intensity profile can then be positioned anywhere in the Image
Display window.
Additional buttons in the Z Stack mode
Start button
Triggers the scan of a stack.
XYscan button
Triggers a single XY-scan
XYcont button
Triggers continuous XY-scan.
Line Sel button
To prepare the Range function, a cutline is created in the scanned XYframe to determine the position at which the XZ-scan through the
specimen is to be produced. Using the mouse, the line for the XZ-scan
can be positioned anywhere in the scan frame. The cutline can be
defined either as a straight line or free shape curve.
Range button
Produces an XZ-scan through the specimen within the limits determined
in Num Slices and Interval; the cutline is determined via the Line Sel
function.
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
5.5.4.2
Carl Zeiss
Frame
When the Frame button is activated, a frame of
variable size is scanned pixel by pixel and line by
line. The laser beam is moved over the specimen
line by line.
The scan parameters and the channels (single
detector, META detector) are set via the Mode
and Channels buttons, and the laser settings can
be checked again or changed.
(1)
Mode
When the Mode button is activated, the
Objective Lens, Image Size & Line Step Factor,
Speed, Pixel Depth, Scan Direction & Scan
Average and Zoom, Rotation & Offset panels
are displayed in the Scan Control window.
Objective Lens, Image Size & Line Step Factor
panel
• Open the Objective list box and select the
objective to be used via a click of the mouse
(identical to Microscope Control). When using
immersion oil objectives, make sure to perform
immersion as required.
Fig. 5-54
Scan Control window - Mode/Frame
• Select the Frame Size from the default sizes via the buttons 128, 256, 512, 1024, 2048, or enter the
required values via the keyboard. Recommended setting to start with: 512 x 512 pixels.
–
It is also possible to enter different values for X and Y. The value for Y is freely selectable between
1 and 2048 pixels (integers). The value for X must always be an integral multiple of 4. The
maximum value for X is also 2048 pixels.
Select the Line Step size between 1 and 10. Only
every n-th line is scanned. The lines in between are
interpolated. This fast scan mode is called Step
Scan.
Fig. 5-55
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Objective Lens, Image Size & Line
Step Factor panel
5-81
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
Carl Zeiss
LSM-FCS
Speed panel
Fig. 5-56
• Select the Scan Speed from the 13 preset
steps via slider or input box. Recommended: 7
for the first scan. A click on the Max button
sets the maximum speed for the current zoom.
Speed panel
–
The Scan Speed determines on the Pixel Time. In the case of different image formats, the Pixel
Time is constant for the same Scan Speed, but the Scan Time is different.
–
Pixel Time: dwell time of the laser beam on the pixel
–
Scan Time: duration of the acquisition for the entire frame
–
The minimum Pixel Time of 0.64 µs is only achieved at resolutions 512 x n and above, the
maximum Pixel Time of 204.8 µs only with frame sizes larger than 1024 x n.
–
A longer Pixel Time for even smaller frame sizes is possible; maximum: 6553.6 µs.
Fast XY only for fast image acquisition during parameter setup.
Pixel time and scan time will be shown.
Fast XY = speeds 8 – 13 (depending on zoom), average = 1, max. resolution: 512 x 512 pixels.
Speed:
1 ... 8
9
10
11
12
13
Zoom:
0.7
1.0
1.4
2.4
3.2
5.6
Note that Lambda Scan mode can only be performed with specifications from speed 1 ... 10 at
512 x 512 pixels.
Pixel Depth, Scan Direction & Scan Average
panel
• Select 8 Bit or 12 Bit Data Depth, i.e. 256 or
4096 gray values.
• Select the Unidirectional or Bi-directional
Scan Direction.
Fig. 5-57
–
–
5-82
Pixel Depth, Scan Direction &
Scan Average panel
–
Unidirectional: The laser scans in one
direction only, then moves back with beam
blanked and scans the next line.
Bi-directional: The laser also scans when moving backwards, i.e. the Scan Time is halved.
The pixel shift between forward and backward movement (double image) resulting from bidirectional scanning must be corrected via the Scan Corr X and Y sliders. Zero° rotation requires
correction in the X-direction, 90° rotation must be corrected in the Y-direction. If the image was
rotated, correction is required in both coordinates. Correction is performed on-line in the
continuous scan mode (Cont. button). The size of the shift depends on the Scan Speed. For
automatic scan correction, click on the Auto button.
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Carl Zeiss
• Select the Line or Frame mode for averaging.
• Select the desired scan average method Mean or Sum in the Method selection box.
• Select the desired scan average from the available values 2, 4, 8 and 16 in the Number selection box
or Continues (only for Frame average mode).
The greater the number of averages selected for Mean average Method, the better the image
quality will be; the scanning time will be prolonged accordingly.
Averaging can be performed in different ways, depending on whether the Mean or Sum method has
been activated.
If you are using the Mean method, the image information is generated by adding up all scans pixel by
pixel and then calculating the mean value.
In the Sum method, the pixel values of all scans are only added up, without a mean value being
calculated.
To create the image information using the Line average mode, each line (depending on the setting) is
scanned 2, 4, 8 or 16 times during Scan Average, and then the average value per pixel is calculated. This
minimizes noise interference during the scanning procedure.
If the Frame average mode is used to create the image information, the complete frame is scanned 2, 4,
8 or 16 times, depending on the setting. The average value is recalculated after each frame scan.
The Frame average mode also permits continuous averaging.
• For this, select the Continuous option in the Number selection box.
If you have selected the Continuous option, the Finish button for ending continuous averaging is
displayed instead of the Cont. button. Use the Single button in this case to start continuous scanning.
When you click on the Finish button, the scan currently in progress will be completed before the process
is stopped.
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5-83
Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Zoom, Rotation & Offset panel
In this panel, the scan range is set for zoom,
rotation and offset in relation to the field of view
of the microscope. The diagonals of the outer
square on the right-hand side correspond to the
field of view of the microscope.
Fig. 5-58
Zoom, Rotation & Offset panel
The inner square contained in it (rectangle in the
case of differently set frame size) represents the
scan range and immediately shows the changes
made to zoom, rotation and offset.
The blue line at the top of the scan range is helpful for orientation when the scan range is rotated in the
direction of the field of view.
• Set the desired zoom factor via the slider (Zoom) or by clicking on the arrow buttons.
–
The zoom factor can be set continuously in the range from 0.7 to the maximum of 8, and is
displayed in the relevant input box. The value 0.7 corresponds to factor 1, and value 8 to factor
11, related to the field of view. From zoom factor 5.6, the magnification will be empty, and the
zoom factors will be displayed in red in that case. Clicking on button 1 enables immediate
resetting to the zoom factor 1.
–
Recommended setting to start with: Zoom 1.
• To rotate the scan area, use the slider (Rotation) or click on the arrow buttons.
–
Clicking on button 0 enables immediate resetting to 0°.
–
Recommended setting to start with: Rotation 0°.
• Move the scan area by clicking on the 4 arrow buttons (Offset).
–
The offset of the scan area from the center of the field of view is displayed online in µm for
X and Y.
–
A click on the center button will recenter the scan area to the field of view.
–
Clicking, holding and drawing the rectangle with the mouse permits the scan area to be moved
directly within the field of view.
–
Recommended setting to start with: Offset X = 0, Y = 0
During the scan procedure, the functions Objective change, Speed, Scan Corr, Zoom,
Rotation and Offset can be influenced online.
By clicking on the Reset button the scan zoom is set to 1 and the XY offsets are set to the zero position
and the ratio angle is set to 0°.
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
(2)
Carl Zeiss
Channels
If the Channels button is activated, the Channel
Settings and Excitation of Track ... panels are
displayed in the Scan Control window.
Channel Settings panel
In the Channel Settings panel, the channels (incl.
META channels if present and ratio channels)
defined in the Configuration Control window
are listed track by track as selectable buttons.
Depending on the selected Channels button (e.g.
ChS1-T1), the currently used settings of Pinhole,
Detector Gain, Amplifier Offset and Amplifier Gain
are displayed.
• The slider near Pinhole enables you to change
the pinhole diameter of the relevant channel.
–
The pinhole diameter is indicated in µm,
Optical Slice and Airy Units. The Airy value
depends on the aperture of the objective,
excitations and the emission wavelength.
–
A small pinhole diameter will increase the
depth of focus, but reduce the light intensity
received by the PMT photomultiplier.
–
When you vary the pinhole diameter, an
Optical Slice value is displayed. For optimum
depth resolution, Airy values should be
small, but in fluorescence applications not
below 1.0 to keep the intensity loss within a
reasonable limit.
–
A click on the 1 button sets the pinhole to a diameter of 1 Airy unit. A click on the Max button
sets the pinhole diameter to the maximum.
Fig. 5-59
Scan Control window – Channels
• The sliders (and the relevant arrow buttons) near Detector Gain, Ampl. Offset and Ampl. Gain
enable you to set the photomultiplier of the selected channel during continuous scanning.
–
Detector Gain: Setting of the high voltage of the PMT photomultiplier - setting of image contrast
and brightness (values available between 80 and 1250)
–
Amplifier Offset: Setting of the electronic offset - background of the image can be set (values
available between -2 and 0.1)
–
Amplifier Gain: Amplification factor (values available between 1 and 3)
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Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
The parameters Detector Gain, Ampl. Offset and Ampl. Gain are described in section
Pinhole / Detector Gain / Ampl. Offset / Ampl. Gain (page 5-392) in the context of image
optimization. In case the Lambda Mode has been chosen in the Configuration Control
window only the META channel ChS is displayed in the Channel settings of the Scan Control
window.
The parameters of a ratio channel are set in a
separate dialog box.
• Click on the button of a ratio channel (e.g. R1).
The dialog box for the setting of the ratio
parameters is displayed.
Clicking on the required tabs enables you to
choose from five formulas (Type 1 to 5) for ratio
calculation. The relevant decimal values can be
entered in the input boxes via the keyboard. The
entered values remain unchanged even after
switchover to another formula and can be
reactivated any time.
The formula type activated last is always used for
ratio formation during the scan procedure. If the
input box does not contain any value at all or no
suitable value, the useful value last used will be
activated.
The ratio channels are displayed in the Image
Display window (see Fig. 5-63).
• Select the required formula and enter the
relevant values.
Fig. 5-60
5-86
Channel Settings panel of a
Ratio Channel
Letters can be entered into the formula fields
which will be valued as 1; it is also possible to
make no entry, which will also be valued as 1, but
will not be displayed.
Set by min/max (in Scan Control window Channels mode) allows the definition of the
display scaling according to the expected minimal
and maximal values.
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Carl Zeiss
Excitation panel
• In the Excitation panel you can select other
lasers and vary laser intensities (in the same way
as in the Laser Control or Configuration
Control window) and you can program the
AOTF for different laser lines.
By clicking on the Laserline button the
Wavelength Switch Control window opens. If
more laser lines than AOTF positions occur the
AOTF can be programmed for various lines.
Fig. 5-61
Excitation of Track ... panel
• Select the required laser lines in the selection
boxes and confirm the selection with a click on
Store.
• Click on the Close button to close the Wavelength Switch Control window.
If bi-directional scanning with12-bit technology, several channels and scan speeds of 9 or 10 are
used at the same time, a data jam can occur and difficulties can therefore arise if 233 MHz PC’s
(or lower) are used. All parameters under Channels can be varied online.
Acquisition of a frame
Once you have set up your parameter as defined in the above section, you can acquire a frame image of
your specimen.
• Click on the Single button in the Scan Control
window. The system will automatically start the
acquisition of a frame. The individual channels
and the overlay image can be viewed by
changing to the Split xy mode. This button is
located on the right-hand side of the Image
Display window.
The following scan image shows the result with
two defined tracks plus the Ratio channel and the
overlay (see Fig. 5-63). The appropriate Channel
Settings panel in the Scan Control window is
shown in Fig. 5-62.
Fig. 5-62
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Channel Settings panel for two
defined tracks plus Ratio channel
5-87
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
Carl Zeiss
LSM-FCS
1st track:
Ratio channel:
- Ch1-T1
- R1
- Ch3-T1
- ChD-T1
2nd
track:
- Ch1-T2
- Ch3-T2
Overlay
Fig. 5-63
5-88
Image Display window with two tracks plus ratio track (Split
xy mode)
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Carl Zeiss
In case the Lambda Mode has been chosen in the Configuration Control window, the following scan
image shows the result of the Lambda Stack.
Fig. 5-64
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Image Display window with a Lambda Stack
5-89
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
Carl Zeiss
(3)
LSM-FCS
Z Stack
This function permits a series of XY-images to be
produced in different focus positions (Z slices).
When the Z Stack button is pressed, the
Z Settings button is automatically activated and
the Z Settings panel is displayed in the Scan
Control window. However, it is possible at all
times to switch over to setting / changing the scan
parameters or the PMT photomultipliers and lasers
via the Channels and Mode buttons.
The additional XYscan, XYcont, Line Sel and
Range buttons are available on the right-hand
side of the Scan Control window, and the
labeling of the Single button changes to Start.
The Z Stack function is deactivated by clicking
again on the Z Stack button.
Fig. 5-65
Scan Control window - Z Settings
Z Settings panel - overview
The parameters of the Z Stack to be created are defined and displayed online in the Z Settings panel.
Stack Z Size:
The dimension of the Z Stack in µm. The stage (nosepiece) is moved in such a way that
the stack size, dependent on the refractive index, is achieved optically.
Focus Position: The current Z position. If the refractive index (Refr. Corr.) changes, the value of the
focus position in relation to the "0" also changes (online).
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Z Slice:
Opens
the
Optical
Slice
window.
The Optical Slice window
contains two buttons (Optimal
Interval: ... µm and Optimal
Pinhole Diameter) to allow the
setting of the optimum interval
and the optimum pinhole
diameter of fluorescence stacks.
Both values influence each other
and depend on the objective
used.
Fig. 5-66
Carl Zeiss
Optical Slice window
In the case of a fixed pinhole diameter, half the value of the smallest pinhole diameter
used is taken to determine the optimum interval. Accordingly, the pinhole diameter to
be used in the case of a preset interval is determined by doubling the value of the
selected interval.
The Optical Slice window displays the following information:
Black: Stack Z Size (µm) = intervals x (number of slices - 1)
Optimal Interval = depending on the objective used and the pinhole diameter setting
Red and other colors: Presentation of the actual data set by the operator helps to
optimize stack creation.
Tabs
Z Sectioning:
Tab for setting of Number of Slices, Interval and Current Slice via slider / arrow
button.
Mark First/Last: Tab for determination of the Z-value for the first and last XY-image of the stack,
combined with manual focusing or Stage control.
Hyperfine
Z Sectioning:
Tab for production of a Z Stack using the optional HRZ 200 fine focusing stage.
First:
Scanning / Display of the beginning (first XY-image) of the stack.
Mid:
Scanning / Display of the center (XY-image in the center) of the stack.
Last:
Scanning / Display of the end (last XY-image) of the stack.
Refr. Corr.:
Considers the different refractive index between the immersion medium of the
objective (n') and the embedding medium of the specimen (n), which can be set
between 0.5 and 3 via the slider / arrow buttons
Ratio =
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n
n'
5-91
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
Carl Zeiss
LSM-FCS
X:Y:Z=1:1:1
Clicking on this button will set the Z-interval in such a way that the voxel has identical
dimensions in the X-,Y- and Z-directions (cube).
Auto Z Corr.
This function permits the set values of the scan parameters Detector Gain, AOTF,
Ampl. Offset and Ampl. Gain (as measure for the brightness level) to be varied
between two freely selectable slices of a stack to be recorded. During the scan
procedure, the interim values of these three parameters are automatically linearly
interpolated between the initial and end values (see page 5-98).
The parameters of a Z Stack can be defined using the Z Sectioning tab, the Mark First/Last tab or - if
the optional HRZ 200 fine focusing stage is connected - the Hyperfine Z Sectioning tab:
Z Sectioning tab
Fig. 5-67
5-92
Scan Control window Z Sectioning tab activated
Num Slices:
Entry of the number of sections
(single
XY-images)
to
be
recorded with the stack via the
slider / arrow buttons. The entry
does not influence the interval.
Interval:
Entry of the step width (Zdistance between the single XYimages)
via
slider / arrow
buttons. The entry has no
influence on Num Slices.
Current Slice:
Display of the current position of
the slice within the stack.
Change
of
position
via
slider / arrow keys. Reset of the
current slice position in the
center of the stack by clicking on
the C button. Of course, the
borders of the stack are also
changed if the current slice
position is changed.
Keep Interval:
The interval remains constant
when the stack limits or number
of slices are changed.
Keep Slices:
The number of slices remains
constant when the stack limits
or interval are changed.
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Fast Z Line:
Carl Zeiss
Not available for frame mode. Fast Z scan for overviews (only for Line scan mode). The
stack size is retained; the interval is adapted depending on the scan speed.
The optimum stack size is determined with the help of the Line Sel and Range functions:
• Click on the Line Sel button.
–
An XY-scan of the current slice is performed. The cutline is displayed in the image center. The Line
toolbar is displayed on the right-hand side of the Image Display window.
The Line toolbar permits you to define the position, shape, width and color of the cutline in the Image
Display window.
The following function buttons are available:
Arrow selection button: Activates the mouse pointer for the selection and positioning of
the cutline in the Image Display window and for changing its length.
Length change: Click on the drag point and keep the mouse button pressed. Drag the point
and release the mouse button.
Shifting: Click on the line and keep the mouse button pressed. Shift the complete line and
release the mouse button.
Line arrow button: Generation of a straight cutline in any direction in the Image Display
window.
Opened free shape curve button: Generation of an open, free shape curve (spline) in the
Image Display window. The first click sets the starting point, each further click adds a line
segment. A click with the right mouse button ends the process.
Line button: Selecting the line width of the cutline.
Color button: Selecting the color of the cutline.
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5-93
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
Carl Zeiss
LSM-FCS
Click on the Line arrow button or the Opened free shape curve button in the Line toolbar.
• Define a straight line or a free shape curve (spline) as the cutline for the XZ scan.
Cutline
Fig. 5-68
5-94
Image Display window with cutline displayed
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Carl Zeiss
• Then click on the Range button.
–
The XZ-scan will be performed and displayed in the Image Display window. At the same time, the
position of the current slice is shown with a green line and the positions of the first and last slice
with two red lines.
Red upper Z range limit
Fig. 5-69
Green cutline (current slice)
Scan Control window and Image Display window
Red lower Z range limit
• Moving the green line (current slice) enables you to change the current focus position (moving the
stage or nosepiece in the process). The stack limits are also changed, while interval and Num Slice
remain unchanged.
• Shifting one of the red lines enables you to change the stack size; in that case, the interval size is
matched, and the Num Slice remains constant.
–
Changing the values of Num Slice, Interval and Current Slice in the Z Sectioning tab will, of
course, also change the positions of the red and green lines in the Image Display window.
• A click on the Start button will start the recording of the Z Stack.
–
The settings of the entire Scan Control window (Mode, Channels, Z settings) will be used when
the stack is produced.
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5-95
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
Carl Zeiss
LSM-FCS
Mark First/Last tab
The determination of the optimum stack size is
performed here via focusing during a continuous
scan.
• Click on the XYcont button.
Fig. 5-70
Mark First/Last tab
–
A continuous XY-scan of the set focus
position will be performed.
–
If you have reduced the scan speed or have
set image averaging, you should use the fast
scanning mode to find the lowest and
highest points of focus. These settings are
made under Mode in the Scan Control
menu, or directly via the FAST XY button.
• Use the manual focusing drive or the Stage and Focus Control window (see Stage, page 5-134) to
focus on the upper position of the specimen area where the Z Stack is to start.
• Click on the Mark First button to set the upper position of the Z Stack.
• Then focus on the lower specimen area where the recording of the Z Stack is to end.
• Click on the Mark Last button to set this lower position.
• The Num Slices slider enables you to set the number of slices. The limits of the Z Stack remain
constant, the interval is matched accordingly.
• Click on the Start button to start the recording of the Z Stack.
In case the upper and lower limits of the stack have been switched round, automatic matching will be
performed by the software, since the nosepiece of the Axiovert 200 M always moves from top to
bottom.
Setting via Range is not possible via the Mark First/Last function, i.e. the lines cannot be
shifted.
The Fast Z Line functions is not available in frame mode.
When you change from Mark First/Last to Z Sectioning or vice versa, the values are updated
in the Z Sectioning tab.
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Carl Zeiss
Hyperfine Z Sectioning tab
Activation of this tab is only possible if the
HRZ 200 fine focusing stage or piezo objective
focusing device has been connected.
The HRZ 200 or piezo objective focusing device
can be controlled via software (see Stage, page 5134).
The accuracy of the HRZ 200 or piezo objective
focusing device regarding the step width in the Zdirection lies in the range of 10 nm.
Fig. 5-71
Hyperfine Z Sectioning tab
The HRZ 200 or piezo objective focusing device allows stacks to be produced considerably quicker than
via the focus of the microscope stand.
The focus position remains unchanged.
• Clicking on the additional Leveling button moves the HRZ 200 to the zero position, while the motor
focus moves into the opposite direction at the same time, i.e. the position of the object in relation to
the objective remains unchanged. This function is used to set defined initial conditions.
• The Calibration slider must normally be left in the default position 0. Calibration is required only if
the examined image field is located clearly outside the center of the specimen carrier on the HRZ 200.
Calibration is not required for the motorized stage. In that case, the Calibration function
cannot even be activated (see Annex: Hints on the use of the HRZ 200 or piezo objective
focusing device).
• Use the slider or the arrow keys to set the number of slices for the Z Stack.
• Use the slider or the arrow keys to set the size of the interval.
Num Slices and Interval can be varied independently of each other within the HRZ 200 work range of
±100 µm. When change is made to Z Sectioning, or vice versa, values are also taken over, provided
they are within the HRZ 200 or piezo objective focusing device work range.
If a larger range is set for the Z Stack under Z Sectioning or Mark First/Last, the Interval is matched
accordingly when changing to Hyperfine Z Sectioning, while Num Slice remains constant.
• Use XYcont, Line Sel and Range to determine the parameters of the Z Stack (identical to Z
Sectioning).
If the green line (Current Slice) is shifted after the creation of Range, the focus position will change (the
HRZ 200 or piezo objective focusing device remains in the center position). The red lines (stack limits) can
only be changed symmetrically to the Current-Slice position within the HRZ 200 or piezo objective
focusing device work range.
Since the HRZ 200 moves from bottom to top during the creation of the Z Stack, top and
bottom of the Axiovert 200 M have been switched round.
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5-97
Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Auto Z Corr.
The function Auto Z Correction allows a linear variation of Detector Gain, Ampl. Offset, and Ampl.
Gain values between the different slices of a stack.
• Click on the Auto Z button, the Auto Z
Brightness Correction window opens.
The buttons Set A and Set B permit definition of
two distinct gain / offset / AOTF settings at two
different Z positions A and B.
Pressing the Move A and Move B buttons permits
the defined Z-position to be directly approached.
The Enable test check box permits simulation of
the value changes for Detector Gain, Ampl.
Offset, Ampl. Gain and Attenuation in the Scan
Control window without the scanners being in
operation.
Fig. 5-72
Z Brightness level control window
If a Z Stack is performed and the Auto Z Brightness Correction window is opened this
correction is automatically performed equal whether the Enable test box is enabled or
disabled.
• Use the focusing drive to set the Z-position where the brightness level correction is to be started.
• In the Scan Control window, set the initial values for Detector Gain, Ampl. Offset and Ampl.
Gain. If required, start the continuous scan procedure for this purpose. Click on the Set A button.
• Use the focusing drive to set the Z-position where the brightness level correction is to be ended.
• Set the end value for Detector Gain, Ampl. Offset and Ampl. Gain in the Scan Control window.
Click on the Set B button.
• If required, check the change of the set values by activating Enable test.
After the start of the scan procedure, the brightness level values are linearly interpolated between the
defined references A and B.
Note that the total Z range where the interpolation takes place can exceed the Z reference A
and B.
5-98
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Carl Zeiss
Acquisition of a Z Stack
Once you have set up your image as defined in the above section, you can collect a series of confocal
images through the different focal planes of your specimen.
• Click on the Start button on the Scan Control window. The system will automatically start the
creation of a Z Stack. Be careful not to bump the air table or the microscope until Z sectioning is
completed. Each successive Z Slice can be viewed by changing to the Gallery Mode. This button is
located on the right-hand side of the image.
Fig. 5-73
Image Display window of a Z Stack
A black bar will be shown under the image and will move from left to right, showing that the LSM-FCS is
in the process. The laser will automatically stop scanning when the Z Stack is completed.
The entire stack of images can be saved using the Save or Save As buttons on the right-hand side of
the image.
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5-99
Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
(4)
LSM-FCS
Use ROI (Region Of Interest)
Performance of the Frame and Z Stack scan
modes can be limited to one or several freely
definable sections within the the Image Display
window using the Use ROI function.
The laser scans the entire line length, but is limited
in the Y-direction by the ROIs. The Scan Time is
therefore reduced.
Definition and activation of the ROIs to be used is
performed via the Edit ROI function (Acquire
subordinate toolbar).
If no ROI has been activated, the Use ROI button
is not available.
Fig. 5-74
Image Display window created via
the Use ROI function
• Click on the Edit ROI button in the Acquire
subordinate toolbar to open the Edit ROI
window.
• Define one or several ROIs as required or select an existing ROI from the ROI Lists panel (see Edit
ROI, page 5-108).
• The selected ROI is automatically activated when the Edit ROI window is closed with a click on Close.
• Click on the Use ROI button in the Scan Control window to perform the scan procedure in the
defined ROI exclusively.
Only the regions of interest defined before are visible in the new scanning image, the other areas remain
dark.
The Scan Time is updated when ROIs are used.
• Clicking on the Use ROI button again will deactivate the function.
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
5.5.4.3
Carl Zeiss
Line
In the Line mode, fluorescent or reflected light
along a freely definable line is displayed in the
form of an intensity profile.
All the possibilities of creating an image (Frame,
Z Stack) are also available in the Line mode.
The Line and Frame buttons are activated
alternately and exclude each other.
If the Line button has been selected, the Line Sel
(selection) button also appears on the right-hand
side of the Scan Control window. It permits
positioning of the line to be scanned as required
within the Image Display window (Frame in XYplane).
• Set all the parameters for the Scan procedure
(Mode and Channels or Z Settings) in the
same way as for the scanning of a frame or a
Z Stack.
• Then click on the Line Sel button.
–
A frame will be scanned and the currently
selected scan line and its intensity profile will
be displayed. The Line toolbar is displayed
on the right-hand side of the Image
Display window.
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Fig. 5-75
Scan Control window - Mode/Line
5-101
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
Carl Zeiss
Fig. 5-76
LSM-FCS
Image Display window after activation of the Line Sel button
The Line toolbar permits you to define the position, shape, width and color of the scan line in the Image
Display window.
The scan line can be defined either as a straight line or a free shape curve (spline).
5-102
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Carl Zeiss
The following function buttons are available in the Line toolbar:
Arrow selection button: Activates the mouse pointer for the selection and positioning of
the scan line in the Image Display window and for changing its length.
Length change: Click on the drag point and keep the mouse button pressed. Drag the point
and release the mouse button.
Shifting: Click on the line and keep the mouse button pressed. Shift the complete line and
release the mouse button.
Line arrow button: Generation of a straight scan line in any direction in the Image Display
window.
Opened free shape curve button: Generation of an open, free shape curve (spline) in the
Image Display window. The first click sets the starting point, each further click adds a line
segment. A click with the right mouse button ends the process.
Line button: Selecting the line width of the scan line.
Color button: Selecting the color of the scan line.
(1)
Defining a straight line as the scan line
• Activate the
Line arrow button of the Line toolbar. Click on the spot in the frame at which the
line is to start and keep the mouse button pressed.
• Then drag the line to its desired end position and let go off the mouse button again.
The position of the line in the image can be changed as follows:
Arrow selection button. To change the position in the X/Y-direction, click on the line
• Activate the
and keep the mouse button pressed.
• Then move the lines to the desired position and let go off the mouse button again.
• To change the rotation direction or the length of the line, click on the start or end point of the line
and keep the mouse button pressed.
• Change the rotation direction and / or the length of the line as required and let go off the mouse
button again.
The intensity profile for the defined line is displayed on-line.
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5-103
Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
After release of the mouse button, the relevant intensity profile along the drawn line will be displayed. In
the Zoom, Rotation & Offset panel, the current, changed angle and the offset in X and Y are
displayed.
• When the Line Sel button is pressed again, a frame will be scanned in such a way that the selected
line lies exactly in the center of the Y-axis again and is parallel to the X-axis.
The position of the Line (rotation and offset) can also be changed directly in the Zoom,
Rotation & Offset panel of the Scan Control window.
In the Line mode, Line Stacks can also be recorded over a defined period of time (see Time
Series, page 5-114).
Line Scan is only possible in the unidirectional mode.
(2)
Defining a free shape curve (spline) as the scan line
• Activate the Free shape curve button
of the Line toolbar.
• Draw the your shape curve (spline) in the Image Display window using the mouse. The first click sets
the starting point, each further click adds a line segment. A click with the right mouse button ends
the line definition.
The scanner represented by a white line immediately begins with the on-line tracing of the defined free
shape curve. The laser excitation remains inactive in this process.
If the defined free shape curve becomes too complicated or the selected Scan Speed is too high, the
following message appears in the status bar of the Image Display windows:
Maximum scanner speed exceeded!
• In this case, reduce the Scan Speed set in the Scan Control window.
• If the generated contour and the line traced by the scanner are not in coincidence, reduce the Scan
Speed by a further amount.
If no sufficient coincidence of the two lines can be achieved by the reduction of the scan speed, you
have to calibrate the scanner position signal.
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Carl Zeiss
• Click on the Maintain button in the Main
menu and then on the Spline button.
–
The Calibrate Spline Scan window is
opened.
• Bring the generated contour and the scanner
line to coincidence by varying the amplitude or
offset values for X and Y.
• If necessary, match the free shape curve to the
scanner line.
• Then click on the Single or Cont. button to
execute the scan process, with the laser
activated.
Fig. 5-77
Calibrate Spline Scan
A Line scan is performed along the defined
freehand shape curve, and the intensity profile is
displayed at the bottom of the Image Display
window.
Fig. 5-78
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Image Display window after definition of a freehand shape
curve for the line scan process
5-105
Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Irrespective of the length of the defined freehand shape curve, the intensity profile is always calculated
(by interpolation) and displayed in accordance with the pixel value set under Line Length (2048 pixels
maximum).
• Click on the Stop button to terminate the scan procedure.
As soon as the free shape curve is modified, the laser excitation is deactivated and the scanner
again starts to trace the newly generated free shape curve.
(3)
Selecting the width and color of the scan line
• Line color and width can be set via the Line and Color buttons of the Line toolbar.
(4)
Line Stack
The intensity profile of a defined straight line or free shape curve can also be recorded as a Z Stack. To
do this, proceed in the same way as for the Frame Stack.
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
5.5.4.4
Carl Zeiss
Spot
In the Spot mode fluorescent or reflected light
occurring from a single voxel xyz is detected. In
this mode a spot can be defined by two
perpendicular lines in the Image Display window.
In the spot mode the Z Stack button is not
available. After definition of the spot position the
only possible scan mode is a time series of a spot.
Fig. 5-79
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Scan Control window - Mode/Spot
5-107
Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
5.5.4.5
LSM-FCS
Camera control
The use of this function permits the control of the
external CCD-camera settings.
(1)
Open / Close the Scan control window
for camera control
• In the Configuration Control
activate the Camera button.
window,
• Click on the Scan button in the Acquire
subordinate toolbar of the main menu.
• Click on the Close button
(2)
Fig. 5-80
5-108
Scan Control window - Mode,
settings for camera control
Function description
Mode button
Displays
the
selected
objective, frame size and
pixel depth.
Frame Size
Selects between square
formats or free defined
frame sizes.
Format
Selects between a range of
default camera resolutions.
The 5x5 binning mode can
be used for focusing in
realtime.
Data Depth
Sets the pixel depth.
Zoom/Offset
Shifts a subregion in the
frame.
Reset
Resets the frame/subregion
to default value selected in
Format.
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OPERATION IN EXPERT MODE
Acquire Menu
Scan Control
LSM-FCS
Channels button
Displays
channels
settings.
Exposure time
Sets the exposure time of
the camera.
Find
Starts a prescan and sets
the
exposure
time
automatically.
In case of a camera
multitracking, only one
channel should be selected
in Configuration Control in
order to speed up the find
function.
Fast X/Y
Starts a fast online scan
mode, e.g. for focusing.
Also, the 5x5 binning mode
can be used (to be set in
Mode / Format).
Single
Starts a single image
acquisition (The Image
Display window appears.).
Continuous
Starts acquisition of a series
of images (The Image
Display window appears.).
Crop
the
and
Carl Zeiss
activated
possible
Defines a ROI for camera
acquisition in the Image
Display window. Note that
this is just a Crop function,
while the whole sample is
illuminated. Rotation of the
ROI is not possible.
Fig. 5-81
Scan Control window - Channels,
settings for camera control
Info button
Shows the acquisition parameters in the Image Display window.
Close
Close the Scan Control window.
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5-109
OPERATION IN EXPERT MODE
Acquire Menu
Edit ROI (Region Of Interest)
Carl Zeiss
5.5.5
LSM-FCS
Edit ROI (Region Of Interest)
A scan image allows certain areas (ROIs) to be defined. Only these areas of interest will be scanned. The
laser beam will be switched on only in these areas via Acousto-Optical Tunable Filters (AOTF). Definition
and activation of the ROIs for the scan procedure is performed in the Edit ROI window.
5.5.5.1
Open / Close the Edit ROI window
• Click on the Edit ROI button in the Acquire subordinate toolbar of the Main menu. The Edit ROI
window appears on the screen and the ROIs defined last are visible in the Image Display window.
• Click on the Close button in the Edit ROI window. The Edit ROI window is closed and the ROIs
disappear from the Image Display window.
Fig. 5-82
Edit ROI window and Image Display window with ROIs
The Use ROI status display (button) in the Scan Control window shows whether the ROI mode is
activated or not. If ROIs shall not be taken in consideration during scanning, the Use ROI button must be
deactivated prior to the scanning procedure.
When Edit ROI is activated and the first ROI is drawn in the Image Display window, the Use ROI is
activated automatically.
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Edit ROI (Region Of Interest)
LSM-FCS
5.5.5.2
Carl Zeiss
Function description
The following functions are available on the right side of the Edit ROI window:
Close button
The Edit ROI window is closed.
Remove button
An entry marked in ROI Lists (stored ROI configuration) is deleted.
Add to Lists button
The Add ROI List window is opened.
(1)
ROI Lists panel
In the ROI Lists panel, all the currently defined and
stored ROI configurations are shown.
• Click on the ROI configuration which you want
to use for the scan procedure.
–
The selected ROI configuration is highlighted
in blue and displayed in the opened Image
Display window.
Fig. 5-83
ROI Lists panel
• To produce a new ROI configuration, an already
stored configuration can be activated, changed
and stored under a new name using the Add
to List button.
• To delete a stored ROI configuration from the
list, click on its name first (highlighted in blue)
and then on the Remove button.
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OPERATION IN EXPERT MODE
Acquire Menu
Edit ROI (Region Of Interest)
(2)
LSM-FCS
Interactive ROI Definition panel
In the Interactive ROI Definition panel, the
parameters of the ROI configuration just selected
from the ROI Lists panel are displayed.
Furthermore, it contains all the functions required
for the creation of ROIs.
The X and Y values for Center Position and
Dimension can be edited.
• Activate the relevant text box with a mouse
click and enter the new value via the keyboard.
Fig. 5-84
Interactive ROI Definition panel
• If you click outside the edited text box, the new
value will be taken over and the ROI figure be
shifted to the new position.
The upper part of the panel gives an overview of all the individual figures stored under the selected
name according to type, position within the Image Display window (in pixels) and greatest dimension in
X and Y (in pixels). The origin of the position indication lies in the left top corner of the Image Display
window.
Check box (e.g.: 1 - 4): Clicking on this check box allows a ROI to be deactivated. The tick
disappears from the check box, as does the relevant marked area from the scanning image.
Clicking on the check box again will reactivate the ROI.
Arrow button: Activation of the mouse button to change the size or move the ROIs in the
Image Display window.
Rectangle button: Draw of a rectangle in the Image Display window; click and keep
mouse button pressed, drag the rectangle in any direction, let go off the mouse button to
end the procedure.
Bezier button: Draw of a bezier figure in the Image Display window; first click sets the
starting point, each additional click adds a line, double-click on the starting point closes the
figure and ends the procedure.
Ellipse button: Draw of an ellipse in the Image Display window; first click sets the center
point, displayed line permits determination of the extension, second click sets the first
dimension, then the second dimension and the rotation direction can be determined, third
click sets the second dimension and direction and ends the procedure.
Circle button: Draw of a circle in the Image Display window; click and keep the mouse
button pressed to set the center point, drag the diameter, let go off mouse button again to
end the procedure.
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Edit ROI (Region Of Interest)
LSM-FCS
Carl Zeiss
Polyline button: Draw of a polyline figure in the Image Display window; first click sets the
starting point, each further click adds a line, double-click on the starting point closes the
figure and ends the procedure.
Recycle bin button: All the ROIs dragged to the scanning image are deleted. If an area
outline was marked before, this area is now deleted in the scanning image.
Auto / Color button: A defined color from the list of colors can be assigned to the ROIs. In
that case, the same color is assigned to all the individual figures. In the Auto position, the
outlines of the dragged ROIs are automatically colored differently.
Line button: This button allows you to determine the line thickness of the area outline. This
is for display purposes only. The scanned line is not effected.
Fit Frame Size to bounding Rectangle of all ROIs check box: If this check box is ticked,
the scan procedure is displayed only within a rectangle which is defined by the greatest
extension in X and Y of all the individual figures together, i.e. the pixel number and the data
quantity of the Image Display window are reduced.
• In the toolbar of the Interactive ROI Definition panel, click on the symbol of the area you want to
use to mark the region of interest in the scanning image. Five different area symbols are available in
the form of buttons.
• Click on the marking area and keep the mouse button pressed to drag the area into the region of
interest in the scanning image. The marking area will be numbered automatically and entered in the
Interactive ROI Definition panel with its position and dimension parameters and the appropriate
number.
• The dragged marking area is marked by clicking on its outline; its size can be changed by clicking on
the marking points. Clicking on the area edge beside the marking points allows repositioning of the
area on the scanning image.
The digits of the ROIs can be shifted independently of the contours of the figure.
• If you have framed all the required ROIs in
accordance with steps 2 to 4, you can store
these ROIs under any required name via the
Add to Lists button.
• The Add ROI List window will appear. Enter
any required name to store the ROIs and click
on the OK button.
• This stored ROI configuration appears in the
ROI Lists panel of the Edit ROI window.
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Fig. 5-85
Add ROI List window
5-113
Carl Zeiss
OPERATION IN EXPERT MODE
Acquire Menu
Time Series
5.5.6
LSM-FCS
Time Series
The Time Series Control window allows the
definition of parameters for time series.
The Time Series function offers the following
options for the creation of image series:
–
Definition of break times between 0.1 ms and
10 hours.
–
Determination of the number of steps from 1
to 10,000 for one scanning procedure.
–
Setting of markers.
–
Interruption of time control via pause function,
and resume of the time series function.
–
Triggering of time series via:
–
numeric input
–
external trigger pulses
–
time (of the PC)
5.5.6.1
Fig. 5-86
Time Series Control window
Open / Close the
Control window
Time
Series
• Click on the Time Series button in the Acquire
subordinate toolbar of the Main menu.
The Time Series Control window appears on the
screen.
• Click on the Close button to close the Time
Series Control window.
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Time Series
LSM-FCS
5.5.6.2
Carl Zeiss
Function description
The following functions are available on the right-hand side of the Time Series Control window:
Close button
Closes the Time Series Control window.
New button
Opens a new Image Display window.
Start T button
Starts the Time Series.
Start B button
Starts the Time Series in combination with a bleach procedure. Bleach
procedure must be defined first in the Bleach Control window.
Stop button
Stops the entire Time Series. A current scan is interrupted.
Pause button
Interrupts the Time Series. Button labeling is changed to Resume. A
current scan is performed until the end. When the button is pressed
again, the Time Series is immediately continued with the next scan
procedure.
Bleach button
Starts a Bleach procedure without a Time Series. Bleach procedure must
be defined first in the Bleach Control window.
Mean ROI button
Creates a Time Series with the intensity values of the Frame or the
default ROIs. An average value is formed of the intensity values of the
Frame or the ROIs determined in the relevant scan procedure. These
average values are displayed in an extended Image Display window as a
function of the time which has passed.
The status line, in which the phases of the current Time Series or notes for the user are displayed, is in
the lower part of the Time Series Control window.
(1)
Start Series panel
In this panel, the parameters for the start of the
time series are set.
Fig. 5-87
Start Series panel
The following functions are available:
Manual button
The time series is started manually with a click on the Start T or Start B
button.
Trigger button
The time series is started via a trigger signal from Trigger Control.
Time button
The time series is started when the set time is reached. The internal
computer time applies.
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Time Series
Carl Zeiss
LSM-FCS
Time input box
Input of the time for the start of the time series (Time button activated).
Trigger in list box
Selection of the trigger key (1-4) with which the start is to be triggered
(Trigger button activated).
Trigger out list box
Selection of the trigger keys (1-4) for the out signal.
(a)
Start via Trigger
For the start via trigger control (Trigger button
activated), first determine the trigger key which is
to trigger the start of the Time Series.
Fig. 5-88
Start Series panel
• Open the Trigger in list box with a click on the
arrow button.
• Choose one of the trigger keys 1 to 4 (e.g. Trigger1).
It is also possible to trigger an out signal via trigger control.
• Open the Trigger out list box with a click on the arrow button.
• Choose one of the trigger keys 1 to 4 (e.g. Trigger1).
In this example, the scan procedure is triggered on pressing key 1 of the trigger control, and an out
signal is given at the same time.
When starting a Time Series via Trigger, the Start T or Start B button must be pressed first.
Waiting for Trigger will then be displayed in the status line.
Then the relevant trigger key on the Trigger Control must be pressed to start the first scan
procedure of the Time Series.
(b)
Start via Time
For the start via the time set on the PC (Time
button activated), the start time must be entered
first in the Time input box.
Fig. 5-89
Start Series panel
• Click in the Time input box to open it.
• Enter a start time via the keyboard. Then click
outside the input box once to close it again.
When starting a Time Series via the time, the Start T or Start B button must also be pressed in
this case. Waiting for Start Time will be displayed in the status line.
The Time Series is started when the starting time has been reached.
The starting time for the Time Series can be changed online.
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Time Series
LSM-FCS
(2)
Carl Zeiss
Stop Series panel
In this panel, the parameters for the end of the
Time Series are set and the number of cycles is
determined.
Fig. 5-90
Stop Series panel
The following functions are available:
Manual button
The time series is finished manually with a click on the Stop button.
Trigger button
The time series is finished via a trigger signal.
Time button
The time series is finished when the set time has been reached. The
internal computer time applies as the set time.
Number input box /
arrow keys / slider
Determination of the number of images acquired or image stacks for the
time series.
Time input box
Input of the time for the end of the time series (Time button activated).
Trigger in list box
Selection of the trigger keys (1-4) with which the end is to be triggered
(Trigger button activated).
Trigger out list box
Selection of the trigger keys (1-4) for the out signal.
• Use the slider near Number to select the images or image stacks for the time series.
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Time Series
Carl Zeiss
(a)
LSM-FCS
Stop via Trigger
To end the Time Series via Trigger Control
(Trigger button activated), first determine the
trigger key which is to end the Time Series.
Fig. 5-91
Stop Series panel
• Open the Trigger in list box with a click on the
arrow button.
• Choose one of the trigger keys 1 to 4 (e.g.
Trigger2).
It is also possible to trigger an out signal via Trigger Control.
• Open the Trigger out list box with a click on the arrow button.
• Choose one of the trigger keys 1 to 4 (e.g. Trigger2).
In this example, the Time Series is ended on pressing key 2 of the Trigger Control, and an out signal is
given at the same time.
If the entered number of cycles has been processed without a trigger impulse having been given
to end the procedure, the Time Series is finished.
If a trigger signal is given before the cycles have been processed, the Time Series will only be
interrupted. Waiting for Trigger will be displayed in the status line. The Time Series can now
be continued via a new trigger signal or ended via Stop.
(b)
Stop via Time
To end the Time Series via the time set on the PC
(Time button activated), the end time must first be
entered in the Time input box.
• Click on the Time input box to open it.
Fig. 5-92
Stop Series panel
• Enter the end time via the keyboard. Then click
outside the input box once to close the box.
The Time Series is interrupted when the end time has been reached.
If the entered Number of cycles has been processed, the Time Series is finished.
If the number of cycles has not yet been processed, the Time Series is only interrupted. Waiting
for Start Time is displayed in the status line. The Time Series can now be continued by entering
a new start time, or finished via Stop.
The end time for the Time Series can be changed online.
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Time Series
LSM-FCS
(3)
Carl Zeiss
Time Delay / Time Interval panels
Depending on the settings in the Time Series tab
(see Options menu, Settings), the time series
interval is defined either as a Time Delay or Time
Interval. Accordingly, either the Time Delay
panel or the Time Interval panel is displayed in
the Time Series Control window.
Time Delay is the interval between the end of one
scan process and the beginning of the next.
Fig. 5-93
Time Delay panel
Fig. 5-94
Time Interval panel
Time Interval is the interval between the
beginning of one scan process and the beginning
of the next.
The Time Delay (or Time Interval) panel permits
the intervals to be activated and changed.
The following functions are available:
Time delay or
Time Interval list box
List of the stored sets of time delays or Time Intervals for time series.
Apply button
Application of the sets of delays for time series selected in the list box.
Store button
Storage of sets of delays for time series.
Delete button
Deletion of sets of delays for time series from the list box.
Time buttons
Activation of the time for the time series set for the relevant button.
Time input box /
arrow buttons / slider
Determination of the cycle time for the currently activated Time button.
Unit buttons
Selection of time units: min, sec or ms.
Trigger in list box
Selection of the trigger key (1-4) to be used to activate the Time button
for the delay time.
Trigger out list box
Selection of the trigger key (1-4) for the out signal.
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Acquire Menu
Time Series
LSM-FCS
• The delay time or time interval to be used during the Time Series is set to a default value by activating
a Time button.
For this purpose, the relevant time must be assigned to the Time button first.
• Activate a Time button with a click of the mouse.
• Set the required delay time or time interval via the slider (arrow keys or input box) near Time. The set
time is displayed online on the button. Select the time unit by clicking on the relevant button near
Unit.
You can assign different times to all the six Time buttons and store this assignment either as a set of
delays or of time intervals.
• Enter a name in the Time Delay list box or Time Interval list box and click on Store to store the set
of delays.
If required, a set of delays or time intervals can be activated again quickly.
• Open the list box with a click on the arrow button and select the required set with a click of the
mouse.
• Then click on the Apply button to activate the set. The stored delays are assigned to the Time
buttons.
Sets of delays or Sets of time intervals which are no longer required can be deleted.
• Open the list box and select the required set.
• Click on the Delete button. The set will be removed.
The Time buttons can also be activated via keys 1 to 4 of the Trigger Control.
• Click on the required Time button.
• Open the Trigger in list box with a click on the arrow button.
• Choose one of the trigger keys 1 to 4 (e.g. Trigger3).
It is also possible to trigger an out signal via Trigger Control.
• Click on the required Time button.
• Open the Trigger out list box with a click on the arrow button.
• Choose one of the trigger keys 1 to 4 (e.g. Trigger3).
In this example, the relevant Time button is activated on pressing key 3 of the Trigger Control, and an
out signal is given at the same time.
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Time Series
LSM-FCS
Carl Zeiss
The delays or time intervals can be changed online with a click on another Time button. The
new delay will be applied immediately.
A change of the delay during a Time Series is displayed in the Image Display window if the
Gallery button (Display toolbar) is activated.
(4)
Marker panel
The setting of a marker permits information about
the moment in the current time series and any
required comment to be assigned to the current
scan. The time indication is set automatically, while
comments must be defined before.
The markers (red squares) are visible in the Image
Display window if the Gallery button (Display
toolbar) is activated.
On storage of the image, all the markers, including
the time indication and the comments, are stored
along with the image contents.
Fig. 5-95
Marker panel
The following functions are available:
Marker list box
List of the stored combinations of markers.
Apply button
Application of the marker combinations selected from the list box.
Store button
Storage of a combination of markers.
Delete button
Deletion of a combination of markers from the Marker list box.
Set 1-7 button
Setting of a marker during the scan procedure.
Edit Text input box (1-7)
Entry of the comments for the marker.
Trigger in list box (1-7)
Selection of the trigger key (1-4) with which the marker is to be set.
Trigger out list box (1-7)
Selection of the trigger key (1-4) for the out signal.
• A marker for the current scan is set by clicking on one of the Set 1 to 7 marker buttons.
The assignment of any required comment for the marker must be performed as follows:
• Click in the Edit Text box of the required marker key (e.g.: Set 1) to open the editing box.
• Enter the comments via the keyboard. Then click outside the editing box to close this box again.
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OPERATION IN EXPERT MODE
Acquire Menu
Time Series
LSM-FCS
You can assign comments of any required length to all the seven Set buttons and store this assignment
as a combination of marker keys.
• Enter a name in the Marker list box and click on Store to store the combination.
If required, a combination of markers can be activated again quickly.
• Open the Marker list box with a click on the arrow button and select the required combination with a
click of the mouse.
• Then click on the Apply button to activate the combination. The relevant comments are displayed in
the Edit Text boxes of the Set buttons.
