User Manual

cellSens
LIFE SCIENCE IMAGING SOFTWARE
 Any copyrights relating to this manual shall belong to Olympus Soft Imaging Solutions GmbH.
We at Olympus Soft Imaging Solutions GmbH have tried to make the information contained in this
manual as accurate and reliable as possible. Nevertheless, Olympus Soft Imaging Solutions
GmbH disclaims any warranty of any kind, whether expressed or implied, as to any matter
whatsoever relating to this manual, including without limitation the merchantability or fitness for any
particular purpose. Olympus Soft Imaging Solutions GmbH will from time to time revise the
software described in this manual and reserves the right
to make such changes without obligation to notify the purchaser. In no event shall Olympus Soft
Imaging Solutions GmbH be liable for any indirect, special, incidental, or consequential damages
arising out of
purchase or use of this manual or the information contained herein.

No part of this document may be reproduced or transmitted in any form or by any means,
electronic or mechanical for any purpose, without the prior written permission of Olympus Soft
Imaging Solutions GmbH.

© Olympus Soft Imaging Solutions GmbH

All rights reserved

Printed in Germany
Version 510_UMA_cellSens16_Han_en_00

Olympus Soft Imaging Solutions GmbH, Johann-Krane-Weg 39, D-48149 Münster,

Tel. (+49)251/79800-0, Fax: (+49)251/79800-6060
 Contents
1. About the documentation for your software................................................... 4
2. User interface .................................................................................................... 5
2.1. Overview - User interface......................................................................................................... 5
2.2. Overview - Layouts................................................................................................................... 6
2.3. Document group....................................................................................................................... 7
2.4. Tool Windows........................................................................................................................... 8
2.5. Working with documents .......................................................................................................... 9
3. Configuring the system .................................................................................. 12
4. Acquiring single images ................................................................................. 14
5. Acquiring image series ................................................................................... 18
5.1. Time stack .............................................................................................................................. 18
5.2. Time Lapse / Movie ................................................................................................................ 20
5.3. Acquiring movies and time stacks.......................................................................................... 21
5.4. Z-stack.................................................................................................................................... 24
5.5. Acquiring a Z-stack................................................................................................................. 26
6. Acquiring fluorescence images ..................................................................... 28
6.1. Multi-channel image ............................................................................................................... 28
6.2. Before and after you've acquired a fluorescence image........................................................ 31
6.3. Defining observation methods for the fluorescence acquisition............................................. 33
6.4. Acquiring and combining fluorescence images...................................................................... 38
6.5. Acquiring multi-channel fluorescence images........................................................................ 44
7. Creating stitched images................................................................................ 50
8. Processing images.......................................................................................... 57
8.1. Fluorescence Unmixing.......................................................................................................... 58
9. Measuring images ........................................................................................... 63
9.1. Measuring images .................................................................................................................. 65
9.2. Measuring intensity profiles.................................................................................................... 71
9.3. Measuring the colocalization .................................................................................................. 74
 About the documentation for your software

1. About the documentation for your

software
The documentation for your software consists of several parts: the installation
manual, the online help, and PDF manuals that are installed together with your
software.
Where do you find The installation manual is delivered with your software. There, you can find the
which information?
system requirements. Additionally, you can find out how to install and configure
your software.
In the manual, you will find both an introduction to the product and an explanation
of the user interface. By using the extensive step-by-step instructions you can
quickly learn the most important procedures for using this software.
In the online help, you can find detailed help for all elements of the program. An
individual help topic is available for every command, every toolbar, every tool
window and every dialog box.
New users are advised to use the manual to introduce themselves to the product
and to use the online help for more detailed questions at a later date.
Writing convention In this documentation, the term "your software" will be used for the cellSens
used in the
software.
documentation
Example images that During the installation of your software some example images have been
are automatically
installed, too. These example images might be of help to you when you
installed
familiarize yourself with the software. Regarding the information as to where the
example images are located, please refer to the online help.
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 User interface

2. User interface
2.1. Overview - User interface
The graphical user interface determines your software's appearance. It specifies
which menus there are, how the individual functions can be called up, how and
where data, e.g. images, is displayed, and much more. In the following, the basic
elements of the user interface are described.
Note: Your software's user interface can be adapted to suit the requirements of
individual users and tasks. You can, e.g., configure the toolbars, create new
layouts, or modify the document group in such a way that several images can be
displayed at the same time.
Appearance of the user
interface

The illustration shows the schematic user interface with its basic elements.

(1) Menu bar You can call up many commands by using the corresponding menu. Your
software's menu bar can be configured to suit your requirements. Use the Tools
> Customization > Start Customize Mode... command to add menus, modify, or
delete them.
Further information is available in the online help.
(2) Document group The document group contains all loaded documents. These can be of all
supported document types.
When you start your software, the document group is empty. While you use your
software it gets filled - e.g., when you load or acquire images, or perform various
image processing operations to change the source image and create a new one.
(3) Toolbars Commands you use frequently are linked to a button providing you with quick
and easy access to these functions. Please note, that there are many functions
which are only accessible via a toolbar, e.g., the drawing functions required for
annotating an image. Use the Tools > Customization > Start Customize Mode...
command to modify a toolbar's appearance to suit your requirements.
(4) Tool windows Tool windows combine functions into groups. These may be very different
functions. For example, in the Properties tool window you will find all the
information available on the active document.
In contrast to dialog boxes, tool windows remain visible on the user interface as
long as they are switched on. That gives you access to the settings in the tool
windows at any time.