Combinations which are no longer required can be deleted.
• Open the Marker list box and select the required combination.
• Click on the Delete button. The combination will be removed.
The marker buttons can also be activated via keys 1 to 4 of the Trigger Control.
• Click on the required Set button.
• Open the Trigger in list box with a click on the arrow button.
• Select one of the trigger keys 1 to 4 (e.g. Trigger4).
It is also possible to trigger an out signal via Trigger Control.
• Click on the required Set button.
• Open the Trigger out list box with a click on the arrow button.
• Select one of the trigger keys 1 to 4 (e.g. Trigger4).
In this example, the relevant Set button is activated on pressing key 4 of the Trigger Control, a marker is
set in the Scan and an out signal given at the same time.
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Time Series
LSM-FCS
5.5.6.3
Carl Zeiss
Time Series of a Frame
• Set the relevant parameters for time control in the Start Series, End Series and Time Delay panels.
• Start the Time Series with a click on the Start T or Start B button.
• If you use Trigger Control, confirm the relevant Trigger key to start the Time Series with the first scan
procedure.
• Use the Set 1 to Set 7 buttons to set markers during the scanning procedure which will allow you to
evaluate interesting scanning images later.
Time end will finish time series even if you have created a program which would exceed the
time end.
Bleach times will be added.
No break is possible during bleaching.
If you want to integrate a bleaching procedure in a time series, start must be triggered via
Start B. The bleaching procedure must be defined first in the Bleach Control window (see
page 5-129).
If a time series is interrupted before its programmed end, the programmed number of images
will be taken over in the database. However, only those images are stored which were created
before interruption of the time series. This is due to the fact that the original image parameters
are to be taken over via the Reuse function.
If a stop time for time series is entered via the Trigger button or the Time button, the
recording of the series will not be definitely finished. It is possible to either continue the series
via new settings of Trigger and Time or to definitely finish the time series via the Stop key.
The following example of a scanning image was taken using the Time Series function. Both the time
and the markers set during the scanning procedure are projected in the image series in different colors.
If the cursor is moved to a marker position in the scanning image, the relevant information on the image
detail is automatically provided in an additional window.
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OPERATION IN EXPERT MODE
Acquire Menu
Time Series
Carl Zeiss
LSM-FCS
Colored
marker positions
Fig. 5-96
Image Display window of a Time Series Scan
The image markers have different colors with the following meaning:
–
red:
manually set marker with time indication and comments
–
blue:
automatically set marker with change of delay
–
green:
automatically set marker at the beginning and at the end of a bleaching procedure
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Time Series
LSM-FCS
5.5.6.4
Carl Zeiss
Time Series of a frame over Z Stack
• First, set all parameters required for recording a Z Stack in the Scan Control window.
• Then set the parameters required for recording the time series in the Time Series Control window
(identical procedure as for the time series of a frame).
• Start the time series by clicking on Start T.
–
Complete stacks are now recorded at the defined time intervals. The result is displayed in the form
of the combined Image Display window of the stack and time series (4D).
Fig. 5-97
Image Display window of a Z Stack and a Time Series Scan
The additional Z, Time and Z + Time buttons are available in the Gallery toolbar of the Image Display
window.
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OPERATION IN EXPERT MODE
Acquire Menu
Time Series
Carl Zeiss
LSM-FCS
When you click on the Z button, the individual frames of the Z Stack are displayed for the selected Time
Slice. When you click on the Time button, the individual frames of the time series are displayed for the
selected Z Slice.
For Z Stacks over the time (4D) following offline functions will be enlarged:
– Slice (Z slider and Time slider)
– Gallery (Z, Time and Z + Time buttons)
– 3D (slider for single time index)
To select the Z or Time Slices, use the appropriate sliders which are displayed if the Slice button in the
Image Display window has been activated.
When you click on the Z + Time button, all individual frames will be displayed.
5.5.6.5
Time Series of a frame over a Z Stack over Lambda
• Activate the META mode sheet in the configuration control and set the relevant parameters.
• Set the relevant parameters for the Start Series, End Series and Time Delay panels.
• Start the time series with a click on the Start T or Start B button.
Fig. 5-98
5-126
Image Display window of a Time Series over Lambda
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Time Series
LSM-FCS
5.5.6.6
Carl Zeiss
Time Series with Mean ROI
• Set all the parameters in the same way as for Time Series of a frame.
• Then click on the Mean ROI button in the time series frame.
A mean intensity profile of the defined ROIs is created as a function of time.
Fig. 5-99
Image Display window of a Time Series with Mean ROI
The Image Display window of the Mean ROI function is structured differently than that of a frame.
On the left-hand side of the Image Display window, the intensity time profiles per ROI are displayed
graphically.
The Select and Display toolbars, which are also available in the standard Image Display window, are
positioned in the center.
The Scan Mean of ROIs toolbar with further function elements is additionally displayed on the righthand side. The major purpose of these function elements is to vary the display of the recorded
Mean ROI.
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OPERATION IN EXPERT MODE
Acquire Menu
Time Series
Carl Zeiss
LSM-FCS
By selecting the appropriate options (see Options menu, Settings – Scan Mean of ROIs) you can
activate the following additional functions:
–
Display of the live image in the Image Display window of the Mean ROI function (used ROIs
only)
–
Scan of the complete image (if Live Image has been activated)
–
Saving of the complete time series (if Live Image has been activated)
The following functions are available:
Display of the data of the ROIs used for the creation of the MeanROI
(identical to the Edit ROI window). If the check box of a ROI is
deactivated, the ROI's intensity values are no longer displayed in the
Intensity-Time diagram.
1 button: Intensity values for ROI and Channels are displayed in a
diagram. Chan button: Intensity values are displayed separately for each
channel used. ROI button: Intensity values are displayed separately for
each ROI used. Mono button: Switches between color and monochromic
display of intensity profiles.
Automatic button: Automatic scaling of the display of Intensity-Time
diagrams. Time Range button: Display of Intensity-Time diagrams is
scaled depending on the Time Range set in the input box shown on the
left. Number Times button: Display of Intensity-Time diagrams is scaled
depending on the Number Cycle set in the input box shown on the left.
Show Image button: Shows the scan image in the Image Display
window to the side of the intensity diagram. This button is active only if
the Live Image option is activated. Copy Table button: The table of
intensity values is copied to the clipboard. Show Table button: The table
of intensity values is displayed at the bottom left of the Image Display
window. Save Table button: The table of intensity values can be stored
as a text file.
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Acquire Menu
Edit Bleach
LSM-FCS
5.5.7
Carl Zeiss
Edit Bleach
The use of this function permits the setting of
bleaching parameters for spot, line or frame
bleaching.
5.5.7.1
Open / Close the Edit Bleach
window
• Click on the Edit Bleach button in the Acquire
subordinate toolbar of the Main menu. The
Bleach Control window appears on the screen.
• Click on the Close button to close the Bleach
Control window.
Fig. 5-100
5.5.7.2
Bleach Control window
Function description
The following functions are available on the right-hand side of the Bleach Control window:
Close button
The Bleach Control window is closed.
Bleach button
Starts the bleaching procedure.
Stop button
Ends the bleaching procedure.
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OPERATION IN EXPERT MODE
Acquire Menu
Edit Bleach
Carl Zeiss
(1)
LSM-FCS
Settings panel
The Settings panel allows you to determine when
and how the bleaching process shall be done (only
works in connection with time series).
Furthermore, all the settings of the Bleach
Control window can be stored, reactivated or
deleted in this panel.
Fig. 5-101
Settings panel
Bleach after number scans: If this check box is ticked , the bleaching procedure is automatically
performed in combination with a time series. Under Scan Number, you
must enter after how many scanning procedures bleaching is to be
performed.
Scan Number:
Bleach repeat
after number scans:
Number of Scans in a time series, after performance of which the
bleaching procedure shall be started.
If this check box is ticked , the bleaching procedure is automatically
performed in combination with a time series. Under Scan Number, you
must enter after how many scanning procedures bleaching is to be
repeated.
Scan Number:
Number of Scans in a time series, after performance of which the
bleaching procedure shall be repeated.
Different Z Position:
If this check box is ticked , you can set the current stage position as the
one in which the bleaching will be done by clicking the Mark Position Z
button. This function is only available using the Line or Frame scanning
mode.
Different XY
Spot Bleach Position:
5-130
If this check box is ticked , you can set a different XY position for spot
bleaching. This function is only available using the Spot scanning mode.
Click on the Spot Select button in the Scan Control window. A new
image is produced and two crosshairs appear in the image. The red
crosshair marks the spot that will be imaged. The green crosshair marks
the spot that will be bleached. Move the center of the crosshairs to the
desired positions and perform bleaching.
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OPERATION IN EXPERT MODE
Acquire Menu
Edit Bleach
LSM-FCS
Carl Zeiss
Proceed as follows to store the entire settings of the Bleach Control window:
• Enter a name in the Settings list box and click on Store to store the settings.
If required, stored settings for the bleaching procedure can be reactivated quickly.
• Open the Settings list box with a click on the arrow button and select the required name with a click
of the mouse.
• Then click on the Apply button to activate these settings. The Bleach Control window will be
updated accordingly.
Settings which are no longer required can be deleted.
• Open the Settings list box and select the required name.
• Click on the Delete button. This stored setting will be removed.
The bleaching procedure can also be activated via keys 1 to 4 of the Trigger Control.
• Open the Trigger in list box with a click on the arrow button.
• Select one of the trigger keys 1 to 4 (e.g. Trigger4).
It is also possible to trigger an out signal via trigger control.
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5-131
OPERATION IN EXPERT MODE
Acquire Menu
Edit Bleach
Carl Zeiss
(2)
Fig. 5-102
Bleach Parameter panel
LSM-FCS
Bleach Parameter panel
The Bleach Parameter panel allows you to
determine how often the bleaching process shall
be performed, and to select the area for bleaching
in the scan image via the Define Region button.
• Enter the number of iterations of the bleaching
procedure in the Iterations input box.
• Click on the Define Region button.
–
The Bleach Regions window appears.
The definition of bleach regions corresponds to the
Edit ROI function and is performed in the same
way (see section Edit ROI, page 5-108).
ROIs already defined with Edit ROI are also
available in the Bleach Regions window. They can
be activated directly, modified - if required - and
stored under a new name.
ROIs newly defined in the Bleach
Regions window will then also be
available in the Edit ROI window.
• Define the required bleach regions in the scan
image or use an existing ROI.
Fig. 5-103
5-132
Bleach Regions window
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OPERATION IN EXPERT MODE
Acquire Menu
Edit Bleach
LSM-FCS
(3)
Carl Zeiss
Excitation of Bleach Track panel
In the Excitation of Bleach Track panel you can
select the lasers and laser intensities for bleaching.
The setting of the lasers for the bleaching
procedure corresponds to that for the scanning
procedure and must be performed accordingly (see
Laser Control, Configuration Control and Scan
Control).
Fig. 5-104
Excitation of Bleach Track panel
• Select the required laser wavelength and its intensity under Excitation.
• If required, switch the relevant laser to On (Laser button).
(4)
Start / End a bleaching procedure
• The bleaching process will be started via the Bleach button. However, it is also possible to start the
bleaching process via the Bleach button in the Time Series Control window or to combine it with a
time series.
When a trigger key is activated to start the bleaching procedure, the Waiting for Trigger message first
appears in the status line of the Bleach Control window. In that case, the bleaching procedure is started
after activation of the relevant trigger key.
• The bleaching process can be finished via Stop in the Bleach Control window.
Stop does not only stop the bleaching process, but the entire scanning process.
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5-133
OPERATION IN EXPERT MODE
Acquire Menu
Stage
Carl Zeiss
5.5.8
LSM-FCS
Stage
The following software description applies to
systems which are equipped with a motorized
stage.
This window enables you to activate both the
motor focus and the scanning stage.
The Focus Position and Stage Position panels
include the function keys for the performance of
defined moves and the display of the current Z and
X, Y positions.
By use of an LSM 510 META scanhead on
an Axiovert 200 MOT sideport system
care should be taken when moving the
motorized XY scanning stage to the
maximum positions, so that fingers are
not bruised between scan head and
stage.
5.5.8.1
Fig. 5-105
Stage and Focus Control window
Open / Close the Stage and Focus
Control window
• Click on the Stage button in the Acquire
subordinate toolbar of the Main menu. The
Stage and Focus Control window appears on
the screen.
• Click on the Close button in the Stage and
Focus Control window to close this window.
5.5.8.2
Function description
The following functions are available on the right-hand side of the Stage and Focus Control window:
Close button
The Stage and Focus Control window is closed.
Start button
Starts the tile scanning procedure.
Stop button
Ends the scanning procedure.
5-134
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OPERATION IN EXPERT MODE
Acquire Menu
Stage
LSM-FCS
(1)
Carl Zeiss
Focus Position panel
Focus buttons (Z Moves)
Clicking on the Up arrow button moves the
specimen stage / nosepiece upwards.
Clicking on the Z button sets the current Zposition to zero.
Clicking on the Down arrow button moves the
specimen stage / nosepiece downwards.
Fig. 5-106
Focus Position panel
Focus Step slider
0.1 µm is the smallest value which can be set, and 100 µm the highest.
Clicking on the arrow keys changes the step size by 1 µm.
Pressing the CTRL key and clicking changes the step size by 0.05 µm.
Pressing the Shift key and clicking changes the step size by 10 µm.
Work button
Pressing the Work button moves the specimen stage / nosepiece back to the Work position. This is the
position last set before the Load button was pressed.
Load button
Clicking on the Load button lowers the specimen stage / nosepiece to make it easier for you to change
the specimen (or objective).
Focus Wheel check box
Clicking on this check box activates / deactivates the focus wheel of the microscope.
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5-135
OPERATION IN EXPERT MODE
Acquire Menu
Stage
Carl Zeiss
LSM-FCS
Use of the optional HRZ 200 fine focusing stage or piezo objective focusing device
The HRZ Step slider is used to set the step width of the fine focusing stage.
Use the arrows of HRZ to move the fine focusing stage upwards or downwards in steps.
As soon as the focus position is changed (via handwheel or software), the HRZ 200 stage is automatically
leveled.
A click on the L button moves the HRZ 200 fine-focusing stage in the center position of its travel range
and the focus position is reset accordingly. Therefore, the same Z-level remains visible (the current
position is not set to zero).
The motor focus of the stand is operated in the same way via the relevant buttons. Moving into the
Work or Load position is always performed via the motor focus and not via the HRZ stage.
Please see the annex for further information on the HRZ 200 fine focusing stage: Hints on the
use of the HRZ 200 fine focusing stage.
(2)
Stage Position panel
The Stage Position panel shows a symbolic
specimen carrier in the left upper.
The buttons for moving to a position and mark it
are below or on its right.
The Current Position display for X and Y is
below.
Below that, you will find the Marks selection box
of marked positions and the possibility to activate
and delete them.
Moving the scanning stage
Fig. 5-107
Stage Position panel
The scanning stage can be moved using the
joystick, or software-controlled using the Stage
XY buttons, or manually.
Stage XY buttons
Clicking on the arrow buttons moves the stage in X or Y direction.
Clicking on the Center button moves the stage in the XY = 0 position.
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OPERATION IN EXPERT MODE
Acquire Menu
Stage
LSM-FCS
Carl Zeiss
XY Step slider
1 µm is the smallest value which can be set for XY movement, and 100 µm the highest.
Manual check box
This check box activates / deactivates the motor control of the stage and the joystick, if available.
If Manual is active, the scanning stage can be moved manually via the knurled screws. The Move To
and Center function buttons in Stage Position are without a function. The Current Position is
updated. You can zero the display via ZERO and mark manually set positions (Mark pos.).
The scanning stage cannot be moved via the software or the joystick.
If Manual is deactivated, the scanning stage can be moved via the software or the joystick. All the
functions of the Stage Position window are available.
Current Pos(ition) field
Current Pos displays the currently set stage position in relation to the zero position.
Marks selection box
Clicking on the arrow button displays the table of the session-related marked specimen areas. The table
includes the ordinal number, the X-position and the Y-position. Click on the appropriate mark to select it
for operation.
Move To button
Clicking on the Move To button moves the stage to the position selected before from the Marks
selection box.
Remove
The Remove command enables a selected position to be deleted from the table. The position then also
disappears from the specimen carrier display.
The selected position is deleted, the position with the next number in sequence moves up one
number.
Remove All
The Remove All command deletes all the entries marked in the current session.
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5-137
OPERATION IN EXPERT MODE
Acquire Menu
Stage
Carl Zeiss
LSM-FCS
Speed selection box
Clicking on the arrow key displays the table of the available speeds for stage movement. Click on the
appropriate speed to select it for operation.
Zero button
Zeros the Current Position display and thus sets the currently set stage position to 0 in relation to X and
Y. The already marked object areas thus receive new X and Y-coordinates.
Mark Pos. button
Mark Pos. allows the Current Position to be marked. This marked position is then stored in the Marks
selection box in sequence. The marked position is shown on the specimen carrier with a cross and its
ordinal number.
HRZ Zero button
Zeros the Current Position display and thus sets the currently set stage position to 0 in relation to X and
Y. The already marked object areas thus receive new X and Y-coordinates.
(3)
Fig. 5-108
Tile Scan window
Tile Scan panel
This function permits a frame to be created as an
overview image of the specimen with a maximum
size of 4096 x 4096 pixels. According to settings,
such a frame is divided in XY-tiles of 1 x 1 to the
maximum of 15 x 15. A tile of special interest
(target) can then be selected for scanning.
The application of the Tile Scan function requires
an objective with a minimum magnification factor
of 2.5x.
Tiles Numbers X / Y input box
Input of the number of tiles for X or Y from which the frame is to be composed.
Tile Size X / Y display
Display of the size of a single tile in µm (corresponds to the value selected in the Scan Control window).
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OPERATION IN EXPERT MODE
Acquire Menu
Stage
LSM-FCS
Carl Zeiss
Frame Size X / Y display
Display of the frame size of the tile scan for X or Y. Specification in pixels and µm.
Move To button
If the Move To button is activated, a rectangle with a target allowing the selection of the region of
interest is positioned in the center of the scanned frame. Click and hold down the left mouse button to
drag the rectangle to the required specimen area. When you release the mouse button, the stage moves
to the selected position.
Mark button
If the Mark button is activated, marks previously set in the Tile Scan image are displayed, and further
marks can be added at spots of special interest by a mouse click in the Tile Scan image. By activating the
Move To button, the stage can be moved to the individual marks set in Tile Scan in the same way as it is
moved to the marks set in the Stage Position panel.
• Set the number of tiles for the frame in the Tiles Numbers X / Y input boxes of the Tile Scan
window.
–
The resulting frame size is displayed on-line.
• Click on Start.
–
The overview frame is scanned and displayed on the screen in a new Image Display window.
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5-139
OPERATION IN EXPERT MODE
Acquire Menu
Stage
Carl Zeiss
Fig. 5-109
LSM-FCS
Image Display window of a Tile Scan
• Activate the Move To button.
• In the Tile Scan image, move the target to the required spot of the frame (dragging with the mouse).
–
The microscope stage then travels to the selected position.
Or:
• Activate the Mark button.
• Set a mark at the spot of interest by clicking with the mouse in the Tile Scan image. A cross with the
consecutive number of the mark is displayed in the Tile Scan image. The new mark is also displayed in
the specimen carrier (Stage Position panel) and included in the Marks selection box.
• Select the mark in the Marks selection box and click on the Move To button in the Stage Position
panel. The stage moves to the selected position.
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OPERATION IN EXPERT MODE
Acquire Menu
VIS, FCS and LSM Buttons
LSM-FCS
Carl Zeiss
• Then click on the Single button in the Scan Control window to scan the selected area as a single
image.
–
The single image is scanned and displayed in a new Image Display window.
Overlay functions cannot be activated in the Tile Scan Image Display window.
The created overview frame can then be stored like any other scan image. If a stored overview
frame is opened again, the rectangle with target will appear again. However, it can be deleted
using the Overlay function.
5.5.9
VIS, FCS and LSM Buttons
The VIS, FCS and LSM buttons are included in the Acquire subordinate toolbar of the Main menu.
They switch the beam path and indicate which beam path has been set in the binocular tube of the
microscope:
–
VIS:
observation via the eyepieces of the binocular tube, lasers are off
–
FCS:
screen observation via laser excitation using the FCS measurements and software
evaluation
–
LSM:
screen observation via laser excitation using the LSM image acquisition and software
evaluation
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5-141
OPERATION IN EXPERT MODE
Process Menu
Add
Carl Zeiss
5.6
LSM-FCS
Process Menu
• In the Main menu toolbar, click on Process.
–
This opens another, subordinate toolbar in the Main menu.
Process subordinate toolbar
Fig. 5-110
Process menu
The functions of the Process menu permit already stored scan images to be subsequently linked and
processed using mathematical functions and algorithms.
5.6.1
Add
The Add function links two channels each of one
or two images into a new channel through
addition. The channel created in this way can be
stored via the Save As function. (This also works
with extracted META channels.)
5.6.1.1
Open / Close the Add window
• Click on the Add button in the Process
subordinate toolbar of the Main menu.
–
This opens the Add window.
• Click on the Close button to quit the Add
window.
Fig. 5-111
5-142
Add window
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OPERATION IN EXPERT MODE
Process Menu
Add
LSM-FCS
5.6.1.2
Carl Zeiss
Source panel
In the Source 1 panel, the first image source for
the addition process is determined. The current
image is displayed in the display box of the image
selection box.
Proceed as follows to select an image via the
image selection box:
Fig. 5-112
Source 1 panel
• Click on the arrow button. The image selection box is opened and all the currently loaded images are
displayed in a minimized form.
• Click on the required image. This image will then appear in the display box of the image selection box
and has been selected as Source 1.
Use the Click into window button to directly select the opened image:
• Click on the Click into window button first and then double-click on the relevant Image Display
window. The selected image will then be displayed in the display box of the image selection box and
has been activated as Source 1.
The channel which is to be used for the Add operation is selected via the Channel selection box:
• Click on the arrow button. The Channel selection box is opened and shows all the recorded channels
of the relevant image.
• Click on the required channel to activate it.
In the Source 2 panel, the second image source for the addition process is determined. The procedure is
identical to that for Source 1.
• Select the image for Source 2 and the relevant channel.
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5-143
OPERATION IN EXPERT MODE
Process Menu
Add
Carl Zeiss
5.6.1.3
LSM-FCS
Destination panel
In the Destination panel, it is determined in
which Image Display window the Add operation
is performed, and the data format which the
newly created image shall have.
Fig. 5-113
Destination panel
The Add operation can be performed in an already
opened window or in a new Image Display
window.
• Click on the arrow button of the image selection box to open this box.
• Click on the relevant image if the Add operation shall be performed in an existing Image Display
window.
or
• Click on New Image 8 bit or New Image 12 bit to use a new Image Display window.
You can also use the Click into window button for image selection.
Clicking on the New 8 bit or New 12 Bit button enables you to determine directly and quickly
whether the new image is to be created in the 8-bit or 12-bit format.
If an existing Image Display window is used to perform the Add function, you must determine whether
an existing channel shall be overwritten with the Add operation or whether a new channel shall be
added.
• In the Channel selection box, click on the channel which shall be overwritten, or click on New for a
new channel.
5.6.1.4
Fig. 5-114
Add panel
Add panel
In the Add panel, the currently set formula for the
Add operation is displayed. The editable input
boxes permit the formula to be changed with any
numeric values.
• Click in the required input box and enter the relevant value.
• Click on the Apply button to perform the operation in the activated window or a new Image
Display window.
• The new image can then be stored via the Save As function.
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OPERATION IN EXPERT MODE
Process Menu
Subtract
LSM-FCS
5.6.1.5
Carl Zeiss
Preview panel
The Preview function enables you to preview the
result of the defined Add operation in a preview
window.
Fig. 5-115
Preview panel
Preview check box with a click
• Activate the
of the mouse. The Add - Preview Image
Display window is displayed with the operation
result.
• Deactivate the Preview check box to close the Add - Preview Image Display window.
After a change of the formula in the Add panel, click in the Add - Preview Image Display
window for an update.
5.6.2
Subtract
The Subtract function links two channels each of
one or two images into a new channel by
subtraction. The channel created in this way can
be stored via the Save As function.
5.6.2.1
Open / Close the Subtract window
• Click on the Subtract button in the Process
subordinate toolbar of the Main menu.
–
This opens the Subtract window.
• Click on the Close button to quit the Subtract
window.
5.6.2.2
Performance
function
of
the
Subtract
This function is performed in the same way as the
Add function (see Add, page 5-142). The only
difference is that the mathematical formula is
based on subtraction.
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Fig. 5-116
Subtract window
5-145
OPERATION IN EXPERT MODE
Process Menu
Multiply
Carl Zeiss
5.6.3
LSM-FCS
Multiply
The Multiply function permits two channels each
to be linked into a new channel by multiplication.
The channel created in this way can be stored via
the Save As function.
5.6.3.1
Open / Close the Multiply window
• Click on the Multiply button in the Process
subordinate toolbar of the Main menu.
–
This opens the Multiply window.
• Click on the Close button to quit the Multiply
window.
5.6.3.2
Fig. 5-117
5-146
Multiply window
Performance of the Multiply
function
This function is performed in the same way as the
Add function (see Add, page 5-142). The only
difference is that the mathematical formula is
based on multiplication.
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OPERATION IN EXPERT MODE
Process Menu
Ratio
LSM-FCS
5.6.4
Carl Zeiss
Ratio
The Ratio function permits two channels to be
linked into a new channel by the creation of a
ratio. The channel created in this way can be
stored via the Save As function.
5.6.4.1
Open / Close the Ratio window
• Click on the Ratio button in the Process
subordinate toolbar of the Main menu.
–
This opens the Ratio window.
• Click on the Close button to quit the Ratio
window.
5.6.4.2
Performance of the Ratio function
This function is performed in the same way as the
Add function (see Add, page 5-142).
However, three different formulas can be used for
ratio creation, each of which can be activated by
clicking on the button.
Fig. 5-118
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Ratio window
5-147
OPERATION IN EXPERT MODE
Process Menu
Copy (Channel)
Carl Zeiss
5.6.5
LSM-FCS
Copy (Channel)
The Copy function permits one channel each of an
existing image to be copied and stored as a new
image.
The selection of Source, Channel and Destination
is made in the same way as in the Add function
(see Add, page 5-142).
5.6.5.1
Fig. 5-119
Open / Close the Copy window
Copy window
• Click on the Copy button in the Process
subordinate toolbar of the Main menu.
–
This opens the Copy window.
• Click on the Close button to quit the Copy
window.
5.6.5.2
Performance of the Copy function
• Select Source, Channel and Destination and then click on the Apply button.
–
The image of the copied channel is then displayed in a new window or in the Image Display
window activated for it.
• The new image can be stored via the Save As function.
For Z Stacks or Time Series, the entire series of the selected channel is copied.
5.6.6
Duplication (Image)
This function permits images (including Z Stacks and Time Series) to be duplicated completely.
• If several images have been opened, select the image to be duplicated.
• Click on the Dup button in the Process subordinate toolbar of the Main menu.
–
The selected image is duplicated and displayed in a new Image Display window.
• Use the Save As function to store the image under a new name.
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OPERATION IN EXPERT MODE
Process Menu
Filter
LSM-FCS
5.6.7
Carl Zeiss
Filter
The filter function permits the subsequent
processing of scanned images via the integrated
Lowpass, Sharpness and Median filters.
Furthermore, User-defined filters can be installed
by the user. User-defined filters can be stored,
reloaded and removed.
5.6.7.1
Open / Close the Filter window
• Click on the Filter button in the Process
subordinate toolbar of the Main menu.
–
This opens the Filter window.
• Click on the Close button to quit the Filter
window.
5.6.7.2
Image panel
In the Image panel, the image or channel to be
processed is selected.
Fig. 5-120
Filter window
The currently selected image is displayed in the
image selection box.
Proceed as follows to select an image via the image selection box:
• Click on the arrow button. The image selection box is opened and all the currently loaded images are
displayed in a minimized form.
• Click on the required image, which will then appear in the display box of the image selection box and
will be available for filtering.
You can also use the Click into window button to select the image.
• Open the Channel selection box with a click on the arrow button and select the channel to be
processed.
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5-149
OPERATION IN EXPERT MODE
Process Menu
Filter
Carl Zeiss
5.6.7.3
LSM-FCS
Filter List and Edit panel
In the Filter List panel, the filters and the matrix size (Kernel Size) are selected.
The matrix of the selected filter and the set filter parameters Factor, Divisor and Offset are displayed in
the Edit panel.
(1)
Kernel Size
The size of the filter matrix can be modified here.
The effect of a filter increases along with the
matrix size. However, this also increases the time
required for filtering.
• Select the required matrix size by clicking on
one of the selection buttons 3 x 3, 5 x 5 or
7 x 7.
(2)
Fig. 5-121
Filter List and Edit panel
(Lowpass)
Lowpass filter
With the lowpass filter, the gray value of each
center pixel is replaced with the average value of
the surrounding neighbor pixels. The viewed
neighbor pixels are defined by a square. The
modified pixel now is the center pixel of the filter
matrix.
Image noise will be reduced by the application of
the lowpass filter. The cutoff of regions will blur.
Local maxima will be flattened. The dynamic range
will be reduced considerably.
This filter permits the matrix size to be modified
only in the 3 preset steps.
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OPERATION IN EXPERT MODE
Process Menu
Filter
LSM-FCS
(3)
Carl Zeiss
Sharpness filter
With the sharpness filter, the original image is
filtered with a lowpass filter first. The result of this
filtering is then subtracted from the original image.
This will improve image sharpness.
The matrix size can be modified in the 3 preset
steps.
Furthermore, divisor values ranging from 1 to 78
can be entered. The higher the divisor value, the
lower the image sharpness.
(4)
Fig. 5-122
Filter List and Edit panel
(Sharpness)
Fig. 5-123
Filter List and Edit panel
(Median)
Median filter
With the median filter, the gray value of each
center pixel is replaced with the median value of
the surrounding neighbor pixels. The viewed
neighbor pixels are defined by a square. The
modified pixel now is the center pixel of the filter
matrix.
The median value is defined as the middle value
(not average) of all the gray values sorted in
ascending order within a matrix.
Image noise will be reduced by the application of
the median filter. The cutoff of regions will slightly
blur. Local maxima will be flattened. The dynamic
range will be reduced considerably.
The settings of this filter can not be modified.
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5-151
OPERATION IN EXPERT MODE
Process Menu
Filter
Carl Zeiss
(5)
LSM-FCS
User-defined filter
The User-defined function permits you to create
your own filters. In addition to the Kernel Size,
the parameters Factor, Divisor and Offset can be
modified here.
The filter result can be subtracted from the original
image via the Subtract from Source check box.
Proceed as follows to store User-defined filters:
• Click on the Add To List button and enter a
name in the Add Filter To List window. The
name will be included in the Filter List.
Proceed as follows to activate stored, Userdefined filters:
• Click on the name of the filter in the Filter List.
The filter will then be activated immediately.
Proceed as follows to delete User-defined filters:
• Click on the name of the filter in the Filter List
and then on the Remove button. The filter will
be deleted.
Fig. 5-124
Filter window (User-defined filter)
• After selection of the required filter, click on the
Apply button to start the filter procedure.
–
Filtering will be performed and displayed in
the current Image Display window.
• In the case of images with several channels,
activate the xy button in the Display image
toolbar to display all the channels. Each channel
must be filtered separately.
Fig. 5-125
5-152
Add Filter to List panel
• Use the Save As function to store the newly
created image.
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OPERATION IN EXPERT MODE
Process Menu
Contrast
LSM-FCS
5.6.7.4
Carl Zeiss
Preview panel
The Preview function allows you to have the result
of the Filter operation displayed as a preview
image.
Fig. 5-126
Preview panel
Preview check box with a click of the mouse. The Filter - Preview Image Display
• Activate the
window with the filter result will be displayed.
• Deactivate the Preview check box to close the Filter - Preview Image Display window.
After a change of the filter settings, click in the Filter - Preview Image Display window once
to update it.
5.6.8
Contrast
The Contrast function permits the subsequent modification of contrast and brightness of the stored
image.
• Open the image to be processed and click on the Contrast button.
–
The function is performed with firmly set parameters and the result is displayed in a new Image
Display window. The procedure can be repeated as often as required.
• The newly created image can be stored using the Save As function.
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5-153
OPERATION IN EXPERT MODE
Process Menu
Interpolate
Carl Zeiss
5.6.9
LSM-FCS
Interpolate
This function permits the continuous contrast and
brightness change in a stack or Time Series
through interpolation between the starting and
end values. This permits the subsequent
compensation of specimen bleaching which
occurred during image recording. Interpolation can
be defined for the entire image or only for
individual channels.
5.6.9.1
Open / Close
the
Interpolate
Brightness and Contrast window
• Click on the Interpolate button in the Process
subordinate toolbar of the Main menu (also see
page 5-142).
–
Fig. 5-127
Interpolate Brightness and Contrast
window
This opens the Interpolate Brightness and
Contrast window.
• Click on the Close button to quit the window.
5.6.9.2
Image panel
The image to be processed is selected in the
Image panel.
Fig. 5-128
Image panel
The currently selected image is shown in the
display box of the image selection box.
Proceed as follows to select a series via the image
selection box:
• Click on the arrow button. The image selection box will be opened and all the currently loaded
images will be displayed in a minimized form.
• Click on the required image, which will then appear in the display box of the image selection box and
has been selected for the interpolation procedure.
You can also use the Click into window button for image selection.
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OPERATION IN EXPERT MODE
Process Menu
Interpolate
LSM-FCS
5.6.9.3
Carl Zeiss
Interpolation panel
In the Interpolation panel, the parameters for the
interpolation procedure are set.
• Use the Start Image slider to select the slice at
which the interpolation procedure shall start.
Clicking on the First button permits the fast
selection of the first slice in the series.
• Use the Brightness and Contrast sliders to set
the image brightness and contrast for the first
slice (Start Image).
• Use the End Image slider to select the slice at
which the interpolation procedure shall end.
Clicking on the Last button permits the fast
selection of the last slice in a series.
Fig. 5-129
Interpolation panel
• Use the Brightness and Contrast sliders to set
the image brightness and contrast for the last
slice (End Image).
• Use the available Channel buttons (e.g.: Ch1) to select the channel for interpolation or click on the All
button if the entire image is to be interpolated.
• Having set the parameters, click on the Apply button. Interpolation will be performed in a new
Image Display window.
• The newly created image (series) can be stored using the Save As function.
If you activate the Overwrite Source Images check box, interpolation will be performed in the
current Image Display window.
If you activate the Ignore Images less than "Start Image" and greater than "End Image"
check box, only the slices lying between Start Image and End Image will be taken into
consideration for interpolation. Otherwise, brightness and contrast will also be changed for the
other slices.
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OPERATION IN EXPERT MODE
Process Menu
Channel Shift
Carl Zeiss
5.6.9.4
Fig. 5-130
Preview panel
• Activate the
–
LSM-FCS
Preview panel
The Preview function enables you to see the
result of interpolation for one slice each in a
preview window.
Preview check box with a click of the mouse.
The Interpolate C&B - Preview Image Display window will be displayed. At the same time, the
Slice slider with the relevant input box and arrow keys and the two buttons Start and End are
displayed in the Preview panel.
• Use the slider or input box / arrow keys to set the slice which shall be displayed in the preview
window.
• Clicking on the Start or End button permits the fast activation of the Start Image or End Image for
previewing.
• Deactivate the Preview check box to close the Interpolate C&B - Preview Image Display window.
5.6.10
Channel Shift
The Channel Shift function is used to produce a
congruent image with relation to the pixels of the
various channels.
This pixel correction function
important in UV applications.
5.6.10.1
Open / Close
window
the
is
particularly
Channel
Shift
• Click on the Shift button in the Process
subordinate toolbar of the Main menu.
Fig. 5-131
Channel Shift window
–
This opens the Channel Shift window.
• Click on the Close button to quit the window.
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OPERATION IN EXPERT MODE
Process Menu
Channel Shift
LSM-FCS
5.6.10.2
Carl Zeiss
Image panel
• Click on the arrow button. The image selection
box will be opened and all the currently loaded
images are displayed in a minimized form.
• Click on the required image, which will then
appear in the display box of the image selection
box and has been selected for the Shift
function.
Fig. 5-132
Image panel
Fig. 5-133
Shift panel
Fig. 5-134
Preview panel
You can also use the Click into window
button for image selection.
5.6.10.3
Shift panel
• Select the channels required for processing in
the Shift box by clicking on the Ch1 or Ch3
will appear in the button
buttons. A tick
when the channels are activated.
and
buttons to
• Use the scrollbar or the
select the pixel shift in the horizontal and
vertical direction.
• Click on the Apply button to activate the
setting.
5.6.10.4
Preview panel
• If Preview is activated, a preview of the shift
is shown in a separate Image Display window.
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OPERATION IN EXPERT MODE
Process Menu
Channel Shift
Carl Zeiss
LSM-FCS
The following image shows the result of a pixel shift via the Shift function. This image change can be
stored in the image database via the Save or Save As buttons.
Fig. 5-135
Image Display window with channel shift
For applications requiring 3- or 4-channel scanning, proceed in the same way as described for the 1- or
2-channel mode.
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OPERATION IN EXPERT MODE
Process Menu
Unmix
LSM-FCS
5.6.11
Carl Zeiss
Unmix
The Unmix functionality permits to extract the emission of single fluorescence dyes (e.g. GFP only, YFP
only etc.) from the overall emission band of strongly overlapping multifluorescence signal intensities by a
pixelwise linear unmixing procedure .
Mathematically, experimental fluorescence spectra of monolabelled samples are taken as an external
reference. Up to 8 different reference signals can be varied in this least-square-fit based algorithm to
produce an 8 channel multifluorescence stack without any partial overlap between the channels.
5.6.11.1
Open / Close the Unmix window
• Click on the Unmix button in the Process subordinate toolbar of the Main menu.
–
This opens the Linear Unmixing window.
• Click on the Close button to quit the Unmix window.
5.6.11.2
Source panel
In the Source panel the image source for the linear unmixing process has to be defined.
This has to be a Lambda Stack, a Lambda Stack Z series or a Lambda Stack T series.
Proceed as follows to select an image via the image selection box:
• Click on the arrow button. The image selection box is opened and all the currently loaded images,
stacks, time series with a Lambda dimension are displayed in a minimized form.
• Click on the required image. This image will then appear in the display box of the image selection box
and has been selected.
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Carl Zeiss
5.6.11.3
OPERATION IN EXPERT MODE
Process Menu
Unmix
LSM-FCS
Definition of Channels panel
In the Channels panel the number of reference spectra (number of fluorescence channels) can be
selected from the channel selection boxes.
• Select the references fluorescence dye spectra which are present in the sample with the check boxes.
• If necessary change the colors of the relevant fluorescence channel.
• If no predefined reference spectra exist, please define reference signals via the Save to Spectra DB
button in the Display - Mean functionality (see page 5-379).
• After definition of the required reference spectra set click on the Apply button.
• A new window with the resulting channels of the unmixing procedure opens immediately.
Try to avoid saturation of fluorescence signals in the stack to be unmixed.
To get the highest quality unmixing results, please define an extra background channel, if
possible.
Handling of Reference Spectra
Fig. 5-136
5-160
–
Reference Spectra used for Linear Unmixing
are stored with result images
–
ReUsable and storable via Load from Image
and Write to DB buttons
–
New file type *.umx for Save and Load of
combinations (configurations) of Reference
Spectra
Handling of Reference Spectra
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OPERATION IN EXPERT MODE
Process Menu
Unmix
LSM-FCS
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Fig. 5-137
Image Display window before unmixing
Fig. 5-138
Image Display window after unmixing
12/02
Carl Zeiss
5-161
OPERATION IN EXPERT MODE
Process Menu
Ion Concentration
Carl Zeiss
5.6.12
LSM-FCS
Ion Concentration
The use of this function (option) permits the
calibration of ion concentrations in physiological
experiments.
(1)
Open / Close the Ion Concentration
window
Click on the Ion Conc button in the Process
subordinate toolbar of the main menu.
Click on the Close button.
(2)
Fig. 5-139
Function description
Ion Conc button
Activates the Ion
Concentration
menu.
Source window
Selects input
images
to
processed.
Destination window
Select output and
pixel
depth
of
processed image.
Ion Concentration window
of
be
Calibration window
Sets the six different calibration options, according to the dyes used
(single wavelength, ratiometric) and required method.
Show Curve button
Shows resulting calibration curve.
Image scaling window
Sets min. and max. concentration.
Preview window
Activates Preview function.
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OPERATION IN EXPERT MODE
Process Menu
Ion Concentration
LSM-FCS
(3)
Carl Zeiss
Single wavelength dyes – offline Calibration
• Subtract background/autofluorescence image from raw images to obtain
• Perform equation- or titration calibration (compare F with a calibration curve -> titration calibration or
put F values in calibration formula)
Raw images
Background/autofluorescence image
Calibration type
Scaling of calibrated images
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5-163
Carl Zeiss
(4)
OPERATION IN EXPERT MODE
Process Menu
Ion Concentration
LSM-FCS
Ratiometric Dyes
• Fura-2, Indo-, SNARF, Cameleon, Ratiometric Pericam, Phluorin,...
• Display fluorescence ratio R over time
• Display fluorescence ratio R corrected for background/autofluorescence over time
• Calculate absolute ion concentrations (pixel by pixel) via titration calibration (known ion
concentrations applied to the cells – in situ – or in solutions – in vitro or equation calibration where
possible [Fura-2, Indo-, SNARF]
• Calculation of R eliminates artifacts and uncertainties caused by
–
inhomogenous dye distribution
–
photobleaching
–
may be applied with moving cells
(5)
Ratiometric Dyes - Online ratio
R(t1) = F1(t1) / F2(t1), R(t2) = F1(t2) / F2(t2) ...
F1(t1) / F2(t1)
F1(t2) / F2(t2)
F1(tn) / F2(tn)
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OPERATION IN EXPERT MODE
Process Menu
Ion Concentration
LSM-FCS
(6)
Carl Zeiss
Ratiometric Dyes - Calibration
• Subtract background/autofluorescence images from raw images to obtain
Rkorr [=F1-F1Background)/(F2-F2Background)]
when calibration reference is not obtained with the experimental sample (in situ)
• Calculate ratio R
• Perform equation- or titration calibration (compare R with a calibration curve -> titration calibration or
put R values in calibration formula)
Raw images
Background/autofluorescence image pair
Calibration type
Scaling of calibrated images
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5-165
Carl Zeiss
(7)
OPERATION IN EXPERT MODE
Process Menu
Ion Concentration
LSM-FCS
Ratiometric Dyes - Equation Calibration (Grynkiewicz)
Fura-2, Indo-1,..
KD (dissociation constant) taken from literature
Rmin: derived from ion-free state of the dye (e.g. 0 Ca2+)
Rmax: derived from ion-bound state of the dye (e.g. saturated with Ca2+)
Fmin2 and Fmax2 are the minimum and maximum fluorescence intensities at wavelength 2
Rmin, Rmax, Fmin2 and Fmax2 may be determined in the cells under investigation (in situ) or in solutions (in
vitro)
Calibration parameters may be saved and reloaded (*.cal)
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OPERATION IN EXPERT MODE
Process Menu
Ion Concentration
LSM-FCS
(8)
Carl Zeiss
Options for Calibration Image Selection (equation- or titration calibration)
• Click into image window.
• Select source channel(s).
• Optional background subtraction
• Optional calculation of parameters from overlay region(s)
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OPERATION IN EXPERT MODE
Process Menu
3D DeConVolution (DCV)
Carl Zeiss
5.6.13
LSM-FCS
3D DeConVolution (DCV)
The 3D Deconvolution option is used for the resolution enhancement of fluorescence image stacks.
5.6.13.1
Background
When a three-dimensional object is reproduced by an optical system the resulting image of the object
does not correspond exactly to the object's actual form. The image of the object is "distorted" as it
passes through the optical system. In physical terms the actual object is convolved by the optical system's
Point Spread Function (PSF).
The Point Spread Function describes how the light of a point object is distorted by the optical system.
This "convolution" makes the image appear grainy and structures in the image seem blurred. This effect
is most prominent in the axial (Z-)direction as each lens is optimized for the two-dimensional image of
the object.
If the PSF is known it is possible to use mathematical algorithms to undo this distortion. The image of the
object is deconvolved using the PSF and the actual form is reconstructed:
The effect of 3D deconvolution can be demonstrated impressively on objects with a known form. As a
rule fluorescent beads are used for this purpose. The following figure shows the 3D deconvolution of an
image stack with a fluorescent bead with a diameter of 1 µm.
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OPERATION IN EXPERT MODE
Process Menu
3D DeConVolution (DCV)
LSM-FCS
Carl Zeiss
As the resolution of an optical system is
significantly lower in the axial direction than in the
lateral (X/Y-)direction, the greatest improvement in
resolution can be achieved in the Z-direction.
The Z Stack must meet the following requirements:
–
At least two-fold oversampling in xyz (z: half
of optimal interval button)
–
High signal-to-noise ratio
–
Detector gain < 500 V
Calculation is either made for one channel of the
opened image which must first be selected
accordingly, or for all channels of a stack.
Calculation is started via Apply and can be
stopped using the ESC key, if required.
5.6.13.2
Fig. 5-140
Image of a fluorescent bead with a
diameter of 1µm before
deconvolution (A,B) and after
deconvolution (C,D)
Fig. 5-141
3D Deconvolution window
Open / Close the 3D Deconvolution
window
• Click on the DCV button in the Process
subordinate toolbar of the Main menu (also see
page 5-142).
–
This opens the 3D Deconvolution window.
• Click on the Close button to quit the window.
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OPERATION IN EXPERT MODE
Process Menu
3D DeConVolution (DCV)
Carl Zeiss
5.6.13.3
Fig. 5-142
Source panel
LSM-FCS
Source panel
The image to be processed is selected in the
Source panel. The currently selected image is
shown in the display box of the image selection
box. Proceed as follows to select a image via the
image selection box:
• Click on the arrow button. The image selection box will be opened and all the currently loaded
images will be displayed in a minimized form.
• Click on the required image, which will then appear in the display box of the image selection box and
has been selected for the interpolation procedure.
You can also use the Click into window button for image selection.
5.6.13.4
Deconvolution panel
The Deconvolution panel contains the two tabs
Method and PSF.
(1)
Method tab
The Method tab permits selection between the
calculation methods Nearest Neighbour, Inverse
and Iterative.
Fig. 5-143
Method tab
(a)
Nearest Neighbor
The Nearest Neighbor method is the simplest and
fastest algorithm which in principle corresponds to
a 3D sharpness filter.
(b)
Inverse Filter
The regularized inverse filter generally achieves better results than the Nearest Neighbor algorithm. It is
well suited to process several image stacks for a preselection of images for the use of the iterative highend methods.
©
Constrained Iterative
The best image quality is achieved using the Constrained Iterative Maximum Likelihood Algorithm.
Increasing the resolution in the image, especially in the Z-direction, is only possible with this method. Due
to the complex mathematical method, depending on the image size and the PC being used the
calculation can take up to several hours.
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OPERATION IN EXPERT MODE
Process Menu
3D DeConVolution (DCV)
LSM-FCS
Carl Zeiss
In the Inverse method, the Restoration Effect slider permits the noise-to-signal ratio to be selected
between the settings Weak (low noise) and Strong (pronounced noise).
Activation of the Auto detect check box will start a routine for the automatic determination of the noise
level in the entire image part of the Z Stack (not available in the Nearest Neighbour method). If Auto
detect is enabled, the Restoration Effect slider is disabled.
The Iterative method permits (in addition to the parameters of the Inverse method) the maximum
number of iterations to be entered between 1 and 200 under Maximum Iterations and the Auto Stop
function to be activated / deactivated. The Auto Stop function interrupts the calculation depending on
the set image improvement (delta between last but one and last cycle in %), no matter whether the
value under Maximum Iterations has been achieved or not.
The Nearest Neighbour method permits entry of the Number of Neighbours and the Sharpness in
Focus value in addition to the Restoration Effect.
(2)
PSF tab
In the 3D Deconvolution option a theoretical point
spread function (PSF) is calculated from the
systems settings (objective data, wavelengths,
pinhole diameter).
The PSF data are displayed in the Method tab. In
the case of wavelengths above 700 nm, the NLO
button is automatically enabled.
The displayed values are always taken over by the
system data, but can be edited subsequently for
simulation purposes.
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Fig. 5-144
PSF tab
5-171
OPERATION IN EXPERT MODE
3D View Menu
3D DepthCod (Color Coded Depth Map)
Carl Zeiss
5.7
LSM-FCS
3D View Menu
The 3D View functions serve to record and play back series of images for 3D display of microscopic
structures.
• In the Main menu toolbar, click on 3D View.
–
This opens another, subordinate toolbar in the Main menu.
3D View subordinate toolbar
Fig. 5-145
3D View menu
5.7.1
3D DepthCod (Color Coded Depth
Map)
By means of the Depth Coding function, the
depth information contained in a sequence can be
colored with the colors of the rainbow, in which
case "blue " stands for front and "red" stands for
rear.
A stack of images must be available.
5.7.1.1
Open / Close the Depth Coding
window
• Click on the DepthCod button in the 3D View
subordinate toolbar of the Main menu.
Fig. 5-146
Depth Coding window
–
This opens the Depth Coding window.
• Click on the Close button to quit the window.
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OPERATION IN EXPERT MODE
3D View Menu
3D DepthCod (Color Coded Depth Map)
LSM-FCS
5.7.1.2
Carl Zeiss
Source panel
In the Source panel, the image source is selected. The currently selected image is displayed in the display
box of the image selection box. Proceed as follows to select an image via the image selection box:
• Click on the arrow button. The image selection box will be opened and all the currently loaded
images are displayed in a minimized form.