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 User interface

(5) Status bar The status bar shows a lot of information, e.g., a brief description of each
function. Simply move the mouse pointer over the command or button for this
information.
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2.2. Overview - Layouts

To switch backwards and forwards between different layouts, click on the right-
hand side in the menu bar on the name of the layout you want, or use the View >
Layout command.
Which predefined For important tasks several layouts have already been defined. The following
layouts are there?
layouts are available:
Acquire images ("Acquisition" layout)
View and process images ("Processing" layout)
Measuring images ("Count and Measure" layout)
Generate a report ("Reporting" layout )
In contrast to your own layouts, predefined layouts can't be deleted. Therefore,
you can always restore a predefined layout back to its originally defined form. To
do this, select the predefined layout, and use the View > Layout > Reset Current
Layout command.
What is a layout? Your software's user interface is to a great extent configurable, so that it can
easily be adapted to meet the requirements of individual users or of different
tasks. You can define a so-called "layout" that is suitable for the task on hand. A
"layout" is an arrangement of the control elements on your monitor that is optimal
for the task on hand. In any layout, only the software functions that are important
in respect to this layout will be available.
Example: The Camera Control tool window is only of importance when you
acquire images. When instead of that, you want to measure images, you don't
need that tool window.
That's why the "Acquisition" layout contains the Camera Control tool window,
while in the "Count and Measure" layout it's hidden.
Which elements of the
user interface belong to
the layout?

The illustration shows you the elements of the user interface that belong to the
layout.
(1) Toolbars
(2) Tool windows
(3) Status bar
(4) Menu bar
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 User interface

2.3. Document group

The document group contains all loaded documents. These can be of all
supported document types.

The illustration shows left, a schematic representation of a user interface. On the

right, the document group is shown enlarged.

Document group in the user interface

You will find the document group in the middle of the user interface. In it you will
find all of the documents that have been loaded, and naturally, all of the images
that have been acquired also. Also the live-image and the images resulting from,
e.g., any image processing function, will be displayed there.

Document bar in the document group

The document bar is the document group's header.

For every loaded document, an individual field will be set up in the document
group. Click the name of a document in the document bar to have this document
displayed in the document group. The name of the active document will be
shown in color. Each type of document is identified by its own icon.

Buttons in the document bar

At the top right of the document bar you will see several buttons.

Button with a Click the button with a hand on it to extract the document group from the user
hand interface. In this way you will create a document window that you can freely
position or change in size.
If you would like to merge two document groups, click the button with the hand in
one of the two document groups. With the left mouse button depressed, drag the
document group with all the files loaded in it, onto an existing one.
You can only position document groups as you wish when you are in the expert
mode. In standard mode the button with the hand is not available.

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 User interface

Arrow button
The arrow buttons located at the top right of the document group are, to begin
with, inactive when you start your software. The arrow buttons will only become
active when you have loaded so many documents that all of their names can no
longer be displayed in the document group. Then you can click one of the two
arrows to make the fields with the document names scroll to the left or the right.
That will enable you to see the documents that were previously not shown.
Button with a cross Click the button with a cross to close the active document. If it has not yet been
saved, the Unsaved Documents dialog box will open. You can then decide
whether or not you still need the data.

Navigation bar in the image window

Multi-dimensional images have their own navigation bar directly in the image
window. Use this navigation bar to determine how a multi-dimensional image is
to be displayed on your monitor, or to change this.
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2.4. Tool Windows

Tool windows combine functions into groups. These may be very different
functions. For example, in the Properties tool window you will find all the
information available on the active document.
Which tool windows are shown by default depends on the layout you have
chosen. You can, naturally, at any time, make specific tool windows appear and
disappear manually. To do so, use the View > Tool Windows command.
Manually displayed or hidden tool windows will be saved together with the layout
and are available again the next time you start the program. Resetting the layout
using the View > Layout > Reset Current Layout command will have the result
that only the tool windows that are defined by default will be displayed.

Position of the tool windows

The user interface is to a large degree configurable. For this reason, tool
windows can be docked, freely positioned, or integrated in document groups.
Docked tool windows Tool windows can be docked to the left or right of the document window, or
below it. To save space, several tool windows may lie on top of each other. They
are then arranged as tabs. In this case, activate the required tool window by
clicking the title of the corresponding tab below the window.

Freely positioned tool You can only position tool windows as you wish when you are in the expert
windows
mode.
You can at any time float a tool window. The tool window then behaves exactly
the way a dialog box does. To release a tool window from its docked position,
click on its header with your left mouse button. Then, while keeping the left
mouse button depressed, drag the tool window to wherever you want it.

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 User interface

Integrating a tool You can only add tool windows to a document group when you are in the expert
window into a
document group
mode.
You can integrate certain tool windows in the document group, for example, the
File Explorer tool window. To do this, use the Document Mode command. To
open a context menu containing this command, rightclick any tool window's
header.
The tool window will then act similarly to a document window, e.g., like an image
window.
Use the Tool Window Mode command, to float a tool window back out of the
document group. To open a context menu containing this command, rightclick
any tool window's header.

Buttons in the header

In the header of every tool window, you will find the three buttons Help, Auto
Hide and Close.

Click the Help button to open the online help for the tool window.
Click the Auto Hide button to minimize the tool window.
Click the Close button to hide the tool window. You can make it reappear at any
time, for example, with the View > Tool Windows command.

Context menu of the header

To open a context menu, rightclick a tool window's header. The context menu
can contain the Auto Hide, Document Mode, and Transparency commands.
Which commands will be shown, depends on the tool window.
Additionally, the context menu contains a list of all of the tool windows that are
available. Every tool window is identified by its own icon. The icons of the
currently displayed tool windows appear clicked. You can recognize this status
by the icon's background color.
Use this list to make tool windows appear.
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2.5. Working with documents

You can choose from a number of possibilities when you want to open, save, or
close documents. As a rule, these documents will be images. However, your
software supports several more document types. You will find a list of supported
documents in the online help.

Saving documents
You should always save important documents immediately following their
acquisition. You can recognize documents that have not been saved by the star
icon after the document's name.
There are a number of ways in which you can save documents.
1. To save a single document, activate the document in the document group
and use the File > Save As... command.
2. Use the Documents tool window.
Select the desired document and use the Save command in the context
menu. For the selection of documents, the standard MS-Windows
conventions for Multiple Selection are valid.

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 User interface

3. Use the Gallery tool window.

Select the desired document and use the Save command in the context
menu. For the selection of documents, the standard MS-Windows
conventions for Multiple Selection are valid.
4. Save your documents in a database That enables you to store all manner of
data that belongs together in one location. Search and filter functions make it
quick and easy to locate saved documents. Detailed information on inserting
documents into a database can be found in the online help.
Autosave and close 1. When you exit your software, all of the data that has not yet been saved will
be listed in the Unsaved Documents dialog box. This gives you the chance to
decide which document you still want to save.
2. With some acquisition processes the images will be automatically saved
when the acquisition has been completed. You will find an overview of the
acquisition processes that are supported in the online help.
3. You can also configure your software in such a way that all images are saved
automatically after image acquisition. To do so, use the Acquisition Settings
> Saving dialog box.
There, you can also set that your images are automatically saved in a
database, after image acquisition.

Closing documents
There are a number of ways in which you can close documents.
1. Use the Documents tool window.
Select the desired document and use the Close command in the context
menu. For the selection of documents, the standard MS-Windows
conventions for Multiple Selection are valid.
2. To close a single document, activate the document in the document group
and use the File > Close command. Alternatively, you can click the button
with the cross . You will find this button on the top right in the document
group.
3. Use the Gallery tool window.
Select the desired document and use the Close command in the context
menu. For the selection of documents, the standard MS-Windows
conventions for Multiple Selection are valid.
Closing all documents To close all loaded documents use the Close All command. You will find this
command in the File menu, and in both the Documents and the Gallery tool
window's context menu.
Closing a document To close a document immediately without a query, close it with the [Shift] key
immediately
depressed. Data you have not saved will be lost.

Opening documents
There are a number of ways in which you can open or load documents.
1. Use the File > Open... command.
2. Use the File Explorer tool window.
To load a single image, doubleclick on the image file in the File Explorer tool
window.
To load several images simultaneously, select the images and with the left
mouse button depressed, drag them into the document group. For the
selection of images, the standard MS-Windows conventions for Multiple
Selection are valid.

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 User interface

3. Drag the document you want, directly out of the MS-Windows Explorer, onto
your software's document group.
4. Use the Database > Load Documents command to load documents from the
database into your software. Further information is available in the online
help.
Generating a test If you want to get used to your software, then sometimes any image suffices to
image
try out a function.
Press [Ctrl + Shift + Alt + T] to generate a color test image.
With the [Ctrl + Alt + T] shortcut, you can generate a test image that is made up
of 256 gray values.
Using sample images During the installation of your software some sample images have been installed,
too. You can find a sample image for each image type. Regarding the information
as to where the example images are located, please refer to the online help.
These example images might be of help to you when you familiarize yourself with
your software.