• Click on the required image, which will then be shown in the display field of the image selection box
and be available for the following operation.
The Click into window button enables you to select the opened image directly:
• Click on the Click into window button first and then double-click on the relevant Image Display
window. The selected image will then be shown in the display box of the image selection box.
Select the channels to be processed via the Channel selection box:
• Click on the arrow button to open the selection box. Click on the required channel to activate it.
5.7.1.3
Depth Coding panel
• On the Depth Coding panel you can set the desired parameters. Activate the Scale Bar check box
if you want a color scale to be shown.
(1)
Depth Coding tab
Mode Front View:
The image is viewed from
the front / above when this
option is activated.
Mode Rear View:
The image is viewed from
the rear / below when this
option is activated.
Threshold:
All brightness values below
the Threshold (range: 0 to
255) are ignored or treated
like 0 when determining the
depth and the display.
Fig. 5-147
Depth Coding tab
Contrast:
Defines the factor with which the contrast of the overlaid series affects the
contrast of the depth-coded color.
Brightness:
Defines the factor with which the brightness of the overlaid series affects the
brightness of the depth-coded color.
Display Scale Bar:
Displays a colored scale in the image.
Display Grey level: The depth information is displayed in gray levels.
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OPERATION IN EXPERT MODE
3D View Menu
3D DepthCod (Color Coded Depth Map)
Carl Zeiss
(2)
Transparency tab
Mode Maximum:
Fig. 5-148
Transparency tab
Mode
Keep Maximum:
LSM-FCS
The color is defined by the Z
position of the brightness
value.
Mode Transparent: The transparent projection is
built up from the rear to the
front. The color is defined
by the Z position at which
the original was last higher
than or equal to Threshold.
Activating this option modifies the specification governing calculation of the
projection.
Threshold:
Pixel value at which the ramp rises (variable from 0 to 100 %).
Ramp:
Slope of the ramp (variable from 0 to 100 %; 0 % corresponds to a vertical rise).
Maximum Opacity: Degree of visibility at the top corner of the ramp (variable from 0 to 100 %;
0 corresponds to the bottom edge in the diagram).
5.7.1.4
Preview panel
The Preview function permits you to regard the
influence of parameter changes in an Image
Display window.
• After finding the optimum adjustment using
the Preview function, you have to generate
the final version of the image using the Apply
button.
–
Fig. 5-149
5-174
The system then generates a color-coded
depth map for the selected channel.
Depth Coding image
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OPERATION IN EXPERT MODE
3D View Menu
Projection
LSM-FCS
5.7.2
Carl Zeiss
Projection
By means of the Projection function, one single
projection or a series of projections can be
calculated after rotation of the data package
about the X, Y or Z axis.
A stack of images must be available.
5.7.2.1
Open / Close the Projection
window
• Click on the Projection button in the 3D View
subordinate toolbar of the Main menu.
–
This opens the Projection window.
• Click on the Close button to quit the window.
5.7.2.2
Fig. 5-150
Projection window
Source panel
• Select the image for the projection operation from the image selection box.
5.7.2.3
Projection panel
• On the Projection panel, set the parameters needed for the animation: Turning Axis, First Angle,
Number Projections and Difference Angle in the Projection tab and the Mode parameters in the
Transparency tab.
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OPERATION IN EXPERT MODE
3D View Menu
Projection
Carl Zeiss
(1)
LSM-FCS
Projection tab
Turning Axis X/Y/Z: Selects the axis about which
the data package is to be
rotated.
First Angle:
Rotation angle in degrees.
Number Projections: Number of projections for a
sequence (variable from
0 to 100).
Fig. 5-151
Projection tab
Difference Angle:
Angle increment
sequence.
of
a
The number keys permit the direct selection of preset values for Number Images and
Difference Angle. If the Panorama button is pressed, a panorama sequence of the image
series is computed.
(2)
Transparency tab
Mode Maximum:
Fig. 5-152
Transparency tab
Mode
Keep Maximum:
The color is defined by the Z
position of the brightness
value.
Mode Transparent: The transparent projection is
built up from the rear to the
front. The color is defined
by the Z position at which
the original was last higher
than or equal to Threshold.
Activating this option modifies the specification governing calculation of the
projection.
Threshold:
Pixel value at which the ramp rises (variable from 0 to 100 %).
Ramp:
Slope of the ramp (variable from 0 to 100 %; 0 % corresponds to a vertical rise).
Maximum Opacity: Degree of visibility at the top corner of the ramp (variable from 0 to 100 %;
0 % corresponds to the bottom edge in the diagram).
Brightness:
5-176
The image can be brightened again by modifying the value (from 0.2 to 5).
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3D View Menu
Projection
LSM-FCS
5.7.2.4
Carl Zeiss
Preview panel
The Preview function permits you to regard the
influence of parameter changes in an Image
Display window.
Fig. 5-153
Preview panel
Fig. 5-154
Projection image
The Slice slider enables you to select the slice
which shall be displayed in the Preview Image
Display window.
• After finding the optimum adjustment using
the Preview function, you have to generate
the final version of the image using the Apply
button.
–
The projection appears. The computation
can be followed in the image or by the
progress bar.
The computed 3D sequence can be
animated with the Anim button in the
Select toolbar.
In addition, the Animate window
appears, in which you can influence the
direction and speed of 3D image rotation
(see section 5.15.8, page 5-295).
You can browse through the rotation sequence manually with the Slice button in the Select toolbar and
the Slice slider.
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OPERATION IN EXPERT MODE
3D View Menu
Projection
Carl Zeiss
LSM-FCS
• To view the computed 3D sequence as a gallery on the screen, click on the Gallery button in the
Display toolbar.
Fig. 5-155
5-178
Projection image (Gallery)
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3D View Menu
Stereo
LSM-FCS
5.7.3
Carl Zeiss
Stereo
Stereoscopic images can be generated in a variety
of ways by means of the Stereo function.
A stack of images must be available.
5.7.3.1
Open / Close the Stereo Images
window
• Click on the Stereo button in the 3D View
subordinate toolbar of the Main menu.
–
This opens the Stereo Images window.
• Click on the Close button to quit the window.
Fig. 5-156
5.7.3.2
Stereo Images window
Source panel
• Select the image for the projection operation from the image selection box.
• Select the channel to be used from the Channel selection box.
5.7.3.3
Stereo Images panel
• In the Stereo Images panel, set the parameters needed for stereoscopic viewing: Mode, Basic
Angle, Right Left Angle, Number Images and Difference Angle in the Projection tab and the
Mode parameters in the Transparency tab.
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OPERATION IN EXPERT MODE
3D View Menu
Stereo
Carl Zeiss
(1)
LSM-FCS
Projection tab
Mode
Red / Green Image: This displays a stereo image
for red / green anaglyph observation using red / green
spectacles.
Mode
Split Images:
Fig. 5-157
Projection tab
This displays a pair of stereo
images
for
observation
through a stereoscope. Colored stereo images are also
possible.
Basic Angle:
Direction angle at which the specimen is viewed; 0° from the front, 180° from the
rear.
Right Left Angle:
Angle between right and left (red and green) image.
Number Images:
Number of 3D images (slices).
Difference Angle:
Angle increment of a sequence.
The number keys permit the direct selection of preset values for Number Images and
Difference Angle. If the Panorama button is pressed, a panorama sequence of the image
series is computed.
5-180
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OPERATION IN EXPERT MODE
3D View Menu
Stereo
LSM-FCS
(2)
Carl Zeiss
Transparency tab
Mode Maximum:
The color is defined by the
Z position of the brightness
value.
Mode Transparent: The transparent projection is
built up from the rear to the
front. The color is defined
by the Z position at which
the original was last higher
than or equal to Threshold.
Fig. 5-158
Mode
Keep Maximum:
Transparency tab
Activating this option modifies the specification governing calculation of the
projection.
Threshold:
Pixel value at which the ramp rises (variable from 0 to 100 %).
Ramp:
Slope of the ramp (variable from 0 to 100 %; 0 % corresponds to a vertical rise).
Maximum Opacity: Degree of visibility at the top corner of the ramp (variable from 0 to 100 %;
0 corresponds to the bottom edge in the diagram).
Brightness:
5.7.3.4
The image can be brightened again by modifying the value (from 0.2 to 5).
Preview panel
The Preview function permits you to regard the
influence of parameter changes in an Image
Display window.
Fig. 5-159
Preview panel
The Slice slider enables you to select the slice
which shall be displayed in the Preview Image
Display window.
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5-181
OPERATION IN EXPERT MODE
3D View Menu
Stereo
Carl Zeiss
LSM-FCS
• To start computation of the stereoscopic image,
click on the Apply button.
–
The image is built up twice (once each for
the red and green colors), resulting in a
stereoscopic image.
The stereoscopic effect can only be seen
with the aid of red / green 3D goggles.
The red lens is to be used for the right
eye and the green lens for the left eye.
The presentation can be modified by
selecting the Split Images (Mode) option
in the Projection tab of the Stereo
Images panel.
Fig. 5-160
Stereoscopic image (red / green)
• By clicking on the Apply button, the two stereo pairs are presented side by side and can be viewed
without red / green 3D goggles.
Fig. 5-161
5-182
Stereoscopic image (split)
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Stereo
LSM-FCS
5.8
Carl Zeiss
Analyse FCS Menu
• In the Main menu toolbar, click on Analyse FCS.
–
This opens another, subordinate toolbar in the Main menu.
Fig. 5-162
Analyse FCS menu
Analyse FCS
subordinate
toolbar
For preparing and measuring, it is recommended to call up and use the tools of the subordinate toolbar
in the following order:
–
–
–
–
Method.
Carrier.
Measure.
Optimize (if necessary).
The VIS, FCS and LSM buttons switch the beam path of the microscope (VIS for viewing, FCS for FCS
measurements via laser excitation with monitor observation, LSM for LSM laser operation with monitor
observation).
For the measuring process, the FCS button in the toolbar subordinate to the Analyse FCS item must be
activated.
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5-183
OPERATION IN EXPERT MODE
Analyse FCS Menu
Select Method
Carl Zeiss
5.8.1
LSM-FCS
Select Method
The Method function shows all existing measure methods for selection or deletion.
• Click on the Method button in the Analyse FCS subordinate toolbar of the Main menu.
–
This opens the Method Selection ... window (Fig. 5-163). The name of the currently selected
method will appear in the status bar of the window.
Fig. 5-163
Method Selection ... window
• You can select the analysis method you want to use by double-clicking on the corresponding list
entry. After the double click the analysis method is selected and the Method Selection ... window
will be closed automatically.
• Alternatively you can select the analysis method by a single click – which will highlight the selected
method – and a subsequent click on the Select button. The window will then be closed
automatically.
• The Delete button allows you to delete a method from the database. To do so, click on the method
entry in the list and then click the Delete button. A window will pop up requesting confirmation of
this action. If you confirm, the method will really be deleted from the database.
Use this function with extreme care! You might lose valuable data!
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Select Method
LSM-FCS
Carl Zeiss
• After a click on the Info button a window will pop up which shows a detailed description of the
analysis method (Fig. 5-164).
All parameters will load with this method.
Fig. 5-164
Detailed description of the analysis method open
• Click on the Copy All button to copy the details to the clipboard. Afterwards you can insert the
details in a WINDOWS application (e.g. Winword) and save it.
• Click the Print ... button to print the details table. This opens the Print Setup window. Set the print
parameters and start the print function by clicking OK. The Print Setup window is closed
automatically.
• Click the Close button to close the Method Details ... window.
• The Close button of the Method Selection ... window allows you to leave the dialog without any
action.
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5-185
OPERATION IN EXPERT MODE
Analyse FCS Menu
Select Carrier
Carl Zeiss
5.8.2
LSM-FCS
Select Carrier
The Carrier function opens the Carrier Position
window. It is used for selecting the carrier and the
sample, for orienting the sample in x- and ydirection and for setting in Z position.
5.8.2.1
Opening / Closing
Position window
the
Carrier
• Click on the Carrier button in the Analyse FCS
subordinate toolbar of the Main menu
(Fig. 5-162).
–
The sample select window (Carrier Position
window) pops up (Fig. 5-165).
Before the sample can be positioned
make sure that the microscope stand is
set up for FCS measurements and that
the appropriate laser(s) is (are) switched
on.
Fig. 5-165
5-186
Carrier Position window
•
Click on the Laser button to open the Laser
Control window (for settings see Config FCS
menu, page 5-208).
•
Close the Carrier Position window by clicking
the Close button.
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Select Carrier
LSM-FCS
5.8.2.2
(1)
Carl Zeiss
Function description
X, Y (Stage) panel
In the X, Y (Stage) panel you can select the
sample carrier and orient the sample carrier in xand y-direction.
Selecting the sample carrier
• Select the sample carrier you are currently using
in the Sample Carrier selection box of the X, Y
(Stage) panel.
Selecting a chamber at the sample carrier
A scheme of the selected sample carrier is shown
in the center part of the panel. The lines of the
chambers are marked with letters, and the
columns with numbers.
Fig. 5-166
X, Y (Stage) panel
Fig. 5-167
X, Y (Stage) panel
(Sample drives unlocked)
The chamber to be used for the X and Y alignment
of the stage can be selected at a click of the
mouse. It then appears with a black frame and is
displayed in the Current Stage Position display
field (left field, e.g. A - 2). The current position of
the mouse pointer is shown in the right field.
• Select the chamber for X and Y alignment of
the stage.
Orienting the Sample in x and y
• Now click on the Orientation button in the
X, Y (Stage) panel (Fig. 5-167).
• Select one of the chambers of the sample for
orientation in x and y.
–
The selected chamber is displayed in yellow
with a black frame.
• Position the center of the selected chamber
over the center of the objective by clicking the
arrow keys with the mouse or by using the
control panel's joystick (if available).
• When you have finished, click the Orientation
button once more. The stage is now oriented.
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Carl Zeiss
OPERATION IN EXPERT MODE
Analyse FCS Menu
Select Carrier
LSM-FCS
• Now you can move any chamber into the measurement position by clicking the appropriate field on
the sample carrier.
The selected chamber is then automatically approached via the motorized microscope stage. When the
defined position has been reached, the chamber is displayed in the Carrier Position window in yellow
with a black frame.
Moving the microscope stage
• Move the stage in the appropriate position by clicking on the arrow keys.
Each mouse click moves the microscope stage in the appropriate direction by one step.
• Set the required step width via the Step slider or the relevant input box.
The travel speed of the microscope stage can be set to steps 1, 2 and 3.
Step 2 is preferably used for standard positioning.
Step 1 (slow speed) should only be used for very precise positioning, since positioning for longer paths
requires more time.
Step 3 is suitable if large vessels are used, since precise center positioning only plays a minor role in such
cases.
• Select the required travel speed of the microscope stage via the Speed selection box.
When the required chamber is approached by activation of the arrow keys, the relevant
chamber is displayed in white with a black frame, since normally the defined position cannot be
set in this way.
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Select Carrier
LSM-FCS
(2)
Carl Zeiss
Z (Focus) panel
The functions of the Z (Focus) panel allows you to position the focus in Z direction. Following function
elements are available:
Z-position display field
Displays the current Z position.
Store button
Stores the current Z position.
Reuse ... button
Moves the z drive to the stored Z position.
Work button
Moves the stage / nosepiece back to the Work position. This is the
position last set before the Load button was pressed.
Load button
Lowers the stage / nosepiece to make it easier for you to change the
sample carrier (or objective).
+200 µm button
Moves the Z drive for +200 µm per mouse click (upwards).
-200 µm button
Moves the Z drive for –200 µm per mouse click (downwards).
Zoom button
(on the right-hand side
of the Carrier Position
window)
Opens Camera window for zoom function.
The Z-position stored by activation of the Store button is a relative parameter and is only valid
during the current session. Accordingly, this position can be approached in a defined way only
during this session via Reuse.
Since the sample is just a solution in many cases and has no structure, we cannot “focus“ the instrument
by looking at sample features. Cover slip reflection has to be used instead to find the glass- / solution
interface.
Proceed as follows:
• Make sure that the front lens of the water immersion objective is wetted by a drop of water. Use
fluorescence-free double-distilled water, since otherwise the immersion water will cause background
fluorescence and deteriorate the correlation signal.
• Choose a chamber by clicking on one in the
sample carrier drawing in the X, Y (Stage)
panel of the Carrier Position window.
• The camera window shows interference fringes
caused by the laser light (Fig. 5-168).
Fig. 5-168
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Camera image far away from the
lower cover slip surface
5-189
OPERATION IN EXPERT MODE
Analyse FCS Menu
Select Carrier
Carl Zeiss
LSM-FCS
• Move the objective cautiously upward by
turning the Z focus knob of the microscope
stand. A lens moving upward will be indicated
by increasing numbers in the position field to
the left of the camera window.
• If the focus position approaches the lower cover
slip surface, the camera window looks similar to
(Fig. 5-169).
Fig. 5-169
Camera image near the lower cover
slip surface
• Continue cautiously moving upwards. If the
camera image looks like (Fig. 5-170), you are
close to the lower cover slip surface.
Fig. 5-170
Fig. 5-171
5-190
Lower cover slip surface
•
Having cautiously moved over a short distance
the cover slip reflection should look as shown
in (Fig. 5-171). The upper cover slip surface has
been found.
•
Store the reached position using the Store
button.
•
Now you can position the detection volume
into the sample in a well-defined way: if you
use one of the C-Apochromat water
immersion objectives, position the detection
volume 150-200 µm deep into the sample to
get rid of disturbing interface effects. The
easiest way of doing this is by clicking the
+200 µm button.
Upper cover slip surface
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Measure Method
LSM-FCS
5.8.3
Carl Zeiss
Measure Method
The Measure function opens the Method
Measurement ... window. It is used for setting
the measurement parameters and for starting the
measurement.
5.8.3.1
Opening / Closing the
Measurement ... window
Method
• Click on the Measure button in the Analyse
FCS subordinate toolbar of the Main menu
(Fig. 5-162).
–
•
The Method Measurement ... window will
appear on the screen (Fig. 5-172). The name
of the currently used method will appear in
the status bar of the window.
Close the Method Measurement ... window
by clicking the Close button.
Fig. 5-172
5.8.3.2
Method Measurement ... window
Function description
The following functions are available on the right-hand side of the Method Measurement ... window:
Close button
The Method Measurement ... window is closed.
Start button
Starts the measuring procedure.
Stop button
Ends the measuring procedure.
Laser button
Opens the Laser Control window.
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5-191
OPERATION IN EXPERT MODE
Analyse FCS Menu
Measure Method
Carl Zeiss
(1)
Fig. 5-173
Task Description panel
LSM-FCS
Task Description panel
In the Task Description panel you can enter the
task name for the measuring procedure.
The Comment field allows you to store a
comment on the measurement you are
performing.
• Enter a task name and a comment (if necessary)
for operation.
The task name will be used as file name when
saving the measured data. It must therefore meet
the WINDOWS conventions for file names.
(2)
Sample panel
The samples to be measured can be selected by
mouse in the schematic drawing of the sample
carrier. Activated chambers will be highlighted in
the drawing. In the Sample Carrier display box,
the selected sample carrier is shown for
information.
• Select the chambers and the work direction for
the measuring procedure using the mouse.
Individual chambers can be selected / deselected
by clicking on them with the mouse.
Alternatively, a whole block of chambers can be
selected / deselected by drawing a frame which
contains the centers of the selected or deselected
chambers.
If multiple samples are selected, the work direction
can be chosen as well.
Fig. 5-174
Sample panel
Following functions are available:
LSM check box
If LSM is activated, the Sample panel changes to define positions in an
opened LSM scan image.
Select All button
Selects all chambers of the sample carrier.
Clear All button
Deselects all chambers of the sample carrier.
Row radio button
If Row is chosen, measurement is performed row by row.
Column radio button
If Column is chosen, measurement is performed column by column.
Meander check box
If Meander is chosen, measurement of the row or column is made in
meanders.
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Measure Method
LSM-FCS
(3)
Carl Zeiss
Sample panel with activated LSM check
box
If LSM is activated, you can define positions for
FCS measurements in a just scanned LSM image
(see also section 5.18).
You can either press the Crosshair button or
check the LSM box (Fig. 5-175). If the Crosshair
button is pressed, a Crosshair will appear in the
Scan Image. If the LSM box is checked, the
Sample Carrier display box is activated (Fig.
5-175).
In the Sample Carrier display box it is shown that
the Define Positions in LSM Image mode is
activated.
The Positions list shows the numbers and
coordinates of the selected positions in the LSM
scan image (Fig. 5-176).
Fig. 5-175
Select button:
Activates
the
cursor
(crossline) to allow the
definition of positions in the
scan image. Shows or hides
just selected positions if
activated or not activated.
Add Pos button:
Adds the position of the
cursor to the Positions list.
A crossline is set at the
selected cursor position in
the image. To select further
positions click at the
appropriate position in the
image and then on Add
Pos. If more than one
position is selected the
crosslines
are
getting
current numbers.
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Sample panel
5-193
OPERATION IN EXPERT MODE
Analyse FCS Menu
Measure Method
Carl Zeiss
Fig. 5-176
LSM-FCS
Del Pos button:
Deletes
the
selected
(highlighted) position in the
Positions list and the
corresponding crossline in
the scan image.
Del List button:
Deletes all positions in the
Positions list and all
crosslines in the scan image.
Mark Pos button:
Marks the selected positions
as overlay elements in the
scan image. The crosslines
are also visible in the scan
image if the Select button
is deactivated or the
Method
Measurement
window is closed.
Sample panel for defining positions
(4)
Auto Save panel
The Auto Save panel of the window enables you
to instruct the program to save the results
automatically during the measurement process
without human intervention.
Fig. 5-177
Auto Save panel
• Activate the Auto Save check box via mouse
click.
• The directory for the automatically saved files can be chosen as well in the Directory selection box.
The Task Name specified in the Task Description panel is normally used as file name. For checking
purposes, it is then displayed in the Filename display box.
5-194
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Measure Method
LSM-FCS
5.8.3.3
Carl Zeiss
Measurement procedure
After setting the parameters the measurement can be run.
• Click on the Start button on the right-hand side of the Method Measurement ... window to start
the measurement and data analysis process.
–
The FCS data evaluation window will appear.
Fig. 5-178
FCS data evaluation window
The further procedure will be explained in section 5.17, Data Evaluation and Result Presentation
for FCS Measurements.
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5-195
OPERATION IN EXPERT MODE
Analyse FCS Menu
Optimize Method
Carl Zeiss
5.8.4
LSM-FCS
Optimize Method
The Optimize function opens the Method
Optimization ... window. It is used for optimizing
an existing method or for creating a new one.
5.8.4.1
Opening / Closing the
Optimization ... window
Method
• Click on the Optimize button in the Analyse
FCS subordinate toolbar of the Main menu
(Fig. 5-162).
–
•
The Method Optimization ... window will
appear.
Close the Method Optimization ... window
by clicking the Close button.
5.8.4.2
Function description
An analysis method has three major sections:
Fig. 5-179
Method Optimization ... window
(Instrumentation subwindow)
–
Instrument settings
–
Analysis Procedure settings
–
Result Handling parameters.
These major sections are available in separate subwindows by clicking the appropriate button in the
Method Optimization ... window.
The following functions are available on the right-hand side of the Method Optimization ... window:
Close button
The Method Optimization ... window is closed.
Save button
Saves the optimized or new created measurement method.
Start button
Starts the measuring procedure for optimizing.
Stop button
Ends the measuring procedure.
Count rate button
Opens the Count rate window.
X,Y,Z-Scan button
Opens the X,Y,Z-Scan window for performing a X,Y,Z-scan.
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Optimize Method
LSM-FCS
5.8.4.3
Carl Zeiss
Instrumentation
• Click on the Instrumentation button on top of the Method Optimization ... window (Fig. 5-179).
–
(1)
The Instrumentation subwindow appears on the screen.
Method Description panel
In the Method Description panel, an existing
method name for the measuring procedure can be
edited, or a new one entered. In the Category
filed, a category name for the method can be
entered.
The Comment field allows you to store a
comment on the method you are creating.
• Enter a name, a category and a comment for
the method.
(2)
Fig. 5-180
Method Description panel
Fig. 5-181
Beam Path panel
Beam Path panel
In the Beam Path panel you can select a saved
beam path configuration. The selected beam path
will be shown as a drawing.
If a two-channel beam path is selected, you can
activate / deactivate both the channels to be used
and the calculation of the cross correlation.
In the Pinhole Adjustment display box, the date
of the last pinhole adjustment is shown.
• Select a corresponding beam path from the
Beam Path list box.
• Activate / deactivate the channels and the cross
correlation function via the appropriate check
boxes.
It is not possible to define a new beam path in this menu. If a new beam path is required, open
the Beam Path Definition menu via the Define Beam Path button (section 5.9.4, page
5-218) in the Config FCS subordinate toolbar of the Main menu.
Once a new beam path is ready, it can be immediately used in the Method Optimization ...
window.
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5-197
OPERATION IN EXPERT MODE
Analyse FCS Menu
Optimize Method
Carl Zeiss
(3)
LSM-FCS
Pinhole Diameter panel
In the Pinhole Diameter panel you can set the
pinhole diameters for channel 1 and 2 using the
sliders or the input boxes.
Fig. 5-182
Pinhole Diameter panel
• It is recommended to set the pinhole diameter
to a size corresponding to the used excitation
wavelength:
458 nm
488 nm
514 nm
543 nm
633 nm
(4)
Fig. 5-183
Excitation panel
66 µm
70 µm
74 µm
78 µm
90 µm
Excitation panel
The Excitation panel allows to activate / deactivate the wavelengths via check boxes and to set
the requested laser attenuation (%) using the
sliders and by choosing the power attenuation in
the selection box.
The selection box allows you to choose from four possibilities of setting the laser power:
Laser power / 1: power defined by AOTF slider.
Laser power / 10: 10 fold reduced laser power.
Laser power / 100: 100 fold reduced laser power.
Laser power / 1000: 1000 fold reduced laser power.
Furthermore you can open the Laser Control window via Laser button to switch on / off the necessary
lasers.
• Activate / deactivate the laser line via mouse click.
• If necessary switch on / off the lasers using the Laser button. When finished, close the Laser Control
window.
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Optimize Method
LSM-FCS
Carl Zeiss
• Set the laser attenuation using the sliders. For
this purpose, open the Count rate window by
clicking the Count rate button on the righthand side of the Method Optimization ...
window.
A good starting point is to set the intensity in
such a way that a count rate between 50 kHz
and 200 kHz is obtained.
• For most dyes, the Counts/Molecule setting
should be optimized in a second step to a value
just under its maximum by changing the laser
power.
If carriers of different slide thickness are employed
the Counts / Molecule setting should be optimized
by using the correction ring on the lens.
The correction ring is turned counterclockwise or
clockwise until a maximum value is obtained. The
correction ring should also be used for adjusting
the Counts / Molecule setting whenever the
immersion media is changed. This is especially
important in cases where the refractive index of
the immersion media is different from that of the
sample.
Fig. 5-184
Count rate window
• When finished, close the Count Rate window
using the Close button.
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5-199
Carl Zeiss
OPERATION IN EXPERT MODE
Analyse FCS Menu
Optimize Method
5.8.4.4
LSM-FCS
Analysis Procedure
• Click on the Analysis Procedure button on
top of the Method Optimization ... window.
–
(1)
The Analysis Procedure
appears on the screen.
subwindow
Times panel
In the Times panel you can set the values for the
Bleach Time, Measure Time and Repeat Count.
Bleach Time:
Bleach Time is the time prior to
the measuring procedure during
which the laser already has an
effect on the sample. The
Bleach Time is taken into
consideration only once (at the
beginning of a measuring cycle).
Measure Time: Measure Time is the period of
one measurement.
Repeat Count:
Fig. 5-185
Method Optimization ... window
(Analysis Procedure subwindow)
The Repeat Count value
determines the number of
measurements, i. e. how often
Measure Time is to be
performed in a row.
• Set the Bleach Time.
Start with the Bleach Time of 0 s. If the signal decreases during the subsequent measurement, this
might be caused by bleaching, as a remedy, you can prebleach the sample before measurement by
setting the Bleach Time to a non-zero value. If the Bleach with this laser power check box was
not activated, bleaching occurs with the laser power defined by the AOTF setting under
Instrumentation in the Method Optimisation. If the box is checked, the laser power defined by
the slider is used.
• Set the Measure Time.
If you do not have any idea about the behavior of your sample, start with a measurement time of
about 20 seconds. Depending on the signal-to-noise ratio of your correlation curve, decrease or
increase the measurement time. Apart from that, set the measurement time to the correct value right
from the beginning.
• Set the Repeat Count.
Start with Repeat Count set to 1. If you want to obtain information about the variation of your fitted
values later on, increase the repeat count to produce a measurement series.
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Optimize Method
LSM-FCS
(2)
Carl Zeiss
Kinetics panel
In the Kinetics panel you can set kinetics
parameters.
• If a kinetics measurement is required, tick the
Kinetics check box.
Fig. 5-186
Kinetics panel
The input boxes Duration and Number permit
entry of the overall duration of the measurement
and the measuring cycles to be performed during
this period. Measuring cycle means that all the
selected chambers are measured once in the
determined order.
Depending on these two parameters, the maximum number of chambers to be measured is calculated
automatically and displayed under Maximum possible number of sample positions (standard
conditions): .... If 0 is displayed, the measurement cannot be performed, and a relevant message is
displayed on the screen if you try to start the measurement nevertheless.
The Shape selection box allows you to choose from three possibilities of distributing the measuring
cycles (Number) to the overall measuring time (Duration):
const: The period between two successive measuring cycles is constant.
lin:
The period between two successive measuring cycles is increased linearly, i.e. it is doubled each
time.
log: The period between two successive measuring cycles is increased logarithmically.
• Enter the duration (Duration input box) of your kinetics measurement and the number (Number
input box) of individual correlation measurements you want to perform.
• In the Shape selection box, set one of the shape options const, lin or log.
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Optimize Method
Carl Zeiss
(3)
LSM-FCS
Sample panel
In the Sample panel you can select the chamber
for the optimization procedure (only one is
selectable).
• Click on the chamber you want to use for
sample development.
–
The motorized microscope stage moves the
selected chamber to the defined position
under the objective.
• If you want to move to other positions within a
chamber, use the arrow buttons to move the
microscope stage.
• Set the size of the steps to be performed via
the Step slider (or the input box).
Fig. 5-187
Sample panel
The travel speed of the microscope stage can be set to steps 1, 2 and 3.
Step 2 is preferably used for standard positioning.
Step 1 (slow speed) should only be used for very precise positioning, since positioning for longer paths
requires more time.
Step 3 is suitable if large vessels are used, since precise center positioning only plays a minor role in such
cases.
• Select one of the three travel speeds from the Speed selection box.
• By activating the LSM check box, you can define the positions in a LSM image in the same way as
described in section 5.8.3, page 5-193.
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Optimize Method
LSM-FCS
5.8.4.5
Carl Zeiss
Result Handling
• Click on the Result Handling button on top of
the Method Optimization ... window.
–
(1)
The Result Handling subwindow appears
on the screen.
Fit panel
The Fit panel of the menu allows you to fix various
model parameters, which otherwise will be a fit
result. The fixation of the number of Components
in this model is mandatory, while the fixation of all
other parameters is optional.
It is generally accepted that non-linear fitting
procedures yield more reliable results when the
number of free parameters is low. It is
recommended to fix parameters which are known
from
independent
measurements.
Good
candidates for fixing are diffusion times of the free
dye and the free (i. e. not bound) partner, which
had been determined in previous measurements.
• Enter the model parameters to be fixed in the
input boxes.
(2)
Fig. 5-188
Method Optimization ... window
(Result Handling subwindow)
Fig. 5-189
Data Handling panel
Data Handling panel
The Data Handling panel allows you to select if
you want as an average the mean values of the
correlation curves or the mean of the fit results by
choosing the appropriate fit procedure in the
Average selection box. In the first case, the single
values of the different correlation curves are
averaged and the new curve fitted. In the second
case, the fitted parameters of each correlation
curve are averaged.
The Dust-Filter selection box allows you to activate an electronic dust filter that will be active in
operation during the measurement. The threshold in % is set by the slider. All measurement points
within a binned count rate time window having a deviation of more than the specified value from the
average count rate will be cut out and not used for the correlation analysis.
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5-203
OPERATION IN EXPERT MODE
Analyse FCS Menu
Optimize Method
Carl Zeiss
LSM-FCS
Please note that the cut off count rate is defined as the exceeding of the average count rate
during a certain measurement period. Thu, the consecutive fast succession of low peaks might
accumulate the same count rate as one high peak within a certain period of time and hence,
the cut off is not defined by the peak height but rather by the counts / binning time.
(3)
Fig. 5-190
Display panel
Display panel
In the Display panel you can tick which result
curves (Count Rate, Correlation Curve, Count
Rate Histogram and Pulse Density Histogram)
will be displayed when the measurement runs. The
meaning of these curves is explained in the
Measurement window.
• Select the appropriate options via mouse click.
(4)
Auto Save panel
The Auto Save panel allows you to determine
which of the resulting curves will be saved
automatically.
• Select the appropriate options via mouse click.
Fig. 5-191
Auto Save panel
(5)
Fig. 5-192
Auto Print panel
Auto Print panel
In the Auto Print panel you can determine
whether or not you want to have an automatic
protocol printout (with varying degree of detail).
• Select the appropriate radio buttons via mouse
click.
5-204
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OPERATION IN EXPERT MODE
Analyse FCS Menu
Optimize Method
LSM-FCS
5.8.4.6
Carl Zeiss
X,Y,Z-Scan for positioning the focus within structured samples
To make the positioning of the detection volume easier, the ConfoCor 2 is provided with a X,Y,Z-Scan
possibility.
This means, that the focus is moved in either the X, Y, or Z direction over a preselected distance while
the count rate in one or both of the detection channels as defined by the channel check boxes is
recorded. A maximum (or minimum) in the resulting curve (Fig. 5-194) usually indicates a region of
interest. The focus can be positioned to this region by a simple mouse click.
• Click on the X,Y,Z-Scan button on the right-hand side of the Method Optimization ... window
–
The X,Y,Z-Scan operating window appears on the screen (Fig. 5-193).
Fig. 5-193
X,Y,Z-Scan operating window
For X,Y,Z-Scanning proceed as follows:
• If you use the Axiovert 100, first switch to the visual or TV mode using the tube sliders on the
microscope stand so that you are able to view the object (the label near the tube sliders will show the
correct tube slider position) or press the VIS button (Axiovert 200).
• Position the object in X and Y.
• Find the upper cover slip surface (interface between the glass and the cell) by carefully focusing the
objective.
• Switch to the FCS mode by clicking on the FCS button in the Analysis subordinate toolbar.
The next steps depend on whether you already have a functional measurement method for your
particular problem or not.
• If you already have a functional method: Select the method first using the Select Method button in
the Analyse FCS subordinate toolbar of the Main menu.
• If you do not have a functional method: Create a new method.
• Select the Channel, Start and End position and the Number of positions.
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5-205
OPERATION IN EXPERT MODE
Analyse FCS Menu
Optimize Method
Carl Zeiss
LSM-FCS
• Clicking on Start opens a new window showing a count rate graph (Fig. 5-194).
Fig. 5-194
Z-Scan result window
When the scan has been finished, a vertical red line appears at the y-axis at the left-hand side. This line
can be dragged with the mouse when the left button is held down.
• Position the vertical red line where the focus should be placed by using the mouse.
• Then close the window with Close. The Refresh Positions button will actualise the current position
if pressed.
The data of the X,Y,Z-Scan result window can be
copied to the clipboard via the context menu or
saved directly as an ASCII file.
Fig. 5-195
Context menu of the
X,Y,Z-Scan result window
• Click in the X,Y,Z-Scan result window with the
right mouse button to open the context menu.
• Select the line Copy data to clipboard with a click of the mouse if you want to insert the data into
other WINDOWS programs directly via the clipboard. The Paste function enables you to insert these
data directly into the required program.
• Select Export data to file to save the data in an external ASCII file (.txt).
5-206
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OPERATION IN EXPERT MODE
Analyse FCS Menu
VIS, FCS and LSM Buttons
LSM-FCS
5.8.5
Carl Zeiss
VIS, FCS and LSM Buttons
The VIS, FCS and LSM buttons are included in the Analyse FCS subordinate toolbar of the Main menu.
They switch the setting of the beam path of the microscope:
–
VIS:
observation via the eyepieces of the binocular tube
–
FCS:
screen observation via laser excitation using the FCS measurements and software
evaluation
–
LSM:
screen observation via laser excitation using the LSM image acquisition and software
evaluation
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5-207
OPERATION IN EXPERT MODE
Config FCS Menu
Laser Control
Carl Zeiss
5.9
LSM-FCS
Config FCS Menu
• In the Main menu toolbar, click on Config FCS.
–
This opens another, subordinate toolbar in the Main menu.
Config FCS subordinate toolbar
Fig. 5-196
Config FCS menu
5.9.1
Laser Control
The Lasers panel shows the types, excitation
wavelengths and operating statuses of the lasers
available.
The subordinate laser settings panel shows the
relevant and currently set Maximum Power,
Wavelength, Status, Tube Current and
Output [%] values of the current laser. The
buttons On, Off and Standby permit the current
laser to be set in the required status, and the laser
intensity (Output) can be set using the slider or
the input box. The name of the selected laser
(Argon, HeNe1 or HeNe2) is displayed in the
headline of this setting panel for checking.
Fig. 5-197
Laser Control window
5.9.1.1
Opening / Closing the Laser Control window
• Click on the Laser button in the Config FCS subordinate toolbar.
– This opens the Laser Control window, which shows all lasers connected to the system.
When the setting of the required lasers has been finished, the Laser Control window can be closed
again.
• Click on the Close button to close the Laser Control window.
– The Laser Control window will be closed.
5-208
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OPERATION IN EXPERT MODE
Config FCS Menu
Laser Control
LSM-FCS
5.9.1.2
Carl Zeiss
Function description
Lasers panel (upper)
List of available lasers, including the display of relevant wavelengths and
switching status.
Selection of the laser to be switched on / off and setting of the laser
output is performed in the subordinate setting panel.
Laser settings panel (lower)
Switch on / off the required laser or set Standby operation.
Display Maximum Power, Wavelength, Status and Tube Current (only
Enterprise and Argon) of the relevant laser.
Set the laser output for Argon.
5.9.1.3
Settings
• Click on the desired laser on the (upper) Lasers panel.
–
This highlights the selected laser.
On the lower panel of the Laser Control window, activate the laser as follows:
This applies to the Ar-multiline laser:
• Click on the Standby button.
–
Wait for the laser to heat up, until the Status ready - Standby message appears.
• Click on the On button.
–
Status ready - On appears.
• Use the Output [%] slider to set the laser power which is ideal for the measurement job.
Thus, the laser needed for image acquisition is available.
• Set output between 25 and 100 % of the maximum tube current.
–
Optimum operation is at 8 A (lowest laser noise). However, the laser life is reduced if the laser is
constantly operated at 8 A. Therefore, 8 A should be used only if this is absolutely necessary.
This applies to HeNe lasers:
• After selecting the laser, click on the On button.
–
The required laser for image acquisition is now available.
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5-209
OPERATION IN EXPERT MODE
Config FCS Menu
Micro
Carl Zeiss
5.9.2
LSM-FCS
Micro
The Axiovert Control (Micro button) window
permits motorized functions (objective and
reflector change settings) and the illumination
mode (transmitted light) of the connected
microscope to be controlled via the software.
Without any difference to software control, these
microscope functions can also be operated directly
on the stand via the relevant controls. In that case,
any changes are recorded by the software and
displayed in the relevant windows / panels.
5.9.2.1
Open the Axiovert Control window
• Click on the Micro button.
–
This opens the Axiovert Control window
on the screen.
After conclusion of the conventional setting of the
connected microscope, the Axiovert Control
window can be closed again.
• Click on the Close button in the Axiovert
Control window.
Fig. 5-198
Axiovert Control window
5.9.2.2
Function description
–
The Microscope Control window will be
closed.
Transmitted Light button
Transmitted light is switched on / off via ON button in the Transmitted
Light frame, setting of light intensity can be varied via input box or
slider. 3200 K color temperature for photo documentation can be
switched on via 3200 K button in the Transmitted Light frame. The
transmission light control potentiometer on the stand is disabled via the
Remote button. By clicking on the Close button the Transmitted Light
frame is closed.
Condensor button
Numerical aperture of the condensor is set via input box or slider. Turret
position selected from graphical pop-up menu (only for motorized
condensors). By clicking on the Close button the Condensor frame is
closed.
Objective button
Objective can be selected via graphical pop-up menu.
Reflector button
Push and click, reflector cube can be selected via graphical pop-up menu.
Tube Lens button
Push and click, tube lens can be selected via graphical pop-up menu.
Reflected Light button
The shutter is switched on and off.
5-210
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OPERATION IN EXPERT MODE
Config FCS Menu
Micro
LSM-FCS
(1)
Carl Zeiss
Reflected-light observation (Epi-fluorescence)
• Turn on the HBO 50 power supply switch.
• Click on the check box for Reflected Light
On.
• In the Reflector Turret list box, select the desired filter set by clicking on it.
–
The filter is automatically moved into the beam path to enable observation in epi-fluorescence.
• In the Tubelens list box, select the desired tube lens by clicking on it.
• Swing the required objective for FCS measurements into the working position. This is performed by
selecting the objective in the Objective selection box in the Axiovert Control window.
We recommend to use the C-Apochromat 40x/1.2 W corr on account of its optimized optics.
• Click on the FCS button on the right-hand side of the Analyse FCS subordinate toolbar in the Main
menu (Fig. 5-162), to select the FCS detection head.
(2)
Transmitted-light observation
• Click on the check box for Transmitted Light
On.
• Activate the condensor function in the Condensor panel.
• Swing the required objective for FCS measurements into the working position. This is performed by
selecting the objective in the Objective selection box in the Axiovert Control window.
• Select Light Remote or 3200 K or set the transmitted light intensity via slider.
• Click on the FCS button on the right-hand side of the Analyse FCS subordinate toolbar in the Main
menu (Fig. 5-162), to select the FCS detection head.
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5-211
OPERATION IN EXPERT MODE
Config FCS Menu
Adjust Pinhole
Carl Zeiss
5.9.3
LSM-FCS
Adjust Pinhole
The pinhole is adjusted using a dye solution. The
general approach is to move the pinhole cyclically
in x, y and z until the intensity maximum is found.
For each excitation wavelength a suitable dye must
be used. We recommend:
–
Rhodamine 6G for 458, 488 and 514 nm
–
Tetra-Methyl-Rhodamine (TMR) for 543 nm
and
–
Cy 5 for 633 nm.
It is also recommended to work with a relatively
concentrated solution (10-5 mol) and low laser
power to achieve smooth intensity curves.
5.9.3.1
Open / Close
the
Adjustment window
Pinhole
• Click on the Pinhole button in the Config FCS
subordinate toolbar of the Main menu.
Fig. 5-199
Pinhole Adjustment window
–
This opens
window.
the
Pinhole
Adjustment
• Click on the Close button to quit the window.
5.9.3.2
(1)
Function description
Beam Path panel
Here you can select the beam path for pinhole
adjustment. The date of the last pinhole
adjustment is shown in the Last Adjustment
display box.
• Select the beam path in the selection box.
Fig. 5-200
5-212
Beam Path panel
–
The selected beam path appears in a
drawing.
–
For the definition of a new beam path, see
section 5.9.4, page 5-218.
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OPERATION IN EXPERT MODE
Config FCS Menu
Adjust Pinhole
LSM-FCS
(2)
Carl Zeiss
Adjust Coordinates panel
The Adjust Coordinates panel is used to adjust
the pinholes. Adjustment can be performed
manually or automatically. Manual adjustment is
not recommended, since the relevant procedure is
very complex.
Fig. 5-201
Adjust Coordinates panel
Following function are available:
Pinhole
Selection of the channel for adjustment.
Diameter
Setting of pinhole diameter to the used wavelength (see page 5-216).
Auto Adjust buttons:
X fine / X coarse
Starts the automatic coarse or fine adjustment for X direction depending
on the activation / deactivation of the Coarse check box.
Y fine / Y coarse
Starts the automatic coarse or fine adjustment for Y direction depending
on the activation / deactivation of the Coarse check box.
Z coarse
Starts the automatic coarse adjustment for Z. Since fine Z adjustments
would be meaningless, they have not been included as a feature.
Coarse check box
If ticked, the coarse adjustment for X or Y is performed, if not ticked, the
fine adjustment for X or Y is performed.
Position input boxes
Display / input of the pinhole position in X, Y and Z.
(3)
Excitation panel
The
Excitation
panel
allows
you
to
activate / deactivate the wavelengths via check
boxes and to set the laser attenuation (%) using
the sliders. Furthermore you can open the Laser
Control window via Laser button to switch
on / off the necessary lasers (see section 5.9.1,
page 5-208).
Fig. 5-202
Excitation panel
The Laser Power display box allows you to reduce
the overall power by factors of 10, 100 and 1000.
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5-213
OPERATION IN EXPERT MODE
Config FCS Menu
Adjust Pinhole
Carl Zeiss
(4)
LSM-FCS
Carrier Position panel
In the Carrier and the Position display boxes, the
carrier and the currently selected position are
shown.
Fig. 5-203
Carrier Position panel
If the carrier or the position have to be changed
for adjustment use, the Carrier button to open
the Carrier Position window (see section 5.8.2,
page 5-186).
(5)
Count rate window
• Click on the Count rate button on the righthand side to open the Count rate window.
In two Channel and one Correlation panels, the
Count rate window shows the values for Count
rate, Correlation or Counts / Molecule depending
on the activated Display button.
The Channel panels and the Correlation panel
feature a big display field for the count rate and,
at the underside, a scale allowing the measuring
range for the displayed count rate to be read from
a colored bar. A click on one of the four arrow
buttons permits the measuring range display to be
narrowed or widened.
The buttons in the lower range of the Count rate
window have the following functions:
Fig. 5-204
5-214
Unit:
Selecting the unit Hz or kHz.
Average:
Averaging after 1 s, 10 s or Off.
Display:
Selecting one of the display
modes Count rate, Correlation
or Counts / Molecule.
Count rate window
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OPERATION IN EXPERT MODE
Config FCS Menu
Adjust Pinhole
LSM-FCS
(6)
Carl Zeiss
FCS Hardware Settings window
• Click on the Coll. button to open the FCM
Hardware settings window. The slider allows
you to set the Collimator position.
If the Collimator setting was changed, the Pinhole
Adjustment window will inform about the Old
and New positions, as long as no Pinhole
alignment was done in x, y, or z.
As soon as one Pinhole alignment was done and
the OK button was clicked, only the current value
will be displayed.
Fig. 5-205
FCM Hardware Settings
The Collimator setting will be stored with the
respective method and will be automatically used
when the method is called up.
5.9.3.3
To adjust the pinhole proceed as follows
• Mount a cuvette with a suitable dye solution and orient the sample stage as described in section
5.8.1, page 5-184.
• Click the Pinhole button in the Config FCS toolbar subordinate of the Main menu. The Pinhole
Adjustment window will appear (Fig. 5-199).
In the Beam Path panel of the Pinhole Adjustment window you will see a drawing of the current
beam path including a line which indicates when this beam path had last been adjusted.
• Select the beam path you want to adjust in the Beam Path selection box in the Beam Path panel of
the Pinhole Adjustment window.
• Select the test sample. Click on the Carrier button close to the bottom of the window. The Carrier
Position window will appear. Then select the sample by mouse click.
• Use the slider in the Excitation section of the menu to set the laser intensity to minimum.
• Click on the Count Rate button at the right-hand side of the Pinhole adjustment window. This will
open the Count Rate window.
• Then cautiously increase the laser intensity until a count rate of about 100 kHz is indicated.
• Close the Count Rate window.
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5-215
OPERATION IN EXPERT MODE
Config FCS Menu
Adjust Pinhole
Carl Zeiss
LSM-FCS
• Set the pinhole diameter to a size corresponding to the used excitation wavelength:
458 nm
488 nm
514 nm
543 nm
633 nm
66 µm
70 µm
74 µm
78 µm
90 µm
• Select the pinhole you want to adjust by clicking on the corresponding Ch1 or Ch2 radio button in
the Adjust Coordinates panel of the window.
In the case of a cross correlation beam path, adjust the two pinholes subsequently in two runs.
Each of the pinhole axe must be adjusted separately. If the Coarse check box is activated, the
corresponding axis buttons will be labeled X Coarse and Y Coarse respectively and the pinhole will
travel over the maximum range for each axis.
If the Coarse check box is deactivated, the buttons will be labeled X Fine and Y Fine respectively. In this
case the pinhole travel will be limited to speed up adjustment.
If the pinholes are adjusted for the first time, the coarse adjustment must be performed first
and then the fine adjustment, each time for X, Y and Z (only coarse).
For subsequent readjustments, the fine adjustment is normally sufficient.
• Perform the pinhole adjustment in the following sequence:
–
X fine; Y fine
–
Z coarse
–
X fine; Y fine
5-216
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OPERATION IN EXPERT MODE
Config FCS Menu
Adjust Pinhole
LSM-FCS
Carl Zeiss
If one of the above buttons, e.g. X Coarse or X Fine or Z Coarse etc., is clicked, the instrument will
behave as follows:
–
The Automatic Pinhole Adjustment window will appear (Fig. 5-206). It is possible to exit the
process using the Cancel button. When the process is finished, quit with OK.