Activating documents in the document group

There are several ways to activate one of the documents that has been loaded
into the document group and thus display it on your monitor.
1. Use the Documents tool window. Click the desired document there.
2. Use the Gallery tool window. Click the desired document there.
3. Click the title of the desired document in the document group.
4. To open a list with all currently loaded documents, use the [Ctrl + Tab]
shortcut. Leftclick the document that you want to have displayed on your
monitor.
5. Use the keyboard shortcut [Ctrl + F6] or [Ctrl + Shift + F6], to have the next
document in the document group displayed. With this keyboard shortcut you
can display all of the loaded documents one after the other.
6. In the Window menu you will find a list of all of the documents that have been
loaded. Select the document you want from this list.
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 Configuring the system

3. Configuring the system

Why do you have to After successfully installing your software you will need to first configure your
configure the system?
image analysis system, then calibrate it. Only when you have done this will you
have made the preparations that are necessary to ensure that you will be able to
acquire high quality images that are correctly calibrated. When you work with a
motorized microscope, you will also need to configure the existing hardware, to
enable the program to control the motorized parts of your microscope.
When do you have to You will only need to completely configure and calibrate your system anew when
configure the system?
you have installed the software on your PC for the first time, and then start it.
When you later change the way your microscope is equipped, you will only need
to change the configuration of certain hardware components, and possibly also
recalibrate them.
Switching off your When you use the MS-Windows Vista operating system: Switch the hibernation
operating system's
mode off.
hibernation mode
To do so, click the Start button located at the bottom left of the operating
system's task bar.
Use the Control Panel command.
Open the System and Maintenance > Power Options > Change when the
computer sleeps window. Here, you can switch off your PC's hibernation mode.

Process flow of the configuration

To set up your software, the following steps will be necessary:
Select the camera and the microscope

Specifying which hardware is available

Configuring the interfaces

Configuring the specified hardware

Calibrating the system

Select the camera and The first time you start your software after the installation has been made, a
the microscope
quick configuration with some default settings will be made. In this step you need
only to specify the camera and microscope types, in the Quick Device Setup
dialog box. The microscope will be configured with a selection of typical
hardware components. Further information is available in the online help.
Specifying which Your software has to know which hardware components your microscope is
hardware is available
equipped with. Only these hardware components can be configured and
subsequently controlled by the software. In the Acquire > Devices > Device List
dialog box, you select the hardware components that are available on your
microscope.
Configuring the Use the Interfaces dialog box, to configure the interface between your
interfaces
microscope or other motorized components, and the PC on which your software
runs.

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 Configuring the system

Configuring the Usually various different devices, such as a camera, a microscope and/or a
specified hardware
stage, will belong to your system. Use the Acquire > Devices > Device Settings...
dialog box to configure the connected devices so that they can be correctly
controlled by your software.
Calibrating the system When all of the hardware components have been registered with your software
and have been configured, the functioning of the system is already ensured.
However, it's only really easy to work with the system and to acquire top quality
images, when you have calibrated your software. The detailed information that
helps you to make optimal acquisitions, will then be available.
Your software offers a wizard that will help you while you go through the
individual calibration processes. Use the Acquire > Calibrations... command to
start the software wizard.
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13
 Acquiring single images

4. Acquiring single images

You can use your software to acquire high resolution images in a very short
period of time. For your first acquisition you should carry out these instructions
step for step. Then, when you later make other acquisitions, you will notice that
for similar types of sample many of the settings you made for the first acquisition
can be adopted without change.

Acquiring a single image

1. Switch to the "Acquisition" layout. To do this, use, e.g., the View > Layout >
Acquisition command.

2. In the Camera Control tool window, click the Live button.

Selecting the objective 3. On the Microscope Control toolbar, click the button with the objective that
you use for the image acquisition.
Switching on the live- 4. Go to the required specimen position in the live-image.
image
Setting the image 5. Bring the sample into focus. The Focus Indicator toolbar is there for you to
quality
use when you are focusing on your sample.
6. Check the color reproduction. If necessary, carry out a white balance.
7. Check the exposure time. You can either use the automatic exposure time
function, or enter the exposure time manually.
8. Select the resolution you want.
Acquiring and saving
an image
9. On the Camera Control tool window, click the Snap button.
 The image you have acquired will be shown in the document group.
10. Use the File > Save As... command to save the image. Use the
recommended TIF file format.
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 Acquiring single images

Behavior of the live window

The behavior of the live window depends on the acquisition settings in the
Acquisition Settings > Acquisition > General dialog box.
Prerequisite For the following step-by-step instructions, the Keep document when live is
stopped option is selected, and the Create new document when live is started
check box is cleared.

Switching the live-image on and off without acquiring

an image
1. Make the Camera Control tool window appear. To do this, use, e.g., the View
> Tool Windows > Camera Control command.

2. Click the Live button in the Camera Control tool window.

 A temporary live window named "Live (active)" is created in the
document group.
 The live-image will be shown in the live window.
 You can always recognize the live modus by the changed look of the

Live button in the Camera Control tool window.

3. Click the Live button again.

 The live mode will be switched off.
 The active live-image will be stopped.
 The live window's header will change to "Live (stopped)". You can save
the stopped live-image located in the live window just as you can every
other image.
The live window may look similar to an image window, but it will be handled
differently. The next time you switch on the live mode, the image will be
overwritten. Additionally, it will be closed without a warning message when your
software is closed.

Switching to the live-image and acquiring an image

1. Make the Camera Control tool window appear. To do this, use, e.g., the View
> Tool Windows > Camera Control command.

2. Click the Live button in the Camera Control tool window.

 A temporary live window named "Live (active)" will be created in the
document group.
 The live-image will be shown in the live window.
 You can always recognize the live modus by the changed look of the

Live button in the Camera Control tool window.

3. Click the Snap button.

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 Acquiring single images

 The live mode will be switched off. The live window's header will change
to "Live (stopped)".
 At the same time, a new image document will be created and displayed
in the document group. You can rename and save this image. If you
have not already saved it when you end your software, you will be asked
if you want to do so.

Displaying the live-image and the acquired images

simultaneously
Task You want to view the live-image and the acquired images simultaneously. When
you do this, it should also be possible to look through the acquired images
without having to end the live mode.
1. Close all open documents.
2. Open the Acquisition Settings > Acquisition > General dialog box.
To do so, click, e.g., the Acquisition Settings button on the Camera
Control tool window.
3. There, make the following settings:
 Choose the Keep document when live is stopped option.
 Clear the Create new document when live is started check box.
 Select the Continue live after acquisition check box.
4. Switch to the live mode. Acquire an image, then switch the live mode off
again
 Both of the image windows "Live (stopped)" and "Image_<Serial No.>"
are now in the document group.
 The "Live (stopped)" image window is active. That's to say, right now you
see the stopped live-image in the document group. In the document bar,
the Name "Live (stopped)" is highlighted in color.
5. Split the document group, to have two images displayed next to each other.
That's only possible when at least two images have been loaded. That's why
you created two images in the first step.
6. Use the Window > Split/Unsplit > Split/Unsplit Document Group (Left).
 This command creates a new document group to the left of the current
document group. In the newly set up document group, the active
document will be automatically displayed. Since in this case, the active
document is the stopped live-image, you will now see the live window on
the left and the acquired image on the right.
7. Start the live mode.
 In the document group, the left window will become the live window Live
(active)". Here you see the live-image.
8. Activate the document group on the right. To do so, click, for example, the
image displayed there.

9. Click the Snap button.

 The acquired image will be displayed in the active document group. In
this case, it's the document group on the right.
 After the image acquisition has been made, the live-image will
automatically start once more, so that you'll then see the live-image
again on the left.

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 Acquiring single images

 While the live-image is being shown on the left, you can switch as often
as you want between the images that have up till then been acquired.

You can set up your software's user interface in such a way that you can view the
live-image (1) and the images that have up till then been acquired (2), next to
one another.
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 Acquiring image series

5. Acquiring image series

5.1. Time stack
What is a time stack?

You can combine a series of separate images into one image. In a time stack all
frames have been acquired at different points of time. A time stack shows you
how an area of a sample changes with time. You can play back a time stack just
as you do a movie.
A standard image is two dimensional. The position of every pixel will be
determined by its X- and Y-values. With a time stack, the time when the image
was acquired is an additional piece of information or "dimension" for each frame.
The frames making up a time stack can be 8-bit gray-value images, 16-bit gray-
value images, or 24-bit true-color images.
Note: A time stack can also be an AVI video. You can load and play back the AVI
file format with your software.

How do I recognize a time stack?

You can immediately recognize the different image types by the icon which
appears in front of the image name in the document group, or in the Documents
tool window. When it is a time stack, this icon will be supplemented by a small
clock. A time stack that is made up of true-color images has, e.g., this icon .
In the Properties tool window, you can use the Frame Count entry to find out how
many individual images are contained in any given image.
A time stack will automatically have its own navigation bar directly in the image
window. Use this navigation bar to browse through the frames making up a time
stack, or to play back the time stack like a movie.
Further information on the navigation bar is available in the online help.

Creating time stacks

There are different ways in which you can generate a time stack.
To acquire a time stack, use one of the two acquisition processes Time Lapse or
Movie.
Use the Image > Combine Frames… command, to have several individual
images combined into a time stack. A detailed description of the Combine
Frames dialog box can be found in the online help. .

Displaying time stacks

A time stack contains much more data than can be displayed on your monitor.
A time stack will automatically have its own navigation bar directly in the image
window. Use this navigation bar to determine which of the frames from a time

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 Acquiring image series

stack is to be displayed on your monitor. You can also play back a time stack just
as you would a movie.

Alternatively, you can also use the Dimension Selector tool window, to determine
how a time stack is to be displayed on your monitor, or to change the display.
Hiding the navigation You can also hide the navigation bar. To do this, use the Tools > Options...
bar
command. Select the Images > General entry in the tree view. Clear the Show
image navigation toolbar check box.

Saving time stacks

When you save time stacks, you will, as a rule, use the VSI file format. Only
when you use this file format is there no limit to the size a time stack can be.
When you save smaller time stacks, you can also use the TIF or the AVI file
format. With any other file format you will lose most of the image information
during saving.
Use the File > Save As... command.