–
In the Count Rate/Position diagram, the black line corresponds to the measured intensity (count
rate). The red line corresponds to the intensity curve fitted from it to find the optimum pinhole
position.
Fig. 5-206
Automatic Pinhole Adjustment
• Repeat the procedure for all axes.
• You will find peaks for x-Coarse, and y-coarse, in any case. If you fail to find the peak in z-coarse
proceed as follows. Go to the FCM Hardware settings window by clicking the Coll. button. Move
slider one step up, if peak is expected to the right; move slider one step down, if peak is suspected to
the left. Repeat x-, y-, z-alignment and repeat changing Collimator setting, until z-peak is within the
window.
• A click with the right mouse button on the diagram opens the context menu. Selection of the option
Copy to clipboard or Export data to file permits the data to be copied to the clipboard or saved
directly in an ASCII file.
• When the whole pinhole adjustment is completed, close the dialog with Close.
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5-217
OPERATION IN EXPERT MODE
Config FCS Menu
Define Beam Path
Carl Zeiss
5.9.4
LSM-FCS
Define Beam Path
The Beampath function allows new / existing beam path configurations to be created / edited and
saved. The Delete function enables you to delete existing configurations.
5.9.4.1
Open / Close the Beam Path Definition window
• Click on the Beampath button in the Config FCS subordinate toolbar of the Main menu.
–
The Beam Path Definition window is opened.
• When the beam path definition is finished, click on the Close button to quit the Beam Path
Definition window.
HFT
NFT
EM
Fig. 5-207
Beam Path Definition window
HFT (main beam splitter)
The main beam splitter (HFT) reflects the specified laser lines and allows the resulting fluorescence
spectrum to pass through.
NFT (secondary beam splitter)
The secondary beam splitter (NFT) splits the fluorescence spectrum onto the various detection channels.
EM (emission filter)
The emission filter (EM) is used to narrow the fluorescence spectrum. The bandpass (BP) allows the range
within the specified border wavelengths to pass through. The longpass (LP) allows the range above the
specified border wavelength to pass through.
5-218
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OPERATION IN EXPERT MODE
Config FCS Menu
Define Beam Path
LSM-FCS
5.9.4.2
Carl Zeiss
Function description
Beam Path selection box
Selection of a new (scratch) or existing beam path for editing.
Pinhole Adjustment display box
Display of the last pinhole adjustment of existing configurations.
Activation / deactivation of a main dichroic beam splitter (HFT) or
secondary dichroic beam splitter (NFT) through selection from the
relevant list box (Fig. 5-207).
Activation / deactivation of an emission filter (EM) through selection
from the relevant list box (Fig. 5-207).
Excitation check boxes field
Selection of the wavelength to be used.
Ch1 / Ch2 input boxes
For entering of a special comment (e.g. used dye).
Close button
The Beam Path Definition window is closed.
Save button
For saving the new or edited configuration.
Delete button
Deletion of an existing configuration.
5.9.4.3
Define a new beam path
configuration
• First tick the laser or lasers (in case of cross correlation) you need for excitation of your
sample.
• Then choose a (main) dichroic beam splitter
which should correspond to the chosen laser(s).
Click on the Beam Splitter symbol
and a
list box will appear. Then click on the beam
splitter you need.
• Select the second beam splitter in a similar way
to the first one.
–
For cross correlation, choose the NFT
488/633 beam splitter.
–
Auto correlation measurements with an
excitation wavelength of 633 nm should be
carried out in detection channel 1. In this
case, select Plate in the second beam
splitter position.
–
All the other auto correlation measurements will be carried out in detection channel 2. To work
with this channel, select a Mirror in the second beam splitter position.
B 45-0006 e
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Fig. 5-208
Beam Splitter list box open
5-219
OPERATION IN EXPERT MODE
Config FCS Menu
Define Beam Path
Carl Zeiss
LSM-FCS
• Click on the Emission Filter symbol(s) to get
the list box allowing you to select the emission
filters you need.
• It is recommended to give the channel a name
which is easy to remember.
Fig. 5-209
Emission Filter list box open
5.9.4.4
Save a new beam path
configuration
• Click the Save button to save the new beam
path.
–
Fig. 5-210
Save Beam Path window
The Save Beam Path window appears.
• Choose a memorable name for the new beam
path which you are likely to remember.
• Click on OK to save the new configuration.
Before you can use a newly defined beam path, you have to adjust the pinhole(s) for this
particular beam path.
5.9.4.5
Delete a beam path configuration
A no longer required configuration can be deleted as follows:
• Select the configuration to be deleted from the Beam Path selection box.
• Click on the Delete button. Confirm the deleting in the following window by clicking OK.
• A beam path can only be deleted if it is not used by any method. If you intend to delete such a beam
path, you will have to delete the corresponding method first.
• When you are finished, close the Beam Path Definition window by clicking on Close.
5-220
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OPERATION IN EXPERT MODE
Config FCS Menu
Define Carrier
LSM-FCS
5.9.5
Carl Zeiss
Define Carrier
The Carrier function allows new / existing sample
carriers to be created / edited and saved. The
Delete function enables you to delete existing
sample carriers.
5.9.5.1
Open / Close the Carrier Definition
window
• Click on the Carrier button in the
Configuration subordinate toolbar of the
Main menu.
–
Fig. 5-211
Define Carrier window
The Carrier Definition window is opened.
• When the beam path definition is finished, click on the Close button to quit the Define Beam Path
window.
5.9.5.2
Function description
Sample Carrier selection box
Selection of a new or existing sample carrier for editing.
Columns - Number input box
Setting of the number of columns for the sample carrier.
Columns - Distance input box
Setting of the distance between the chambers of the column.
Rows - Number input box
Setting of the number of rows for the sample carrier.
Rows - Distance input box
Setting of the distance between the chambers of the row.
Close button
The Carrier Definition window is closed.
Save button
For saving the new or edited sample carrier.
Delete button
Deletion of an existing sample carrier.
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5-221
Carl Zeiss
5.9.5.3
OPERATION IN EXPERT MODE
Config FCS Menu
Define Carrier
LSM-FCS
Define a new sample carrier
• In the Sample Carrier selection box, use the (new) entry or select the sample carrier to be edited.
• Enter the number of chambers and the distance (mm) between the chambers in the appropriate
Column and Row input boxes.
5.9.5.4
Save a new sample carrier
• Click on the Save button to save the new
sample carrier.
–
Fig. 5-212
Save Sample Carrier window
The Save Sample Carrier window appears
on the screen.
• Choose a memorable name for the new sample
carrier which you are likely to remember.
• Click on OK to save the new sample carrier.
5.9.5.5
Delete a beam path configuration
A no longer required sample carrier can be deleted as follows:
• Select the sample carrier to be deleted from the Sample Carrier selection box.
• Click on the Delete button. Confirm the deleting in the following window by clicking OK.
• When you are finished, close the Carrier Definition window by clicking on Close.
5-222
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OPERATION IN EXPERT MODE
Macro Menu
Macro Language
LSM-FCS
5.10
Carl Zeiss
Macro Menu
The macro function permits the recording, running and editing of command sequences and their
allocation to buttons in the Macro menu.
• In the Main menu toolbar, click on Macro.
–
This opens another, subordinate toolbar in the Main menu.
Macro subordinate toolbar
Fig. 5-213
Macro menu
5.10.1
Macro Language
"Visual Basic for Applications", called VBA in the following, is used as the Macro language. This
language is well known through its widespread use as Macro language in the "Microsoft Word for
Windows" and "Microsoft Excel for Windows" products. Experience with "Microsoft Visual Basic"
would also be beneficial for macro-programming of the LSM-FCS.
An Integrated Development Environment, called IDE in the following, is available for the editing and
debugging of macros. IDE includes an "online help program" where the VBA language is described in
detail.
Macros are stored in project files. One project file can include several macros.
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5-223
OPERATION IN EXPERT MODE
Macro Menu
Macro Control
Carl Zeiss
LSM-FCS
5.10.2
Macro Control
5.10.2.1
Open / Close the Macro Control
window
• Click on the Macro button in the Macro
subordinate toolbar of the Main menu.
–
This opens the Macro Control window.
• Click on the Close button to quit the window.
5.10.2.2
Edit Macro function
This function allows you to manage project data.
Macros can be recorded, stored, performed,
edited and, if required, deleted.
Fig. 5-214
Macro Control window
• Press the Edit Macro button to switch to the
Macro and Recording panels.
(1)
Macro panel
New button:
Creates a new project.
Load button:
Opens an existing project.
Save button:
Stores the project on the hard
disk.
Save As button: Stores an existing project under
a new name.
Unload button: Removes the selected macro
from the Macros list.
Fig. 5-215
5-224
Macro panel
Edit button:
Allows macros to be edited and
debugged.
The
editor
(Microsoft Visual Basic) is
automatically located at the
beginning of the relevant macro.
Run button:
Runs a macro.
Step button:
Opens the editor for line-by-line
editing / debugging.
Delete button:
Deletes the selected macro.
Editor button:
Opens the editor. Displays the
processed area of the macro
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OPERATION IN EXPERT MODE
Macro Menu
Macro Control
LSM-FCS
Carl Zeiss
edited last.
Macros are stored and managed in project files (*.lvb). Before you can record or edit a macro, you have
to create a project as follows:
• Press the New button to create a project file.
–
A new project is created and displayed in the Project selection box (e.g.: LSM 150503). The
project name is automatically default, but can be edited afterwards.
To activate an existing project, proceed as follows:
• Press the Load button.
–
The Open window will be opened.
• Select the relevant project file (data extension:
*.lvb) from the Macros list box. Click on the
Open button.
–
The project file will be opened and the
macros contained in it are displayed in the
Macros selection box of the Macro Control
window.
Fig. 5-216
Open window
Recorded macros are stored in main memory first. Before the macros can be assigned to the buttons in
the Macro submenu, the project must be stored on the hard disk.
• Press the Save button under the project name in the Macro Control window and determine the file
name in the Project selection box, if required.
(2)
Recording panel
Before recording a command sequence, you can
enter the name for the macro to be created in the
Rec Name input box of the Recording panel.
Start button:
Starts recording.
Cancel button:
Cancels
the
procedure.
Stop button:
Stops recording.
Edit On Stop:
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recording
Fig. 5-217
Recording panel
On stopping the recording
procedure, the macro editor
is automatically opened at
the relevant position.
5-225
Carl Zeiss
OPERATION IN EXPERT MODE
Macro Menu
Macro Control
LSM-FCS
Proceed as follows to record a macro:
• Enter a name for the macro to be created under Rec Name in the Recording panel.
• Click on the Start button to start recording the macro.
• Then perform the operations to be stored, e.g.:
–
Click on the Find button in the Scan Control window. A Find scan will be performed.
–
Click on the New button in the Scan Control window. A new Image Display window will be
opened.
–
Click on the Single button in the Scan Control window. A Single scan will be performed.
• Then click on the Stop button to end the recording. (Cancel enables you to cancel recording)
–
If recording was successful, the entered Rec Name will then also be available in the Macros list
box of the Macro panel. The new macro is automatically assigned to the current project. It is
possible to assign as many macros as required to a project.
• Click on the Save button to store the new macro.
Proceed as follows to perform a macro:
• Select the required macro from the Macros list box of the Recording panel.
• Click on the Run button to start performing the macro.
Provided that a macro is linked to a button in the Macro subordinate toolbar, you only need to
click on this button to perform the macro.
Proceed as follows to delete a macro:
• Select the required macro from the Macros list box of the Recording panel.
• Click on the Delete button. The macro will be removed from the list.
Proceed as follows to edit a macro:
• Select the required macro from the Macros list box of the Recording panel.
• Click on the Edit button. The Microsoft Visual Basic editing window will be opened.
• Make the required changes (also see the notes on page 5-228).
5-226
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OPERATION IN EXPERT MODE
Macro Menu
Macro Control
LSM-FCS
5.10.2.3
Carl Zeiss
Assign Macro to Button function
This function permits stored macros to be linked
with one button each in the Macro subordinate
toolbar.
• Press the Assign Macro to Button button to
switch to the Define Buttons panel.
Define Buttons panel
Proceed as follows to link a macro to a button of
the Macro subordinate toolbar:
• Select the button number from the Button
selection box.
• Enter the button labeling in the Text editing
box.
• Select the name of the project file from the
Project box using the ... button
Fig. 5-218
Macro Control window
• Select the macro name from the Macros box.
• Press the Apply button to assign the relevant
macro to the specified button in the Macro
toolbar.
Proceed as follows to delete the linking between a button in the Macro subordinate toolbar and a
macro:
• Select the button number from the Button selection box.
• Press the Delete button to delete the linking.
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Carl Zeiss
5.10.2.4
OPERATION IN EXPERT MODE
Macro Menu
Macro Control
LSM-FCS
Editing and debugging of macros
The Edit button activates IDE (Integrated Development Environment) which allows macros to be edited
and debugged. Under the Help - Contents and Index menu item, IDE contains detailed "online" help
on its operation and on the VBA macro language. Therefore, only a few hints are provided in the
following:
You should activate the required toolbars. We would recommend you to activate the Debug toolbar via
the View - Toolbars -Debug menu item.
The following buttons in the toolbar can help you when debugging macros:
Starts running the command lines.
Stops running the command lines.
Interrupts processing of the command lines (pause).
Sets a breakpoint in the line with the text cursor.
Processes a command line and steps into subprocedures.
Processes a command line and steps over subprocedures.
Exits the subprocedure (step out).
Displays the value of the marked expression (Watch). If nothing is marked, the value of the
variable above the text cursor is displayed.
Activates the Watch window in which values of variables and expressions can be displayed. For
this, text is marked in the code window and dragged into the Watch window. Variables can be
modified in the Watch window.
In the left-hand edge of the code window you will find an arrow beside the current command line. A
new current command line can be determined by moving the arrow via the mouse. This makes it
possible to skip command lines or to process command lines several times.
5-228
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OPERATION IN EXPERT MODE
Macro Menu
Overview of available Macros (all LSM releases)
LSM-FCS
5.10.3
Carl Zeiss
Overview of available Macros (all LSM releases)
Documentation files (*.rtf, *.doc) of advanced macros will be located in the macro directory.
Name
Description
AOTFfit.lvb
Linearize laser attenuation (AOTF or mechanical)
AOTFfitlin.lvb
New method to linearize laser attenuation (AOTF or
mechanical)
Autofocus.lvb
Automatic focusing according to a set configuration
Bleach.lvb
Bleaching of a rectangular area or a line; combines old macros
BleachRectangle.lvb, BleachLine.lvb and Spot.lvb
CameraColor.lvb
(also Button in Maintain)
Color balance of Axiocam HRc
Centerv28-30.lvb
Centers the field of view around the position marked with the
cross tool;
CopyPasteOverlays28-30.lvb
Copies actual overlay drawing into the clipboard and pastes
the drawing into a selected image window
CopyPasteRoi.lvb
Copies drawing element of overlay into clipboard and pastes
it into other selected windows
CopyRoisToOverlay28-30.lvb
Copies ROIs to overlay drawings; both can be viewed and
measured at the same time;
CpCanTrace.lvb
Checks communication of PC with CAN-Bus/net
CpDsp.lvb
Checks communication of PC with DSP
DeleteMultiTimeRecipies.lvb
Deletes all available Multiple Time Series set ups
Distance.lvb
Example macro for measurement
Distance28-30.lvb
Release 3.0: measures the distance using the mouse;
DivideThroughReferenceImage.lvb
- divide complete time series through a single image/part of
the series
- duplicate a single image or part of a time series to this
series.
EventPollPeriod.lvb
Not documented
FastModeSwitch.lvb
Store settings from "Scan-Control" and reuse.
FileExport.lvb
Exports one or more selected images according to the set file
format in one go; Exports image intensity values in ASCI
format;
GDC_calib.txt
Description
HotKey.lvb
Shift focus with a button and start Single-Scan
KSPlastv25.lvb
KS software macro
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5-229
OPERATION IN EXPERT MODE
Macro Menu
Overview of available Macros (all LSM releases)
Carl Zeiss
LSM-FCS
Name
Description
Kundenmacro32.doc
Documentation
Lambdatrans.lvb
Time series alternating between lambda and transmission
mode
LsmHWAdmin.lvb
Maintain)
(also
Button
in
Direct service hardware access (password protected)
LsmHWAdminEx.lvb
Calibration service macro (password protected)
LsmHWAdminMeta.lvb
Calibration for META channel
LsmTime.lvb
Triggered Time scan Macro
Macro_Description.pdf
Description
MCS30.lvb
Lsm.mac
Control of spectrometer
MetaExport.lvb
Export of META image files including all channels as tif, bmp...
ModifySeries30.lvb
Modifies Z Stacks and Time Stacks like Rotation of the stacks,
being mirrored, Conversion of time stacks into z-stacks and
vice versa;
ModifySeries30.rtf
MultiProfile.lvb
StitchArt macro (Software option)
MultiStack.lvb
Similar to StitchArt, but generates stacks only (no profiles) and
uses the settings in scan control.
MultiTime28-32.lvb
Set up of time series experiments including repeated imaging,
bleaching and autofocusing with defined configurations at
multiple locations and for various views at each location
(Software option)
MultiTime28-32.rtf
OptimizeGDCV3_0.lvb
OptimizeGDCV3_2.lvb
Optimize the max. peak power of fiber coupled Ti:Sa lasers
(Release 3.0/3.2)
Parameters.lvb
Check the scan parameters
Pixel28-30.lvb
Displays and stores the mean intensity values of each line of
one or more channels of one or more images; data from each
line are stored as a txt file in the current folder;
Profile.lvb
Displays the pixel values along a line
Profilev28-30.lvb
Opens VBA editor for profile
Reboot.lvb (also Button in Maintain)
Service reset of scan-module (password protected)
Scalebar30.lvb
Indication of self defined intensity levels assigned to a ROI as
scale bar in the image; also attaches tick marks and
concentration values to the grayscale/color wedge.
Scalebar30.rtf
SetFind.lvb (also Button in Maintain)
Sets properties of the Find function
Spline.lvb (also Button in Maintain)
Calibration of a spline-scan
5-230
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OPERATION IN EXPERT MODE
Macro Menu
Overview of available Macros (all LSM releases)
LSM-FCS
Carl Zeiss
Name
Description
TileScanRotation.lvb
Defines rotation of a tile-scan
TimeSeriesShutter.Lvb
Close laser shutter in time series on a LSM 5 PASCAL
Trigger.lvb
Trigger test
TuneNLOLaser32.lvb
Change wavelength of a tunable Ti:Sa laser and run excitation
series
Remarks:
–
During installation, default macros will be installed according to their type either in AIM\, AIM\HWT or
AIM\Macros\. Self generated Macros will be in AIM\Macros.
–
In case of a new installation, old macros will be stored in AIM\Macros\BackupMacros or AIM\Backup\,
to avoid problems with identical names of existing and new macros.
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5-231
OPERATION IN EXPERT MODE
Macro Menu
Sample Macros
Carl Zeiss
5.10.4
LSM-FCS
Sample Macros
The LSM-FCS software package includes e. g. the Distance, Profile, Spot and Multiple Time Series
sample macros.
They can be easily executed by clicking on the relevant button in the Macros subordinate toolbar.
During the execution of a macro, the Stop Macros window is always displayed on the screen. This
enables a macro to be stopped any time by pressing the Stop button.
The functions of the sample macros are explained below.
5.10.4.1
Distance macro
This macro permits measurement of the distance of a line created in the scan image.
• Click on the Distance button in the Macro subordinate toolbar.
–
An XY scanning image of the specimen is recorded and displayed. At the same time, the Mouse
position test window appears on the screen.
• Then draw a line over the distance to be measured by clicking and holding down the mouse button.
The click of the mouse sets the starting point, releasing the mouse sets the end point of the line.
–
After release of the mouse button, the length of the line in the scanning image is displayed (in
µm).
–
Any required number of lines can be defined in the image. The previous line is deleted.
• A click on the Exit button in the Mouse position test window will end the macro.
5.10.4.2
Profile macro
This macro permits the gray values of a line created in the scanning image to be determined pixel by
pixel.
• Click on the Profile button in the Macro subordinate toolbar.
–
An XY scanning image of the specimen is recorded and displayed. The Profile window is shown
on the screen.
• Then click and hold down the mouse button to draw a line in the scanning image for which the gray
values shall be determined.
–
The current numbers of the pixels of the created line to which the relevant gray value is assigned
now appear in the Profile window.
–
At the same time, the distance of the created line is displayed in µm for checking.
–
Any required number of lines can be defined in the image. The previous line is deleted.
• A click on Cancel will end the macro.
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OPERATION IN EXPERT MODE
Macro Menu
Sample Macros
LSM-FCS
5.10.4.3
Carl Zeiss
Bleach macro
This macro permits the specimen to be excited with the laser as required along a line created in the
scanning image.
• Click on the Bleach button in the Macro subordinate toolbar.
–
An XY scanning image of the specimen is recorded and displayed. The Spot Scan window is
shown on the screen.
• Click on the Select Excitation Line button.
• Create a free-hand line (spline) in the scanning image over the area to be excited by clicking and
holding down the mouse button.
• Then determine the duration of the excitation by moving the Exposure Time slider.
• Click on the Excite button to trigger the excitation procedure.
• A click on Exit will end the macro.
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5-233
Carl Zeiss
OPERATION IN EXPERT MODE
Macro Menu
Sample Macros
5.10.4.4
LSM-FCS
Multiple Time Series macro, Rev. 3
The Multi Time Series program is designed to
provide flexible programming of automated time
dependent experiments.
–
A pre-program time delay
–
A pre-bleaching functionality
–
An autofocus functionality
–
A number of different, single time series (called
a block) either at the same position of a sample
or at different positions (if a motorized stage is
available)
–
A time series of a tile scan
–
A time series of a tiled Z stack
The basic programming unit is a single Time Series
Block in line, frame or Z-stack mode In each block
user can define configuration for the data
acquisition (single or multi-track), the number of
images and the time interval/delay between
images.
User can activate an optional autofocus function
before each block, pre-program time interval
before each block (from the beginning of the
previous block to the beginning of the current
block), and/or execute bleach track with arbitrarily
specified bleach ROI's, laser line(s) and power.
Fig. 5-219
5-234
Multiple Time Series main dialog
box
The autofocus function can be executed using
specified single-track configuration, or with the
configuration used for imaging (using the first
channel of the first track).
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OPERATION IN EXPERT MODE
Macro Menu
Sample Macros
LSM-FCS
Carl Zeiss
One can define the z-offset, the distance in the z direction from the position where the autofocus finds
the reference feature in focus (position of maximum intensity - e.g. position of the cover slip reflection in
the reflected light configuration) to the position, which is moved into focus plane when the image
acquisition begins (e.g. into the tissue).
The image acquisition is done with the configuration specified for the given block.
Autofocus search parameters: Z-offset as well as the Z-range (the distance in the z direction over which
the autofocus function searches for the plane of maximum intensity) can be set independently for
different stage locations (on the systems with the motorized stage).
5.10.4.5
StitchArt macro
The StitchArt software option for LSM 510 MAT and LSM 5 PASCAL MAT permits the automated 3D
measurement of large samples:
- Highly resolved topographies of 10 times larger scan field
- Extra large profiles of more than 10 times of length of scan field
Example:
9 x 9 stacks
Single scan field size
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3.6 mm x 3.6 mm
Epiplan Neofluar 20x/0.5
5-235
OPERATION IN EXPERT MODE
Macro Menu
Sample Macros
Carl Zeiss
(1)
LSM-FCS
Multiple Profile Mode
Maximum format:
Variable overlap:
Scan Speed:
Scan time:
Height difference DZ:
Total height Z:
Auto alignment:
16384 pixels x 2048 slices
10 ... 50 % of single profile length
8 (fixed)
20 ... 210 seconds (depending on Z)
0.1 ... 0.6 µm [in steps of 0.1]
50 µm ... 1 mm
Cross-correlation in X, Y & Z
Maximum profile lengths:
Objective:
5x
10x
20x
50x
100x
Single X Profile (0.7):
2.6 mm
1.3 mm
650 µm
260 µm
130 µm
Multiple Profile (1.0):
30.0 mm
15.0 mm
7.5 mm
3.0 mm
1.5 mm
(2)
Multiple Stack mode
Single stack format (X):
Number of stacks:
Variable overlap:
Scan Speed:
Scan time:
Height step DZ:
Total height Z:
Auto alignment:
4x4 ... 512x512 pixels
1x1 ... 16x16
10 ... 50 % of single image size
5 ... 8
1 minute ... 10 hours
0.5 ... 100 microns
4 mm
Cross-correlation in X & Y
Maximum stack sizes:
Objective:
5x
10x
20x
50x
100x
Single
XYZ 2.6 mm
Stack (0.7):
1.3 mm
650 µm
260 µm
130 µm
Multiple
(1.0):
13.3 mm
6.7 mm
2.6 mm
1.3 mm
Stack 26.7 mm
Adjustment functionality:
Find Focus
Find Gain
Adjust scan mirrors:
Adjust spherical objective error:
Autofocus by fast Z line
Auto Brightness & Contrast
To XY direction of MOT stage
On a plane mirror
Macro VBA programming is described in chapter 6.
5-236
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OPERATION IN EXPERT MODE
Options Menu
Export RM Function
LSM-FCS
5.11
Carl Zeiss
Options Menu
The Options menu permits performance of the following functions:
–
User-Defined Examination Methods of the Expert Mode are made available to the Routine Mode.
–
Deletion of Routine Mode examination methods.
–
Display of a current list of dyes with preferred wavelengths for the scanning procedure.
–
Display / modification of the user-accessible program Settings of the LSM-FCS software.
• In the Main menu toolbar, click on Options.
–
This opens another, subordinate toolbar in the Main menu.
Options subordinate toolbar
Fig. 5-220
Options menu
5.11.1
Export RM Function
This function permits User-defined Examination
Methods created in the Expert Mode to be
made available to the Routine Mode.
• Load a Frame, Stack or Time Series image
which
was
scanned
using
optimized
parameters.
It is also possible to export the
examination method of a currently
displayed image which has just been
scanned.
Fig. 5-221
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Image Display window
5-237
OPERATION IN EXPERT MODE
Options Menu
Load RM Function
Carl Zeiss
LSM-FCS
• Click on the Options button of the Main menu
and then on Export RM (see Fig. 5-220).
–
The Export to Routine Mode window
appears.
• Enter any name for the method to be taken
over.
• If required, enter a brief description and
additional comments under Description and
Notes.
Fig. 5-222
Export to Routine Mode window
• Click on Ok. (Cancel allows you to cancel the
procedure).
–
The method used for image acquisition is
taken over in the Routine Mode.
The exported User-Defined Examination Method must be activated before it can be used in
the Routine Mode (see Routine Mode, chapter 5).
If you want to change from the Expert Mode to the Routine Mode and vice versa, close all the
windows first.
It's not possible to export ratio channels and bleach parameters. All methods will be loaded in
the Routine Mode with Zoom 1, Rotation 0, Offset 0 and without defined ROIs.
5.11.2
Load RM Function
The Load RM (Routine Mode) function permits the deletion of Standard Examination Methods and
User-defined Examination Methods from the database of the Routine Mode.
• Click on the Options button of the Main menu and then on Load RM (see Fig. 5-220).
–
The Routine Mode Database is opened.
The Routine Mode database is identical to the database of the Expert Mode, though not all the functions
are available. The listed methods cannot be loaded as an image.
• To delete an Examination Method of the Routine Mode, select the method from the Form, Gallery
or Table display of the Routine Mode Database.
• Then click on the Delete button.
–
5-238
The selected method is deleted from the database and is no longer available in the Routine Mode.
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OPERATION IN EXPERT MODE
Options Menu
Dye DB Function
LSM-FCS
5.11.3
Carl Zeiss
Dye DB Function
The Dye DB function is for information only and permits access to the database contained in the system,
including a list of suitable dyes for fluorescence microscopy.
The database contains a comparison of tables of dyes, optimum excitation wavelengths and maxima of
emission wavelengths.
• Click on the Dye DB button in the Options subordinate toolbar.
–
The Dye database will be opened and displayed on the screen.
Fig. 5-223
Dye database window
• Click on the Close button to exit the Dye database.
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5-239
OPERATION IN EXPERT MODE
Options Menu
Spectra DB Function
Carl Zeiss
5.11.4
LSM-FCS
Spectra DB Function
Spectrums created with the Lambda Mode can be stored in the Spectra Database.
• Click on the Spectra DB button in the Options subordinate toolbar.
–
The Spectra Database will be opened and displayed on the screen.
Fig. 5-224
Spectra Database window
• Click on the Close button to exit the Spectra Database.
5-240
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OPERATION IN EXPERT MODE
Options Menu
LSM Settings Function
LSM-FCS
5.11.5
Carl Zeiss
LSM Settings Function
The Settings function permits the individual setting and matching of software settings with regard to
the following points:
–
–
–
–
–
–
–
–
Autosave
Database General
Database Table Viewer
Database Gallery Viewer
Import / Export
Scan Information
Image Status Display
Print Status Display
5.11.5.1
–
–
–
–
–
–
–
–
Recording / Reuse
Timeseries
Scan Mean of ROIs
Temporary Files
Program Start
Shutdown
Image Display Toolbars
Save
Open / Close the Settings for user : ... window
• Click on the Settings button in the Options subordinate toolbar of the Main menu.
–
This opens the Settings for user : ... window.
• Click on the OK button to quit the window. The last settings will be taken over. Cancel enables you
to cancel the procedure, with any changes you made not being taken over.
Fig. 5-225
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Settings for user : ... window
5-241
OPERATION IN EXPERT MODE
Options Menu
LSM Settings Function
Carl Zeiss
5.11.5.2
LSM-FCS
Autosave
This tab permits activation or deactivation of
automatic data storage. Only one option can be
selected at a time.
(1)
Fig. 5-226
(2)
Autosave tab
No Autosave
On activation of this option, the Autosave
function is switched off. Save and Save As give
the same dialogues.
Use LSM image database and auto increment image name
On activation of this option, newly recorded or modified images are stored by Save automatically and
assigned to the name or defined in this function. The image name is automatically created using a base
name and a serial number. For this, a base name must be entered in the Base image name input box,
and a starting value for the serial number in the Counter value input box. The Database selection box
permits selection of the directory in which the data will be stored.
(3)
Export to Attofluor format
On activation of this option, newly recorded or modified images are stored by Save in the Attofluor
format. The displayed Experimental directory selection box permits selection of the directory in which
the data will be stored.
(4)
Export to Metafluor format
On activation of this option, newly recorded or modified images are stored by Save in a subdirectory in
the MetaFluor format. An existing higher layer of folders must be selected for the subdirectory from the
Base directory selection box. Furthermore, a name for the subdirectory must be entered in the
Subdirectory base name input box. The starting value for the images then created, to which a
continuous number is automatically assigned, is set in the Subdirectory counter input box.
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OPERATION IN EXPERT MODE
Options Menu
LSM Settings Function
LSM-FCS
5.11.5.3
Carl Zeiss
Database General
This tab permits the basic starting settings for the
use of databases.
(1)
Start with "Form"
On opening of a database, the Form option is
displayed first.
(2)
Fig. 5-227
Database General tab
Start with "List"
On opening of a database, the List option is displayed first.
(3)
Start with "Gallery"
On opening of a database, the Gallery option is displayed first.
(4)
Show first recordset at opening of database
On opening of a database, the first recordset is displayed.
(5)
Show first recordset at opening of database
On opening of a database, the middle recordset is displayed.
(6)
Show first recordset at opening of database
On opening of a database, the last recordset is displayed.
(7)
Use separate path for "Create" and "Open"
This option permits the path to be changed when the Open or New database function is used.
(a)
Save most recently used path at exit and reuse at next program start
On activation of this option, the path setting last used is automatically selected again in the Open
Database or Create New Database window.
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5-243
Carl Zeiss
(b)
OPERATION IN EXPERT MODE
Options Menu
LSM Settings Function
LSM-FCS
Use the following path at program start
On activation of this option, the path for the Open Database or Create New Database window can
be entered directly in the relevant selection box, or selected by clicking on the ... button in the Choose
Directory window. This path will then always be set when a database is opened or created.
Clicking on the User default path button firmly sets the C:\users\default\ path.
5.11.5.4
Database Table Viewer
The Database Table Viewer tab permits the
definition of the columns for the table display of a
database. This only requires the relevant check box
to be activated with a click of the mouse.
Fig. 5-228
Database Table Viewer tab
On activation of the Automatic column width
calculation option, the column width is calculated
automatically.
On activation of Save and use interactive
column width setting, the column width in the
database can be matched as required. The
individual setting will be retained when the
database is closed.
5.11.5.5
Database Gallery Viewer
The Database Gallery Viewer tab permits the
image information to be displayed in the Gallery
mode of the database to be activated by clicking
on the relevant check box.
Fig. 5-229
5-244
Database Gallery Viewer tab
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OPERATION IN EXPERT MODE
Options Menu
LSM Settings Function
LSM-FCS
5.11.5.6
Carl Zeiss
Import / Export
Use separate path for "Import" or "Export"
This option permits the change of the path setting
for use of the Import or Export function (File
menu).
(1)
Save most recently used path at exit and
reuse at next program start
Fig. 5-230
Import / Export tab
On activation of this option, the path used last is
automatically selected again in the Import Images
or Export Images and Data window.
(2)
Use the following path at program start
On activation of this option, the path for the Import Images or Export Images and Data window can
be entered directly in the relevant selection box, or selected by clicking on the ... button in the Choose
Directory window. This path will then always be set when the Import / Export function is used.
Clicking on the User default path button firmly sets the C:\users\default\ path.
5.11.5.7
Scan Information
This tab permits the setting of which scan
information shall be displayed in the Scan
Information window (see Window pull-down
menu of the Main menu, page 5-272f).
Activation / deactivation of the information to be
displayed is performed with a click of the mouse.
Fig. 5-231
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Scan Information tab
5-245
OPERATION IN EXPERT MODE
Options Menu
LSM Settings Function
Carl Zeiss
5.11.5.8
LSM-FCS
Image Status Display
This tab permits selection of which image
information is displayed on opening of an image
or on activation of the Info button of the Image
Display window. Furthermore, you can determine
which information will be displayed in the Image
status bar.
Fig. 5-232
Image Status Display tab
On activation of the Show status display upon
opening of a new image display check box, the
image information is automatically displayed
immediately after opening of the Image Display
window (Info button is activated).
5.11.5.9
Print Status Display
This tab permits selection of which information is
displayed in print preview.
On activation of the Print Status Information
check box, the status information will be printed.
Fig. 5-233
Print Status Display tab
5.11.5.10 Recording / Reuse
The parameters to be taken into consideration for
the use or load of a recording configuration are set
in this tab.
As an option, you can also determine whether the
objective setting shall be taken over when the
Reuse function is used.
Fig. 5-234
5-246
Recording / Reuse tab
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OPERATION IN EXPERT MODE
Options Menu
LSM Settings Function
LSM-FCS
Carl Zeiss
5.11.5.11 Time series
In the Timeseries tab, you can determine whether
the time for the recording of a time series is set as
Time Delay or as Time Interval.
Time Delay is the interval between the end of one
scan process and the beginning of the next.
Time Interval is the interval between the
beginning of one scan process and the beginning
of the next.
Fig. 5-235
Timeseries tab
Fig. 5-236
Scan Mean of ROIs tab
You can select the unit for Mean of ROIs
diagrams.
5.11.5.12 Scan Mean of ROIs
The Mean of ROIs tab permits the presetting of
the Image Display window for the optional
MeanROI function (time series) to be changed
with regard to scaling and display mode of the
intensity time diagrams.
(1)
Diagram Scaling
The following settings are possible by activating
one of the option buttons:
–
Automatic diagram scaling
–
Fixed time range for diagram time scale; input of the time range in seconds via input box
–
Fixed number of cycles for diagram time scale; input of the time range in number of cycles via
input box
(2)
Initial diagram types
The following settings are possible by activating the relevant option button:
–
One diagram
–
Channels diagram
–
ROIs diagram
On activation of the Black graphs
black (monochrome).
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check box, the intensity profiles in the diagram are displayed in
5-247
OPERATION IN EXPERT MODE
Options Menu
LSM Settings Function
Carl Zeiss
(3)
LSM-FCS
Live image
If you activate the Also display the live image check box, the live image will be additionally displayed
in the Image Display window of the Mean of ROI function during the Mean of ROI scan.
On activation of the Also display the live image check box, two further options become available in
the Scan Mean of ROIs tab:
–
Scan the whole image check box
–
Save the whole time series check box
(a)
Scan the whole image
If you activate this check box, the complete live image will be scanned; only the defined ROIs will be
scanned if the check box is deactivated.
(b)
Save the whole time series
If you activate this check box, the complete Time Series will be scanned; only the Mean of ROI series will
be scanned if the check box is deactivated.
5.11.5.13 Temporary Files
The Temporary Files tab permits determination
of the directory in which temporary files are
stored.
(1)
Fig. 5-237
Temporary Files tab
Use "TEMP" environment variable
Temporary files are stored in the TEMP standard
directory of the computer's hard disk.
(2)
Use the following path
The directory for temporary files can be selected
by clicking on the ... button in the Choose
Directory window.
5-248
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OPERATION IN EXPERT MODE
Options Menu
LSM Settings Function
LSM-FCS
Carl Zeiss
5.11.5.14 Program Start
The Program Start tab permits selection of a
track configuration via the Startup configuration
selection box, which will be loaded automatically
when the Expert Mode is started.
check
On activation of the Don't show logo
box, the initial screen with the Zeiss logo will not
be displayed after the start of the LSM-FCS
software.
Fig. 5-238
Program Start tab
Fig. 5-239
Shutdown tab
Fig. 5-240
Image Display Toolbars tab
5.11.5.15 Shutdown
The Shutdown tab allows you to determine, by
activation of the Lasers off on Exit check box,
that the lasers are automatically switched off
when the LSM-FCS software is exited.
Allow lasers to cool for five minutes before
switching of the system.
5.11.5.16 Image Display Toolbars
The Image Display Toolbars tab enables you to
determine the window toolbars which shall be
automatically displayed when an Image Display
window is opened.
Furthermore, the color mode (color / mono), to
which the image display will switch when the
Color Palette function is opened / closed, can be
determined.
(1)
Display Windows Toolbars
On activation of the relevant check box, the following window toolbars are automatically displayed when
an Image Display window is opened: Channels, Zoom, Slice, Overlay.
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5-249
OPERATION IN EXPERT MODE
Options Menu
LSM Settings Function
Carl Zeiss
(2)
LSM-FCS
Color Palette
Switch to "mono" on activation of a palette
If this check box is activated, the Mono(chrome) image display mode is switched automatically when
a palette is selected in the Color Palette window.
Switch to "color" on deactivation of a palette
If this check box is activated, the Color image display mode is switched automatically when a palette
is deactivated in the Color Palette window.
5.11.5.17 Save
The Save tab permits the presetting for the
storage of scanned or processed images to be
changed.
Activation of one of the three option buttons
enables you to determine the database directories
to which stored images are assigned:
Fig. 5-241
–
Save tab
–
Image files to subdirectory of the database
–
Database and image files to the same
directory
At "Create Database" automatically create a subdirectory with the same name as the specified
database and create the database and image files in that subdirectory
If the Save prompt at closing modified windows check box is activated, you are automatically asked
on closing a changed Image Display window whether the image shall be stored.
If the Warning before overwrite existing recordsets check box is activated, this question is asked
automatically on storing an image under a new name if an image file with this name already exists in the
database.
If the Remember "Name", "Description" and "Notes" in the save dialog check box is activated, the
Name, Description and Notes text boxes of the Save Image and Parameter As window show the
text for the image last saved. You can edit the text boxes as required for the new image to be saved.
If the Remember "Name", "Description" and "Notes" in the save dialog check box is deactivated,
the three text boxes are blank when the Save Image and Parameter As window is opened.
5-250
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OPERATION IN EXPERT MODE
Options Menu
FCS Settings Function
LSM-FCS
5.11.6
Carl Zeiss
FCS Settings Function
The FCS Settings function permits the individual setting and matching of software settings for FCS
measurements with regard to the following points:
–
–
Open / Save
Raw Data
5.11.6.1
–
–
Measure
LSM+ConfoCor
Open / Close the Settings for user :
... window
• Click on the Settings button in the Options
subordinate toolbar of the Main menu.
–
This opens the Settings for user : ...
window.
• Click on the appropriate tab to change this
option.
• Click on the OK button to quit the window.
The last settings will be taken over. Cancel
enables you to cancel the procedure, with any
changes you made not being taken over.
5.11.6.2
Fig. 5-242
Settings for user : ... window
(Open / Save tab activated)
Open / Save tab
Ticking the Save prompt at closing modified windows option box will set the following procedure: if
the field is ticked and you exit a window in which some settings were changed a dialog will ask you to
save these changes.
Ticking the Calculate mean value for ConfoCor 1 files option box will give you a calculation of the
mean of opened ConfoCor 1 files in the last row. You can choose in the Average display box if you
would like to Average the correlation results (every measurement point of the single correlation
curves is averaged) or Average the fit results (values of parameters obtained from the single
correlation curves are averaged).
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5-251
OPERATION IN EXPERT MODE
Options Menu
FCS Settings Function
Carl Zeiss
5.11.6.3
LSM-FCS
Raw Data tab
When you tick the Save raw data during
measurement check box, the raw data (photon
trace) will be stored on disk. This option is used
when access to the raw data is required to analyze
the data in a different way than by calculating the
correlation functions.
The raw data file structure is described in
Appendix A.
Fig. 5-243
Raw Data tab
• In the Directory field, the directory will be set
where the data should be saved. When the
button is clicked, a WINDOWS NT directory selection dialog will open.
• In the Base File Name field, the base file name is set.
• The maximum number of files can be set (up to 100).
Each single measurement is stored in one raw data file. When the set limit is reached, the oldest
file will be overwritten and the data in this file are lost. It is the responsibility of the operator to
save the data.
5.11.6.4
Fig. 5-244
Measure tab
Measure tab
This option allows you to move the stage during
measurement. In this case the stage will be moved
forth and back during the entire measurement
time. This option is useful during rare event
detection when large, slowly moving objects in
extremely small concentrations have to be
registered. Since these aggregates are diffusing
very slowly without such movement the
measurement times would be prohibitively long. It
should be taken into account that, in this case, the
data stream is analyzed with the coincidence
analysis rather than by correlation analysis.
If the Move stage during measure check box is checked, the following settings are possible in the
dialog:
–
Travel distance of the stage in millimeters:
Only integer numbers of millimeters are possible.
–
Speed of the stage:
Only the three preset stage speeds 0.48 mm/s, 4.81 mm/s and 24.01 mm/s are possible.
–
Travel direction:
The stage can be moved along the x- or y-axis of the stage.
5-252
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OPERATION IN EXPERT MODE
Options Menu
FCS Collimator Settings Function
LSM-FCS
5.11.6.5
Carl Zeiss
LSM+ConfoCor tab
This function permits the lateral matching of the
ConfoCor 2 beam path with that of the LSM
through entry of the relevant offset values.
Determination of these values and their setting will
be performed by service staff during start-up of
the system.
5.11.7
Fig. 5-245
LSM+ConfoCor tab
Fig. 5-246
Collimator tab
FCS Collimator Settings Function
This function permits the setting of the collimator
for FCS measurements.
Under normal conditions, the collimator settings
should not be changed.
In some cases when the pinhole is close to the
limit of the pinhole range for a certain wavelength
it might be useful to alter the collimator settings
carefully.
This function should be used with
extreme care. An unsuitable collimator
setting can render the pinhole adjustment
impossible.
• To change the collimator setting, click the FCS Colli. button in the Options toolbar subordinate of
the Main menu (Fig. 5-220).
• The Collimator tab will appear.
• Now the new collimator settings can be entered either by drawing the slider, clicking on the arrows or
directly entering a new number.
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5-253
OPERATION IN EXPERT MODE
Maintain Menu
Scanner
Carl Zeiss
5.12
LSM-FCS
Maintain Menu
The Maintain menu contains additional modules to check and guarantee the interference-free operation
of all the software and hardware components of the LSM-FCS.
• In the Main menu toolbar, click on Maintain. This opens another subordinate toolbar in the Main
menu.
Maintain subordinate toolbar
Fig. 5-247
Maintain menu
5.12.1
Scanner
The Scanner function is used for scanner
calibration and involves a manipulation of the
LSM-FCS hardware.
Fig. 5-248
Scanner Calibration window
5.12.1.1
Calibration with Speed 1-10 (electr., unidirectional / bidirectional) and Speed 11-13
(electr., only unidirectional)
(1)
Preliminary notes
The electrical calibration has to be performed every 2-3 months. For electrical calibration no laser
scanning is performed and for that reason no calibration sample is needed.
(2)
Calibration conditions
Before the calibration process can be started, the system has to be in operation for at least one hour.
5-254
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OPERATION IN EXPERT MODE
Maintain Menu
Scanner
LSM-FCS
(3)
Carl Zeiss
Calibration procedure
• Click on the Scanner button in the Maintain subordinate toolbar of the Main menu.
–
This opens the Scanner Calibration window.
• Click on the Speed 1-10 (electr.) or Speed 11-13 (electr.) button respectively.
• For electrical calibration of speeds 11-13 the appropriate zoom factors have to be applied (11 : zoom
≥ 2.5, 12 : zoom ≥ 3.6, 13 : zoom ≥ 5.6).
• Activate the Display Graphics check box enables to check the progress of the calibration process on
the Progress Status bar.
–
During successful calibration process, the status button is of green color, in case of an error it
switches to red. The progress of the calibration process is indicated by the Progress Status bar.
The calibration process is completed, when the indicator button is grayed.
• Click on the Calibrate button to start the automatic scanner calibration.
• Confirm warning information with OK.
• Click on the Close button to close the Scanner calibration window.
The More function is for servicing purposes only and can only be performed by authorized personnel. Its
access is therefore password-protected.
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5-255
OPERATION IN EXPERT MODE
Maintain Menu
Scanner
Carl Zeiss
5.12.1.2
(1)
LSM-FCS
Calibration with Speed 11-13 (optical, bidirectional)
Preliminary notes
The optical calibration of scan speeds 11-13 (bidirectional) can only be performed at systems with
complete hardware level of Release 2.8.
The optical calibration procedure has to be repeated every two weeks in normal use and after long delay
times of the system.
The minimum duration of the calibration process is 10 minutes. However, it can last up to a maximum
time of 40 minutes depending on the performance of the scanner in use and the actual tuning
conditions.
If the optical calibration is successfully finished, there is no need to start the electrical calibration for
unidirectional scanning of speed 11-13 (in opposite, the electrical calibration would overwrite the much
more accurate values of the optical calibration procedure).
With speed 11, 12 and 13, scanning is performed at scanner frequencies of 868, 1042 and 1306 Hz,
respectively.
In bidirectional scanning, the total line frequencies are 1736, 2084 and 2612 Hz, the image recording
times for 512 lines are 0.29, 0.25 and 0.20 seconds.
(2)
Calibration conditions
• The microscope stand has to be placed totally vibration-free.
– Note, that even power units on the granite plate or inappropriate situated cables can cause
vibrations.
• Before the calibration process is started, the system has to be in operation for at least one hour
(better: two hours).
– Otherwise, the tuning results will be incorrect and the forward / backward image contents do not
match with each other.
• The longest available laser wavelength of the system has to be applied.
• A 80/20 neutral beamsplitter and a None position in the emission filter wheel of either Channel 1 or
2 has to be used in the Scan Configuration window.
• A Plan-Neofluar 10x/0.3 or Epiplan Neofluar 10x/0.3 objective lens has to be used.
• A special sample (see Fig. 5-249) with two identical but 90 degrees rotated gratings (one for each
scan direction) has to be used as a calibration standard.
• The pinhole has to be completely opened.
• A dynamic range of 8 bit has to be used.
(3)
Calibration procedure
• It is advantageous to perform the electrical calibration before starting the optical calibration process.
For a first scan of the calibration standard start with scan speed 12 and zoom 3.6 (unidirectional).
• Focus on the calibration standard and adapt the dynamic range of the detector on the sample.
– Optimize Detector Gain and Ampl. Offset values in the Channels sheet of the Scan Control
window by means of the Range Indicator mode.
5-256
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OPERATION IN EXPERT MODE
Maintain Menu
Scanner
LSM-FCS
Fig. 5-249
Carl Zeiss
Calibration standard
• The calibration standard has to be positioned as indicated in Fig. 5-250.
• Before starting the calibration procedure, change to the bidirectional scan mode in the Scan Control
window.
• Scan Corr. X and Scan Corr. Y in the Mode sheet of the Scan Control window has to be set to
zero.
• Click on the Scanner button in the Maintain subordinate toolbar of the Main menu.
– This opens the Scanner Calibration window.
Fig. 5-250
Correct orientation of calibration sample for X scanner calibration
• Click on the Speed 11-13 (optical) button.
• Activate the Display Graphics check box enables the graphical display of the calibration process.
• Click on the More (Less) button to display the Speed Selection menu.
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5-257
Carl Zeiss
OPERATION IN EXPERT MODE
Maintain Menu
Scanner
LSM-FCS
• With activated Auto Calibration box, all
speeds (index) and axis are calibrated one after
another automatically.
–
Goal of the calibration procedure is the
achievement of a minimum of the forwardbackward-difference (blue line in the
graphical display) and the best possible
linearity (black line in graphical display
monitors the linearity deviation). Both lines
(blue and black) should be as straight as
possible and as close as possible to the Zero
level (red line in graphical display). Green
line represents the driver voltage.
• Click on the Calibrate button to start the
scanner calibration procedure.
• Confirm warning information with OK.
–
Fig. 5-251
Scanner Calibration window
The procedure starts with Speed 11 and the
X-scanner. When the Auto calibration for
Speed 11 is finished successfully the
procedure continuos with Speed 12 (higher
acoustic frequency) and larger zoom.