Converting time stacks

Breaking down time Use the Image > Separate > Time Frames menu command, to have a time stack
stacks into individual
broken down into selected individual images.
images

Reducing the number It is possible that, within a time stack, only a short period of time interests you.
of frames within a time
Use the Extract command, to create a new time stack that only contains a
stack
selection of frames, from an existing time stack. In this way, you will reduce the
number of frames within a time stack to only those that interest you. You will find
this command in the context menu in the tile view for time stacks. Further
information on this command is available in the online help.
Converting time stacks When you save a time stack in another file format as TIF or VSI, the time stack
while saving them
will also be converted. The time stack will then be turned into a standard true-
color image. This image shows the frame that is at that moment displayed on the
monitor.

Processing time stacks

Image processing operations, e.g., a sharpen filter, affect either the whole image
or only a selection of individual images. You can find most of the image
processing operations in the Process menu.
The dialog box that is opened when you use an image processing operation is
made up in the same way for every operation. In this dialog box, select the Apply
on > Selected frames and channels option, to determine that the function only
affects the selected frames.
Select the Apply on > All frames and channels option, to process all of the
individual images.
Select the individual images that you want to process, in the tile view. Further
information on mthis image window view can be found in the online help. Look
through the thumbnails and select the images you want to process. In the tile
view, the standard MS-Windows conventions for multiple selection are valid.
An image processing operation does not change the source image's dimensions.
The resulting image is, therefore, comprised of the same number of separate
images as the source image.
00011

19
 Acquiring image series

5.2. Time Lapse / Movie

Both the Time Lapse and the Movie acquisition processes document the way a
sample changes with time. What is the difference between the two processes?
When is it better for me Use the "Time Lapse" acquisition process in the following cases:
to acquire a time stack?
 Use the "Time Lapse" acquisition process when processes that run
slowly are to be documented, e.g., where an acquisition is to be made
only every 15 minutes.
 Use the "Time Lapse" acquisition process when, while the acquisition is
in progress, you want to see the frames that have already been acquired,
for example, to check on how an experiment is progressing. To do this,
click the Tile View button in the navigation bar in the image window.
 Use the "Time Lapse" acquisition process when you want to use those of
your software's additional functions that can only be saved in the VSI or
TIF file format.
For example, to measure objects, to insert drawing elements such as
arrows, or a text, or to have the acquisition parameters for the camera
and microscope that you've used, available at any time in the future.
 Use the "Time Lapse" acquisition process when the important thing is to
achieve an optimal image quality, and the size of the file is no problem.
Saving a time stack as You can also save a time stack as an AVI file, at a later date. To do this, load the
an AVI
time stack into the document group, select the File > Save as... command, and
select the AVI file type. Make, if necessary, additional settings in the Select AVI
Save Options dialog box.
When is it better for me Use the "Movie" acquisition process in the following cases:
to acquire a movie?
 Use the "Movie" acquisition process when processes that run very
quickly are to be documented (the number of acquisitions per second is
considerably higher with movies than with time stacks).
 Use the "Movie" acquisition process when you want to give the movie to
third persons who do not have this software (AVI files can also be played
back with the MS Media Player).
 Use the "Movie" acquisition process when keeping file sizes small is of
great importance.
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20
 Acquiring image series

5.3. Acquiring movies and time stacks

With your software you can acquire movies and time stacks.

Acquiring a movie
You can use your software to record a movie. When you do this, your camera will
acquire as many images as it can within an arbitrary period of time. The movie
will be saved as a file in the AVI format. You can use your software to play it
back.
1. Switch to the "Acquisition" layout. To do this, use, e.g., the View > Layout >
Acquisition command.
Selecting the objective 2. On the Microscope Control toolbar, click the button with the objective that
you want to use for the movie acquisition.
Selecting the storage 3. In the Camera Control tool window's toolbar, click the Acquisition Settings
location
button.
 The Acquisition Settings dialog box will open.
4. Select the Saving > Movie entry in the tree structure.
5. Decide how you want to save the recorded movies after the acquisition
process is finished. Select the Filesystem entry in the Automatic save >
Destination list to automatically save the movies you have acquired.
 The Base field located in the Directory group shows the directory that will
currently be used when your movies are automatically saved.
6. Click the [...] button next to the Base field to alter the directory.
Selecting the  The AVI file format is preset in the File type list. This is a fixed setting
compression method
that cannot be changed.
7. Click the Options... button if you want to compress the AVI file in order to
reduce the movie's file size.
8. From the Compression list, select the M-JPEG entry and confirm with OK.
Please note: Compressing the movie is only possible if the selected
compression method (codec) has already been installed on your PC. If the
compression method has not been installed the AVI file will be saved
uncompressed.
The selected compression method must also be available on the PC that
is used for playing back the AVI. Otherwise the quality of the AVI may be
considerably worse when the AVI is played back.
9. Close the Acquisition Settings dialog box with OK.
Setting the image 10. Switch to the live mode, and select the optimal settings for movie recording,
quality
in the Camera Control tool window. Pay special attention to setting the
correct exposure time.
 This exposure time will not be changed during the movie recording.
11. Find the segment of the sample that interests you and focus on it.