• If necessary slightly reposition the sample and
click on the Next button.
• Do not focus or change scan parameters during
calibration procedure!
• After calibration of X-axis the orientation of the
calibration grid in the calibration window
changes from horizontal to vertical orientation.
The second grid of the calibration sample has to
be selected and again positioned as indicated in
Fig. 5-250.
• Click on the Next button and continue with Yscanner calibration.
• If the calibration for all speeds and scanners has
finished successfully, quit the scanner
calibration window by pressing the Close
button.
• If the Auto Calibration procedure can not be
performed successfully after several tries,
calibrate the scanner manually.
Fig. 5-252
5-258
Scanner Calibration window
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OPERATION IN EXPERT MODE
Maintain Menu
Scanner
LSM-FCS
Carl Zeiss
• If the Auto Calibration box is disabled, the speed index and the scanner (X-axis or Y-axis) has to be
chosen manually. The calibration cycle only contains one speed index and one scanner. For calibration
of all speeds (11-13) and both scanners, the manual calibration procedure has to be repeated several
times with changing axis and index parameters (Start with scanner X and speed 11, continue with
scanner X and speed 12, then X and speed 13, ...). The position of the calibration standard has to be
controlled at each new speed index. A repositioning of the calibration sample is required after scanner
change (for X-scanner calibration horizontal grid, for Y-scanner vertical grid).
(4)
Important notes and hints
The tuning procedure runs automatically to a large extent without any problems. However, several errors
can occur. That’s why it is strongly recommended to observe the complete calibration process.
If a status error message occurs or the calibration procedure is not finished properly, this can have the
following reasons:
No optimal positioning of the calibration standard.
Indication: One end of blue and black line jumps a bit forward and backward because software
recognizes sometimes the outer line of the grating and sometimes not.
• Stop the calibration procedure.
• Check the focus of the calibration sample.
• Check if the yellow horizontal line crosses the scale pattern of the calibration standard properly.
• (If necessary) Shift the calibration standard by half a scale unit (No scale tick but a gap has to be
situated directly on the edge of the image).
• Restart the calibration procedure.
No optimal setting of Detector gain and Amplifier Offset.
Indication: The forward-backward-difference shows a lot of peaks and changes significantly from
image to image.
• Stop the calibration procedure.
• Calibrate speed 12 (unidirectional) electrically.
• Optimize gain & offset: Ticks of the grid have to have intensity values of 250 ... 255 (just before
red color in Range Indicator palette). Minimum intensity values have to be between 1 ... 5 (no
blue parts occur in the image by applying Range Indicator palette).
• Restart the optical calibration procedure.
Sliders of Scan Corr. X and Scan Corr. Y in the Mode sheet of the Scan Control window were
not set to zero.
Indication: Calibration process does not converge.
• Stop the calibration procedure.
• Set both values to zero.
• Restart the calibration procedure.
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5-259
Carl Zeiss
OPERATION IN EXPERT MODE
Maintain Menu
Scanner
LSM-FCS
Non-regularities of the scanner feedback.
Indication: The ticks on the outer sides of the grid vary about more than 1 tick width between
consecutive images (in the middle of the calibration process, the linearity is optimized and
the problem starts to occur).
• Stop the calibration procedure.
• Call the LSM service hotline.
If optical calibration comes not to a successful end, please contact your service hotline.
Scanner calibration in LSM 5 Software,
Release 3.0
In LSM 5 Software, Release 3.0, the do full
calibration (Measure spectral response)
checkbox must be set at the first calibration
procedure Speed 11-13 (optical).
Fig. 5-253
5-260
Scanner Calibration window
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OPERATION IN EXPERT MODE
Maintain Menu
Objective
LSM-FCS
5.12.2
Carl Zeiss
Objective
This function permits changed objectives to be
activated and the parfocality to be set without
having to exit the software.
5.12.2.1
Objective change
• Change the required objective in the nosepiece.
• Click on the Objective button in the Maintain
subordinate toolbar of the Main menu.
–
The Objective Control window appears on
the screen. The Objective button is
activated in accordance with the presetting,
and the Objective panel is displayed in the
Objective Control window.
• Click on the graphical button of the relevant
nosepiece mount (Position).
–
Fig. 5-254
Objective Control window
Fig. 5-255
Objective Control window
The Change Objective window appears.
All available objectives are listed in the Potential
Objectives directory of the Change Objective
window.
• Select the new objective by double clicking
from the list in the Potential Objectives
directory.
• Click on Close to exit the Change Objective
window.
(1)
Add Objective
This function permits new objectives to be added
to the database.
For this, proceed as follows:
• Click on the Add Objective button on the
Change Objective window.
–
The Create new Objective window is
opened.
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5-261
OPERATION IN EXPERT MODE
Maintain Menu
Objective
Carl Zeiss
LSM-FCS
• Enter the data of the new objective in the
Create new Objective window, then click on
the Apply button.
The new objective is stored in the database and
included in the Change Objectives window. You
can now activate the new objective as a favorite
objective using the procedure described above.
Fig. 5-256
(2)
If you have activated the Non Zeiss
check box, objectives from other
manufacturers can also be included in the
database.
Create new Objective window
Remove Objective
You can only remove objectives in the Favorite Objectives and the User Defined Objectives
directories.
• To remove an objective from the database, select it with a click of the mouse in the Change
Objective panel and then click on Remove Objective. The new objective appears in the User
Defined Objectives directory.
• Click on Close to close the Create new Objective window.
5.12.2.2
Focus speed change
• Change the required objective in the nosepiece.
• Click on the Objective button in the Maintain
subordinate toolbar of the main menu.
Fig. 5-257
5-262
–
The Objective Control window appears on
the screen. The Focus Speed has to be
activated in the Objective Control window.
–
The focusing speed of the relevant objective
can be selected by using either the slider or
the input box in 40 steps.
Focus Speed window
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OPERATION IN EXPERT MODE
Maintain Menu
Objective
LSM-FCS
5.12.2.3
Carl Zeiss
Parfocality Correction
The parfocal setting is performed via screen
dialogs in successive panels.
• Click on the Parfocal Correction button.
–
The Parfocal Adjustment panel appears.
• Start the setting with the objective of the
highest
magnification
factor
(reference
objective). Proceed in accordance with the
displayed instructions.
• Click on Start.
–
The next dialog is displayed in the Parfocal
Adjustment panel.
• Focus on your slide object.
• Click on the Next step button.
• Perform these steps for each objective.
Fig. 5-258
Objective Control window
Fig. 5-259
Objective Control window
• Click on the Close button to exit the Objective
Control window and accept the settings.
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5-263
Carl Zeiss
OPERATION IN EXPERT MODE
Maintain Menu
Pinhole Adjustment
5.12.3
LSM-FCS
Pinhole Adjustment
In the Pinhole and Collimator window, the
pinholes and collimators are optimally aligned and
adjusted to the used beam path (configuration).
The position of the pinhole (X-Y-Z-coordinates) in
relation to the detector makes a major
contribution to image optimization.
In all existing standard configurations, the pinholes
have already been adjusted at the factory. These
settings are taken over for active operation when a
standard configuration is loaded.
If you want to create a setting that differs from
the standard configurations, adjust the pinhole as
follows.
5.12.3.1
Open / Close the Pinhole &
Collimator Control window
• Click on the Pinhole button in the Maintain
subordinate toolbar of the Main menu.
–
Fig. 5-260
5-264
Pinhole & Collimator Control
window
This opens the Pinhole & Collimator
Control window.
• Click on the Close button to quit the window.
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OPERATION IN EXPERT MODE
Maintain Menu
Pinhole Adjustment
LSM-FCS
5.12.3.2
(1)
Carl Zeiss
Function description
Pinhole panel
No further software function can be activated and
executed during pinhole adjustment.
Pinhole /
Description field:
Diameter;
Pos. X; Y; Z slider:
Selection of pinholes (PH1
to PH4) to be adjusted via
the Pinhole selection box,
display of the relevant
channel in the Description
field.
Setting of diameter, X-, Yand Z-position of the
pinhole in relation to the
beam path (Z-position can
be set only for PH1) using
the slider or arrow buttons,
status display for setting
procedure: green for ready
and red for busy.
Fig. 5-261
Pinhole panel
Store current
Position button:
Storage of the current pinhole setting.
Move to stored
Position button:
Pinhole setting is reset to the position last stored.
Adjust
Automatically
button:
Automatic pinhole adjustment.
Fast Adjust
mode check box:
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If this check box is activated, the pinhole adjustment is only performed in a limited
area. Used for readjustment.
5-265
OPERATION IN EXPERT MODE
Maintain Menu
Pinhole Adjustment
Carl Zeiss
(2)
Collimator panel
Collimator
Description field:
Positions field:
Fig. 5-262
Collimator panel
Store Current
Position button:
Move To Stored
Position button:
Move to Optimal
Position button:
5-266
LSM-FCS
Selection of the collimator
(IR / VIS or UV / VIS) to be
adjusted via Collimator
selection box, display of
selected collimator in the
Description field.
Setting of collimator position using the slider or arrow buttons; the display to
the right of the slider indicates the current position,
status display for setting
procedure: green for ready
and red for busy.
Stores the current
collimator position.
the
Sets the collimator to the
stored value.
Starts
the
automatic
collimator
adjustment.
Available for most common
objectives.
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OPERATION IN EXPERT MODE
Maintain Menu
Pinhole Adjustment
LSM-FCS
5.12.3.3
Carl Zeiss
Pinhole and collimator adjustment
Adjustment of the LSM-FCS pinholes can be performed manually or automatically.
If several channels are used to produce the image, all the used pinholes must be adjusted separately.
(1)
Manual pinhole and collimator adjustment
The position of the pinhole relative to the detector in terms of X-Y-Z coordinates contributes substantially
to image optimization.
Requirements to make pinhole position changes visible immediately:
–
The image must be scanned by the continuous scan method.
–
Select a fast scanning speed.
–
Measurement with Average Number 1 only (no averaging of several measurements).
–
On the Channel Settings panel (click on Channels button in the Scan Control window), select
the pinhole diameter so as to have the best possible image contrast.
• Click on the Pinhole button in the Maintain
subordinate toolbar.
• Select the pinhole to be adjusted from the
Description list box.
• Use the Diameter slider to set the smallest
possible size which produces a good, highcontrast image.
–
This setting changes the pinhole diameter.
–
The Z Slice display box simultaneously
displays the depth resolution corresponding
to the pinhole diameter.
Image optimization can be effected with
the Range Indicator or in the Line-Scan
mode.
Fig. 5-263
Pinhole panel
• Optimize the pinhole position in X, Y and Z (Z only for PH1) relative to the PMT using the X, Y and Z
sliders to maximum image brightness.
• Click on the Save Current Position button to save the pinhole adjustment.
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5-267
Carl Zeiss
OPERATION IN EXPERT MODE
Maintain Menu
Pinhole Adjustment
LSM-FCS
• Removing the Current Positions slider in the Collimator panel allows the collimator to be adjusted
to maximum image brightness. Optimum collimator adjustment obtained in this way can be stored by
clicking on the Save Current Position button.
• Click on the Stop button to stop the continuous scan.
Please do not make any program manipulations while the automatic pinhole adjustment is
running (status display is red - busy).
(2)
Automatic pinhole and collimator adjustment
The automatic adjustment allows the LSM-FCS pinholes to be used with any combination of beam
splitters.
• Click on the Adjust Automatically button.
–
The Requirements for
window will then appear.
Adjustment
• Meet the requirements listed in the
Requirements for Adjustment window and
press the OK button.
Fig. 5-264
Requirements for Adjustment
window
–
Pinhole
adjustment
will
then
run
automatically. The adjusting procedure takes
approx. 3 min.
–
The
determined
data
are
stored
automatically and will be available for all
further examinations using the same
configuration.
• Click on the Move to Preadjust button in the
Collimator panel. Optimum positioning of the
collimator will be performed. The Default
button enables the collimator to be set back to
the factory-adjustment.
• Activate the Fast Adjust Mode check box for a
faster readjustment.
5-268
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OPERATION IN EXPERT MODE
Maintain Menu
DSP (Digital Signal Processor)
LSM-FCS
Carl Zeiss
A change of the pinhole diameter made manually in the Pinhole panel will not be activated in
the Scan Control window. Therefore, changes must always be made in the Channel Settings
panel of the Scan Control window.
A filter change in Autoadjust is not displayed in the Config. Control window.
Configuration 1 is equipped in such a way that pinhole adjustment for channel 1 can only be
made with λ = 488 nm, NFT 545, NFT 610 or NFT 570.
Please remember that the Z-coordinate for channel 1 is not optimized during the automatic
pinhole adjustment. Subsequent optimization can be performed via the Move to Preadjust
button in the Collimator panel of the Pinhole & Collimator Control window.
Please do not make any program manipulations while the automatic pinhole adjustment is
running (status display is red - busy).
The optimum setting of the collimator must be performed separately for each track via the Move to
Preadjust button in the Pinhole & Collimator Control window. If several tracks are activated
(Recording), an average value of the positions valid for the various tracks will be set on pressing the
Preadjust button. When all the tracks have been defined and are active (only the ticked tracks will be
included in the calculation), press the Move to Preadjust button.
5.12.4
DSP (Digital Signal Processor)
The DSP function is used to display the current
performance of the system processor for checking
purposes.
Fig. 5-265
DSP Performance window
• Click on the DSP button in the Maintain
subordinate toolbar of the Main menu.
–
This opens the DSP Performance window.
• Click on the
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button to close the DSP Performance window.
12/02
5-269
Carl Zeiss
OPERATION IN EXPERT MODE
Maintain Menu
Set Find
5.12.5
LSM-FCS
Set Find
This function permits the preset parameters of the
Find function (see Scan Control, page 5-72) ) to
be matched individually.
• Click on the Set Find button in the Maintain
subordinate toolbar of the Main menu.
–
The Auto B&C Control window appears on
the screen.
• Change the settings for the Upper Threshold
of Data Depth and Gain Correction using the
relevant sliders.
The settings can be made individually for each
detection channel or for all channels together.
Fig. 5-266
Auto B&C Control window
For experiments with increasing of fluorescence
over the time it's necessary to reduce the Upper
Threshold of Data Depth for the Find function.
Each slider should be used separately.
• Click on the C button to set the value for the Gain Correction to 100 %.
• If required, activate the Set Amplifier Gain to 1.0 check box.
• Click on the Find button to start sample scanning with the current settings.
• Clicking on Default enables you to activate the default settings again.
• If required, activate Auto B&C for all tracks checkbox for Find function.
5.12.6
Spline
This function permits calibration of the Scanner
position signals. This is required for the use of
spline curves in the Line scanning mode (see
section 5.5.4, (3) Line, page 5-101f).
Fig. 5-267
5-270
Calibrate Spline Scan window
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OPERATION IN EXPERT MODE
Maintain Menu
DSP Trace
LSM-FCS
5.12.7
Carl Zeiss
DSP Trace
The DSP Trace function is for servicing purposes only and may only be performed by authorized
personnel. Its access is therefore password-protected.
5.12.8
Parameter
The Parameter function is for servicing purposes only and may only be performed by authorized
personnel. Its access is therefore password-protected.
5.12.9
Reboot
The Reboot function is for servicing purposes only and may only be performed by authorized personnel.
Its access is therefore password-protected.
5.12.10
HW/Admin
The HW/Admin function is for servicing purposes and may only be used by authorized service
personnel. Its access is therefore password-protected.
5.12.11
Test Grid
The TestGrid function is for servicing purposes only and may only be performed by authorized
personnel. Its access is therefore password-protected.
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5-271
OPERATION IN EXPERT MODE
Window Menu
Full Screen
Carl Zeiss
5.13
LSM-FCS
Window Menu
The Window menu includes the additional functions Full Screen, Close All Image Windows, Toolbar
and Scan Information which are not available from a toolbar.
Fig. 5-268
5.13.1
Window pull-down-menu
Full Screen
This function shows the active Image Display window in full screen size.
• Activate the image to be shown in full size by clicking on the image content.
• Click on the term Window in the menu bar of the Main menu.
–
The Window menu (pull-down) will be opened.
• Click on the Full Screen line.
–
The image will be displayed in full screen size.
• Click in the image to show it again as an Image Display window in normal size.
5.13.2
Close All Image Display Windows
This function closes all the opened Image Display windows.
• Open the Window menu.
• Click on the Close All Image Windows line.
–
All the opened Image Display windows will be closed.
In the Options menu in the function Settings in Save tab at position Save prompt at closing
modified windows it can be determined whether a prompt is shown on Closing of All Image
Display Windows or not.
5-272
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OPERATION IN EXPERT MODE
Window Menu
Toolbar
LSM-FCS
5.13.3
Carl Zeiss
Toolbar
This function activates / deactivates (alternately) the toolbar and the subordinate toolbar of the Main
menu.
• Open the Window menu.
• Click on the Toolbar line.
–
The toolbars of the Main menu are displayed / not displayed.
Fig. 5-269
5.13.4
Main menu without toolbars
Scan and System Information
This function opens the Scan Information
window, in which the current scan data are
displayed.
The extent of the data displayed in the Scan and
System Information window depends on the
settings made in the Options menu under
Settings (see page 5-241).
• Open the Window menu.
• Click on the Scan Information line.
–
The Scan and System
window will be displayed.
Information
button to close the Scan
• Click on the
Information window.
In the Options menu in the function Settings in
the change of parameters shown can be
determined in the Scan Information tab.
Fig. 5-270
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Scan Information window
5-273
OPERATION IN EXPERT MODE
Help Menu
Help
Carl Zeiss
5.14
LSM-FCS
Help Menu
The Help menu permits activation of the Help function and of a window containing information on the
installed software version.
Fig. 5-271
5.14.1
Help pull-down menu
Help
• Open the Help menu.
• Click on the Help line to open the online help.
5.14.2
About
• Open the Help menu.
• Click on the About line to open the About
window.
The
About
window
includes
important
information about the software, such as the
software version number, copyright, version
numbers of the various program components and
firmware, and the Dongle number.
• Click on the Close button to close the About
window.
Fig. 5-272
5-274
About LSM-FCS window
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Structure of the Image Display Window
LSM-FCS
5.15
Display and Analysis of Images
5.15.1
Structure of the Image Display
Window
Carl Zeiss
The Image Display window shows the image or
images when they are
–
scanned by any scanning function (see Scan
control and Time series control) or
–
loaded from the image database (see Open
database-Load) or
–
imported by the import function (see Import).
In addition to show images the Image Display
window offers two toolbars for
–
changing the display parameters and save an
image or images (see Select toolbar below)
–
generating new ways of displaying the data as
well as analysis tools (see Display toolbar
below).
Fig. 5-273
Image Display window showing a
single frame image
The Image Display window of the LSM-FCS software corresponds to the basic structure of other
Microsoft ® WINDOWS applications. The Image Display window can be moved as required within the
screen, and its vertical, horizontal and diagonal size can be matched to the current requirements
(identical to Microsoft ® WINDOWS).
The caption at the top of the Image Display window contains the control menu for the Image Display
window (identical to Microsoft ® WINDOWS), the name of the displayed image, and the Minimize,
Maximize and Close buttons.
In the status line at the bottom of the Image Display window, the progress bar of a current scanning
procedure and the parameters used for image display are shown and updated when changed.
On the left-hand side of the Image Display window, an overview of the scan parameter is displayed,
provided that the Info button of the Display toolbar is activated.
The Settings function of the Options subordinate toolbar with the Image display toolbars tab some
of the functions of the Image Display window toolbars can be activated at the opening of a new
Image Display window.
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5-275
Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Structure of the Image Display Window
LSM-FCS
It is possible to display the Chan, Zoom, Slice and Overlay image display toolbars immediately on
opening an Image Display window. The relevant check boxes to be activated in the Image Display
Toolbars tab under Settings (see Options menu).
It is also possible to display the scan parameter of an image (Info button) immediately when an Image
Display window is opened. The data to be displayed can be defined (see Image Status Display tab
under Settings in the Options menu.
The set of functions available at the Image Display window toolbars depends on the type of image
shown. The LSM 5 software handles the following formats:
–
frame (single image and Z Stack of images)
–
frame time series (time series of images and time series with Z Stack of images)
–
line time series (time series of lines and time series with Z Stack of lines)
–
point time series (time series of points)
–
Lambda series (Lambda Stack of images and time series with Lambda Stack of images)
5-276
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Structure of the Image Display Window
LSM-FCS
Carl Zeiss
The following display modes are available for the different acquisition modes:
Image type
Frame
Frame
Frame
Frame
Z Stack
Time
Lambda
xy
xy
xy
xy
xt, tx*
xt, tx
Split xy
Split xy
Split xy
Coded
Split xt,
Split xt
Split tx*
Split tx
Series type
Display
functions
Line
Line
Line
Point
Time
Lambda
Time
Max
xt, tx
Coded
Split xt
Split tx
Ortho*
Ortho
Max
Cut*
Cut
Gallery*
Gallery
Gallery
Gallery
Histo
Histo
Histo
Histo
Histo
Histo
Histo
Profile
Profile
Profile
Profile
Diagr
Diagr
Diagr
Mean
t**
Mean
Lambda
Mean*
Mean
Mean
Lambda
Diagr
2.5 D
2.5 D
2.5 D
Select
Select
Select
3D**
3D**
3D**
2.5 D
2.5 D
2.5D
Topo**
Topo**
Topo**
Prev
Prev
Prev
Prev
Info
Info
Info
Info
Prev
Prev
Prev
Prev
Info
Info
Info
Info
* inactive
** optional
All display functions are exclusive functions. Only one can be active at a given time. To generate different
views of the same image set use the Duplicate function in the Process menu.
During image acquisition all active display functions can be used.
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5-277
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Chan
Carl Zeiss
5.15.2
LSM 510
Select - Chan
This function permits to
–
change the color assignment of channels of images
–
switch individual channels of a multi channel image on/off
–
switch to monochrome display of the image instead of color display
Click on Chan will display the Channels toolbar. Any changes done with this toolbar are effective
immediately.
• Click on the Chan button in the Select toolbar.
–
The Channels toolbar will be displayed on the right-hand side of the Image Display window.
• Click on the Chan button again to remove the Channels toolbar.
Fig. 5-274
5-278
Image Display window; Select - Chan
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Chan
LSM 510
(1)
Carl Zeiss
Assigning another color to a channel
• Click on one of the channels button in the
Channels toolbar (e.g.: Ch1).
–
The color selection box with all the currently
defined colors will appear.
Fig. 5-275
Color selection box
• Click on the required color.
–
(2)
The selected color will be assigned to the current channel, the color selection box is closed and the
displayed image is updated. The control box of the channel button (e.g.: Ch1) also shows the
selected color.
Switching a channel of a multi channel image off or on
• Click on one of the channel buttons in the Channels toolbar (e.g.:Ch1).
–
The color selection box will appear.
• Click on OFF to deactivate the display of the relevant channel.
A newly assigned color or a channel switched off is not taken into consideration during the
following scanning procedure, since the setting in the Configuration Control window always
applies here.
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5-279
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Chan
Carl Zeiss
(3)
LSM 510
Switching to monochrome image display
• Click on the Mono button in the Channels
toolbar.
–
The image will then be displayed in shades
of gray exclusively. If you click on the button
again, the channels will be displayed in color
again.
If you want to view the channels
individually, select the split display by
clicking on Split xy button in the Display
toolbar.
(4)
Defining a new color
• Click on the Colors button to open the
Channel Colors window.
Fig. 5-276
5-280
Channel Colors window
• Define a new color in the same way as in the
Configuration Control window (see page
5-63).
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Zoom
LSM-FCS
5.15.3
Carl Zeiss
Select - Zoom
This function allows to change the zoom factor of an image displayed.
Click on Zoom will display the Zoom toolbar. Any changes done with this toolbar are effective
immediately.
The image can be zoomed by various methods. The zoom function can be performed online.
• Click on the Zoom button in the Select toolbar.
–
The Zoom toolbar will be displayed on the right-hand side of the Image Display window.
• Clicking on the Zoom button again will remove the Zoom toolbar.
Fig. 5-277
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Image Display window; Select - Zoom
5-281
Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Zoom
LSM-FCS
Zoom-Auto
The image is fitted automatically to size of the Image Display window.
Zoom-Resize
Restores the image to its initial size.
Zoom-+
Enlarges the image by factor 2.
Zoom-–
Reduces the image by factor 2.
Zoom 1:1
Restores an image zoomed in any way to its original size.
Zoom-Mouse
Allows you to enlarge / reduce the zoom factor of an image using the
left / right mouse button, provided that the cursor is inside the image.
Zoom-+, Zoom-–, Zoom 1:1 and Zoom-Mouse can only be defined when the Zoom-Auto
function is deactivated.
Slider with display box
5-282
The zoom factor can be set by moving the slider. The display box below
displays the current zoom factor. Factor 1 corresponds to the original
size.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Slice
LSM-FCS
5.15.4
Carl Zeiss
Select - Slice
This function allows to
– select and view individual slices from a Z Stack or a time series, when images where acquired in frame
mode.
The button is grayed, when these conditions are not true.
Click on Slice will display the Slice toolbar. Any changes done with this toolbar are effective
immediately.
• Click on the Slice button in the Select toolbar.
–
The Slice toolbar is displayed on the right-hand side of the Image Display window.
• If you click on the Slice button again, the Slice toolbar is removed.
Fig. 5-278
Image Display window; Select - Slice
Example:
Slice No. 10 from a Z Stack or
time series of 20 slices
• Select the slices using the slider on the right.
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5-283
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Overlay
Carl Zeiss
5.15.5
LSM-FCS
Select - Overlay
This function allows to
– select from a set of drawing functions such as rectangles and arrows
– add a scale bar to the image
– use a set of interactive measurement functions for length, angle and size
The overlay function uses a plane separate from the image plane (the graphics plane) and does therefore
not change the content of the image(s).
The button is only available if the XY or Split XY Display functions are selected. Otherwise it is grayed.
Some of the Display functions such as Ortho or Cut turn the overlay graphics off temporarily.
Any changes done with this function are effective immediately.
The overlay graphics can be stored together with images and can be retrieved from the LSM 5 image
database.
• Click on the Overlay button in the Select toolbar.
– The Overlay toolbar will be displayed on the right-hand side of the Image Display window.
• If you click on the Overlay button again, the Overlay toolbar will be removed.
Provided that the display of the overlay elements has not been deactivated by clicking on the Off button,
the created elements will still be displayed in the Image Display window even after closing of the
Overlay toolbar.
Fig. 5-279
5-284
Image Display window; Select - Overlay
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Overlay
LSM-FCS
Carl Zeiss
The following functions can be used on activation of the buttons in the Overlay toolbar:
Arrow (selection) button: Activation of the mouse button for selection, resizing or
movement of an overlay element in the Image Display window.
Resizing: Click on the handle and hold down the mouse button, drag the handle, release the
mouse button.
Movement: Click on the line and hold down the mouse button, move the entire element,
release the mouse button.
Line button: Creation of a straight line in the Image Display window. Click and hold down
the mouse button, draw a line in any required direction, release the mouse button to end
the procedure.
Rectangle button: Creation of a rectangle in the Image Display window. Click and hold
down the mouse button, draw a rectangle in any required direction, release the mouse
button to end the procedure.
Closed polyline button: Creation of a closed polyline figure in the Image Display window.
The first click sets the starting point, each additional click adds a further line, a click with the
right mouse button closes the figure and ends the procedure.
Open polyline button: Creation of an open polyline figure in the Image Display window.
The first click sets the starting point, each additional click adds a further line, a click with the
right mouse button ends the procedure.
Ellipse button: Creation of an ellipse in the Image Display window. The first click sets the
center point, the displayed line permits the determination of the first dimension, the second
click sets the first dimension, the second dimension and rotation direction can then be
determined, the third click sets the second dimension and direction and ends the procedure.
Closed free-shape curve button: Creation of a closed Bezier figure in the Image Display
window. The first click sets the starting point, each additional click adds a further line, a click
with the right mouse button closes the figure and ends the procedure.
Open free-shape curve button: Creation of an open Bezier figure in the Image Display
window. The first click sets the starting point, each additional click adds a further line, a click
with the right mouse button closes the figure and ends the procedure.
Circle button: Creation of a circle in the Image Display window. Clicking and holding down
the mouse button sets the center point, drag the diameter and release the mouse button to
end the procedure.
Line with arrow button: Creation of a line with arrow in the Image Display window. Click
and hold down the mouse button, drag the line in any required direction, release the mouse
button to end the procedure.
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5-285
Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Overlay
LSM-FCS
Scale button: Creation of a horizontal or vertical scale with default increments in the Image
Display window. Click and hold the mouse button for the starting point, drag horizontal or
vertical scale, release the mouse button to end the procedure.
Gray tones / color shades button: Generates a rectangle with a display of gray tones or
color shades in the image. Color shades are displayed if a palette has been loaded, with
different colors being assigned to the gray tones.
A (Text) button: Creation of a text box in the Image Display window. After clicking on A,
the Text window will be displayed, and text can be entered via the keyboard. The Font ...
button enables you to select the font style and size in the Font window. The entered text
will be displayed in the left upper corner of the Image Display window after clicking on OK
and can be moved to the required position using the mouse. The Text window can also be
activated with a double-click on a created text box, and the entered text can be edited
subsequently.
Recycle bin button: All the overlay elements and dimensions dragged to the scanned image
are deleted. If one overlay element was marked before, this element is now deleted from the
scanned image.
Multiple button: On activation of this button, the overlay function subsequently selected is
performed several times in succession, without the need to activate the function button
again. This function remains selected until the Multiple button is deactivated again.
Measure button: Measurement of the overlay element in the Image Display window. On
activation of the Measure button, the selected overlay element and all the elements created
afterwards are measured and assigned with a measuring value. The measuring value can be
shifted without regard to the overlay element. If of importance, the length and perimeter of
a line figure, the area of a closed figure and the inclination angle of a single line will be
displayed. On deactivation of the Measure button, the measuring value of the selected
element is no longer displayed, and all the elements created afterwards will not be assigned
with a measuring value.
Off button: Deactivation of the display of overlay elements in the Image Display window
(hide overlay). Deactivation of the overlay functions.
Line button: This button allows you to determine the line thickness of the area outline.
Cut button: The image contents of an overlay element are cut out, and the area will then
appear in black.
Copy button: The image contents of a closed overlay element are copied to the clipboard.
5-286
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Overlay
LSM-FCS
Carl Zeiss
Paste button: The image contents of an overlay element copied to the clipboard are inserted
in the current Image Display window and can be positioned anywhere in the image using
the mouse.
Undo button: The last Cut or Paste action can be undone by clicking on the Undo button.
Extract Region button: The region of a Z Stack or 4D-image surrounded by an Overlay
element is extracted and can be displayed and stored separately in a new Image Display
window. This function is only active if an Overlay element is used, that generates a closed
contour.
Color selection box: The colors displayed in the Color selection box can be assigned to the
overlay elements with a click of the mouse. The currently selected color is displayed in the
larger rectangle (left top) of the selection box. A selected color is automatically assigned to
the currently selected overlay element and then to all the elements created afterwards.
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5-287
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Contr
Carl Zeiss
5.15.6
LSM-FCS
Select - Contr
This function allows to
–
change the contrast and brightness of an image
–
change the contrast and brightness of a
channel of an image
–
define interactively a new relationship between
the intensities of pixels in the image memory
and the displayed values of this pixel intensities
on the computer screen
Click on Contr will display the Contrast toolbar.
Any changes done with this toolbar are effective
immediately.
Fig. 5-280
Image Display window;
Select - Contr
Modification done by this function are for display
purposes only. To permanently change the
contrast and brightness of an image use the
function Contrast in the Process menu.
• Click on the Contr button in the Select toolbar.
–
The Brightness and Contrast window will be displayed.
• Change brightness and contrast via the sliders in the Brightness and Contrast window. You can
adjust each channel individually by activating the channel button (e.g.: Ch1), or influence all channels
simultaneously by clicking on All.
• Clicking on the Reset button will reset the
original setting of brightness and contrast.
• Clicking on the Close button will close the
Brightness and Contrast window.
Fig. 5-281
Brightness and Contrast window
Further contrast and brightness parameters can be
activated or deactivated alternately using the
More and Less buttons.
• Click on the More button to display the additional functions.
–
5-288
The Brightness and Contrast window will be enlarged, the labeling of the button changes from
More to Less. If you click on Less, the additional functions are no longer displayed.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Contr
LSM-FCS
Carl Zeiss
Simultaneously with the setting of brightness and contrast, the intensity values of the image can be set
directly in the Intensity Screen via the Ramp, PolyLine, Spline and Gamma functions.
The intensity values can also be set either for all channels together or individually.
If the image has already been changed using the Contrast and Brightness sliders, this setting difference
is displayed in the Intensity Screen by means of the Shape and Result lines.
(1)
Ramp
The intensity is set via two knots in the Intensity
Screen, which allows an intensity line to be
created in the form of a ramp.
The original line form is reset via Reset.
The line form will be retained even when the
additional functions are no longer displayed, and
on closing the Brightness and Control window.
Fig. 5-282
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Brightness and Contrast window
with activated Ramp function
5-289
Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Contr
(2)
LSM-FCS
PolyLine
The intensity is set in the Intensity Screen via a
freely selectable number of knots, which permits
the creation of an intensity line in the form of a
polyline. The number of knots can be selected
from the Number of Knots selection box.
The original line form is reset via Reset.
The line form will be retained even when the
additional functions are no longer displayed or
when the Brightness and Control window is
closed.
Fig. 5-283
Brightness and Contrast window
with activated PolyLine function
(3)
Spline
The intensity is set in the Intensity Screen via a
freely selectable number of knots, which permits
the creation of an intensity line in the form of a
spline. The number of knots can be selected from
the Number of Knots selection box.
The original line form is reset via Reset.
The line form will be retained even when the
additional functions are no longer displayed or
when the Brightness and Control window is
closed.
Fig. 5-284
5-290
Brightness and Contrast window
with activated Spline function
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Contr
LSM-FCS
(4)
Carl Zeiss
Gamma
The intensity is set in the Intensity Screen by
varying the gamma curve (clicking and dragging
with the mouse) or by moving the Gamma slider.
It is possible to set gamma values between 0.1 and
2.0.
The original line form is reset via Reset.
The line form will be retained even when the
additional functions are no longer displayed or
when the Brightness and Control window is
closed.
Fig. 5-285
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Brightness and Contrast window
with activated Gamma function
5-291
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Palette
Carl Zeiss
5.15.7
LSM-FCS
Select - Palette
This function allows to
–
change the palette used for displaying the image(s)
–
define and save new palettes
–
delete palettes by removing them
Click on Palette will display the Palette toolbar. Any changes done with this toolbar are effective
immediately.
The standard palettes No palette, Range indicator, Glow Scale and Rainbow are system palettes and
can not be deleted.
The Range indicator palette is useful to optimize the gain and offset setting of images in the Scan
control window before scanning.
Palettes are stored and retrieved together with the images when archived in the Image Database.
• Click on the Palette button in the Select toolbar.
–
The Color Palette window will be displayed.
• Select the required palette from the Color Palette List panel by clicking on the relevant name.
• If you want to deactivate a palette selected before, click on No Palette in the Color Palette List
panel.
• Click on the Close button to close the Color Palette window.
• A changed image can be stored via the Save As function.
In the Options menu in the function Settings it is possible to switch to Mono automatically when a
palette is activated and to Colour on deactivation of a palette.
In addition it is possible to activate / deactivate Mono in the Channel toolbar.
Some of the handling functions of the Image Display window toolbars can be activated at the opening
of a new Image Display window.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Palette
LSM-FCS
Carl Zeiss
Range
Indicator
Palette
Fig. 5-286
(1)
Image Display window, Select - Palette; Color Palette window and
Add Palette to List window
Editing and storing a palette
A palette is edited by moving the knots in the
Ramp, Polyline and Spline functions (identical to
the setting in the Contrast and Brightness
window, see page 5-288f).
The palette can be set for all colors together or
separately for each color.
• Activate the relevant button: Red, Green, Blue
or All.
Proceed as follows to store an edited palette under
a new name:
• Click on the Add To List button: the Add
Palette To List window will be displayed.
• Enter a name for the palette and click on Ok.
–
The palette will be stored and the name
included in the Color Palette List panel.
Fig. 5-287
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Color Palette window
5-293
Carl Zeiss
(2)
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Palette
LSM-FCS
Delete a palette
Proceed as follows to delete a palette:
• Click on the name of the palette to be deleted in the Color Palette List panel and then on the
Remove button.
–
The palette will be removed from the list.
The standard settings (No Palette, Range Indicator, Glow Scale and Rainbow) cannot be
deleted.
(3)
Import a palette
Proceed as follows to import a palette:
• Click on the Import button. The Import Palette window will be opened.
• Select the required palette (file extension: *.lut) from the relevant directory and click on Open.
–
The palette will be imported and displayed in the Color Palette List panel.
File with the extension *.lut are LSM 310 / 410 palette files.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Anim
LSM-FCS
5.15.8
Carl Zeiss
Select - Anim
This function allows to
• animate frames of a Z Stack or a time series
• specify animation parameters such as range and
animation speed
Click on Anim will display the Animate toolbar.
Any changes done with this toolbar are effective
immediately.
When the image(s) displayed in the Image
Display window is neither a Z Stack nor a time
series this button is grayed and not accessible.
Fig. 5-288
Animate window
• Click on the Anim button in the Select toolbar of the Image Display window of a stack.
–
The Animate window will be displayed and the animation started immediately.
• Click on the Close button to close the Animate window and to stop the animation.
The animation is controlled via the following function elements:
Current Slice slider: Manual movement through the individual slices of a stack by moving
the slider, or by entering the slice number in the input box. Slider can be accessed only,
when the automatic animation is off.
Start slider: The setting of the Start sliders limits the number of slices to be used for the
animation. Previous slices are not taken into consideration for the animation. Can be
changed during automatic animation.
End slider: The setting of the End slider limits the number of slices to be used for the
animation. Subsequent slices are not taken into consideration for the animation. Can be
changed during automatic animation.
Starts the forward motion of the automatic animation. After the last slice has been passed,
restart is made at the first slice.
Starts backward motion of the automatic animation. After the first slice has been passed,
restart is made at the last slice.
Starts the combined forward / backward motion of the automatic animation, i.e. when the
last slice has been reached, the backward motion is activated, and the forward motion is
activated again on reaching the first slice.
Stops the automatic animation.
Move to the first slice.
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5-295
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Anim
Carl Zeiss
LSM-FCS
After each click on this button, backward motion is made by the number of slices set under
Increment.
After each click on this button, forward motion is made by the number of slices set under
Increment.
Move to the last slice.
Speed1 /Speed2 buttons / sliders: Selection between two speeds, change of the relevant
speed via slider or input box.
Increment slider: Reduction of the slices to be displayed by selecting an increment n (step
width) of slices to be taken into consideration for the animation. If n = 3, for example, only
every third slice of the stack will be displayed during the animation.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Reuse
LSM-FCS
5.15.9
Carl Zeiss
Select - Reuse
This function allows to
–
transfer acquisition parameters of an image from the image data base to the Microscope control,
Configuration Control, Scan Control, Time Series Control and Bleach Control windows and
applies those parameters directly on the system.
The acquisition parameters of an image are displayed in the Image Display window and can be viewed
by using the Info function. In the Image Status Display tab in the Settings function of the Options
subordinate toolbar it can be determined what parameters to view with the Info function.
The parameters include the following:
Frame Size, Speed, Data Depth, Scan Direction, Average, Zoom Rotation, Offset, Pinhole diameter,
Detector Gain, Amplifier Offset, Amplifier Gain, Excitation, Beam Path and Scan Mode (Line, Frame,
Stack, Time Series). However, the required objective must be selected by the user.
• Click on the Reuse button. The acquisition parameters of the active image (stack) are applied
immediately to the system.
In the Options menu in the function Settings with the Recording/Reuse tab, it can be determined
whether the objective should also be transferred and set. Setting the microscope objective only works in
microscopes with motorized objective revolvers.
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5-297
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Crop
Carl Zeiss
5.15.10
LSM-FCS
Select - Crop
This function allows to
Fig. 5-289
Image Display window;
Select - Crop
–
interactively define the size and orientation of a
rectangular scan area on the image displayed in
the Image Display window.
–
The defined area is displayed together with the
Zoom, Offset and Rotation parameters in the
Scan Control window in the Mode submenu.
Click on Crop will display the Crop Rectangle in
the Image Display window. Any changes done
with the Crop Rectangle are setting the
parameters immediately. On the next execution of
a scan (Find, Fast xy, Single, Contineous in
Scan Control or Start T or Start B in Time
Series Control) these new scan parameters will
be used.
To reset the crop function and use default values
set Zoom=1, Offset=0 and Rotation=0 in the
Scan Control window in the Mode submenu.
• Click on the Crop button.
–
The Crop Rectangle will appear on the Image Display window.
The Crop Rectangle is controlled via the following functional elements:
Offset
• Click into the crop rectangle, keep the left mouse button pressed and drag the crop rectangle to the
required position. Release the mouse button.
Zoom
• Click on a corner of the crop rectangle, keep the left mouse button pressed and set the required size.
Release the mouse button.
Rotation
• Click on one end of the crosslines, keep the left mouse button pressed and set the required rotation
angle. Release the mouse button. The first line scanned is highlighted in blue.
Side ratio
• Click on any of the intersection points between crossline and crop rectangle, keep the left mouse
button pressed and change the side ratio as required. Release the mouse button.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Copy
LSM-FCS
5.15.11
Carl Zeiss
Select - Copy
This function allows to
–
copy the current displayed image into the clipboard.
Click on Copy will be immediately effective.
From the clipboard images can be incorporated into other programs such as MS Excel, MS Powerpoint or
MS Word.
To export image series, use the Export function in the File menu.
• Click on the Copy button.
–
The content of the Image Display window is copied to the clipboard.
• Start the clipboard application of WINDOWS.
• Select Paste in the Edit menu of the Clipboard application.
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5-299
OPERATION IN EXPERT MODE
Display and Analysis of Images
Select - Save
Carl Zeiss
5.15.12
LSM-FCS
Select - Save
This function allows to
–
save the image(s) of the Image Display window into an Image Database
–
by not showing a dialogue and using the automatic assigned and incremented image name and a
predefined existing Image Database
–
Prerequisite: Autosave is checked in the Settings function with the Autosave tab
Click on Save will be immediately effective.
When the prerequisite is not met, the Save As dialogue is displayed.
In the Options menu in the function Settings with the Autosave tab parameters such as an
automatically incremented filename can be determined and the Autosave activated/deactivated.
5.15.13
Select - Save As
This function allows to
–
save the image(s) of the Image Display window into an Image Database
–
by showing a dialogue
–
use the defaults as defined in the Settings function with the Save tab
Click on the Save As button displays the Save Image and Parameter As window. Changes will be
effective on closing this dialogue.
In the Options menu in the function Settings with the Save tab default parameters such as Name,
Description and Notes can be set.
• Click on the Save button.
–
The Save Image and Parameter As window appears
• Enter text for the image name, description, notes or change the user name.
• Select the Image Database from the list of databases (MDB) or
• Open other Image Databases by selecting Open MDB or
• Create new Image Databases by selecting New MDB.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - xy
LSM-FCS
5.15.14
Carl Zeiss
Display - xy
This function allows to
–
display a single image in frame mode
–
display multi channel images in superimposed mode
The settings of Chan, Zoom, Slice, Overlay, Contr and Palette are applied.
Click on xy will be immediately effective.
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5-301
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Split xy
Carl Zeiss
5.15.15
LSM-FCS
Display - Split xy
This function allows to
–
display the individual channels of a multi channel image as well as the superimposed image
The settings of Chan, Zoom, Slice, Overlay, Contr and Palette apply.
Click on Split xy will be immediately effective.
Channel images
Fig. 5-290
Image Display window, Split xy display
Composite image
This function is useful to optimize the individual channels in a multi channel image acquisition
together with the Range Indicator palette .
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Ortho
LSM-FCS
5.15.16
Carl Zeiss
Display - Ortho
This function allows to
–
display a Z Stack of images in an orthogonal view
–
measure distances in three dimensions
–
generate 2D deconvolution views of the yz and xz plane
The settings of Chan, Zoom, Slice, Overlay, Contr and Palette apply.
Click on Ortho will be immediately effective.
• By clicking on the Ortho button section lines appear in the Display toolbar together with orthogonal
projections in the image. On the right-hand side, the Orthogonal Sections toolbar is shown.
YZ plane (red)
XZ plane (green)
XY plane (blue)
Section
result,
XZ plane
Fig. 5-291
Image Display window, Ortho display
Section result, YZ plane
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5-303
Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Ortho
LSM-FCS
5.15.16.1 Ortho - Select function
• By changing the parameters X, Y and Z in the Orthogonal Sections toolbar, the section plane can be
positioned at any XYZ coordinate of the Z Stack.
The position of section planes can be changed in various ways:
• By moving the sliders on the Orthogonal Sections toolbar.
–
X and Y settings may range from 1 up to the maximum number of pixels scanned (in the example
shown: 512).
–
Z settings may range from 1 to a maximum of n, with n standing for the number of slices
produced in the stack.
• By directly entering the relevant number value in the X-, Y- or Z-input box and pressing the Enter key.
. By
• If you move the cursor into the Image Display window, it changes into a crosslines symbol
dragging this symbol with the mouse you can position the XZ and YZ section planes to any point of
intersection with the XY plane. A click with the left mouse button places the intersection to the
desired position.
• If you move the crosslines symbol
onto the intersection of the red and green section planes, it
changes into the:
symbol. If you now press the left mouse button and keep it pressed you can
reposition both section planes simultaneously.
onto the green section plane, it changes into the
symbol. If
• If you move the crosslines symbol
you now press the left mouse button and keep it pressed, you can reposition the (green) XZ section
plane.
• You can reposition the (red) YZ plane in the same way using the
symbol.
The result of an orthogonal section is visible at the image margin.
–
Section of the XZ plane (green line) through the stack: above the XY image.
–
Section of the YZ plane (red line) through the stack: right of the XY image.
–
Section of the XY plane (blue, slice plane of the stack): center image.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Ortho
LSM-FCS
Carl Zeiss
5.15.16.2 Ortho - Distance function
• Activating the Dist. button permits length measurements in 3D space.
Yellow
measuring lines
(projections)
Fig. 5-292
Image Display window, Ortho display
• Click on the Mark button to set the first XYZ-point for the measurement of the spatial distance.
• Set the second XYZ-point for measurement by moving the X-, Y-, Z-sliders or by moving the green,
red and blue lines in the image.
–
The projections of the spatial distance are shown in the image planes by yellow lines. The actual
spatial distance is calculated and shown in µm below the Select, Dist. and Mark buttons, e.g. 3D
Distance: 55.60 µm.
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5-305
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Ortho
Carl Zeiss
LSM-FCS
5.15.16.3 Ortho - 2D DeConVolution function
The 2D deconvolution function causes orthogonal projection enhancement through the computed
correction of the resolution in the Z-coordinate.
Image enhancement is only effective for the two projections of a fluorescence stack in the Ortho display
or for an XZ-scan in fluorescence, and is also only computed for this.
• Activate the 2D DCV button in the Orthogonal Sections toolbar.
If the Fast button is activated, calculation of the 2D-deconvolution (inverse DCV mode) is performed
immediately.
The 2D DCV settings button can only be activated if a licence for the 3D DCV option has been
purchased. Otherwise this button is grayed.
• Click on the Settings button. The 2D Deconvolution window is opened.
The 2D Deconvolution window contains the Deconvolution panel with the two tabs Method and
PSF.
(1)
Method tab
The Method tab enables you to choose between
the calculation methods Inverse and Iterative.
For more details of explanation of deconvolution
and the calculation methods see section 3D
DeConVolution (page 5-163).
Fig. 5-293
Method tab
In the Inverse method, the Restoration Effect
slider can be used to set the signal-to-noise ratio
between Weak (low noise) and Strong
(pronounced noise).
Activation of the Auto detect check box starts a
routine for the automatic determination of the
noise level in the entire image part of the Z Stack.
If Auto detect is enabled, the Restoration Effect
slider is disabled.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Ortho
LSM-FCS
Carl Zeiss
The Iterative method permits (in addition to the parameters of the Inverse method) the maximum
number of iterations to be entered between 1 and 200 under Maximum Iterations and the Auto Stop
function to be activated / deactivated. The Auto Stop function interrupts the calculation depending on
the set image improvement (delta between last but one and last cycle in %), no matter whether the
value under Maximum Iterations has been achieved or not.
(2)
PSF tab (optional with 3D DCV)
The objective data are displayed in the Method
tab. In the case of wavelengths above 700 nm, the
NLO button is automatically enabled.
The displayed values are always taken over by the
system data, but can be edited subsequently for
simulation purposes.
• Select the required method and determine the
relevant parameters.
The deconvolution calculation is performed
immediately after the 2D Deconvolution window
has been closed, and the image display is updated
(on-line).
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Fig. 5-294
PSF tab
5-307
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Cut
Carl Zeiss
5.15.17
LSM-FCS
Display - Cut
This function allows to
–
display a Z Stack of images at a user defined section plane (= cut plane)
–
improve the image of the section plane by trilinear interpolation
The settings of Chan, Zoom, Slice, Contr and Palette are applied.
Click on Cut will display the Cut toolbar. Any changes done with this toolbar are effective immediately.
The content of the overlay plane is temporarily deleted while the toolbar is displayed.
• Clicking on the Cut button in the Display toolbar opens the Cut toolbar to the right of the Image
Display window.
Positioning
the section
planes
View of
section plane
Fig. 5-295
Image Display window, Cut display
• By varying the parameters X, Y, Z, Pitch and Yaw, you can position a section plane of any orientation
within the stack volume.
• The resulting position of the section plane is shown as a red area below the Trilinear Interpolation
button. At the same time, the result is shown in the Image Display window.
• A click on the Reset All button restores the original position.
• A click on the Trilinear Interpolation button will improve the quality of the image by performing a
3D interpolation of the image.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Gallery
LSM-FCS
5.15.18
Carl Zeiss
Display - Gallery
This function allows to
–
display images (Z Stack, time series, combination of both) side by side in tiled fashion
–
add data relevant to the images displayed (Z Stack slice distance, time of acquisition or wavelength)
–
extract a subset of images from the original stack and store the result as a new image
The settings of Chan, Zoom, Slice, Contr and Palette apply.
Click on Gallery will display the Gallery toolbar. Any changes done with this toolbar are effective
immediately.
• A click on the Gallery button in the Display toolbar not only produces the gallery itself but also the
Gallery toolbar with two buttons: Data button and Subset button.
Gallery
Fig. 5-296
Image Display window, Gallery display
• Clicking on the Data button shows the Z Slice distance, the acquisition time or the wavelength or
combinations.
• Clicking on the color selection button (below the Data button) will open a color selection window
allowing you to choose - at a click of the mouse - in which color the data will be shown in the gallery
display.
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5-309
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Gallery
Carl Zeiss
LSM-FCS
• Clicking on the Subset button opens another
window entitled Subset, in which you can
select images of the set of images displayed.
–
Fig. 5-297
Subset window
A stack consisting of the selected images
only is generated and displayed.
• Select Start Slice and End Slice via the sliders,
the input box or the Click (into window)
button.
• Enter a value for n in the Every n-th Slice
panel.
• If required, activate the Convert 12 bit to
8 bit check box .
• Clicking on the Ok button will generate a new
subset of images.
• Cancel will stop the procedure.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Histo
LSM-FCS
5.15.19
(1)
Carl Zeiss
Display - Histo
Display - Histo - Overview
This function allows to
–
display a histogram (distribution of pixel intensities) of an image
–
show the histogram values in table form
–
copy table to clipboard or save as text file
–
analyze the colocalization between two channels
–
measure area and mean gray value and standard distribution in an area
–
show separate histograms for each channel in a multi channel image
Colocalization is only available in case of a two or multi channel image.
The settings of Chan, Zoom, Slice, Contr and Palette are applied.
Click on Histo will display the Histogram toolbar. Any changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
• Click on the Histo button. The Histogram toolbar will be displayed on the right.
Control of
colocalisation
Control of area
measurement
Control of histogram
display parameters
Fig. 5-298
Image Display window, Histogram display
The Histo button can also be used online during scanning.
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5-311
Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Histo
LSM-FCS
The Histogram toolbar contains the following function elements:
Histogram functions
Skip Black button
Ignore black pixels (gray value 0) in the histogram.
Skip 4 % button
Ignore the lower 4% of the intensities in the histogram.
Skip White button
Ignore white pixels (gray value 255 or 4096) in the histogram.
Step input box
Set the number of intensity steps which shall be displayed in the diagram.
Step 1 corresponds to 256 intensity steps, Step 64 to 4 intensity steps in
case of 8 bit images. Reduction is made by averaging.
Show Image button
Shows the image in the Image Display window beside the histogram.
Show Table button
The histogram is shown as a table at the bottom left of the Image
Display window.
Copy Table button
The histogram table is copied to the clipboard.
Save Table button
The histogram table can be stored as a text file (extension *.txt).
Area function
Area button
Interactive definition of area for size and intensity measurement.
Save Values button
Copies area values to the clipboard (only available if the Area button is
activated).
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Histo
LSM-FCS
(2)
Carl Zeiss
Area function
• Click on the Area button in the Histogram toolbar.
–
The function elements for Area measurement are displayed at the bottom right of the Histogram
toolbar.
Fig. 5-299
Image Display window, Area Measure display
The following function elements are available:
Set the number of intensity steps which shall be displayed in the diagram. Step 1
corresponds to 256 intensity steps, Step 64 to 4 intensity steps in case of 8 bit
images. Reduction is made by averaging.
Threshold low slider with Color selection button: The intensity values below
threshold are not displayed. The removed areas are masked in the color selected in
the Color selection button.
Threshold high slider with Color selection button: The intensity values above
threshold are not displayed. The removed areas are masked in the color selected in
the Color selection button.
Ch1, Ch3 ... buttons: Selection of the channel for which the area measurement is
to be performed.
Display box: Display of the mean value and the standard deviation of the nonmasked area. Area measurements of very small areas (< 10 pixels) give only
approximate values.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Histo
LSM-FCS
Arrow (selection) button: Activation of the mouse button for selection, resizing, or
movement of a mask element in the Image Display window.
Resize: Click on handle and hold down the mouse button, drag the handle, release mouse
button.
Movement: Click on line and hold the mouse button, move the entire figure, release mouse
button.
Closed polyline button: Creation of a polyline figure in the image. The first click sets the
starting point, each additional click adds a further line, a click with the right mouse button
closes the figure and ends the procedure.
Recycle bin button: All the mask elements are deleted. If one element was marked before,
this element is now deleted from the image.
Mask button: Enables the Mask Mode, where the region can be defined with ink.
Rectangle button: Creation of a rectangle in the image. Click and hold down the mouse
button, drag a rectangle in any direction, release the mouse button to end the procedure.
Ellipse button: Creation of an ellipse in the image. The first click sets the center point, the
displayed line permits the determination of the first dimension, the second click sets the first
dimension, the second dimension and the rotation direction can then be determined; the
third click sets the second dimension and the direction and ends the procedure.
Line button: Determines the line thickness of the area outline.
Flood fill button: Fills the overlay element or the scatter diagram with the color selected
under Mask.
Closed free-shape curve button: Creation of a closed Bezier figure in the scatter diagram.
The first click sets the starting point, each additional click adds a further line, a click with the
right mouse button closes the figure and ends the procedure.
Circle button: Creation of a circle in the scatter diagram. Clicking and holding down the
mouse button sets the center point; drag the diameter and release the mouse button to end
the procedure.
Color selection button: The colors displayed in the selection box can be assigned to the
mask elements with a click of the mouse. The currently selected color is always displayed in
the larger rectangle (top left) of the selection box.
Clear Mask button: Removes the color filling from an overlay element or from the scatter
diagram.
• The function can be activated by clicking on one of the geometry buttons, e.g.
(polyline).
• The figure of interest can be marked in the image by cursor control in conjunction with a mouse click.
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Display - Histo
LSM-FCS
Carl Zeiss
• Clicking on the Flood fill button (paint jar) and moving the cursor to the area to be excluded causes
the remaining area to be computed and the result indicated under Area Measure.
Fig. 5-300
Image Display window, Area Measure display
• If you specify a top and bottom intensity threshold, the area lying within this intensity interval can be
computed.
• Specify the thresholds either with the Threshold low and Threshold high sliders, or with the
buttons.
and
Fig. 5-301
Image Display window, Area Measure display
• Click on the xy button of the Display toolbar if you want to return to the original image.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Histo
Carl Zeiss
(3)
LSM-FCS
Colocalisation function
The Colocalisation function permits interactive analysis of two channels of an image by computing a
scatter diagram (colocalisation).
• Click on the Colocalisation button. The scatter diagram is created and displayed beside the image.
How a scatter diagram is generated:
All pixels having the same positions in both images are considered a pair. Of every pair of pixels (P1, P2)
from the two source images, the intensity level of pixel P1 is interpreted as X coordinate, and that of
pixel P2 as Y coordinate of the scatter diagram. The value of the pixel thus addressed is increased by one
every time, up to the maximum number of pixels used. This way, each pixel of the scatter diagram is a
value that shows how often a particular pair of pixels has occurred.
Differences between the images cause irregular spots in the scatter diagram.
Fig. 5-302
5-316
Image Display window, Colocalization display
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OPERATION IN EXPERT MODE
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Display - Histo
LSM-FCS
Scatter diagram
Fig. 5-303
Carl Zeiss
Colocalisation button
Image Display window, Colocalization display
Identical images produce a clean diagonal line running from bottom left to top right, because only pixel
pairs (0,0), (1,1), (2,2) with the same intensity can occur. Differences between the images cause an
irregular distribution in the scatter diagram.
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Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Histo
LSM-FCS
The following function elements are available:
Colocalisation button
Displays the scatter Histogram of the two image channels
Show table button
Adds display of the according data table
Area button
Adds display of the image
Crosshair button
Displays a movable crosshair for different areas in the scatter histogram
Threshold button
Opens the Intensity Threshold window to sets threshold for
colocalisation in the scatter histogram.
Set from Image ROIs button: Sets background threshold from ROI
(Threshold button)
Cut Mask button:
Channel Toggle button:
Invert Mask button: Inverts the mask or the scatter diagram.
Source 1 selection box with Color selection box: Selection of the first
channel to be selected via the selection box, assignment of a defined
color via the Color selection box.
Source 2 selection box with Color selection box: Selection of the second
channel to be selected via the selection box, assignment of a defined
color via the Color selection box.
Mask selection box with Color selection box: Selection of RGB or
Overlay display of the mask, assignment of a defined color via the Color
selection box.
Drawing tools
Allows the selection of ROIs in the Histogram and the Image
Save at drawing tools
Stores ROIs and threshold settings
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Histo
LSM-FCS
Carl Zeiss
Enhanced colocalisation
Scattergram, image display and data table are interactively linked:
Fig. 5-304
Image Display window, Colocalization display
Scattergram and thresholding with crosshair:
Crosshair
Scatterregion 2:
Pixels in channel 1 only
Scatterregion 3:
Colocalizing pixels
Scatterregion 4:
Sub-threshold pixels
(background intensities)
Scatterregion 1:
Pixels in channel 1
only
Fig. 5-305
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Scattergram and thresholding with crosshair
5-319
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Histo
Carl Zeiss
LSM-FCS
Select ROIs in scattergram and view corresponding pixels in image display:
Color
assignment
for pixels in
ROI (RGB or
overlay)
Fig. 5-306
Image Display window, Colocalization display
Select ROIs in image display and view corresponding pixels in scattergram:
Fig. 5-307
5-320
Image Display window, Colocalization display
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Display and Analysis of Images
Display - Histo
LSM-FCS
Carl Zeiss
Quantitative Parameters:
–
No. of pixels in image ROI or scatter region
–
Area / relative area of image ROI or scatter region
–
Mean intensities / SD within image ROI or scatter region
–
Colocalization coefficients
–
Weighted colocalization coefficients
–
Overlap coefficient after Manders
–
Correlation coefficients (R and R )
2
Colocalization coefficients
c1 =
pixelsCh1,coloc
pixelsCh1,total
c2 =
pixelsCh 2,coloc
pixelsCh 2,total
–
Relative number of colocalizing pixels in channel 1 or 2, respectively, as compared to the total number
of pixels above threshold.
–
Value range 0 – 1 (0: no colocalization, 1: all pixels colocalize)
–
All pixels above background count irrespective of their intensity.
Weighted colocalization coefficients
∑ Ch1
=
∑ Ch1
M1
i,total
i
∑ Ch2
=
∑ Ch2
i,coloc
i,coloc
i
M2
i
i,total
i
–
Sum of intensities of colocalizing pixels in channel 1 or 2, respectively, as compared to the overall sum
of pixel intensities above threshold and in this channel.
–
Value range 0 – 1 (0: no colocalization, 1: all pixels colocalize)
–
Bright pixels contribute more than faint pixels
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Histo
Carl Zeiss
LSM-FCS
Correlation coefficient, Pearson´s correlation coefficient
∑ (Ch1
i
Rp =
− Ch1aver ) * (Ch2 i − Ch2 aver )
i
∑ (Ch1
i
i
− Ch1aver ) 2 * ∑ (Ch2 i − Ch2 aver ) 2
i
–
Provides information on the intensity distribution within the colocalizing region
–
Value range -1 to +1
-1,+1: all pixels are found on straight line in the scattergram
0:
pixels in scattergram distribute in a cloud with no preferential direction
Overlap coefficient, overlap coefficient after Manders
(Manders, Verbeek and Aten, J. Microscopy 169:375-382, 1993)
∑ Ch1 * Ch2
i
r=
i
i
∑ (Ch1 ) * ∑ (Ch2 )
2
i
i
2
i
i
–
Another parameter used to quantify colocalization in image pairs
–
Insensitive to differences in signal intensities between the two channels, photo-bleaching or amplifier
settings
–
Value range 0 – 1 (0: no colocalization, 1:all pixels colocalize)
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Display and Analysis of Images
Display - Profile
LSM-FCS
5.15.20
Carl Zeiss
Display - Profile
This function allows to
–
display the intensity distribution of an image along a straight or curved line
–
show the intensity values in table form and copy table to clipboard or save as text file
–
show separate profiles for each channel in a multi channel image
The settings of Chan, Zoom, Slice, Contr and Palette are applied.
Click on Profile will display the Profile toolbar. Any changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
• Click on the Profile button. The Profile toolbar will be displayed.
–
The intensity curves are shown in a graph below the image(s).
• On the Profile toolbar you can select the width and color of the profile line.
Marker 1
and 2
Freely
selectable
line for
intensity
profile
Fig. 5-308
Image Display window, Profile display
• You can place two markers on the profile line to measure differences in intensities and distances in
the XY plane.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Profile
Carl Zeiss
LSM-FCS
• Click on the Diagr. in Image button to see an intensity graph superimposed on the image.
Fig. 5-309
Image Display window, Profile display
The Profile toolbar contains the following function elements:
Arrow (selection) button: Activates the mouse button for selection, resizing or movement of
the profile line in the Image Display window.
Resize: Click on handle and hold down the mouse button, move the handle, release mouse
button.
Movement: Click on line and hold down the mouse button, move the entire line, release
mouse button.
Line with arrow button :(open arrow): Activates the straight profile drawing mode.
Click into the image and hold the mouse button, drag a line in any direction and release the
mouse button to end the procedure.
Open polyline button: Activates the open polyline drawing mode.
The first click into the image sets the starting point, each additional click adds a further line,
right mouse click ends the procedure.
Open free-shape curve button: Activates the Bezier figure drawing mode.
The first click into the image sets the starting point, each additional click adds a point, right
mouse click ends the procedure.
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Display - Profile
LSM-FCS
Carl Zeiss
Line button: This button allows you to determine the line thickness of the profile line. It has
no influence on the way the intensity profile is generated.
Color selection box: The colors displayed in the Color selection box can be assigned to the
overlay profile line with a click of the mouse. The currently selected color is displayed in the
larger rectangle (top left) of the selection box.
Diagr. In Image button: The profile diagram is displayed in the image along the drawn
profile line.
Marker 1 button (red): Activates the red marker for movement in the profile diagram; the
marker shown as a red line in the diagram can now be moved to the right or left of the
diagram using the mouse. The marker in the image display (red circle) follows accordingly.
Marker 2 button (blue): Activates the blue marker for movement in the profile diagram; the
marker shown as a blue line in the diagram can now be moved to the right or left of the
diagram using the mouse. The marker in the image display (blue circle) follows accordingly.
Zoom button: Zoom function for profile diagram. Click and drag a rectangle over the area
to be enlarged in the profile diagram; the selected area is enlarged on release of the mouse
button. The zoom function can be performed several times. A click with the right mouse
button will reset the original size.
Reset Zoom button: Resets the zoom factor of the profile diagram to the original size.
Show Table button: The profile diagram is displayed as a table at the bottom of the Image
Display window.
Copy Table button: The profile table is copied to the clipboard.
Save Table button: The profile table can be stored as a text file (extension *.txt).
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Display - 2.5 D
Carl Zeiss
5.15.21
LSM-FCS
Display - 2.5 D
This function allows to
–
display the two-dimensional intensity distribution of an image in an pseudo 3D mode
–
show the intensity values in profile, grid or filled mode
–
show separate distribution for each channel in a multi channel image
• Click on the 2.5 D button.
–
The Pseudo 3D toolbar is displayed.
The settings of Slice apply. The settings of Chan, Zoom, Contr and Palette are not applied.
Click on 2.5 D will display the Pseudo 3D toolbar. Any changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
The viewing plane of the Image Display window can be rotated, tilted either directly with the mouse or
by the scroll bars on the right-hand side and the bottom of the Image Display window.
(1)
Direct setting in the image
• Click in the image and hold down the mouse button. The perspective is changed by moving the
mouse button in horizontal or vertical direction.
(2)
Setting via scrollbars
• Move the
horizontal scrollbar to rotate the image around the vertical axis. The rotation angle is
displayed in the yellow display box.
• Move the left vertical scrollbar to rotate the image around the horizontal axis. The rotation angle is
displayed in the yellow display box.
The intensity scale can be varied by the scroll bar on the right-hand side of the Image Display window:
• Moving the
right vertical scrollbar enables you to expand or to compress the intensity scale of the
image, while the expansion of this intensity axis ranges between 10 % and 100 % of the X-scale size.
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Display - 2.5 D
LSM-FCS
Carl Zeiss
Type of
pseudo-3D
presentation
Select channel
Select monochrome / color
presentation
Fig. 5-310
Image Display window, 2.5 D display
The Pseudo 3D toolbar contains the following function elements:
Profile button
Profile display (vertical polygon display). Setting of the Profile Distance
between 1 and 20 using the slider.
Grid button
Grid display (horizontal grid display). Setting of the Grid Distance
between 1 and 20 using the slider.
Filled button
Color diagram (filled 3D diagram). Selection between the Mono,
Rainbow and Six Step color palettes.
Channel list box
Permits the selection of a Channel in a multi channel image.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - 3D (Image VisArt)
Carl Zeiss
5.15.22
LSM-FCS
Display - 3D (Image VisArt)
This optional function allows to
–
reconstruct and display 3 D fluorescence image stacks or time series of frames and stacks from the
Image Display window
–
select from a variety of reconstruction modes
The settings of Chan are applied. The settings of Zoom, Slice, Contr and Palette are not applied.
Click on 3D will display the 3D toolbar. Any changes done with this toolbar are effective immediately.
The content of the overlay plane is temporarily deleted while the toolbar is displayed.
• Click on the 3D button. The 3D toolbar will be displayed.
Fig. 5-311
5-328
Image Display window, Profile display
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Display and Analysis of Images
Display - 3D (Image VisArt)
LSM-FCS
Carl Zeiss
The 3D toolbar contains the following function elements:
Shadow projection
Front button
Shadow rendering front view
Back button
Shadow rendering back view
Any View button
Shadow rendering with user defined view
Transparency projection
Basic button
Transparency rendering (voxel based)
Advanced button
Transparency rendering (voxel based) with textures
Surface projection
Basic button
Surface rendering (voxel based)
Advanced button
Surface rendering (triangle based)
Full Resolution button
High accurate surface rendering (triangle based)
Appearance button
Opens the render properties dialog
Series button
Renders a series of 3D image stack or 3D / 4D time series, opens the
Series render dialog
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - 3D (Image VisArt)
Carl Zeiss
LSM-FCS
5.15.22.1 Shadow Projection
With a click on Front, the 3D reconstructed image is displayed in a shadow projection where it is
illuminated at a 45° angle from the front left.
A click on the Back button creates the same projection with illumination from back left.
The zoom wheel to the left of the Image Display window allows continuous zooming of the 3D
reconstructed image.
Fig. 5-312
5-330
Image Display window, 3D display, Shadow projection, Front
view
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Display - 3D (Image VisArt)
LSM-FCS
Carl Zeiss
A click on the Any View button displays the 3D reconstruction image in a shadow projection where the
viewing point can be defined. In addition to the zoom setting, the image can be rotated around the
three orthogonal axes via the relevant setting wheels.
However, the 3D orientation can also be set directly in the Image Display window by clicking, holding
and dragging the 3D reconstructed image with the mouse.
Fig. 5-313
Image Display window, 3D display, Shadow projection,
Any View
The following additional buttons are available in the Any View shadow projection mode:
• After activation of the Frame button (below the image), a bounding box is drawn around the 3D
reconstructed image.
Depending on the used mode and hardware configuration, it can take several seconds until the
3D reconstruction is refreshed on the monitor after reorientation.
• A click on the Coordinate System button displays a colored coordinate system in the Image Display
window, where the X axis is displayed in red color, the Y axis in blue and the Z axis in green.
• A click on the Scale button display an X-,Y- and Z-scale in the Image Display window.
• A click on the Home button resets the display parameters to the default values.
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OPERATION IN EXPERT MODE
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Display - 3D (Image VisArt)
Carl Zeiss
•
LSM-FCS
A click on the Animation button activates the animation mode. The object can be pushed by
dragging in the Image Display window and rotates continuously. Any new push with pressed left
mouse button changes the rotation direction and speed of the animation.
The fastest animation results can be achieved with the advanced surface rendering mode (even
without additional graphics cards).
5.15.22.2 Transparency Projection
The transparency projection generates a transparent 3D reconstructed image.
The elements for image display (zoom, 3D rotation, home, coordinate system, scale, frame and
animation function) are identical to those of the Any View function of the shadow projection and are
operated in the same way.
The transparency projections Basic and Advanced are perspective-type 3D reconstructions, with the
Advanced projection permitting the perspective impression being varied between parallel and centric
projection by changing the View angle. The Advanced projection also offers the possibility of selecting
between fast and precise calculation via the Precise / Fast slider (at the bottom right in the 3D toolbar).
Of course, the precise calculation method is more time consuming.
Fig. 5-314
5-332
Image Display window, 3D display, Transparency projection,
Advanced
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Display and Analysis of Images
Display - 3D (Image VisArt)
LSM-FCS
Carl Zeiss
5.15.22.3 Surface Rendering
The surface rendering generates surface rendered 3D reconstructed images.
The elements for image display (zoom, 3D rotation, home, coordinate system, scale, frame and
animation function) are identical to those of the transparency projection and are operated in the same
way.
The surface projections Basic and Advanced are perspective-type reconstructions of the surface and
differ in the fact that the calculation of the 3D information is based on voxels or triangles.
The Advanced projection permits the View angle to be varied in order to enhance the perspective
impression. It is also possible to select between fast and precise calculation via the Precise / Fast slider
(at the bottom right in the 3D toolbar). Of course, the precise calculation method is more time
consuming.
The Full resolution projection is based on a high precision calculation method for 3D information on
the basis of triangles with maximum resolution.
Depending on the hardware configuration, it can take several seconds until the surface
projection is refreshed on the screen.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - 3D (Image VisArt)
Carl Zeiss
LSM-FCS
5.15.22.4 Appearance (Settings)
The Appearance button opens the 3D Rendering
window.
This window allows settings for Light, Material,
Background and Projection properties to be
defined for all 3D projection modes.
Depending on the selected 3D projection mode,
different setting parameters are displayed.
In the Shadow projection, the parameter
Roughness is also available and can be set
between 0 and 1.
A default setting is permanently available for all
modes.
If individual settings for 3D rendering are to be
used again, they can be stored and loaded when
required.
Proceed as follows to save individual settings:
• Click on the Add to List button.
• Enter a name in the opening Add Shading
Model to list window.
• Click on OK.
Fig. 5-315
3D Rendering window (e.g. Surface
Advanced rendering mode)
–
The settings are saved and the entered name
appears in the Shading Model List
selection box.
• To activate the settings, double-click on the relevant name in the Shading Model List selection box.
Settings which are no longer required can be removed.
• Select the name of the setting to be deleted with a click of the mouse in the selection box and then
click on the Remove button.
–
5-334
The setting is deleted.
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Display - 3D (Image VisArt)
LSM-FCS
Carl Zeiss
5.15.22.5 Series
The Series button opens the Render Series
window. This window allows settings for the axis
to be used for rotation of the 3D reconstructed
images.
• Click on the Series button to open the Render
Series window.
• Select the requested projection mode by
clicking on the relevant option button under
Mode.
• Depending on the activated mode, directly set
the parameters for animation in the Render
Series window and the position of the 3D
image in the Image Display window (zoom,
rotation axes, rendering parameters).
Fig. 5-316
Render Series window
(e.g. Turn around X mode)
• Click on Apply to start the animation
The animation is performed in a separate Image
Display window, which permits the animation to
be saved afterwards.
(1)
Turn around X and Turn around Y mode
In this mode, the image is turned around the X-axis or the Y-axis exclusively.
The values for Number of Views, Difference Angle and First Angle can be selected accordingly (see
section 5.7.2 Projection, page 5-175).
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - 3D (Image VisArt)
Carl Zeiss
(2)
LSM-FCS
Start and End mode
In this mode, the image is reconstructed between
a start position and an end position.
The rotation angles for X, Y and Z and the distance
(zoom) can be determined using the sliders.
The value for Number of View can be varied.
Fig. 5-317
Render Series window
(e.g. Start and End mode)
(3)
Position List mode
In this mode, the image is reconstructed between
any required number of interim positions to be
determined individually.
The rotation angles for X, Y and Z and the zoom
can be determined directly in the image.
Every required interim position is included in the
list of the Render Series window with a click on
the Add Position button.
Clear List permits the contents of the list to be
deleted.
The value for Number of View can be varied.
Fig. 5-318
Render Series window
(e.g. Position List mode)
• Click on the Apply button calculates a spline
along all the defined positions from the list and
starts an animation along this spline track in
space.
(4)
Time series
When the input images is a Z Stack time series, the
reconstructed images are generated for each time
point.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
LSM-FCS
5.15.23
Carl Zeiss
Display - Topo for LSM
This optional function allows to
–
process, display and measure topographic information.
–
use frame Z Stacks
–
and frame Z Stacks over time
The Topo function is mainly used for applications in material research and industry.
The settings of Chan and Zoom are applied. The settings of Slice and Contr are not applied. The
Palette settings are applied in some 3D display modes.
Click on Topo will display the Topography toolbar. All changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
• Click on the Topo button. The Topography toolbar is displayed.
–
The topography of a Z Stack is displayed in the Image Display box of the Image Display
window. The parameter used at the last exit of the Topo function are applied.
Fig. 5-319
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Image Display window, Topography display
5-337
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
Carl Zeiss
LSM-FCS
The Topography toolbar contains the following function elements:
Channels buttons
The selection of a channel to be used.
Generate buttons
The selection of the mode of calculation of the topography image
(maximum, center of gravity, first intensity).
Threshold buttons
The selection of thresholds (intensity, height, Auto Z) to be used.
Filter buttons
The definition of filter procedures (geometrical, frequency cut-off filters)
for smoothing, separation of roughness or waviness.
Geometry buttons
Automated correction procedures, changes of geometry, tilting.
Display buttons
2D (Intensity, z Map, Gradient); Iso-Lines (z Map, Intensity, Black)
3D (Profiles, Grid, Filled, Shaded).
Measure buttons
Diagrams (Profile, z Distribution, Bearing area ratio plot, Slope
distribution); Roughness parameters; Volume parameters.
5.15.23.1 Channel Selection
• Select the channel to be viewed using the relevant button (e. g.: Ch1).
5.15.23.2 Generate Topography
The three buttons provided in the Generate button bar allow you to generate the topography in
different ways:
Maximum
• Click on the Maximum button to calculate the topography surface by finding the maximum intensity
value. If the optical section with the highest intensity value is found, the intensity values of both
neighboring slices are also taken into account, so that a 3 point maximum fit is calculated.
Center
• Click on the Center button to calculate the topography surface by using the center of gravity of all
summed up intensities of the stack for a given xy print.
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Display - Topo for LSM
LSM-FCS
Carl Zeiss
This mode provides better result for smooth surfaces of low intensity or nearly transparent
surfaces. The receiver gain and offset has to be properly tuned and MarkFirst- MarkLastpositions of the stack should be located approximately in the same distance from the real
surface.
First
•
Click on the First button to calculate the topography surface by using the first slice coming from the
top, where the intensity reaches the value defined by the lower intensity threshold.
This mode provides better result for surfaces of semitransparent materials with inclusions of
higher reflectivity or transparent multilayers with subsurface layers of higher signal intensity.
Extended First / Last Mode
1. Definition of an intensity (I) threshold.
2. Starting from top / bottom of a stack to find I = 400.
3. Search of a local maximum one FWHM of actual Z PSF forwards / backwards.
4. Search of the next local maximum one FWHM forwards / backwards from the last max until you have
not found any new local maximum.
5. Last local maximum is taken as surface point.
5.15.23.3 Topography Thresholds
Intensity threshold
Click on the Intensity button to calculate the topography surface by using the lower and the upper
intensity thresholds for image display. Use of this function is recommended to find the real surface in the
case of images with pronounced noise. All image pixels with intensity less or higher than the thresholds
set are ignored for the surface calculation.
• Click on the Intensity button to select the
intensity thresholds for the surface generation.
The Intensity Threshold window appears.
• Set the lower and upper intensity thresholds
using the appropriate sliders.
• Click on Close to
Threshold window.
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close
the
Intensity
Fig. 5-320
Threshold window
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LSM-FCS
Height threshold
Fig. 5-321
Z Threshold window
Click on the Height button to calculate the
topography surface by using the lower and the
upper height thresholds for image display. Use of
this function is recommended to get rid of
unwanted peaks and valleys taken into account for
parameter calculation. All topographic data with
height values less or higher than the thresholds set
are ignored for the display and parameter
calculation. This threshold applies both for 2D as
well as for 3D topography display modes.
• Click on the Height button to select the intensity thresholds for the surface generation. The Z
Threshold window appears.
• Set the lower and upper intensity thresholds using the appropriate sliders.
• Click on Close to close the Z Threshold window.
Auto Z
By clicking on the Auto Z button the surface topography is displayed in the Image Display window in
that way that it is automatically normalized to the lowest and highest Z value of the 3D topography.
•
Click on the Auto Z button. The topography is automatically normalized with respect to the highest
and lowest Z value.
5.15.23.4 Processing by Filtering
(1)
Topography smoothing
The three buttons in the Filter button bar allow activation / deactivation of the filter functions for
surface smoothing.
None button
Median /
Gauss / Aver. button
No filter for input data.
Smoothing of z data using a low-pass Median, Gauss or average filter.
Clicking on this button opens a selection box, where the number of
neighboring pixels to be used for filtering can be specified:
1st row: small smoothing via Median/Gauss filter (Median; 3 x 3; 5 x 5;
7 x 7)
2nd row: medium smoothing via Average (9 x 9; 11 x 11; 15 x 15)
3rd row: pronounced smoothing via Average (25 x 25; 35 x 35; 45 x 45)
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Carl Zeiss
• To investigate the effects of various filter modes, select one of the 3D display modes (Profiles, Grid,
Filled or Shaded) from the Display button bar.
• Click on the Median sub button to set the smoothing of the integrated Median filter.
Or
• Click on a Gauss or Average sub button and select the required degree of smoothing from the
selection box with a click of the mouse.
FFT button:
This function performs a Fast Fourier Transformation (FFT) in the
frequency range, applies highpass or lowpass filtering in the frequency
range and performs the inverse FFT.
• Click on the FFT button, the FFT Filter window opens.
• Click on the arrow in the filter Type select box to choose an adequate filter function:
–
Gauss Lowpass
–
Gauss Highpass
–
Butterworth Lowpass
–
Butterworth Highpass
• Select a position of the Cut off slider to display either the lower frequencies (waviness) with the
lowpass filters or the higher frequencies (roughness) with the highpass filters.
• The Cut off frequencies ranges from 1/1000 of the X dimension of the stack to four times of the X
dimension of the stack. The dimensions of the filtering is given in units of µm.
st
th
• Select a position of the Degree slider. The filter functions can be calculated from 1 order to 5 order
accuracy.
• Click on the Close button closes the FFT Filter window.
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(2)
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
LSM-FCS
Changing the topography geometry
The three buttons in the Geometry button bar allow the surface geometry to be changed.
Inverse button
Inverse surface. Depths change to heights, and vice versa.
Fit button
The following fit modes can be set:
1) No Fit
2) Plane
The topography is tilted in such a way that the mean deviation value
plane is calculated.
3) Cylinder fit correction
A cylinder form is eliminated, determination of micro roughness on
cylindrical surfaces can be performed.
4) Sphere fit correction
A spherical form is eliminated, determination of micro roughness on
spherical surfaces can be performed.
If Cylinder / Sphere fit is chosen, the Manu Tilt button is disabled.
You can display the exact values of the Cylinder / Sphere fit by
opening a context menu in the Image Display box with a click of the
right mouse button and selecting the Show processing parameter
function.
Manu Tilt button
Manual tilt correction.
Clicking on the Manu Tilt button activates the function (enabled). The
sliders for manual tilt correction are displayed on the right and below the
Image Display box. Vary the tilt by adjusting the horizontal and vertical
sliders or the arrows. The tilting angle is varied in steps of 1 degree. By
additional pressing of the Ctrl key, the tilting angle can be varied in steps
of 0.1 degrees. A yellow box showing the tilt angle currently set is
displayed next to each slider for checking purposes. A second click on the
Manu Tilt button ends this function and saves the setting (save and
disabled). The sliders for tilt correction disappear from the display.
You can also change the tilt angle directly in the Image Display box.
Click the left mouse button in the image and hold it down. Moving the
mouse pointer in horizontal or vertical direction tilts the topography by
an axis parallel or vertical to the screen. On releasing the mouse button,
the change of the tilt is stored and the function is deactivated (disabled).
To reset the manual tilt correction, click the Fit button.
• Click on the Inverse button for the inverse display of the topography. Clicking again will reset the
normal display.
• Correct the tilt via the Fit or Manu Tilt functions.
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Carl Zeiss
5.15.23.5 Display Modes
The three buttons in the Display button bar allow stacks to be displayed in the 2D, Iso-Lines or 3D
display mode.
(1)
2D modes
The following 2D modes can be set:
Intensity button: Display of projection of all intensities of the stack (black-and-white
display).
z Map button: Height coded color map with color bar.
Gradient button: Display of height gradient (slope), averaged pixelwise over all neighbors
(black-and-white display).
• Click on the 2D button in the Display button bar.
–
The 2D display mode selected last is activated. At the same time, an additional button bar is
displayed beside the 2D button permitting selection of the required 2D display mode.
• Select the required 2D display mode with a click of the mouse.
(2)
2D Iso-Lines display mode
Iso-Lines are lines which connect points of equal height on the topography.
The following 2D Iso-Lines display modes can be set:
Intensity button: Intensity projection superimposed with colored iso-lines (lines of equal
height).
z Map button: z Map function with black iso-lines.
Black button: White iso-lines on a black background.
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Display - Topo for LSM
Carl Zeiss
LSM-FCS
• Click on the Iso-Lines button in the Display button bar.
–
The Iso-Lines display mode selected last is activated. At the same time, an additional button bar is
displayed beside the Iso-Lines button permitting selection of the required Iso-Lines display mode.
–
Below the Measure button bar, the Line Dist. and Line Offset sliders / input boxes are displayed.
• Select the required Iso-Line display mode by clicking the left mouse button.
The additional function elements of the Iso-Lines display mode have the following meaning:
Line Dist. slider: Changes the distance of the iso-lines.
Line Offset slider: Setting of the height level where the Iso-Lines display starts.
To apply the topography functions to a small portion of the Z Stack image use the Overlay
function (Overlay button) and cut out and store as new topographic evaluation via the Extract
Region function.
(3)
3D display
Topo animations are possible. The following 3D modes can be set:
Profiles button: Profile display.
Grid button: Grid display.
Filled button: Display of color shades.
Shaded button: Surface rendering. Can be combined with LUT. Topo animations are
possible.
• Click on the 3D button in the Display button bar.
–
The 3D display mode selected last is activated. At the same time, an additional button bar appears
beside the 3D button to permit selection of the required 3D display mode.
–
Below the Measure button bar, the Scaling button bar and the Profile Dist. and Fill Level
sliders / input boxes are displayed.
–
The Image Display box contains one horizontal and two vertical scrollbars for the setting of the
image viewing angle.
• Select the required 3D display mode with a click of the mouse.
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Display - Topo for LSM
LSM-FCS
Carl Zeiss
The additional function elements of the 3D display mode have the following meaning:
Profile Dist. slider: Setting of the distance of profiles and the mesh value of the grid.
Fill Level slider: Used to push through a color LUT Look Up Table (e.g.: if the Rainbow
palette is used) in the Profiles / Filled display mode. In combination with the Volume
button, the filling of the flood function level of the topography can be varied for volume
measurements (see the Measurement functions paragraph).
Fill Holes procedure
• Intensity of a missing pixel of a hole has to be interpolated by the distance-weighted intensity of all
surrounding pixels.
• Fill hole algorithm is optimized for short calculation times.
The image viewing angle is set as follows:
Setting directly in the image
• Click in the image and hold down the mouse button. The perspective is changed by moving the
mouse button in horizontal or vertical direction.
Setting via scrollbars
• Move the
horizontal scrollbar to rotate the image around the vertical axis. The rotation angle is
displayed in the yellow display box.
• Move the left vertical scrollbar to rotate the image around the horizontal axis. The rotation angle is
displayed in the yellow display box.
• Moving the
right vertical scrollbar enables you to expand the image in height or to compress it,
while the Z-range between 10 % and 100 % of the X-range is scaled.
You can set the x, y and Z scales to an identical ratio by opening a context menu in the Image
Display box with a click of the right mouse button and selecting the Metric equal ratio
function.
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Carl Zeiss
LSM-FCS
The displayed boxes for rotation angle and relative scaling percentage value z : x ratio permit the setting
of identical perspectives for different images (e.g.: the plot of several topographies).
The Profiles and Filled 3D display modes permit a color palette (e.g.: Glowscale, Rainbow or
User defined) to be loaded or redefined by pressing the Palette button (see page 5-292).
5.15.23.6 Context Menu of the 3D Display
Mode
• Click in the Image Display box with the right
mouse button to open the context menu.
The context menu for the 3D mode currently
selected is displayed.
• Click on the required option with the left
mouse button to execute the function.
Fig. 5-322
Context menu of the
3D display mode (Profiles)
(1)
Metric equal ratio item
This option is available in all of the 3D display
modes.
After activation of the function, the x, y and z
scales are set to an identical ratio.
(2)
Export ... item
This option is available in the Profiles and Grid 3D
display modes.
Use the function to save the Profiles or Grid data
as a text file.
• Open the context menu with a click of the right
mouse button, then click on the option
Export ... with the left mouse button.
–
Fig. 5-323
5-346
Save As window
The Save As window is opened.
• Select the directory where you want the text
file to be stored, enter a file name and click on
Save.
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LSM-FCS
Carl Zeiss
A text file containing the topography in the form
of an XYZ matrix is generated.
Fig. 5-324
(3)
Topography matrix
Copy ... to clipboard item
This option is available in the Profiles and Grid 3D display modes.
After selection of this option, the Profiles or Grid data are copied as an XYZ matrix to the clipboard and
can be inserted in other programs using the Paste command.
(4)
Export x,y,z- triples item
This option is available in the Profiles and Grid 3D
display modes.
Use the function to save the Profiles or Grid data
as a text file.
• Open the context menu with a click of the right
mouse button, then click on the option
Export ... with the left mouse button.
–
The Save As window is opened.
• Select the directory where you want the text
file to be stored, enter a file name and click on
Save.
Fig. 5-325
Save As window
Fig. 5-326
Topography table
A text file containing the topography in the form
of an XYZ table is generated.
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Carl Zeiss
(5)
LSM-FCS
Copy x,y,z- triples to clipboard item
This option is available in the Profiles and Grid 3D display modes.
After selection of this option, the Profiles or Grid data are copied as an XYZ table to the clipboard and
can be inserted in other programs using the Paste command.
Please make sure that the amount of exportable data is adequate to the maximum importing
size of the following software package. To lower the amount of data points, use the profile
distance slider.
(6)
Render properties item
This option is only available in the Shaded 3D
display mode.
Fig. 5-327
Context menu of the
3D display mode (Shaded)
Use this function to vary the illumination
conditions, reflection properties and projection
settings of the topography. You can either select
preset Shading Models or use parameters
specifically defined as required.
The
specifically
defined
parameters
can
subsequently be stored as a Shading Model and
are then available at any time for further use.
Shading Models can also be deleted if no longer
needed.
Load a Shading Model
• Open the context menu with a click of the right
mouse button, then click on the option Render
properties with the left mouse button.
–
Fig. 5-328
3D Rendering window
The 3D Rendering window is opened.
• Click on the name of the required model in the
Shading Model List. The parameters are
immediately set for the current topography.
• Click on Close to close the 3D Rendering
window again.
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Display - Topo for LSM
LSM-FCS
Carl Zeiss
Defining a specific Shading Model
• Open the 3D Rendering window.
• Click on the Define button.
• Change the parameters of the topography
using the appropriate sliders.
• Save the settings by clicking on the Add to List
button. The Add Shading Model to List
window is displayed.
• Enter a name for the model and click on OK.
The model is included in the Shading Model
List.
Light panel
Determines the properties of illumination on a
sample.
Distance
Goes for diffuse and specular, see
visualization.
Azimuth
See visualization. Rise angle of the
"sun".
Elongation
See visualization. North-south
east-west direction of the "sun".
/
Fig. 5-329
3D Rendering window
Background Choose background color.
Material panel
Determines the reflective properties of a sample.
Ambient
Specular
Material properties; how many % of the light component are projected by the
material into which spectral ranges.
Shininess
Goes together with specular light. Shininess equal to 25 % determines diffuse light.
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Display - Topo for LSM
Carl Zeiss
LSM-FCS
Projection panel
Determines the reflective properties of a sample.
View angle
Determines the perspective, 0.0 parallel projection, central projection
Distance
Zoom function, zoom in, zoom out
Fig. 5-330
Render function visualization aid
A zoomed rendering setting permits the zoomed section to be moved via the cursor keys after a click on
the 3D window.
If a change of the 3D image angle follows, centration is made on the center again.
Deleting a Shading Model
• Select the model to be deleted in the Shading Model List, then click on the Remove button. The
model is deleted.
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(7)
Carl Zeiss
Renderer item
This option is only available in the Shaded 3D
display mode.
After selection of the Renderer item, the 3D
Renderer window appears. It allows the selection
of the hardware and software option which shall
be used for the 3D graphics calculation.
OpenGI - Software
The graphics calculation is performed using the
installed software.
OpenGI - Hardware
The graphics calculation is accelerated by using the
installed graphics processor.
Direct3D – Software / Hardware Rasterizer /
Hardware
These options can be used for offline versions of
the LSM 5 software for PC's with the WINDOWS
98 or 2000 operating system (not for WINDOWS
NT).
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Fig. 5-331
3D Renderer window
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OPERATION IN EXPERT MODE
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Display - Topo for LSM
Carl Zeiss
(8)
LSM-FCS
Show processing parameters
After selection of the Show processing parameters function, a reporting of the following applied topo
processing functionality is displayed on the right-hand side of the Image Display window:
–
Mode {calculation mode: Max, Center, First)
–
Threshold (applied intensity threshold)
–
Filter
–
Fit (plane, cylinder / sphere parameters).
Fig. 5-332
5-352
Show processing parameters
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Display - Topo for LSM
LSM-FCS
Carl Zeiss
5.15.23.7 Measurement Functions
The topography measurement functions are activated via the Measure button bar. The measurement
functions can be performed in the 2D or 3D display mode.
Automated convention in height statistics analysis:
Topo Filters
None,
<9x9
median, FFT High
FFT Low
>9x9
Data formats
Primary profile
Roughness
Waviness
2D profile
Pxx
Rxx
Wxx
n.a.
3D topography
SPxx
SRxx
SWxx
n.a.
The following measurement functions are available:
Diagram button: Diagram display. The Profile, z Histo, Curve of tp
and Grad. Histo diagram display modes can be activated via the
Diagram button and deactivated via the Off Diagram button. By
activation of the Diagram button, an additional button bar is displayed
for the selection of the required diagram or for deactivation. The labeling
of the Diagram button changes depending on which diagram display
mode has been activated.
Roughness button: Calculation of the roughness parameters.
Volume button: Calculation of the volume parameters.
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Carl Zeiss
(1)
LSM-FCS
Profile measurement mode in 2D display
• Select the required 2D display of the stack via the 2D button.
• Click on the Diagram button in the Measure button bar. Click on the Profile button in the button
bar displayed afterwards.
–
The Table and Profile button bars are displayed below the Measure button bar.
–
A colored arrow (intersection line of the profile) is displayed in the image and the profile diagram
appears below the image.
• If required, match the size of the Image Display window in order to obtain the complete display of
the profile diagram.
Fig. 5-333
5-354
Image Display window, Topography display: 2D - Profile
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LSM-FCS
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The additional Table / Profile buttons have the following functions:
Show button: The profile is displayed in the form of a table at the bottom below of the
Image Display window.
Copy button: The profile table is copied to the clipboard and can be transferred to other
programs (MS Word or MS Excel) via the Paste function.
Save button: The profile table can be stored as a text file (ASCII).
Arrow (selection) button: Activation of the mouse button for selection, resizing or
movement of the intersection line in the image.
Resizing: Click on the handle and hold down the mouse button, drag the handle, release
the mouse button.
Click on the line and hold down the mouse button, move the entire
Movement:
intersection line, release the mouse button.
Line with arrow button (open arrow): Creation of the intersection line to define the
position of the profile to be produced in the image. Click and hold the mouse button, drag
the line in any required direction, release the mouse button to end the procedure (The profile
diagram changes online.
Line button: This button allows you to determine the line thickness of the intersection line.
Measure button: Activates the Profile measurement mode in the profile diagram. The
required tools are displayed to the right of the profile diagram (see Profile measurement
mode, page 5-356).
z/x=1 button: Sets the z/x ratio in the profile diagram to the value 1. Check: the following
creation of a circle using the relevant tool really results in a circle in the profile display.
Measured angle values correspond to the actual slope of the line displayed.
Color button: Clicking on the Color button opens a color selection box in which the color
for the intersection line can be selected with a click of the mouse.
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Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
LSM-FCS
Profile measurement mode
button, the Profile
If you click on the
window with the tools of the profile measurement
mode appears.
This window can be moved as required over the
entire screen.
Fig. 5-334
Tools of the Profile measurement
mode
The tools of the Profile measurement mode have the following functions:
Zoom button: Zooming of a section of the profile diagram. Click and drag a rectangle over
the area to be enlarged in the profile diagram, release the mouse button to enlarge the
selected area. The zoom function can be performed several times. A click with the right
mouse button resizes the profile.
Marker button: Activation of the marker functions for the intersection line. The red and blue
marker lines in the profile diagram can now be moved using the mouse. After movement of
a marker line in the profile diagram, the relevant marker (red or blue circle) follows along the
intersection line in the 2D and Iso-Lines mode.
Arrow (selection) button: Activation of the mouse button for selection, resizing or
movement of one of the following drawing elements in the profile diagram.
Resizing: Click on the handle and hold down the mouse button, move the handle, release
the mouse button.
Movement: Click on the line and hold down the mouse button, move the entire drawing
element, release the mouse button.
Inclined Line button: Creation of a straight line in the profile diagram. Display of distance,
inclination angle, dxdy and dz. Click and hold down the mouse button, drag the line in any
required direction, release the mouse button to end the procedure.
Free angle button: Creation of a free angle in the profile diagram. Display of the enclosed
angle (max. 180 °). The first click sets the starting point, the second and third clicks define
the angle and the end point.
Rectangle button: Creation of a rectangle in the profile diagram. Display of distance, area,
height and width. Click and hold down the mouse button, drag the rectangle in any required
direction, release the mouse button to end the procedure.
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LSM-FCS
Carl Zeiss
Open Polyline button: Creation of an open polyline figure in the profile diagram. Display of
the length of the line figure. First click sets the starting point, any further click adds another
line, click with the right mouse button ends the procedure.
Closed Polyline button: Creation of a closed polyline figure in the profile diagram. Display
of the perimeter of the figure. First click sets the starting point, each further click adds
another line, a click with the right mouse button closes the figure and ends the procedure.
Open free-hand curve button: Creation of an open Bezier figure in the profile diagram.
Display of the length of the line figure. First click sets the starting point, each further click
adds another line, a click with the right mouse button ends the procedure.
Closed free-hand curve button: Creation of a closed Bezier figure in the profile diagram.
Display of the length of the line figure. First click sets the starting point, each further click
adds another line, a click with the right mouse button closes the figure and ends the
procedure.
Ellipse button: Creation of an ellipse in the profile diagram. Display of the area. First click
sets the center point, the displayed line permits the determination of the first dimension,
second click sets the first dimension, the second dimension and rotation direction can now
be determined, third click sets the second dimension and direction and ends the procedure.
Circle button: Creation of a circle in the profile diagram. Display of radius and area. Clicking
three times to define 3 points on the profile. A circle fit is automatically applied on the
profile.
Recycle bin button: Deletes all drawing elements or the one just selected.
Line width button: Change of the line width of the drawing elements.
Color button: Clicking on the Color button opens a color selection box where the color of
the drawing element can be selected with a click of the mouse.
x1- button: Resets the zoom factor of the profile diagram to its original size.
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OPERATION IN EXPERT MODE
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Display - Topo for LSM
Carl Zeiss
(2)
LSM-FCS
z Histo measurement mode in 2D display
• Click on the Diagram button in the Measure button bar. Click on the z Histo button in the
additional button bar now displayed.
The lower part of the Image Display box shows the 3D height distribution of the topography.
Fig. 5-335
5-358
Image Display window, Topography display: 2D - Histo
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(3)
Carl Zeiss
Curve of tp measurement mode in 2D display
• Click on the Diagram button in the Measure button bar. Click on the Curve of tp button in the
additional button bar now displayed.
–
(4)
The curve of the bearing area ratio as a function of the height is displayed below the image (also
see 3D measurement functions, page 5-365).