21
 Acquiring image series

Switching to the "Movie 12. Select the Movie recording check box (1). The check box can be found below
recording" mode
the Live button in the Camera Control tool window.

 The Snap button will be replaced by the Movie button.

Starting movie
recording

13. Click the Movie button to start the movie recording.

 The live-image will be shown and the recording of the movie will start
immediately.
 In the status bar a progress indicator is displayed. At the left of the slash
the number of already acquired images will be indicated. At the right of
the slash an estimation of the maximum possible number of images will
be shown. This number depends on your camera's image size and
cannot exceed 2GB.

 This icon on the Movie button will indicate that a movie is being
recorded at the moment.
Stopping movie
recording

14. Click the Movie button again to end the movie recording.
 The first image of the movie will be displayed.
 The navigation bar for time stacks will be shown in the document group.
Use this navigation bar to play the movie.
 The software will remain in the "Movie recording" mode until you clear
the Movie recording check box once more.

22
 Acquiring image series

Acquiring a time stack

In a time stack all frames have been acquired at different points of time. With a
time stack you can document the way the position on the sample changes with
time. To begin with, for the acquisition of a time stack make the same settings in
the Camera Control tool window as you do for the acquisition of a snapshot.
Additionally, in the Process Manager tool window, you have to define the time
sequence in which the images are to be acquired.
Task You want to acquire a time stack over a period of 10 seconds. One image is to
be acquired every second.
1. Switch to the "Acquisition" layout. To do this, use, e.g., the View > Layout >
Acquisition command.
Selecting the objective 2. On the Microscope Control toolbar, click the button with the objective that
you want to use for the image acquisition.
Setting the image 3. Switch to the live mode, and select the optimal settings for your acquisition,
quality
in the Camera Control tool window. Pay special attention to setting the
correct exposure time. This exposure time will be used for all of the frames in
the time stack.
4. Choose the resolution you want for the time stack's frames, from the
Resolution > Snap/Process list.
5. Find the segment of the sample that interests you and focus on it.
Selecting the 6. Activate the Process Manager tool window.
acquisition process
7. Select the Automatic Processes option.

8. Click the Time Lapse button.

 The button will appear clicked. You can recognize this status by the
button's colored background.
 The [ t ] group will be automatically displayed in the tool window.
9. Should another acquisition process be active, e.g., Multicolor, click the button
to switch off the acquisition process.
 The group with the various acquisition processes should now look like
this:

Selecting the 10. Clear the check boxes Delay and As fast as possible.
acquisition parameters
11. Specify the time that the complete acquisition is to take, e.g., 10 seconds.
Enter the value "00000:00:10" (for 10 seconds) in the Recording time field.
You can directly edit every number in the field. To do so, simply click in front
of the number you want to edit.
12. Select the radio button on the right-hand side of the field to specify that the
acquisition time is no longer to be changed. The lock icon will
automatically appear beside the selected radio button.
13. Specify how many frames are to be acquired.
Enter e.g., 10 in the Cycles field.
 The Interval field will be updated. It shows you the time that will elapse
between two consecutive frames.

23
 Acquiring image series

Acquiring a time stack

14. Click the Start button.
 The acquisition of the time stack will start immediately.

 The Start Process button changes into the Pause button. A click on
this button will interrupt the acquisition process.

 The Stop button will become active. A click on this button will stop
the acquisition process. The images of the time stack acquired until this
moment will be preserved.
 At the bottom left, in the status bar, the progress bar will appear. It
informs you about the number of images that are still to be acquired.
 The acquisition has been completed when you can once more see the

Start button in the Process Manager tool window, and the progress
bar has been faded out.
 You will see the time stack you've acquired in the image window. Use the
navigation bar located in the image window to view the time stack.
Further information on the navigation bar is available in the online help.
 The time stack that has been acquired will be automatically saved. The
storage directory is shown in the Acquisition Settings > Saving > Process
Manager dialog box. The preset file format is VSI.
Note: When other programs are running on your PC, for instance a virus
scanning program, it can interfere with the performance when a time stack is
being acquired.
00304 12012011

5.4. Z-stack
What is a Z-stack?

You can combine a series of separate images into one image file. A Z-stack
contains frames acquired at different focus positions. A Z-stack is needed, e.g.,
for calculating an EFI image by the Process > Enhancement > EFI Processing...
command.
A standard image is two dimensional. The position of every pixel will be
determined by its X- and Y-values. With a Z-stack, the focus position or the
height of the sample is an additional item of information for every pixel.
The frames making up a Z-stack can be 8-bit gray-value images, 16-bit-gray-
value images or 24-bit-true-color images.

24
 Acquiring image series

How do I recognize a Z-stack?