Grad. Histo measurement mode in 2D display
• Click on the Diagram button in the Measure button bar. Click on the Grad. Histo button in the
additional button bar now displayed.
The lower part of the Image Display box shows the gradient distribution of the topography. Before
creation of the slope diagram, the image should be filtered at least once using a low-pass filter, since
otherwise the rough height gradation of the image will result in a comb-shaped histogram. The RootMean-Square Slope (RMS Slope) parameter is calculated and displayed below the chart. The following
formula is used for calculation:
RDQ =
(5)
(
) (

1
 z xi , y j − z xi −1 , y j
⋅ ∑ ⋅∑ ⋅ 
( N x − 1) ⋅ ( N y − 1) i =1 j =1 
∆x

Nx
Ny
) 
(
2
) (
z x , y − x , y
i
j
i
j −1
+
∆y
 
 
)  
2
 
 
Roughness measurement mode in 2D display (Profile display)
2D Amplitude parameters (Profile Roughness):
Mean height z
Rc
Pc
Wc
Arithmetic mean deviation
Ra
Pa
Wa
Root mean square deviation
Rq
Pq
Wq
Asymmetry
Skewness
Rsk
Psk
Wsk
Sharpness
Kurtosis
Rku
Pku
Wku
Extremes
Highest peak
Rp
Pp
Wp
Lowest valley
Rv
Pv
Wv
Absolute peak to valley
Rt
Pt
Wt
Averaged peak to valley
Rz
Pz
Wz
Maximum peak to valley
Rmax
Pmax
Wmax
FFT High
No, M
FFT L
Dispersion
If choosen filters are
• Click on the Profile button in the Measure button bar.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
Carl Zeiss
LSM-FCS
• Click on the Roughn. button in the Measure button bar.
–
The roughness parameters are calculated and displayed on the left below the image. All roughness
parameters calculated from a 2D profile are named with R.
–
The Copy button is displayed below the right-hand side of the image. This button permits the
roughness parameters to be copied to the clipboard and imported to another program (e.g.: MS
Word or MS Excel) via the Paste function.
The following roughness parameters are calculated (e.g. for a Y-section)
–
Mean height of all profile height values Rc
N
y
1
− Rc =
⋅ ∑ ⋅ z (x, y j )
N y j =1
–
Arithmetic mean deviation of all profile height values Ra
N
[
y
1
− Ra =
⋅ ∑ ⋅ z (x, y j ) − Rc
N y j =1
–
]
Quadratic mean deviation of all profile height values Rq
N
− Rq =
–
Nx, Ny ... number of pixels in X- or Y-direction
[
y
1
⋅ ∑ ⋅ z (x, y j ) − Rc
N y j =1
]
2
Skewness of the distribution of all profile height values RSK
N
RSK =
–
y
1
⋅
⋅ z 3 ( x, y j )
3 ∑
N y ⋅ Rq j =1
Kurtosis of the distribution of all profile height values RKU
N
RKU =
–
y
1
⋅
⋅ z 4 ( x, y j )
4 ∑
N y ⋅ Rq j =1
Maximum peak height RP
RP = z max − Rc
–
Maximum valley depth RV
RV = Rc − z min
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
LSM-FCS
–
Carl Zeiss
Maximum roughness depth Rt (= Peak to Valley / PV)
− Rt = zmax − zmin
maximum height difference of the overall topography along a profile.
Classification of topography in 5 equal area elements (rectangles in the 2D mode)
–
average roughness depth Rz:
− Rz =
zmax 1 − zmin 1 + zmax 2 − zmin 2 + zmax 3 − zmin 3 + zmax 4 − zmin 4 + zmax 5 − zmin 5
5
Averaging of Rt-values of all the 5 single area elements. When combined, both parameters
provide information about the homogeneity of the surface. Big differences are indicative of
pronounced inclination of the overall area or of spikes.
Developed Surface Area Ratio: Σ (surface areaij) / Σ (projected areaij)
The percentage of the 3D surface area (sum off all triangles formed by adjacent data points) to
the 2D surface area produced by projecting the 3D surface onto the threshold plane.
–
maximum roughness depth Rmax:
− Rmax = Max ( zmax 1 − zmin 1 , zmax 2 − zmin 2 , zmax 3 − zmin 3 , zmax 4 − zmin 4 , zmax 5 − zmin 5 )
maximum of Rt-values of all the 25 single area elements.
Both the roughness parameters and the z-histogram can be changed by using filters!
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
Carl Zeiss
(6)
LSM-FCS
Roughness measurement mode in 3D display
3D Amplitude parameters (Topography Roughness):
Mean height z
SRc
SPc
Wc
Arithmetic mean deviation
SRa
SPa
Wa
Root mean square deviation
SRq
SPq
Wq
Asymmetry
Skewness
SRsk
SPsk
Wsk
Sharpness
Kurtosis
SRku
SPku
Wku
Extremes
Highest peak
SRp
SPp
Wp
Lowest valley
SRv
SPv
Wv
Absolute peak to valley
SRt
SPt
Wt
Averaged peak to valley
SRz
SPz
Wz
Maximum peak to valley
SRmax
SPmax
Wmax
FFT High
No, M
FFT L
Dispersion
If choosen filters are:
• Click on the Roughn. button in the Measure button bar.
–
The roughness parameters are calculated and displayed on the left below the image. All roughness
parameters calculated from a 3D topography are named with S.
–
The Copy button is displayed below the right-hand side of the image. This button permits the
roughness parameters to be copied to the clipboard and imported to another program (e.g.: MS
Word or MS Excel) via the Paste function.
The following roughness parameters are calculated:
–
Mean height of all surface height values Sc
N
Nx
y
1
− Sc =
⋅ ∑ ⋅∑ ⋅ z (xi , y j )
N x ⋅ N y i =1 j =1
–
Arithmetic mean deviation of all surface height values Sa
N
[
Nx
y
1
− Sa =
⋅ ∑ ⋅∑ ⋅ z (xi , y j ) − S c
N x ⋅ N y i =1 j =1
5-362
Nx, Ny ... number of pixels in X- or Y-direction
]
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
LSM-FCS
–
Quadratic mean deviation of all surface height values Sq
N
− Sq =
–
Carl Zeiss
[
Nx
y
1
⋅ ∑ ⋅∑ ⋅ z (xi , y j ) − S c
N x ⋅ N y i =1 j =1
]
2
Skewness of the distribution of all surface height values SSK
N
S SK
–
Nx
y
1
=
⋅
⋅
⋅ z 3 (xi , y j )
∑
∑
3
N x ⋅ N y ⋅ S q i =1 j =1
Kurtosis of the distribution of all surface height values SKU
N
S KU =
–
Nx
y
1
⋅
⋅
⋅ z 4 (xi , y j )
4 ∑ ∑
N x ⋅ N y ⋅ S q i =1 j =1
Maximum peak height SP
S P = z max − S c
–
Maximum valley depth SV
SV = S c − z min
–
Maximum roughness depth St (= Peak to Valley / PV)
− S t = z max − z min
maximum height difference of the overall topography.
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5-363
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
Carl Zeiss
LSM-FCS
Classification of topography in 25 equal area elements (rectangles in the 2D mode)
–
average roughness depth Sz:
− Sz =
z max 1 − z min 1 + z max 2 − z min 2 + ⋅ ⋅ ⋅ + z max 25 − z min 25
25
Averaging of Rt-values of all the 25 single area elements. When combined, both parameters
provide information about the homogeneity of the surface. Big differences are indicative of
pronounced inclination of the overall area or of spikes.
–
maximum roughness depth Smax:
− S max = Max ( z max 1 − z min 1 , z max 2 − z min 2 , ⋅ ⋅ ⋅ , z max 25 − z min 25 )
maximum of Rt-values of all the 25 single area elements.
Both the roughness parameters and the z-histogram will be influenced by the use of filters!
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
LSM-FCS
Carl Zeiss
5.15.23.8 3D Measurement Functions
(1)
Volume measurement mode (Flood function)
• Use the 3D button to select the required 3D display of the stack.
• Click on the Volume button in the Measure button bar.
–
The volume parameters are calculated and displayed below the image.
–
The Copy button is displayed below the right-hand side of the image. This button permits the
volume values to be copied to the clipboard and imported to other programs (e.g.: MS Word or
MS Excel) via the Paste function.
• Setting the Fill Level slider enables you to change the height level of the topography. The portion of
the topography lying below the set height level is filled with "water" (blue color) and the volume
parameters are calculated online only for the projecting part of the topography.
To use the Fill Level function, load the Profiles 3D display mode containing the Glowscale
palette, or activate No Palette to obtain optimum display.
• If the Diagram function Curve of tp is also activated, a red marker line shows the position of the
height level in the percentage of contact area curve.
Fig. 5-336
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Image Display window, Topography display: 3D - Volume
5-365
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
Carl Zeiss
LSM-FCS
The following parameters are calculated:
Z:
height level (selectable with the Z-Threshold and Fill Level sliders). The setting of this value
influences the following parameters.
Vm (z):
material volume above chosen height level
Vv (z):
void volume below chosen height level
Smr (z):
material volume ratio
S mr ( z ) =
Svr (z):
Vm ( z )
Vm ( zmin )
void volume ratio
Svr ( z ) =
Vv ( z )
Vv ( zmax )
Au:
surface bearing area of the topography at Z (= projection area of those parts which are
situated above chosen height level)
Smr:
surface bearing area ratio of the topography at Z
percentage of contact area (= Au / ( x * y) * 100 %)
Sda:
true surface = sum of all triangles formed by adjacent data points of the surface
reconstruction
Sdr:
developed surface area ratio:
Σ (surface areaij) - Σ (projected areaij) / Σ (projected areaij) * 100 %
projected area = x * y
The percentage of the 3D surface area (sum of all triangles formed by adjacent data points
of the surface reconstruction) to the 2D surface area produced by projecting the 3D surface
onto the threshold plane.
is equal to base plane (Sdr = 0 %)
absolute flat surface
The increase by which the 3D surface is larger than the basic plane (e. g. 625 % is a 3D
surface which is about 6.25 times larger than the projected basic plane)
⇒
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
LSM-FCS
(2)
Carl Zeiss
Profile measurement mode in 3D display
This function is performed in the same way as in the 2D display mode, with the following exceptions:
are replaced with the buttons
and
and
. Furthermore, the Position slider and
The buttons
the input box (information of the position of the intersection line in pixels) are displayed below the Table
and Profile button bar. Changing the Z-Threshold also results in a change in the profile. In the 3D
image, a red marker line shows the y- and x-position of the displayed profile diagram.
red
marker line
(x position)
Fig. 5-337
Image Display window, Topography display: 3D - Volume
• The position of the marker line (profile intersection line) can be changed by moving the Position
slider in x or y.
• Press the x- or y-button to select the required intersection plane.
(3)
z Histo measurement mode in 3D display
This function is performed in the same way as in the 2D display mode.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
Carl Zeiss
(4)
LSM-FCS
Curve of tp measurement mode in 3D display
This function is performed in the same way as in the 2D display mode.
Before determination of the tp bearing portion, individual peaks (noise, steep slopes) must be eliminated.
The Median filter and perhaps a 3x3 longpass filter can be used for this purpose.
Fig. 5-338
Image Display window, Topography display: 3D – Curve of tp
Shifting the two cursor crosses permits two bearing portions to be given in percent (e.g. Smr1 = 10 %;
Smr2 = 90 %) as default values for which the height difference Rdc is determined automatically.
(5)
Grad. Histo measurement mode in 3D display
This function is performed in the same way as in the 2D display mode.
(6)
Roughness measurement mode in 3D display
This function is performed in the same way as in the 2D display mode.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Topo for LSM
LSM-FCS
Carl Zeiss
5.15.23.9 Export data
–
multiple profiles (Rel. 3.2)
–
single profile
–
parameters
–
topography as matrix
–
topography as triples
5.15.23.10 Topo ReUse
Topo routines can be saved and reloaded as tgp-files (TopoGraphic Parameters).
These files include settings for:
–
reconstruction mode,
–
intensity threshold,
–
filters (including FFT),
–
tilt angles (manual, 3 point fit),
–
fit procedures (plane, cylinder, sphere),
–
inverse and
–
fill holes.
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5-369
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Prev.
Carl Zeiss
5.15.24
LSM-FCS
Display - Prev.
This function allows to
–
–
–
compose images, graphs and text for printing
use any image format
change fonts and line width in graphs via context sensitive menus
The settings of Chan, Zoom, Slice, Contr and Palette apply.
In the Options menu in the function Settings with the Print Status Display tab parameters are
determined and the Print Status Information is activated/deactivated.
Click on Prev will display the Preview window and the Print toolbar. Any changes done with this
toolbar are effective immediately. The content of the overlay plane is temporarily deleted while the
toolbar is displayed.
Assembly of image,
intensity profile and
scan info
Fig. 5-339
(1)
Image Display window, Prev display
Context menu for scan information text
The context menu (right mouse button) allows to vary the output of the scan info.
• Click with the right mouse button. A context menu with the options Color and Font is displayed.
• In the Color menu, you can select a different type color for the scan info, in the Font menu a
different type font and type style.
(2)
5-370
Context menu for Topography Images
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Prev.
LSM-FCS
Carl Zeiss
When transferring a topography to the print preview, you can change the size and shape of type and
scale lines for the 3D graphics and profile measurement results.
(a)
Context menu for 3D graphics
• Click on the right mouse button. A context menu with the options Font enlargement and Line
width enlargement is displayed.
• You can change the type size in the Font enlargement menu and the line width of the scales in the
Line width enlargement menu.
(b)
Context menu for Profile measurement function
• Click on the right mouse button. A context menu with the options Scaling font enlargement,
Marker font enlargement and Overlay font enlargement is displayed.
• You can change the type font in the Scaling font enlargement menu, the size of the marker table
in the Marker font enlargement menu and the type size of the red measurement results in the
Overlay font enlargement.
(3)
Arranging
Preview
and
printing
the
Print
• Click on the Arrange button for optimum
layout of image size and position relative to the
textual information.
• A layout generated with Prev. (Preview) can be
printed by clicking on the Print button in the
Print toolbar.
• Clicking on the Setup button opens the Print
Setup window, in which you can specify print
parameters.
• Click on the slider to change the zoom value of
the selected items.
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Fig. 5-340
Print Setup window
5-371
OPERATION IN EXPERT MODE
Display and Analysis of Images
Display - Info
Carl Zeiss
5.15.25
LSM-FCS
Display - Info
This function allows to
–
display the parameters used during image acquisition of the image(s) displayed in the Image Display
window
–
use any image format
–
remove the info display
The settings of Chan, Zoom, Slice, Contr and Palette are not relevant for this function.
In the Options menu in the function Settings with the Image Status Display tab parameters to shown
are determined.
Click on Info will show the parameters. Click again to hide the info display.
Image parameters
Fig. 5-341
5-372
Image Display window, Info display
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Mode in Time Series
LSM-FCS
5.15.26
Carl Zeiss
Additional Display Mode in Time Series
5.15.26.1 Display - Mean
This function allows to
–
display the intensity time diagram (mean intensity in user defined ROIs over time)
–
use frame time series and frame Z Stack time series as input
–
show the intensity values in table form and copy table to clipboard or save as text file
–
show separate diagrams for each channel in a multi channel image
The settings of Chan, Zoom, Slice, Contr and Palette apply.
Click on Mean will display the Mean of ROIs toolbar. Any changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
To use a similar functionality while scanning use the optional Mean of ROI function with the Time
series control.
• Click on the Mean ROI button.
–
The Mean of ROIs image display toolbar will be displayed on the right. The used ROIs become
visible in the image, and the Intensity-Time diagram is shown on the left of the Image Display
window.
Fig. 5-342
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Image Display window, Mean ROI display
5-373
Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Mode in Time Series
LSM-FCS
The Mean of ROIs toolbar contains the following function elements:
ROIs selection box: Display of the ROIs used during scanning of the time
series and of the other ROIs available in the system.
Add button: Opens the Add ROI List window for the storage of
changed or newly defined ROIs under a new name.
Remove button: Deletes the selected ROI from the ROIs selection box.
ROI data: Display of the data of the ROI selected from the ROIs selection
box. On deactivation of the check box of a ROI, its intensity values from
the Intensity-Time diagram are not displayed.
Arrow button: Activation of the mouse button for resizing or movement
of the ROI in the Image Display window.
Bezier button: Activates the Bezier figure drawing mode. The first click
sets the starting point, each additional click adds a further line, a doubleclick on the starting point closes the figure and ends the procedure.
Circle button: Activates the circle drawing mode. Clicking and holding
down the mouse button sets the center point; drag the diameter and
release the mouse button to end the procedure.
Recycle bin button: All the ROIs to the image are deleted.
Rectangle button: Activates the rectangle drawing mode. Click and hold
down the mouse button, drag the rectangle in any direction, release the
mouse button to end the procedure.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Mode in Time Series
LSM-FCS
Carl Zeiss
Ellipse button: Activates the ellipse drawing mode. The first click sets the
center point, the displayed line permits the determination of the first
dimension, the second click sets the first dimension, the second
dimension and the rotation direction can then be determined; the third
click sets the second dimension and the direction and ends the
procedure.
Polyline button: Activates polyline drawing mode. The first click sets the
starting point, each additional click adds a further line, a double-click on
the starting point closes the figure and ends the procedure.
Line button: This button allows you to determine the line thickness of
the ROI outline.
Color / Auto button: One color from the list of colors can be assigned to
all ROIs. When Auto is pressed, the outlines of all ROIs are automatically
colored differently.
Buttons for diagram display:
1 button: Intensity values for ROIs and channels are shown in one
diagram.
Chan button: Intensity values are shown separately for each channel.
ROI button: Intensity values are shown separately for each ROI.
Mono button: Change between color and monochrome display of the
intensity time diagrams.
Area button: Display of the area of the ROI in the intensity time diagram,
depending on the set threshold values. Area measurements of very small
areas (< 10 pixels) give only approximate values.
Mean button: Display of the mean values of the relevant ROI in the
intensity time diagram.
Ch1 / Ch3 / Ch4 button: Selection of the channel to be used.
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5-375
Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Mode in Time Series
LSM-FCS
Threshold low slider: The intensity values below threshold are not
displayed for the Area function.
Threshold high slider: The intensity values above threshold are not
displayed for the Area function.
Buttons for Table functions:
Copy Table button: The table of intensity values is copied to the
clipboard.
Show Table button: The table of intensity values is displayed on the
bottom left of the Image Display window.
Save Table button: The table of intensity values can be stored as a text
file.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Modes in Lambda Mode
LSM-FCS
5.15.27
Carl Zeiss
Additional Display Modes in Lambda Mode
The following display modes of a Lambda Stack are available:
–
Display - Coded
–
Display - Max
–
Display - Mean
Fig. 5-343
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Image Display window of a Lambda Stack, Display - Mean
activated
5-377
Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Modes in Lambda Mode
LSM-FCS
5.15.27.1 Display - Coded
This function allows to
–
display a Lambda Stack of images in a coded color view. A color palette is automatically assigned to
the individual images which displays the spectral color of the most intense signal of a Lambda Stack
The settings of Chan, Zoom, Slice and Contr apply. The settings of Palette do not apply.
Click on Coded will be immediately effective.
5.15.27.2 Display - Max
This function allows to
–
display a Lambda Stack of images in a maximum intensity projection.
The settings of Chan, Zoom, Slice, Contr and Palette apply.
Click on Max will be immediately effective.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Modes in Lambda Mode
LSM-FCS
Carl Zeiss
5.15.27.3 Display - Mean
This function allows to
–
display the intensity Lambda diagram (mean intensity in user defined ROIs over Lambda)
–
use Lambda Stack, Lambda Stack time series, Lambda Stack Z series and Lambda Stack (Z+time) series
as input
–
show the intensity values in table form and copy table to clipboard or save as text file
–
show separate diagrams for each ROI or all in one diagram
–
generate unmixed multi channel images
The settings of Chan, Zoom, Slice, Contr and Palette apply. The settings of Overlay do not apply.
Click on Mean will display the Mean of ROIs toolbar. Any changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
The Mean of ROIs toolbar contains the following function elements:
ROIs selection box: Display of the ROIs used during scanning of the time
series and of the other ROIs available in the system.
Add button: Opens the Add ROI List window for the storage of
changed or newly defined ROIs under a new name.
Remove button: Deletes the selected ROI from the ROIs selection box.
ROI data: Display of the data of the ROI selected from the ROIs selection
box. On deactivation of the check box of a ROI, its intensity values from
the Intensity-Time diagram are not displayed.
Arrow button: Activation of the mouse button for resizing or movement
of the ROI in the Image Display window.
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5-379
Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Modes in Lambda Mode
LSM-FCS
Bezier button: Activates the Bezier figure drawing mode. The first click
sets the starting point, each additional click adds a further line, a doubleclick on the starting point closes the figure and ends the procedure.
Circle button: Activates the circle drawing mode. Clicking and holding
down the mouse button sets the center point; drag the diameter and
release the mouse button to end the procedure.
Recycle bin button: All the ROIs to the image are deleted.
Rectangle button: Activates the rectangle drawing mode. Click and hold
down the mouse button, drag the rectangle in any direction, release the
mouse button to end the procedure.
Ellipse button: Activates the ellipse drawing mode. The first click sets the
center point, the displayed line permits the determination of the first
dimension, the second click sets the first dimension, the second
dimension and the rotation direction can then be determined; the third
click sets the second dimension and the direction and ends the
procedure.
Polyline button: Activates polyline drawing mode. The first click sets the
starting point, each additional click adds a further line, a double-click on
the starting point closes the figure and ends the procedure.
Crosshair button with user-defined spot size for Lambda Stacks.
Line button: This button allows you to determine the line thickness of
the ROI outline.
Color / Auto button: One color from the list of colors can be assigned to
all ROIs. When Auto is pressed, the outlines of all ROIs are automatically
colored differently.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Modes in Lambda Mode
LSM-FCS
Carl Zeiss
Buttons for Image display:
Single button: Displays only a single slice out of a Lambda Stack.
Coded button: Displays the Lambda Coded mode (see chapter 0).
Max button: Displays the Lambda Max mode (see chapter 5.15.27.2).
Buttons for diagram display:
1 button: Intensity values for ROIs and channels are shown in one
diagram over Lambda.
ROI button: Intensity values are shown separately for each ROI over
Lambda.
2.5 D button: 3D graph of intensity, wavelength and time of ROIs in
Lambda Stacks.
Mono button: Change between color and monochrome display of the
intensity time diagrams over Lambda.
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5-381
Carl Zeiss
OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Modes in Lambda Mode
LSM-FCS
Buttons for Table functions:
Copy Table button: The table of intensity values over Lambda is copied
to the clipboard.
Show Table button: The table of intensity values over Lambda is
displayed on the bottom left of the Image Display window.
Save Table button: The table of intensity values over Lambda can be
stored as a text file.
The toolbar below the intensity Lambda diagram has the following functions:
ChS1 button / slider
Selection of a wavelength range (use the sliders) assigned with the
spectral channel ChS1. This assignment can be used for scanning (when
Apply to hardware is pressed, see Config Control) and /or for
extracting a single or multi channel image from the Lambda Stack (when
Extract channels is pressed). Clicking on the color box in the ChS
buttons opens the color selection box and allows a new color assignment
to be made for this channel.
Channel + / − button
Add (+) or reduce (-) the number of channels displayed and available for
assignment. Up to 8 channels labeled ChS1, ChS2, ChS3 ... ChS8 are
available.
Extract channels button
Generates a new image or multicolor images based on the settings made
in the ChS1, ChS2, ChS3 ... ChS8 buttons. Two or more Lambda
channels are binned to form the channels ChS1 ... 8. The generated
image is displayed in a new Image Display window and is no longer a
Lambda Stack of images.
Linear Unmixing button
Performs the unmixing with the selected spectra.
Apply to hardware button
Sets the parameters for the META detector used in binning mode as
specified in the settings ChS1, ChS2, ChS3 ... ChS8. See Single Track
and Multi Track in Config Control for the effect of the action. The
Lambda Stack setting is not affected by this action.
Save to spectra DB button
The values of the intensity Lambda diagram are saved in a file (extension:
*.rls) in the aim/macro/roi directory. Make sure to have displayed only
data of single dyes in the intensity Lambda diagram when executing this
function.
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Modes in Lambda Mode
LSM-FCS
Carl Zeiss
5.15.27.4 Functionality for Emission Fingerprinting
Extract Channels
–
Sum or mean signals from selected detector
elements
–
Autoscaling of output channels (individually)
Linear Unmixing
–
Autoscaling of output channels
–
Generate an additional "Channel with
Residuals"->
pixel-by-pixel
display
the
difference between fit and original data (for the
channel of the Lambda Stack that shows the
greatest deviation)
–
Ignore negative results
–
Ask for ROI characterizing background spectra
before Linear Unmixing
–
Spectra Display with option Normalize (right
mouse click into spectra graph)
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Fig. 5-344
Extract and Unmix Properties
window
Fig. 5-345
Context menu with Normalize
function
5-383
OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Modes in Lambda Mode
Carl Zeiss
–
Fig. 5-346
5-384
Deselect of individual channels in Lambda Stack
affects:
–
Display in Lambda Coded and Lambda
max modes, if Chan button in Select
toolbar activated
–
Linear unmixing; deselected channels are not
included
Channels toolbar
–
Fig. 5-347
LSM-FCS
Spectra Database with subfolders
Subfolder of Spectra Database
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OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Modes in Lambda Mode
LSM-FCS
Carl Zeiss
5.15.27.5 Automatic Component Extraction
The use of this function permits the automatic search for the individual reference spectra in a lambda
stack.
(1)
Open / Close the Mean of ROI window
• Click on the Mean of ROI button in the image display window.
• Click on another button to select a different function.
Fig. 5-348
Image Display window of a Lambda Stack, Display - Mean activated
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5-385
OPERATION IN EXPERT MODE
Display and Analysis of Images
Additional Display Modes in Lambda Mode
Carl Zeiss
(2)
LSM-FCS
Function description
ACE button
Opens the component number window; after selecting an appropriate
number of labels in your sample, press OK.
Spectra checkboxes
Select required spectra. ACE generated spectra and interactively selected
spectra can be mixed here.
Drawing Tools
For interactive search and selection of spectra in the image display
window.
Linear Unmixing button
Performs the unmixing with the selected spectra.
• Select number of dyes present.
• Click OK.
• View results in graph.
Fig. 5-349
Automatic Component Extraction function
Restriction:
Little to moderate spatial overlap of emission signals
Benefit:
Solution for Emission Fingerprinting in cases where reference spectra are
not accessible via single-labeled controls
5-386
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OPERATION IN EXPERT MODE
Image Optimization
Single Channel
LSM-FCS
5.16
Image Optimization
5.16.1
Single Channel
Carl Zeiss
Described below is the example of the acquisition of an image, using an excitation wavelength of
543 nm and a fluorescence emission range above 560 nm. The HFT 488/543 is used as the main dichroic
beam splitter.
Let the specimen be a thin section through a stem of Convallaria majalis (Lily-of-the-Valley).
5.16.1.1
Requirements
• The suitable laser is switched on.
• The specimen has been positioned and focused
for scanning.
• Click on the LSM button in the Acquire
subordinate toolbar of the Main menu. The
tube slider on the microscope tube is in the
LSM position and the LSM button is activated.
• Click on the Config button in the Acquire
subordinate toolbar of the Main menu.
–
This opens the Configuration Control
window.
• Click on the Single Track button.
Ch3 icon and assign a color to
• Click on the
Channel 3 in the Channel Color Selection
window. Activate channel 3 via check box.
icon of emission filter 3 (before
• Click on the
Ch3) and select the LP 560 filter.
• If required, deactivate all the other channels
(ChS, Ch2, 4, monitor diode, transmission,
R1-2) via check box.
Fig. 5-350
Configuration Control window
icon of the main dichroic beam
• Click on the
splitter and select HFT 488/543.
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5-387
OPERATION IN EXPERT MODE
Image Optimization
Single Channel
Carl Zeiss
LSM-FCS
icon, activate the 543 nm
• Click on the
laser line and click on Line Active . If
required, deactivate other laser lines which are
not needed.
• Use the Transmission slider to set the laser
intensity to approx. 30 % at first.
The Beam Path and Channel Assignment panel
displays the current configuration loaded.
Fig. 5-351
Excitation panel
The set laser intensity must be
subsequently optimized for the current
situation via the Transmission slider.
For overlaying fluorescence and transmitted-light
images, click on the ChD (Transmission) button in
the Beam Path and Channel Assignment panel.
The transmitted light PMT photomultiplier will be
activated.
Of course, all other transmitted light applications
like
– phase contrast
–
differential interference contrast (DIC)
–
polarization contrast (Pol)
–
darkfield
can also be performed.
For the generation of images in
reflection, the main dichroic beam splitter
must be a neutral-density filter.
Standard equipment contains a neutraldensity filter with a division ratio of 80 to
20 % (at 543 nm).
Fig. 5-352
5-388
Configuration Control window
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OPERATION IN EXPERT MODE
Image Optimization
Single Channel
LSM-FCS
Carl Zeiss
• In the Main menu click on the Scan button in
the Acquire subordinate toolbar.
–
This opens the Scan Control window.
• Click on the Mode button.
• For a frame scan, click on the Frame button.
• On the Objective Lens & Image Size panel,
select Objective and Frame size for the scan
(e.g. X 512 / Y 512 scan).
• On the Speed panel, enter a scanning speed of
7, for example, to start with.
• Start with the following settings on the Pixel
Depth, Scan Direction & Scan Average
panel:
Data depth:
Scan direction:
Average:
8 bits
unidirectional
Number: 1
• On the Zoom, Rotation & Offset panel, set a
zoom of 1 and a rotation of 0.
Using the Fast XY button is a convenient
way of creating an overview scan.
Fig. 5-353
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Scan Control window (Mode)
5-389
Carl Zeiss
OPERATION IN EXPERT MODE
Image Optimization
Single Channel
LSM-FCS
• Click on the Channels button.
–
This displays the preset parameters of the
configuration loaded.
• Click on the Find button. Make sure to position
the slider correctly. Then scan while the slider is
in the LSM position.
–
This starts the scanning process.
–
The image is seen to build up gradually in a
new window.
Function Find produces images of
different brightness for different scan
speeds.
Fig. 5-354
5-390
Scan Control window (Channels)
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OPERATION IN EXPERT MODE
Image Optimization
Single Channel
LSM-FCS
Fig. 5-355
Carl Zeiss
Image Display window
As a rule, the first scanned image (Pre-Scan) is not ideal, since the photomultiplier is not matched to the
light output. More often than not, the screen image is dull and needs subsequent optimization.
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5-391
Carl Zeiss
OPERATION IN EXPERT MODE
Image Optimization
Single Channel
5.16.1.2
LSM-FCS
Pinhole / Detector Gain /
Ampl. Offset / Ampl. Gain
• In the Scan Control window, click on the
Cont. button (see Fig. 5-354).
–
This starts a continuous scan.
• Use the Pinhole slider to set the pinhole
diameter in the Scan Control window under
Channels.
Fig. 5-356
5-392
Image Display window with
confocal ROI
–
The pinhole diameter should be so small that
there is still enough variation for the setting
of the detector gain and that sufficient
image information is still available. 1 Airy is a
good value to enable a confocal
fluorescence XY-image to be obtained.
–
A small pinhole diameter will increase the
depth of focus, but reduce the light intensity
received by the PMT photomultiplier (for
reflection mode confocal images start with a
pinhole value of 0.5 Airy Units).
–
The influence of the pinhole diameter on
image creation is shown by the example in
Fig. 5-356. The entire image was first
scanned with too large a pinhole diameter.
The pinhole diameter was then optimized for
a defined ROI. This considerably improved
the display of the specimen structures.
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OPERATION IN EXPERT MODE
Image Optimization
Single Channel
LSM-FCS
Carl Zeiss
• Click on the Palette button in the Select image
processing toolbar.
–
This opens the Color Palette window.
• In the Color Palette List panel, click on the
Range Indicator item.
–
The scanned image appears in a false-color
presentation.
Fig. 5-357
Color Palette window
Fig. 5-358
Image Display window
If the image is too bright, it appears red on the
screen.
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5-393
OPERATION IN EXPERT MODE
Image Optimization
Single Channel
Carl Zeiss
LSM-FCS
If the image is not bright enough, it appears blue
on the screen.
• On the Channel Settings panel of the Scan
Control window, set the PMT (photomultiplier)
gain with the Detector Gain slider.
Fig. 5-359
–
The image should not have more than a
trace of red.
–
This adjustment is very sensitive. Try using
the left and right arrows to make the
adjustment instead of dragging the slider
bar.
Image Display window
• To adjust the black level (background), use the
Ampl. Offset slider so that areas without
picture content just show a trace of blue.
• If necessary, re-amplify brightness with the
Ampl. Gain slider.
Do not change the Ampl. Gain setting
unless the settings made so far are
insufficient for image optimization.
• In the Color Palette List panel of the Color
Palette window, click on No Palette.
–
Fig. 5-360
Image Display window
This deselects the Range Indicator and
activates the new presentation.
• In the Scan Control window, click on the Stop
button.
–
This stops the continuous scan.
If you use the Range Indicator for image optimization, it may happen that the ranges marked
in the Range Indicator will vary when the channel color is changed.
5-394
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OPERATION IN EXPERT MODE
Image Optimization
Single Channel
LSM-FCS
5.16.1.3
Carl Zeiss
Scan Speed, Scan Average and
Pixel Depth
The signal-to-noise ratio can be substantially
improved by reducing the scanning speed to an
acceptable level and averaging over several scans
(i.e. with an average Number greater than 1 for
the Mean average Method in the Scan Control
window).
• Use the Scan Speed slider in the Speed panel
to set the slowest acceptable scanning speed.
–
The corresponding pixel scanning time (Pixel
Time) and the total scanning time (Scan
Time) are shown in the dialog box.
• In the Number text box of the Pixel Depth,
Scan Direction & Scan Average panel enter
the number of measurements to be averaged.
Image optimization can be effected much
faster if you select a smaller frame, since
less data have to be processed.
The greater the number of averages
selected for Mean average Method, the
better the image quality will be; the
scanning time will be prolonged
accordingly.
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Fig. 5-361
Scan Control window
5-395
Carl Zeiss
OPERATION IN EXPERT MODE
Image Optimization
Multiple-channel
LSM-FCS
5.16.2
Multiple-channel
5.16.2.1
Requirements
• The suitable lasers are on.
• The specimen has been positioned and focused
for scanning.
• Click on the LSM button in the Acquire
subordinate toolbar of the Main menu. The
tube slider on the microscope tube is in the
LSM position and the LSM button is activated.
In the following example, 2 Channels shall be
activated for the scanning procedure: one for
488 nm using emission filter BP 505-530 and one
for 543 nm with LP 560. HFT 488/543 is used as
the main dichroic beam splitter, and NFT 635 VIS
and NFT 545 as the secondary dichroic beam
splitter.
• In the Acquire subordinate toolbar, click on the
Config button.
Fig. 5-362
Configuration Control window
–
This opens the Configuration Control
window.
• Click on the Single Track button.
• Activate (in the same way as for the single channel, see page 5-387) channel 2 and channel 3 (Ch2,
Ch3), the indicated emission filters and the main and secondary dichroic beam splitter for the
scanning procedure.
–
The configuration loaded is displayed in the Beam Path and Channel Assignment panel.
• Click on the Scan button in the Acquire subordinate toolbar of the Main menu.
–
This opens the Scan Control window.
• In the Scan Control window, set the parameters in the same way as described for single-channel
presentation.
• Click on the Find button in the Scan Control window.
–
5-396
This starts the scanning process. The scanned image appears in a separate window.
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OPERATION IN EXPERT MODE
Image Optimization
Multiple-channel
LSM-FCS
Fig. 5-363
Carl Zeiss
Scan control and Image Display windows
As a rule, the first scanned image (Pre-Scan) is not ideal, since the photomultiplier is not matched to the
light output. More often than not, the screen image is dull and needs subsequent optimization.
• Click on the Channels button in the Scan Control window.
–
This opens the Channel Settings and Excitation of Track panels.
–
The channels used are color-highlighted.
5.16.2.2
Image Optimization
The image optimization processes
–
setting of pinhole diameter
–
Detector Gain / Ampl. Offset / Ampl. Gain
–
Scanning speed and Average
must be carried out separately for each channel used (see section (1) Single channel, page 5-387).
For the optimum setting of the single channels, Split xy-display must be selected in the Image Display
window to enable the direct viewing of the separate images of the relevant channels.
• Click on the Cont. button in the Scan Control window.
–
This starts a continuous scan.
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5-397
OPERATION IN EXPERT MODE
Image Optimization
Multiple-channel
Carl Zeiss
LSM-FCS
• Click on the Split xy button in the Image Display window toolbar.
–
This displays the separate images scanned in the channels and the composite (overlay) image.
• Now click on the Ch2 button in the Channel Settings panel to optimize Channel 1. Optimization is
performed in the same way as for the single channel and can be monitored online in the relevant
separate image of the channel.
• Then optimize the second channel by clicking on the relevant button (Ch3) in the Channel Settings
panel of the Scan Control window.
Scan image 1
Fig. 5-364
Scan Control and Image Display windows
Scan image 2
Cumulative scan image
Now effect image optimization as explained for the single-channel mode, but separately for each
channel.
• Now click on the xy button of the Display toolbar.
–
The composite scan image of two channels is presented in a common window.
Image optimization can be effected much faster if you select a smaller frame, since less data
have to be processed.
5-398
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OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Structure of the Data Evaluation Window
LSM-FCS
5.17
Data Evaluation and Result Presentation for FCS Measurements
5.17.1
Structure of the Data Evaluation Window
Carl Zeiss
The data evaluation window (Fig. 5-365) of the FCS software part corresponds to the basic structure of
other Microsoft ® WINDOWS applications.
The control line at the top of the data evaluation window contains the control menu for the data
evaluation window (identical to Microsoft ® WINDOWS), the name of the displayed data file, and the
Minimize, Maximize and Close buttons (identical to Microsoft ® WINDOWS).
In the status line at the bottom of the data evaluation window, the progress of a current measuring
procedure are shown online. During the measurement you can stop the procedure by clicking the Stop
button.
The major part of the window contents the measuring results (graph and table) corresponding to the
selected display modes.
To its right, the Display toolbar is always shown in the standard setting. Depending on whether some
buttons in the Display toolbar are activated / deactivated, further toolbars (e.g.: Correlation, Print, Fit)
are displayed / not displayed on the right-hand side of the data evaluation window.
The data evaluation window can be moved as required within the screen, and its vertical, horizontal and
diagonal size can be matched to the current requirements (identical to Microsoft ® WINDOWS).
Fig. 5-365
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Data evaluation window
5-399
Carl Zeiss
5.17.2
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Open / Close the Data Evaluation Window
LSM-FCS
Open / Close the Data Evaluation Window
• Click Start either from the Optimize Method or from the Measure Method window, or open an
existing data file from the database.
–
The Data Evaluation window will appear (Fig. 5-365).
Formats that will be recognized by pressing the Open FCS button include:
–
ConfoCor1 data files. If more than 1 file is selected they will be opened in the same Data
Evaluation window. According to the setting under Options FCS Settings in Open / Save tab
the average of the correlation or the average of the fit results are shown in the last row.
–
ConfoCor2 data files (*.fcs). Each data file will be opened in a separate Data evaluation window.
–
ConfoCor2 raw data files: Each file will be
opened in a different Data evaluation
window. If you open a raw data file the
Options menu for loading raw data files will
appear (see Fig. 5-366). You have several
options to open raw data files.
• You have the possibility to set the integration
time for display in the Count Rate histogram by
typing in or setting with the slider the bin time
in µs in the Pulsedensity Histogram
Integration Time (µs) display box. The Reset
button returns the setting to the default value
of 0.2 µs.
Fig. 5-366
The Options menu for loading
raw data files with dust filter
inactivated
• You have the possibility to select the channels
you want to have displayed by checking the
corresponding Channel 1 and Channel 2
boxes, respectively. If both channels are
activated and you open up an one channel
autocorrelation data file, only the respective
channel will be displayed.
• You can check the Use options for next Raw Data files to open box to automatically select the set
options the next time you load raw data.
• You can apply an electronic dust filter in the Dust Filter display box in the following ways:
–
5-400
Selection of Off (see Fig. 5-366) will inactivate the dust filter.
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OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Open / Close the Data Evaluation Window
LSM-FCS
–
Carl Zeiss
Selection of Auto (see Fig. 5-367) will allow
you to set a threshold intensity in the Dust
Filter Threshold Intensity display box by
typing in a value or selecting a specific value
with the slider. The value (0 … 100) will
define the threshold in percentage. If now
the integrated count rate over a certain
count interval will exceed the average count
rate by that threshold, this special interval is
discarded for correlation analysis. For
example, if the system detects a count rate
in a certain time interval that exceeds the
average count rate by over 30 % and the
threshold was set at 30, this interval is
Fig. 5-367 The Options menu for loading
discarded for correlation analysis. The time
raw data files with dust filter set to
intervals before and after the discarded
automatic
region are separately correlated and the
results averaged. These holds also true, if
more than one region is discarded.
The single regions taken into account and disrupted by discarded regions are separately correlated
and the resulting average will be displayed. Note, that due to built up of an average, this kind of
dust filter does not work for count rates exceeding the average that will come at the beginning of
the measurement time before the system had the possibility to calculate the average count rate.
Also, due to the necessity to average signals over a certain integration time, more than only the
peak area will be discarded. Another outcome of the necessity to average the count rate signal is
that several small peaks following close to each other will be treated as a huge peak and might be
cut out. This means, in the Auto mode accumulated count rates rather than peaks are cut out. For
cross-correlation experiments, any of the regions discarded in either autocorrelation function will
not be used. Cut off regions are framed by stippled boxes and appear greyed in the Count Rate
window.
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5-401
Carl Zeiss
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Open / Close the Data Evaluation Window
–
Fig. 5-368
The Options menu for loading
raw data files with dust filter set
to user defined
Fig. 5-369
5-402
LSM-FCS
Selection of User (see Fig. 5-368) will give
you the possibility to select up to ten regions
that should be discarded for correlation
analysis (for auto and cross-correlation).
Type in the respective start times and end
times in s of the regions you want to
discard. To this end it is recommended to
open the raw data with the dust filter set to
Off, look for the peaks you want to cut out,
and reopen the same raw data file by
pressing the Reload button. Type in the
corresponding boundery values and click ok.
Alternatively, press Select, click with the left
mouse button in the old opened file and
position the two black bars to frame the
region that you want to discard. Press Select
again, which will display the position of the
boundaries. Click ok and the diagram will
reopen. The discarded regions will appear
greyed out (see Fig. 5-369, 5-370). The
regions taking into account and separated
by discarded regions will be separately
correlated and the average will be displayed.
The Reset button will reset all typed in
values to zero.
The Count Rate Trace window with region of interest framed
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OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Open / Close the Data Evaluation Window
LSM-FCS
Fig. 5-370
Carl Zeiss
The Count Rate Trace window with region of interest removed
The Display toolbar on the right allows you to select additional data post-processing actions. These will
be described in detail in the following sections.
• When you are finished, close the Data evaluation window by clicking the Close button in the
Display toolbar.
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5-403
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Open / Close the Data Evaluation Window
Carl Zeiss
5.17.2.1
LSM-FCS
Description of the Raw Data Format
The data format is an exact representation of the dual channel photon trace within the limits given by
the digitalization. This means that the data are recorded without losses within these limits.
The basic idea is to record the time between subsequent pulses from the detector in units of elapsed
clock cycles (run length encoding). However, the format is modified to conserve space at high count
rates and to be capable to handle dual channel data.
Data will be recorded in 16 bit words. Whenever a pulse is detected or the counter counting the clock
cycles overruns 255 (FF hex), a word will be recorded. The word will contain information how many clock
cycles elapsed (1 ... 255) since the last word had been recorded and additionally what happened in the
four cycles of data generation bt1 ... bt4.
The recorded word has the following structure:
Bit
0 (LSB) ... 7
Table 1
meaning
clock counter value (starting at 1) during the triggering event
trigger events are pulse recordings or counter overruns
zero is reserved and only transmitted at the end of the measurement
8
1, if pulse recorded in channel 1 during cycle bt1; else 0
9
1, if pulse recorded in channel 2 during cycle bt1; else 0
10
1, if pulse recorded in channel 1 during cycle bt2; else 0
11
1, if pulse recorded in channel 2 during cycle bt2; else 0
12
1, if pulse recorded in channel 1 during cycle bt3; else 0
13
1, if pulse recorded in channel 2 during cycle bt3; else 0
14
1, if pulse recorded in channel 1 during cycle bt4; else 0
15 (MSB)
1, if pulse recorded in channel 2 during cycle bt4; else 0
Structure of the recorded word
We hope the two examples will make these statements clearer. The tables show part of the running
pulse train (from left to right) divided into clock cycles. "1" in the corresponding box indicates that a
pulse arrived in this cycle. The "counter" row shows the counter readings. The lowest row indicates
when the word has been recorded.
5-404
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OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Open / Close the Data Evaluation Window
LSM-FCS
bt1
CH 1
1
CH 2
counter
bt2
bt3
bt4
bt1
1
Table 2
0
bt2
bt3
bt4
0
0
0
...
1
120 121 122 123
Carl Zeiss
...
0
0
1
2
3
4
...
254 255
⇑
⇑
W1
W2
1
2
Example 1
Example 1
The following words will be recorded:
•
at W1:
high byte: 00011001(bin) = 19 (hex); low byte: 123 (dec) = 7B (hex); resulting word = 197B (hex)
•
at W2:
high byte: 00000000(bin) = 00 (hex); low byte: 255 (dec) = FF (hex); resulting word = 00FF (hex)
bt1
CH 1
bt2
1
bt3
bt4
bt1
1
...
CH 2
counter
bt2
Table 3
0
0
0
1
2
3
4
...
bt4
1
...
120 121 122 123
bt3
1
254 255
0
0
0
⇑
⇑
W3
W4
1
2
Example 2
Example 2
The following words will be recorded:
•
at W3:
high byte: 00010001(bin) = 11 (hex); low byte: 123 (dec) = 7B (hex); resulting word = 117B (hex)
•
at W4:
high byte: 00100100(bin) = 24 (hex); low byte: 255 (dec) = FF (hex); resulting word = 24FF (hex)
The clock runs at a clock rate of 20 MHz. This means a maximum data rate of 10 Mbyte/s. If no pulses
are recorded, the clock rate drops to approx. 155 Kbyte/s according to counter overflows.
The first 30 bytes of the raw data file contain the comment "ConfoCor_2_-_Raw_data_file_1.0" and
have to be ignored.
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5-405
Carl Zeiss
5.17.3
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Display - Correlation
LSM-FCS
Display - Correlation
• If not selected, click on the Correlation button to activate the Correlation display mode.
–
The Correlation display toolbar will appear to the right of the Data Evaluation window
(Fig. 5-365).
Up to four windows with different graphs become visible in the upper left part of the window
depending on the activated Diagrams buttons. By clicking on the appropriate button, the diagram can
be toggled between ON and OFF. The size of the diagrams can be matched as required by moving the
border lines. Click on the border line, hold down the mouse button, move the line in the required
direction; and release the mouse button.
The measuring results are displayed in a table below the diagrams. The width of the columns can also be
changed by moving the border lines. The order of the columns can also be changed. For this purpose,
click on the head line of the relevant column, hold down the mouse button and move the column to the
required position. When the mouse button is released, the column is inserted in the new position.
If more than one line of the table is selected, an appropriate legend can be added to the diagrams by
activating the required Diagram Legend check boxes.
To select a line in the table, click on it with the mouse (multiple choice is possible by additionally pressing
the Shift or Ctrl key). Selected lines are highlighted in color.
After completion of the measurement, the correlation curves are fitted to the model curve using the
parameters provided by the measurement method.
The fitting results are written into the table in the lower part of the window.
Any line of the table can be made the current one by clicking on it. This also means that the
corresponding graphs are shown in the displays.
The appearance of the table can be changed with regard to columns to be displayed and colors of
certain areas by using the Columns function (see section 5.17.12, page 5-423).
The Correlation toolbar contains the following function elements:
Count Rate Trace button. Shows the Count Rate diagram: Here the count
rate(s) vs. running time is displayed. If a cross correlation set-up is used, the count
rate trace for each channel is displayed. If you have set an electronic dust filter
under the Method Optimisation window in Result Handling or loaded Raw
data files with an electronic dust filter activated the cut off regions will be
displayed as greyed out areas if the Dust Filter Information check box in the
Display – Column is checked. Correlation analysis is than performed separately
with all sections of the correlation curves that are disrupted by cut off regions and
the calculated curves are averaged.
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Display - Correlation
LSM-FCS
Carl Zeiss
Correlation Curve button. Shows the Correlation Curve graph: In case of auto
correlation measurement, the developing correlation curve is displayed. In case of
cross correlation measurement, three curves will be shown: the two auto
correlation curves for each of the channels and the cross correlation curve. By
clicking the right mouse button you can choose between displays. Selection of
Use fit limits to restrict view range will display only the part of the correlation
curve that is used for fitting as determined in the Fit window by the Start and
End channels, respectively (see Fig. 5-372). Selection of this option will
automatically activate this form of display for the Fit diagram as well. Selection of
Normalized View will display all checked correlation curves with two defined
values normalized to 1 and 2, respectively (see Fig. 5-373). The position of the bar
more to the right defines the correlation time τn1to which the corresponding
correlation values will be normalized to 1. The difference of G(τn1) to 1 will than be
added to all the other values. Therefore G(τ)new = G(τ)old + (1-G(τn1)). The position of
the bar more to the left defines the correlation time for which the correlation
value will be normalized to 2. All other values (substracted by 1) will than be
multiplied by the correction factor defined by the ratio of 1 to [G(τn2)-1] and
increased by1, hence G(τ)new=[1/ (G(τn2)-1) * (G(τ)old -1)]+1. The bars are only visible
if the Normalization Markers box in the Display – Column is checked.
Selection of none of these options will display the normal correlation curve (see
Fig. 5-371)
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Fig. 5-371
The Correlation Curve window in normal display
Fig. 5-372
The Correlation Curve window in restricted display
Fig. 5-373
The Correlation Curve window in normalized display
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Carl Zeiss
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Display - Correlation
LSM-FCS
Count Rate Histogram button. Shows the Count Rate histogram: To
obtain this histogram, the number of pulses (or: photons recorded from
the detector) in a moving time window are recorded and included in a
histogram. If you load a raw data file, you can determine the size of the
time window (binning time in µs) by changing the default value of 0.2 µs
in the Pulsedensity Histogram Integration Time (µs) display box of
the Options menu for loading raw data files.
Pulse Density button. Shows the Pulse Density histogram: Here the
times elapsed between two subsequent pulses (or: photons recorded
from the detector) are measured and the times are included in a
histogram.
Number check box
When this box is activated, the number of the measurement is displayed
as a legend for the measurements shown in the diagram.
Sample Carrier check box
When this box is activated, the type of the sample carrier of the
measurements shown in the diagram is displayed as a legend.
Chamber check box
When this box is activated, the name of the chamber is displayed as a
legend for the measurements shown in the diagram.
Kinetics Number check box
When this box is activated, the kinetics number is displayed as a legend
for the measurements shown in the diagram.
Channel check box
When this box is activated, the name of the channel is displayed as a
legend for the measurements shown in the diagram.
Dust Filter Information
check box
Normalization Markers
check box
When this box is activated, the boxes defining the cut off regions in the
intensity trace windows will be displayed.
When this box is activated, the normalization markers in the correlation
curve if in the normalization view mode will be displayed.
The zoom function can be used to display certain diagram areas of interest in an enlarged form:
• Use the left mouse button to click on the margin of the area of interest in the diagram, keep the
mouse button pressed and draw a rectangular above the area of interest in any required direction. On
release of the mouse button, the selected area is displayed in an enlarged form in the diagram. The
scaling of axes is matched automatically.
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Display - Correlation
LSM-FCS
Carl Zeiss
The context menu of the relevant diagram can be
used to reset the zoom to its original value.
• Click on the diagram with the right mouse
button.
–
The context menu is opened.
• Click on the line Reset Diagram Zoom with
the left mouse button.
–
Fig. 5-374
Context menu of the
Correlation diagrams
The zoom value is reset.
The diagram display can be manipulated with the context menu as follows:
• Click on the diagram with the right mouse button to open the context menu.
– Selection of Abcissa logarithmic will scale the x-axis logarithmically.
– Selection of Ordinate logarithmic will scale the y-axis logarithmically.
– Selection of Use fit limits to restrict view range will display only the part of the correlation
curve that is used for fitting.
– Selection of Normalized View will give you a context submenu from which you can choose the
following options (see Fig. 5-375):
Select Off to inactivate the
normalization of the curve
Select First Marker (on 2.0) to
normalize the correlation curve at
Fig. 5-375 Subcontext menu of the
the position of the black bar to 2.
Normalized view
Select Second Marker (on 1.0) to
normalize the correlation curve at
the position of the black bar to 1.0.
Select Both Markers (on 2.0 and 1.0) to normalize the correlation curve to 2.0 at the position
of the left black bar and to 1.0 at the position of the right black bar. The position of the bar
more to the right defines the correlation time τn1to which the corresponding correlation value
will be normalized to 1. The difference of G(τn1) to 1 will than be added to all the other values.
Therefore G(τ)norm1 = G(τ) + (1-G(τn1)). The position of the bar more to the left defines the
correlation time for which the correlation values will be normalized to 2. Note that this value is
defined by the arithmetic mean of the correlation values at position defined by the bar and the
correlation values defined by the correlation times just before and after. All other values
(substracted by 1) will than be multiplied by the correction factor defined by the ratio of 1 to
(G(τn2)-1) and increased by 1, hence G(τ)norm2=[1/ (G(τn2-1)-1) * (G(τ)norm1-1)] + 1.
Note that selection of any normalized view will automatically normalize the display in the fit curve as
well. The normalization bars will only be visible, if the Normalization Markers check box is checked.
Not all Options are equally available for all diagrams.
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Carl Zeiss
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Display - Correlation
LSM-FCS
The data (measuring values) of each diagram can be copied to the clipboard or stored directly as an
ASCII file via the context menu.
• Click on the diagram with the right mouse button to open the context menu.
• Select the line Copy data to clipboard with a click of the mouse if you want to insert the data
directly into other WINDOWS programs via the clipboard. The Paste function permits direct insertion
of these data into the required program.
• Select Export data to file to store the data in an external ASCII file (.txt).
The table of measuring rows in the lower part of the Data Evaluation window contains a check box on
the right-hand side of each row. Deactivation of these check boxes will exclude the relevant rows from
all subsequent evaluation procedures.
However, immediate reactivation is possible via the check box, if required.
Deactivated rows can be hidden in the table by activating the check box Hide unused Rows with a click
of the mouse on the Table toolbar (see Display – Columns, page 5-423).
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Display - Fit
LSM-FCS
5.17.4
Carl Zeiss
Display - Fit
• Click on the Fit button to activate the Fit display mode.
–
A Fit menu subordinate will appear (Fig. 5-376).
In general this menu allows you to work with already measured and probably also fitted correlation
curves. The intention is to have the possibility of performing another fit using different fitting parameters
if the automatic fit did not yield optimal results.
Fig. 5-376
Display fit
The upper graph shows the correlation curve with the overlaid fit graph.
The lower graph depicts the fit residuals.
• Use the 1, 2 and 3 buttons to select a one-, two- or three-component model. If a one- or twocomponent model is selected, the parameters not used are grayed.
• Clicking on the Triplet button allows you to change between taking the triplet fraction into account
or not.
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Carl Zeiss
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Display - Fit
LSM-FCS
• An additional button bar (Free or Fix) will appear if you click on the button at the right-hand side of
the parameters.
–
If you click on Free, the parameter will be defined as free. After the fit, the field will contain the fit
result for the parameter.
–
Fix allows you to fix the parameter. This is useful if you know its value from other measurements.
• Start Channel and End Channel enable you to determine which part of the correlation curve should
be fitted to the model. Moving the red or blue line with the mouse permits the parameters Start
Channel and End Channel to be determined directly in the Correlation-Time diagram. The start and
end position of the channels will be displayed as the absolute position (1 – 255) or correlation times
(in µsec) and are represented by the red and blue bar, respectively.
Caution: The numbers indicate which channels are used for correlation. If you want to know
the number of channels omitted from the respective end you have to substract the Start
channel number by 1 and substract from 255 the End channel number, respectively.
The State display box informs you whether the fitting operation has been successful or not. This merely
2
indicates, if the algorithm was able to fit the results. The quality of the fit is displayed in the chi display
2
2
box. The Χ (chi ) value should approach zero for highest quality.
• When you are finished, click the Preview button and the fit procedure will be run for the fit settings
field (on the right-hand side of the diagrams).
–
When the fit is completed, the free parameters will be replaced with the new fitting results and
the fit result graph in the correlation curve diagram will be redrawn.
–
The result table remains unaffected by the Fitting procedure.
• Click on the Apply button to also apply the procedure performed in the fit settings field to the
current (highlighted) measurement in the result table.
• With a click on the Apply to all button, the fit procedure is performed for all activated
measurements (via the check box of the row) in the result table.
The Write to Method function permits the fitted parameters to be transferred directly to an existing or
a new Measuring Method.
• Open the Optimize Method window by clicking the appropriate button in the Analyse FCS
subordinate toolbar of the Main menu.
• Then click on the Write to Method button.
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Display - Fit
LSM-FCS
Carl Zeiss
The parameters of the selected fitting model are assigned to the method selected in the Method
Optimization ... window and stored.
• If the parameters shall be assigned to a new method, click on the Save button and enter a new name
for the method in the appearing Save Method window. Confirm your entry by clicking on OK.
All the table functions in the Fit mode are identical to those in the Correlation mode.
The zoom function can be used to display certain
diagram areas of interest in an enlarged form:
• Use the left mouse button to click on the
margin of an area of interest in the diagram,
keep the mouse button pressed and draw a
rectangular above the area of interest in any
required direction. On release of the mouse
button, the selected area is displayed in an
enlarged form in the diagram. The scaling of
axis is matched automatically.
Fig. 5-377
Context menu of the
Fit diagram
The context menu can be used to reset the zoom to its original value.
• Click on the diagram with the right mouse button to open the context menu (see Fig. 5-377).
• A click on the Reset Diagram Zoom with the left mouse button will reset the zoom value.
The data of the diagram can be copied to the clipboard or stored directly as an ASCII file via the Context
menu.
• Click on the diagram with the right mouse button to open the context menu.
• Select the line Copy data to clipboard with a click of the mouse if you want to insert the data
directly into other WINDOWS programs via the clipboard. The Paste function permits direct insertion
of these data into the required program.
• Select Export data to file to store the data in an external ASCII file (.txt). Only the coordinates of
displayed curves are exported.
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Carl Zeiss
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Display - Fit
LSM-FCS
The display of the diagram can be adapted by use of the context menu.
• Click on the diagram with the right mouse button to open the context menu.
• Select Abcsissa logarithmic or Ordinate logarithmic to scale the x- or y-axis, respectively,
logarithmically.
• Select Use fit limits to restrict view range to display only the part of the correlation curve that is
used for fitting as determined by the Start channel and End channel, respectively (see Fig. 5-380).
Selection of this option will automatically activate this form of display for the Correlation diagram as
well.
Fig. 5-378
5-414
The Correlation Curve window in normal display
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Data Evaluation and Result Presentation for FCS Measurements
Display - Fit
LSM-FCS
Carl Zeiss
• Select Normalized view to display the
correlation curves in a normalized form. Two
bars define the positions of the curve that will
be normalized to 1 and 2, respectively (see Fig.
5-381). You have the option to normalize to 2
Fig. 5-379 Subcontext menu of the
and 1 separately, or to normalize to both
Normalized view
Options are given in the subcontext menu (see
Fig. 5-379). In this case the left bar defines the
position were the curve is normalized to 2,
whereas the right bar specifies the position at
which the curve is normalized to 1.
The bars are only visible if the Normalization Markers box in the Display – Column is checked. Not
that activating the Normalization view in the fit display will automatically activate the
Normalization view in the correlation display as well. The normalization bars are only visible if the
Normalization Markers check box is checked.
Fig. 5-380
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The Correlation Curve window in restricted display
5-415
Carl Zeiss
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Display - Fit
Fig. 5-381
The Correlation Curve window in normalized display
Fig. 5-382
The Correlation Curve window in fit curve display ("View
correlation curve" unchecked)
LSM-FCS
• Selelect View correlation curve will display the Correlation curve in addition to the Fit curve (see Fig.
5-378). If this option is not selected, only the Fit curve is displayed (see Fig. 5-382).
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Display - Fit
LSM-FCS
Carl Zeiss
The zoom function can be used to display certain areas of interest in the Fit deviation diagram in an
enlarged form:
• Use the left mouse button to click on the margin of the area of interest in the Fit deviation diagram,
keep the mouse button pressed and draw a rectangular above the area of interest in any required
direction. On release of the mouse button, the selected area is displayed in the diagram in an
enlarged form. The scaling of axes is matched automatically.
The context menu of the Fit deviation diagram can be used to reset the zoom to its original value.
• Click on the diagram with the right mouse
button.
–
The context menu is opened.
• Click on the line Reset Diagram Zoom with
the left mouse button.
–
Fig. 5-383
Context menu of the
Fit deviation diagram
The zoom value is reset.
The scaling of the G axis (t) of the Fit deviation diagram can be varied in percentage values of the fit
scaling via the context menu.
• Click on the diagram with the right mouse button to open the context menu.
• Select the line Scaling with a click of the
mouse.
–
The Diagram scaling window is opened.
• Enter the required percentage value (from 1 to
100) for the scaling and click on OK.
–
The scaling is changed accordingly.
Fig. 5-384
Diagram scaling window
The data of the diagram can be copied to the clipboard or stored directly as an ASCII file via the context
menu.
• Click on the diagram with the right mouse button to open the context menu.
• Select the line Copy data to clipboard with a click of the mouse if you want to insert the data
directly into other WINDOWS programs via the clipboard. The Paste function permits direct insertion
of these data into the required program.
• Select Export data to file to store the data in en external ASCII file (.txt).
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Carl Zeiss
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Display - Fit
LSM-FCS
The table of measuring rows in the lower part of the Data Evaluation window contains a check box on
the right-hand side of each row. Deactivation of these check boxes will exclude the relevant rows from
all subsequent evaluation procedures.
However, immediate reactivation is possible via the check box, if required.
Deactivated rows can be hidden in the table by activating the check box Hide unused Rows with a click
of the mouse on the Table toolbar (see Display - Columns).
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Data Evaluation and Result Presentation for FCS Measurements
Display - Coincidence
LSM-FCS
5.17.5
Carl Zeiss
Display - Coincidence
• Click on the Fit button to activate the Fit display mode.
–
The coincidence analysis generates a two-dimensional color-coded count rate histogram
(Fig. 5-385).
This is an alternative to the standard correlation analysis during rare event detection. If the FCS is used to
look for small amounts of double-labeled objects (molecules, aggregates etc.) in liquids, the
measurement time to generate a correlation curve which can be evaluated by the standard model may
be prohibitively long. As a rule of thumb, approximately 1000 transitions of labeled aggregates are
required to generate a correlation curve of sufficient quality.
Fig. 5-385
Coincidence diagram window
During the measurement in small time windows the count rates in channel 1 (red) and channel 2 (blue)
are registered and the respective frequencies are calculated. The coordinates of the entry respective to
the x- and y-axes of the diagram are given. Once the coordinates of the cell are found, the value will be
incremented.
If double-labeled aggregates are present, the diagonal of the diagram will be populated.
All the table functions in the Coincidence mode are identical to those in the Correlation mode
.
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OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Display - Preview
Carl Zeiss
5.17.6
LSM-FCS
Display - Preview
• Click on the Preview button to activate the Preview display mode.
–
A print preview and the additional Print subordinate toolbar will appear on the screen (Fig. 5-386).
Fig. 5-386
Preview / Print window
The Preview / Print window shows an overview, as a table and/or a graph, of the results of a
measurements and the relevant measuring parameters of the used measuring method. This result
overview can be printed directly on a connected printer.
For this purpose, the following function buttons are available in the Print subordinate toolbar (at the
right-hand side of the Preview / Print window):
Print button
Starts the print function.
Setup button
Opens the printer setup window to set the print parameters.
Landsc. button
Selects Landscape paper orientation.
Portrait button
Selects Portrait paper orientation.
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Data Evaluation and Result Presentation for FCS Measurements
Display - Preview
LSM-FCS
Carl Zeiss
Compressed button
Selects the Compressed Report preview without diagrams.
Standard button
Selects the Standard Report preview with correlation and fit curves.
Full button
Selects the Full Report preview with all diagrams.
All button
Prints all measurements.
Selection button
Prints selected measurements.
Current button
Prints currently selected measurements only.
To print a report, proceed as follows:
• Select the Compressed, Standard or Full button.
• Select the All, Selection or Current button.
• Click on Landsc. or Portrait for paper orientation.
• If required, change the printer settings using the printer Setup button.
• Click on Print to start the print procedure.
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Carl Zeiss
5.17.7
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Display - Save Data
LSM-FCS
Display - Save Data
• Save allows you to save the whole data set (curves, fitting results, fit parameters) to disk. If Save is
selected, the Save menu appears.
The Save menu offers the possibility of entering the task name from which the file name is derived.
It also offers the possibility of entering a comment in the comment field.
The directory can be chosen.
It is possible to store the data as ANSI text (space-consuming, but readable) or as a binary file.
• OK will confirm the action, Cancel will exit the menu without further action.
5.17.8
Display - Copy Table
• With Copy Table, the whole table will be copied to the WINDOWS NT clipboard. The contents of the
clipboard can be used by most WINDOWS NT applications via the Paste function.
5.17.9
Display - Save Table
• With Save Table, the result table can be written to a white space in a separate text file. If the button
is clicked, a file menu will appear offering the possibility to select a directory and to enter a file name.
5.17.10
Display - Copy Graphs
• Click on the Copy Graphs button to copy the displayed graphs to the clipboard.
–
Using the Paste function of Microsoft WINDOWS, you can paste the graphs into other WINDOWS
applications.
5.17.11
Display - Export
• With Export, the measured data will be exported in a ConfoCor 1-compatible format and can be
handled using the FCS Access Fit program.
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Display - Columns
LSM-FCS
5.17.12
Carl Zeiss
Display - Columns
• With a click on the Columns button, the Table subordinate toolbar can be toggled on and off.
Fig. 5-387
Display Column with Table subordinate toolbar
• By ticking the appropriate Columns check box in the Table subordinate toolbar, it is possible to
include any fitting parameter into the displayed table or remove it from there.
• It is possible to configure the colors for “special situation” highlighting. On clicking the buttons of the
Table subordinate toolbar, a menu will pop up which allows you to choose the desired color.
• By ticking the Order by Correlation check box, the table of measurement results is arranged in the
order of correlation (only useful for more than one correlation type).
• Activate the Hide unused Rows check box if deactivated rows shall be hidden from the table of
measuring values.
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Carl Zeiss
5.17.13
OPERATION IN EXPERT MODE
Data Evaluation and Result Presentation for FCS Measurements
Display - Info
LSM-FCS
Display - Info
• The Info button is used to open the Method Information window in which all the relevant
parameters of the method are displayed.
Fig. 5-388
Method Information window
• With Copy All, the complete parameter set will be copied to the clipboard.
• A click on the Print ... button opens the Print Setup window and allows the parameters of the
method to be printed in the form of a table.
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OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Taking FCS Measurements within a Cell
LSM-FCS
5.18
Combined Application of LSM Scanning and FCS Measurement Procedure
5.18.1
Taking FCS Measurements within a Cell
Carl Zeiss
The simultaneous installation of the LSM 510 and the ConfoCor 2 to the stand of the Axiovert 200 M
permits examination of points selected with the ConfoCor 2 within a single LSM image.
For combined application, proceed as follows:
• Click on the FCS button in the Analyse FCS subordinate toolbar of the Main menu to select the FCS
mode.
• Click on the Method button in the Analyse FCS subordinate toolbar to open the Method Selection
... window. Select the FCS measurement method and click the Select button. Close the window.
• Open the Carrier Position window. Initialize stage and focus. Switch on the lasers using the Laser
button. Close the Laser Control and the Carrier Position windows.
• Click on the VIS button in the Analyse FCS subordinate toolbar
• Select the specimen area to be examined by moving the stage and focus.
• Click on the LSM button in the Analyse FCS subordinate toolbar
• Select the appropriate beam path configuration in the Configuration Control window.
• Open the Scan Control window by clicking the Scan button in the Acquire subordinate toolbar. Set
the scan parameters and click on Single to scan an image of the sample.
• When finished, select the FCS mode in the Analyse FCS subordinate toolbar.
There are two ways of defining the positions, where FCS measurements should be performed. One is
activated in the Method Measurement ... window (treated in section 5.18.1.1) and the other in the
Method Optimization ... window (see section 5.18.1.2).
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Carl Zeiss
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Taking FCS Measurements within a Cell
5.18.1.1
LSM-FCS
Using the Method Measurement ...
Window
With this method you can conveniently choose
several points of interest at which FCS measurements will be performed.
Fig. 5-389
Sample panel for defining positions
in the Method Measurement
• Open the Method Measurement ... window
by clicking the Measure button in the Analyse
subordinate toolbar. Activate the LSM check
box in the Sample panel via mouse click. If the
Piezo Stage Option has been choosen in FCS
Options in Measure tab than checking the
LSM box will automatically activate the usage
of the Piezo Stage. A little display left to the
stage control buttons will appear telling that
the “Piezo stage active”. Note, that it will not
be possible to use the normal stage if the LSM
box is activated in the FCS mode. If you want to
use the normal stage with the LSM box
checked, you have to deactivate the Piezo Stage
in FCS Options before starting the LSM-FCS
software. In the LSM mode, always the normal
stage is active and the Piezo stage cannot be
activated.
–
The Sample panel changes to allow the
definition of positions in the LSM scan
image.
• Activate the Select button via mouse click.
–
The cursor (crossline) appears in the LSM
scan image.
• Move the cursor to the desired position in the
scan image (drag and drop). Click on the Add
Pos button to select this position. The
coordinates of the position appears in the
Positions list of the Sample panel.
Fig. 5-390
5-426
LSM scan image with cursor
(crossline) and 3 selected positions
• In a Z Stack, you can also vary the Z-plane in
which measurement is to be performed. For
this purpose, select the required Z slice in the
stack or set the Z value via the focusing drive of
the microscope.
• Select further positions to be measured in the
same way.
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Combined Application of LSM Scanning and FCS Measurement Procedure
Taking FCS Measurements within a Cell
LSM-FCS
Carl Zeiss
• Click on the Mark Pos button to fix the selected positions as overlay elements in the image.
The X-, Y- and Z- coordinates of the selected positions are only stored in the Positions table
and not in the scanned image (stack). Accordingly, all the marked positions in all the Z-planes
are displayed in the scanned image (Z Stack), no matter in which Z-plane they lie.
• When finished, click on the Start button in the Method Measurement ... window to start the FCS
measurements of the selected positions.
–
The measurement procedure starts and the data and evaluation window appears on the screen.
Please note that the positions selected for FCS measurements in the Method Measurement ...
window using the ConfoCor 2 are approached by moving the microscope stage, since the FCS
beam path is rigidly fixed in one position. When the last selected position has been approached
and measured, the stage will return to the start position.
Possible offsets during scanning with the LSM 510 are compensated by the stage movement in
such a way that the marked points will be hit.
An accuracy of 1 µm can be obtained. We recommend to take measurements not all over the
frame but in one section only to increase the accuracy. Refer to chapter 5.18.3 for more
information.
5.18.1.2
Using the Method Optimization ... Window
When using this method, FCS measurements are performed on a fixed position without table movement
if the Crosshair function is activated or at one defined position if the Position function is used. In the
former case the structure of interest must be moved to the position manually under LSM control prior to
FCS measurements. This positioning allows for a higher accuracy of <1 µm deviation.
• Open the Method Optimization ... window by clicking on the Optimization button in the Analyze
subordinate toolbar. Activate the LSM check box in the Sample panel via mouse click. If the Piezo
Stage Option has been chosen checking the LSM box will automatically activate the usage of the
Piezo Stage. Note, that it will not be possible to use the normal stage if the LSM box is activated. If
you want to use the normal stage with the LSM box checked, you have to deactivate the Piezo Stage
in the Config. Tool before starting the LSM-FCS software. In the LSM mode, always the normal stage
is active and the Piezo stage cannot be activated
–
The Sample panel changes to allow the definition of positions in the LSM scan image or to
activate the Crosshair function.
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Carl Zeiss
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Taking FCS Measurements within a Cell
(1)
LSM-FCS
Using the Crosshair function
In this mode you have to position your sample
manually.
• Activate the Crosshair button via mouse click
and click into the image.
–
Fig. ss23
(2)
Sample panel with activated
crosshair
The crosshair appears in the LSM scan
image. Place the cross at the position, where
the Laser beam is positioned as determined
in section 5.18.3.2. We recommend that
having set the crosshair with the bleaching
procedure as outlined in section 5.18.3.2
you do not close that Scan window. Then
the crosshair will be preserved within the
new scan. The crosshair will be positioned
on the site were you click with the left
mouse button. If you have activated the
Lock button, than the position will be fixed.
• Scan the image continuously and position the
site of interest under the crosshair.
Using the Position function
In this mode, the position will be approached by the scanning table.
• Check the Position box. The Sample panel changes to allow the definition of positions in the LSM
scan image
• Click onto the Select button. Move the cursor to the desired position in the scan image (drag and
drop). Select that position by clicking the Add button. Repeat until all positions are marked, than click
the Mark Position button to store all positions in the overlay.
In the optimization window only the currently selected position will be approached by the
scanning table used for an FCS measurement, not all your defined positions. Select the position
by clicking into the corresponding row of the displayed Positions table.
5-428
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OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Taking FCS Measurements within a Cell
LSM-FCS
Carl Zeiss
• After defining the site of interest, click on the
Start button in the Method Optimization ...
window to start the FCS measurement.
–
The measurement procedure starts and the
data and evaluation window appears on the
screen.
Please note that the position selected for
FCS measurements in the Method
Optimization ... window using the
ConfoCor 2 is not approached by moving
the microscope stage if the Crosshair
function is activated. To find out the
position of the fixed laser beam refer to
chapter 5.18.3. Since no stage movement
is involved, the manual positioning of
your sample will be more precise with an
accuracy of at least 1 µm.
Fig. ss24
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Sample panel for defining positions
in the Method Measurement
5-429
Carl Zeiss
5.18.2
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Performing FCS Measurements on the Cell Membrane
LSM-FCS
Performing FCS Measurements on the Cell Membrane
Due to the shape of the confocal volume, the membrane should not be approached from the side but
instead rather from the top of the cell. Please note that it is better to use the upper membrane for
measurement, since the lower membrane might be too close to the glass bottom surface resulting in
disturbing reflections. You can home in on the membrane either manually or using the Z Scan feature in
the Method Optimization ... window.
5.18.2.1
Manual Focusing on the Membrane
• Acquire an image of the cell.
• Place the focus above the cell and scan continuously.
• Focus slowly down until you see a fluorescence signal.
• Stop focusing and proceed as described in sections 5.18.1.1 and 5.18.1.2.
Due to its high sensitivity ConfoCor2 can still detect signals that cannot be imaged by the LSM. If the
signal is too low for imaging, the membrane can still be detected using the Z Scan feature in the
Method Optimization ... window.
5.18.2.2
Focusing on the Membrane by a Z Scan
The Z Scan measures the count rate at previously defined Z positions. It is performed without table
movement, that is at the fixed position of the laser beam.
• Position the region of interest at the site of the fixed laser beam path as described in chapter
5.18.3.2.
• Optional: Perform a Z Stack that includes the membrane of the cell as outlined in chapter 5.5.4 with
the LSM.
• Open the Method Optimization ... window by clicking on the Optimization button in the Analyze
subordinate toolbar. Activate the LSM check box in the Sample panel via mouse click.
–
The Sample panel changes and shows the position of the Laser beam.
• Activate the Select button via mouse click.
–
The cursor (crossline) appears in the LSM scan image.
• Move the cursor to the appropriate position.
• Select by pressing the Add button.
• Press Mark Pos to fix the position as an overlay element.
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OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Performing FCS Measurements on the Cell Membrane
LSM-FCS
Carl Zeiss
You might also activate the Crosshair
and manually placing your sample at the
position of the laser beam as defined in
section 5.18.3.2.
• Press the Z Scan button. The Z Scan display
shows the current Z position of the laser beam.
(see Fig. 5-391). Note, that after initialization of
the LSM 510 / ConfoCor2 software the position
is the same both for the FCS and LSM mode.
• Check the corresponding channel if not yet
activated.
Fig. 5-391
Z-Scan display window
• Select the start and end position of the Z Stack. If you have acquired a Z Stack in the LSM mode, use
the values defined by the Mark First/Last button. Note that the values defined by the Z Stack in the
LSM mode are not automatically used in the Z Scan of the FCS mode.
• Enter the number of positions. If you have acquired a Z Stack, use the value defined in the
Z Sectioning tab.
• Press the Start button to perform a Z scan. Press the Cancel button if you want to leave the Z Scan
settings.
• After pressing the Start button a Z scan is
performed and the intensity displayed in
dependence of the Z position (see Fig. 5-392).
The red line in the diagram shows the actual
Z position. Peaks show Z levels of high signal
intensity and may correlate to labeled
membranes. Note, that the glass surface will
also give a peak. To determine its position, just
perform a Z Scan at a position where there is
no cell. Place the red line at the peak that
corresponds to the membrane to select this
Z position.
Fig. 5-392
Z-Scan image
• If no clear signal can be detected, or the peak of interest lies too close to the border, close the Scan
window and perform a new Z scan. Choose a different Z range by modifying start and end positions
to values that lie closer to the position at which the signal should be expected.
• If the Z position has been selected, close the Image Display window by clicking on the Close button.
• Press the Start button in the Method Optimization ... window to perform an FCS measurement.
–
The FCS measurement is performed at the same X/Y position as the Z scan.
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5-431
Carl Zeiss
5.18.3
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Compensating Offsets between FCS and LSM
LSM-FCS
Compensating Offsets between FCS and LSM
As described in chapter 5.18.1.1 you can select the positions in the Method Measurement ... window
or the Method Optimization ... window in the Positions mode, at which the FCS measurements
should be performed. Before you use this function you have to adjust the offsets in X,Y between the FCS
and the LSM mode following the description in section 5.18.3.1. For the Method Optimization ... in
the Crosshair mode you locate the position of the beam and place the structure of interest at the
defined site. This procedure is described in section 5.18.3.2. For Z adjustment you have to use the
Method Optimization ... window as described in section 5.18.3.3. In all cases you need a thin layer of
a fluorescent dye, preferentially Rhodamine 6 Green, dried on a glass bottom of for example a Labtec 8well chamber.
5.18.3.1
Alignment in X, Y for the Method Measurement ... and the Method Optimization in
the Positions Mode
You can compensate manually or by using a Macro.
5.18.3.1.1 Manual Adjustment
Do the manual adjustment according to the
following procedure:
•
Obtain an LSM image of the Rh6G layer.
• Set the following LSM parameters in the
Configuration Control (see Fig. 5-393):
Fig. 5-393
5-432
–
Channel 1: select
–
Excitation: 488 nm at 2.1%, tube current
6.1 Amps
–
Main beam splitter: HFT 488
–
Emission filter of channel 1: LP505
LSM beampath for imaging the test
layer of Rh6G
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LSM-FCS
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Compensating Offsets between FCS and LSM
Carl Zeiss
• Set the following LSM parameters in the Scan
Control (see Fig. 5-394):
–
Frame Size: X=512, Y=512
–
Scan Speed: 8
–
Data Depth: 8 Bit
–
Scan direction: ->
–
Mode: Line
–
Method: Mean
–
Number: 4
–
Zoom: 5
–
Rotation:0
Fig. 5-394
Settings at the LSM control
Fig. 5-395
LSM image of the Rh6G layer before
bleaching. Usually some texture is
observed.
• Optimize focus position and Detector Gain
value for a good LSM image (see Fig. 5-395).
• Leave the LSM image window and LSM control
window on the computer screen.
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5-433
Carl Zeiss
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Compensating Offsets between FCS and LSM
LSM-FCS
• Switch to FCS mode.
Fig. 5-396
5-434
In the main toolbar press the FCS button.
–
Click Analyze / Select Method: Select
“LSM-FCS_STANDARD_488-Rh6G”
–
Click Analyze / Measure: activate the
check box LSM.
–
Mark the points in the LSM image that you
want to bleach by positioning the cross and
pressing the Add button. The marked points
will be listed. Click Mark Pos to overlay the
markers in the LSM image (see Fig. 5-396
and Fig. 5-397).
The Method Measurement window
in the LSM mode
•
Fig. 5-397
–
Bleach the marked sites.
–
Click Start. The usual FCS measurement
window appears.
–
Once finished, close the FCS measurement
window without saving.
LSM image of the Rh6G layer with
markers for FCS-LSM adjustment
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LSM-FCS
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Compensating Offsets between FCS and LSM
Carl Zeiss
• Rescan the Rh6G layer.
–
Switch to LSM mode.
–
Click Single in the Scan Control.
–
Once the scan is finished you see the
bleached spots (see Fig. 5-398).
Fig. 5-398
LSM image of the Rh6G layer after
bleaching. Deviations between the
markers and the bleached spots are
noticeable.
Fig. 5-399
LSM image after bleaching with
overlaid distance markers
• Measure the distance between the burned
spots and the markers (see Fig. 5-399).
–
Click on Overlay.
–
Click on the 1 µm button.
–
Draw a line between the center of the
bleached spot to the vertical and horizontal
lines of the marker cross.
• Determine the average X and Y deviation values
between the markers and bleached spots.
Depending on the direction of these deviations
increase or decrease the X and Y values by the
measured mean deviation. Type these X and Y
values into FCS Settings / LSM+ConfoCor.
–
Click Options / FCM Settings /
LSM+Confocor.
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5-435
Carl Zeiss
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Compensating Offsets between FCS and LSM
LSM-FCS
• Type the measured distance correction values
under Options / FCS Settings in the LSM +
ConfoCor tab (see Fig. 5-400).
Fig. 5-400
The Settings for user window with
the LSM+Confocor tab selected
–
If the bleached point is left to the marker:
type distance in X as positive value
–
If the bleached point is right to the marker:
type distance in X as negative value
–
If the bleached point is below the marker:
type distance in Y as positive value
–
If the bleached point is right to the marker:
type distance in Y as negative value
–
Offset Z: no entry
Note that this correction is valid only for
the ConfoCor2 sitting at the sideport.
ConfoCor2 attached at the baseport
requires other algebraic signs.
• Check the correctness of your “FCM Settings”
by repeating the adjustment procedure (see Fig.
5-401).
If the deviations between bleached areas and
markers is marginal, the LSM/FCS superposition
alignment is sufficient to guarantee a precision
of 1 µm. Please note, that precision is higher if
you decide to make a measurement in one
section of the image rather than all over it. In
this case only deviations in that sector should
be taken into account for calculating the offset
values.
Fig. 5-401
5-436
LSM image after bleaching and
correction of offset values
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LSM-FCS
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Compensating Offsets between FCS and LSM
Carl Zeiss
5.18.3.1.2 Adjustment Using the LSM/FCS Macro
Before running the Macro choose the Method “LSM-FCS_STANDARD_488-Rh6G” in the Method file
menue in FCS and set the beam path for the LSM according to 5.18.3.1.1. Please, focus on the
Rhodamine 6 Green layer.
• Activate the Macro toolbar by clicking the
Macro button.
• Choose the Lsm-Fcs macro by clicking the
corresponding button.
• The “Requirements
for
window will be displayed.
Adjustment”
• Click Ok to run the Macro, Cancel to abort.
• Once Ok was chosen, the Lsm-Fcs macro
window will be displayed (see Fig. 5-402).
• Set the Pinhole diameter to 10 and the
Bleach power to 1%. If holes are too weak or
getting to big you should increase or decrease
the Bleach power, respectively.
Fig. 5-402
The Macro "Fcs-Lsm" display before
offset compensation
• Choose the numbers of Bleach spots by checking the corresponding number.
• You have now two possibilities to continue: Stepwise or automatic.
• Stepwise Procedure:
–
Press the Scan Ref button which is highlighted and labeled by a red 1. A scan is performed. Once
finished the Bleach button will be highlighted and labeled with a red 2. The 1 will turn to black.
–
Press the Bleach button. The bleach is performed. The FCS evaluation window will appear. Once
ready, the Rescan button will be highlighted and labeled with a red 3. The 2 will turn to black The
FCS evaluation window will disappear.
–
Press the Rescan button. The image is rescanned. You will see a number of bleached spots
according to your settings which corresponds to the positions defined by the rigid FCS beam path.
You will also see yellow crosses which are the positions of the Laser beam for the LSM. Because
there might be an offset between both, crosses do not necessarily correspond with the bleached
spots. Once ready, the Autoscan button is highlighted and labeled with a red 4. The 3 will turn to
black.
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5-437
Carl Zeiss
Fig. 5-403
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Compensating Offsets between FCS and LSM
LSM-FCS
–
Press the Autoscan button. Once finished,
the 4 will turn black. The program will find
the burned spots which will turn red and
label them by green circles. The distance
between the green circles and yellow crosses
is indicated by the green line and the value
displayed as the R value (see Fig. 5-403). The
program also calculates the deviation in X
and Y, the values which are also displayed
under X and Y offsets (see Fig. 5-404). The
program also calculates the scan drift during
the procedure and gives a warning if the
scan drift was to great.
–
Press the Save button to store the offset
values. Deviations between LSM and FCS will
than be automatically compensated with the
corresponding offsets.
–
Please note that you can do a readjustment
without leaving the Lsm-Fcs Macro.
However, all buttons will be left highlighted
and numbers will stay black. That means
that you have to watch for yourself when
one step is finished and you have to
remember, which is the next step you have
to activate.
–
Press the Help button if you wish to view
the “Requirements for adjustment”
display. Press Close to leave the Macro.
LSM image after performance of
the “Fcs-Lsm” Macro
By activating the Macro a Stop Macro
window will appear. You can leave the
Macro at any time by pressing the Stop
Macro button.
• Automatic Procedure:
Fig. 5-404
5-438
The “Fcs-Lsm” Macro after offset
compensation
–
Press the Auto Adjust button. The Macro
will automatically perform Steps 1. to 4. The
corresponding submenu button will be
highlighted.
–
Once finished, offsets are displayed. Press
Save to store the offsets, Help to view the
“Requirements for adjustment” display or
Close to leave the Macro.
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OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Compensating Offsets between FCS and LSM
LSM-FCS
5.18.3.2
Carl Zeiss
Determination of the Fixed X,Y
Position of the Laser Beam in the
Method Optimization ... Window
in the Crosshair Mode
• Get a LSM image of a Rh6G layer as described
under 5.18.3.1.1.
• Switch to FCS mode.
–
In the main toolbar press the FCS button.
–
Click Analyze / Select Method: Select
“LSM-FCS_STANDARD_488-Rh6G”
–
Click Analyze / Optimization: activate the
check box LSM.
–
Click the Crosshair box (see Fig. 5-405).
• Perform a bleach at the position of the fixed
laser beam.
–
Click Start. The usual FCS measurement
window appears.
–
Once finished, close the FCS measurement
window without saving.
Fig. 5-405
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The Method Optimization ...
window in the LSM mode with the
Crosshair function activated
5-439
Carl Zeiss
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Compensating Offsets between FCS and LSM
LSM-FCS
• Rescan the Rh6G layer.
–
Switch to LSM mode.
–
Click Single in the Scan Control.
–
Once the scan is finished you see a bleached
spot corresponding to the position of the
rigid laser beam (see Fig. 5-406).
• Record the position of the spot.
Fig. 5-406
–
With the crosshair. Place crosshair above the
bleached spot (see Fig. 5-407). We
recommend to use the crosshair for
positioning your cell.
–
With the cursor. Make sure that under
Options / LSM Settings in the Image
Status Display tab the Pixel Intensity box
is checked to activate the display of the
coordinates. The coordinates will than be
displayed at the lower bar of the image.
–
With an overlay arrow. Click Overlay and
activate the Arrow button. Place the
arrowhead in the middle of the spot.
LSM image after bleaching in the
Method Optimization
If the Position box is checked, the
Crosshair function is inactivated.
Fig. 5-407
5-440
LSM image after bleaching with
crosshair
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OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Compensating Offsets between FCS and LSM
LSM-FCS
5.18.3.3
Carl Zeiss
Alignment in Z
• In the LSM mode take an image of the dye layer and focus at the Z level of maximum intensity.
Record the corresponding Z position.
• Switch to FCS mode.
• Open the Method Optimization ... window by clicking the Optimization button in the Analyze
subordinate toolbar. Activate the LSM check box in the Sample panel via mouse click.
–
The Sample panel changes to allow the definition of positions in the LSM scan image.
• Activate the Select button or the Crosshair via mouse click.
–
If you have pressed the Select button the cursor (crossline) appears in the LSM scan image.
Choose a position by moving the cursor and pressing the Add button.
• Press the Z Scan button. The Z-Scan display shows the current Z position of the laser beam. (see Fig.
5-391). Note, that after initialization of the LSM 510 / ConfoCor software the position will be the
same for both the FCS and LSM mode.
• Check the corresponding channel if not yet activated.
• Set the start and end position of the Z Stack around the current position.
• Enter the number of positions.
• Press Start to perform the Z scan.
• A Z scan is performed and the intensity displayed in dependence of the Z position (see Fig. 5-392).
The major peak corresponds to the labeled layer. Place the red line at this peak value to select this
Z position. Record the position and the direction the red bar was moved.
• Calculate the offsets between LSM and ConfoCor2 by subtracting both Z values.
• Type the calculated values under Options / FCS Settings in the LSM + ConfoCor tab (see Fig.
5-400).
–
Offset X: no entry
–
Offset Y: no entry
–
Offset Z: type in the positive or negative number, if you have moved the red line to the right or
left, respectively.
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5-441
Carl Zeiss
OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Piezo Stage
5.18.4
Piezo Stage
5.18.4.1
Activating the Piezo Stage
LSM-FCS
You have the option to mount a Piezo stage onto your normal stage. If you want to have access to the
Piezo stage in the LSM-FCS software, the Piezo stage checkbox in the AIM Configuration tool has to be
checked. To this end double click on the ConfTool icon, select FCS tab and check the corresponding box
(see Fig. 5-408). Βe sure to select on the control unit of the Piezo Stage the closed loop operation. If
closed loop is selected the corresponding LCD will light up. To work with the Piezo stage, it should be
activated under FCS Options in Measure tab by checking the Use Piezo stage if available check box.
The Piezo stage will be automatically used in the FCS-mode when the LSM box is checked in the
Method Measurement or the Optimization Measurement / Analysis Procedure. If you do not
want to use the Piezo Stage with the LSM box checked , you have to deactivate the check box.
Fig. 5-408
Activating the Piezo Stage in the Conf.Tool
Note: If you haven't chosen the closed loop operation mode, the Piezo stage will not work
properly in the LSM-FCS Software.”
The Piezo stage allows for 2D translational motion in the x and y direction with a maximum motion of 80
µm in each direction. The resolution is 1 nm. Positioning accuracy using the Piezo Stage is 10 nm and
less.
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OPERATION IN EXPERT MODE
Combined Application of LSM Scanning and FCS Measurement Procedure
Piezo Stage
LSM-FCS
5.18.4.2
Carl Zeiss
Working with the Piezo stage
Open Method Optimization / Analysis procedure (see Fig. 5-409) or Method Measurement
and check the LSM box. In the FCS mode the
Piezo stage is automatically used.
Any stage movement done with the stage control
will address the Piezo stage.
Movement by the joystick will overrule the Piezo
Stage and the normal stage will be addressed.
The position of the Piezo stage will be centered in
respect to the normal stage whenever the Piezo
stage to 0 button was pressed.
The position in x and y are displayed in the Stage
Position display box.
If the Piezo stage is activated the x, and y-Scans in
the X,Y,Z-Menu will be done by the Piezo stage.
Note: The numbers displayed will not
correspond to the positions in the LSM
mode, if the Piezo stage is activated. If
the standard stage is used, the numbers
will be the same.
Note: The Piezo stage will only be active
in the FCS mode. In the VIS and LSM
mode the normal stage will be used.
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Fig. 5-409
Method optimization window
(Analysis Procedure subwindow)
with LSM box checked and Piezo
stage active
5-443
OPERATION IN EXPERT MODE
Shut-Down Procedure
Exiting the LSM-FCS Program
Carl Zeiss
5.19
LSM-FCS
Shut-Down Procedure
Never shut down the computer by its main switch while your LSM-FCS program is still active, or
else you will lose the currently set operating parameters and the images just scanned.
In the Settings for user dialog window, which can be activated with the Options / Settings
buttons, activate Laser off or Exit in the Shutdown tab. The lasers will then automatically be
switched off when you exit the LSM-FCS program.
5.19.1
Exiting the LSM-FCS Program
• Close all open windows of the LSM-FCS program by clicking on the closing icon
corner of each window.
in the top right
–
This closes the respective window and removes the respective icons from the taskbar.
–
After all dialog windows have been closed, the LSM-FCS Switchboard window appears.
Fig. 5-410
LSM-FCS Switchboard menu
• Click on the Exit button.
–
This terminates the LSM-FCS program.
–
The monitor screen shows the desktop of the WINDOWS NT operating system.
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OPERATION IN EXPERT MODE
Shut-Down Procedure
Shut Down the WINDOWS Operating System
LSM-FCS
5.19.2
Carl Zeiss
Shut Down the WINDOWS Operating System
• Move the cursor to the bottom margin of the screen.
–
This opens the taskbar containing the Start button.
• Click on the Start button of the taskbar.
–
This opens a pop-up menu.
• Click on the Shut Down item.
Fig. 5-411
Start menu
Taskbar
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5-445
OPERATION IN EXPERT MODE
Shut-Down Procedure
Turning Power Off
Carl Zeiss
–
LSM-FCS
This opens the Shut Down Windows
window, in which you can select between
Shut down, Restart and Login.
• Unless already set by default, click on Shut
down the computer?
• Click on the Yes button.
The screen now displays the message
Fig. 5-412
Shut Down window
Shutdown in Progress - Please wait while the
system writes unsaved data to the disk.
About 20 seconds after WINDOWS NT has been run down, the Shutdown Computer window appears
which tells you that you can now turn off your computer.
5.19.3
Turning Power Off
Please bear in mind that a cooling phase of at least 5 minutes is required between switching off
of the laser via the software and switching off of the entire system via the REMOTE CONTROL
main switch or the Power Supply switch of the Enterprise UV laser.
Throw the REMOTE CONTROL main switch and the power supply switch of the Ar Laser to
position "OFF" after 5 minutes.
–
This puts your LSM-FCS microscope system, including the computer, off power.
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OPERATION IN EXPERT MODE
Software and Hardware Options
Software
LSM-FCS
5.20
Carl Zeiss
Software and Hardware Options
This section describes optional software and hardware configurations. Depending on your configuration,
the content of dialogue and function may differ.
5.20.1
Software
The following software packages for Release 3.0 are available:
–
Software "Physiology evaluation“
–
Software “Topography evaluation”
–
Software “Macro Recorder and Editor”
–
Software "3D for LSM“
–
Software “Multiple Time Series”
–
Software “Image VisArt”
–
Software “Deconvolution”
–
Software "StitchArt"
If your configuration does not include the "Physiology evaluation“ software package, the following
functions are not available:
–
Mean of ROI scan button in Time Series Control
–
Mean of ROI button in the Image Display window
If your configuration does not include the “Topography evaluation” software package, the following
functions are not available:
– Topo button in the Image Display window after acquisition of image stacks
If your configuration does not include the “Macro Recorder and Editor” software package, the following
functions are not available:
– New, Save and Save as buttons in the Macro Control window
–
Edit, Step, Delete, Editor buttons in the Macro Control window
If your configuration does not include the “3D for LSM” software package, the following separate
application is not available:
– 3D for LSM
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Carl Zeiss
OPERATION IN EXPERT MODE
Software and Hardware Options
Software
LSM-FCS
If your configuration does not include the “Multiple Time Series” software package, the following
function is not available:
– Macro: “Advanced Time Series”
If your configuration does not include the “Image VisArt” software package, the following function is
not available:
– 3D button in the Image Display window
If your configuration does not include the “Deconvolution” software package, the following functions
are not available
– DCV Settings button in the Ortho function of the Image Display window
–
DCV button in the Process menu
If your configuration does not include the "StitchArt" software package, the following function is not
available
– Macro: "StitchArt"
5-448
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OPERATION IN EXPERT MODE
Software and Hardware Options
LSM-FCS
5.20.2
Carl Zeiss
Hardware
Depending on whether the following hardware components are available or not, the content of the
screens may differ:
–
HRZ 200 fine focusing stage
–
Piezo objective focusing device
–
X-Y scanning stage DC 4 × 4 or DC 100 × 90, each with MCU 28
–
Depending on the configuration the scan head equipment may differ in filters, beamsplitters and the
number of photomultiplier
–
Transmitted-light PMT
–
Monitor diode
–
Programmable AOTF
If your configuration does not include the HRZ 200 fine focusing stage, the following functions are not
available:
–
Hyperfine Z Sectioning in the Z Stack function in the Scan Control window
–
HRZ parameters in the Stage and Focus Control window
If your configuration does not include the X-Y scanning stages DC 4 × 4 or DC 100 × 90, each with
MCU 28, the following functions are not available:
–
Stage Position and Tile Scan functions in the Stage and Focus Control window
If your configuration does not include scan head META, monitor diode and/or transmitted light PMT, the
following functions may differ:
–
Context and accessibility of the Config Control window
If your configuration does not include programmable AOTF, the following functions are not available:
–
Laserline in the Config Control window and Channels in the Scan Control window
If your configuration does not include an AxioCam, the following functions are not available:
–
Camera in the Config Control window, Scan Control window
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OPERATION IN EXPERT MODE
System Configuration Tool
Carl Zeiss
5.21
LSM-FCS
System Configuration Tool
Transparent configuration of system hardware
Start by double-click on the ConfTool.exe in directory /AIM
Configures:
–
Scanning stages, focus accessories, AxioCam
–
Microscope and microscope accessories (lamps, reflector cubes ...)
–
Substitutes direct editing of system databases
Benefits:
–
Fast and easy integration of new hardware
–
Optional rotation/flip of scanned images according to image orientation in visual observation
Fig. 5-413
5-450
Configuration Tool menu
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OPERATION IN EXPERT MODE
Courses on How to Operate the System in an Optimized Way
LSM-FCS
5.22
Carl Zeiss
Courses on How to Operate the System in an Optimized Way
Carl Zeiss is offering training courses on how to operate the system in an optimized way.
Courses are held in our application center in Jena, Germany.
Check out
www.zeiss.de/lsm
for the latest dates and ask your Zeiss representative for a quotation on courses.
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OPERATION IN EXPERT MODE
Carl Zeiss
5-452
LSM-FCS
B 45-0006 e
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