You can immediately recognize a multi-dimensional image by its icon which
appears in front of the image name in the document group or in the Documents
tool window. When it is a Z-stack, this icon will be supplemented by a small Z .
In the Properties tool window, you can use the Frame Count entry to find out how
many individual images are contained in any given image.
A Z-stack image will automatically have its own navigation bar directly in the
image window. Use this navigation bar to browse through the frames making up
a Z-stack, or to play back the Z-stack like a movie. Further information on this
navigation bar is available in the online help.

Creating a Z-stack
There are different ways in which you can generate a Z-stack.
1. To acquire a Z-stack, use the "Z-Stack" acquisition process.
2. Use the Image > Combine Frames… command, to have several
separate images combined into a Z-stack.

Displaying a Z-stack
A Z-stack contains much more data than can be displayed on your monitor.
A Z-stack image will automatically have its own navigation bar directly in the
image window. Use this navigation bar to determine which of the frames from a
Z-stack is to be displayed on your monitor. You can also play back the Z-stack
just as you would a movie.

Alternatively, you can also use the Dimension Selector tool window, to set how a
Z-stack is to be displayed on your monitor, or to change the display.

Saving a Z-stack
Please note: Z-stacks can only be saved in the TIF or VSI file format. Otherwise
they loose a great deal of their image information during saving.

Converting a Z-stack
Breaking down Z- Use the Image > Separate > Height Frames menu command, to have a Z-stack
stacks into separate
broken down into selected frames.
images

Reducing the number It is possible that, within a Z-stack, only a short Z-range interests you. Use the
of frames within a Z-
Extract command, to create a new Z-stack that only contains selected frames,
stack
from an existing Z-stack. In this way, you will reduce the number of frames within
a Z-stack to only those that interest you. You can find this command in the
context menu in the tile view for Z-stacks.
Converting Z-stacks When you save a Z-stack in another file format as TIF or VSI, the Z-stack will
while saving them
automatically be converted. The Z-stack will then be turned into a standard true-
color image. This image shows the frame that is at that moment displayed on the
monitor.
00012

25
 Acquiring image series

5.5. Acquiring a Z-stack

With the Z-Stack acquisition process, you acquire a series of frames one after
the other, a Z-stack.

Acquiring a Z-stack
Example: You want to acquire a Z-stack. The sample is approximately 50 µm
thick. The Z-distance between two frames is to be 2 µm.

1. Switch to the "Acquisition" layout. To do this, use, e.g., the View > Layout >
Acquisition command.
Selecting the objective 2. On the Microscope Control toolbar, click the button with the objective that
you want to use for the image acquisition.
Setting the image 3. Switch to the live mode, and select the optimal settings for your acquisition,
quality
in the Camera Control tool window. Pay special attention to setting the
correct exposure time. This exposure time will be used for all of the frames in
the Z-stack.
4. Search out the required position in the sample.
Selecting the 5. Activate the Process Manager tool window.
acquisition process
6. Select the Automatic Processes option.

7. Click the Z-Stack button.

 The button will appear clicked. You can recognize this status by the
button's colored background.
 The [ Z ] group will be automatically displayed in the tool window.
8. Should another acquisition process be active, e.g., Multi Channel, click the
button to switch off the acquisition process.
 The group with the various acquisition processes should now look like
this:

Selecting the 9. Select the Range entry in the Define list.

acquisition parameters
10. Enter the Z-range you want, in the Range field. In this example, enter a little
more than the sample's thickness (= 50 µm), e.g., the value 60.
11. In the Step Size field, enter the required Z-distance, e.g., the value 2, for a Z-
distance of 2 µm. The value should correspond with your objective's depth of
focus.
 In the Z-Slices field you will then be shown how many frames are to be
acquired. In this example, 31 frames will be acquired.
12. Find the segment of the sample that interests you and focus on it. To do this,
use the arrow buttons in the [ Z ] group. The buttons with a double arrow
move the stage in larger steps.
Acquiring an image
13. Click the Start button.

26
 Acquiring image series

 Your software now moves the Z-drive of the microscope stage to the
start position. The starting positions lies half of the Z-range deeper than
the stage's current Z-position.
 The acquisition of the Z-stack will begin as soon as the starting position
has been reached. The microscope stage moves upwards step by step
and acquires an image at each new Z-position.
 You can see the acquired Z-stack in the image window. Use the
navigation bar located in the image window to view the Z-stack. Further
information on the navigation bar is available in the online help.
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27
 Acquiring fluorescence images

6. Acquiring fluorescence images

6.1. Multi-channel image
What is a multi-channel image?
A multi-channel image combines a series of monochrome images into one
image. The multi-channel image usually shows a sample that has been stained
with several different fluorochromes. The multi-channel image is made up of a
combination of the individual fluorescence images. You can have the individual
fluorescence images displayed separately or also as a superimposition of all of
the fluorescence images.

At the top of the illustration you can see the individual fluorescence images (1).
Below, you can see the superimposition (2) of the separate fluorescence images.
The separate images' The separate images making up a multi-channel image can be 8-bit gray-value
image type
images, or 16-bit gray-value images.
Combination with a A multi-channel image can be combined with a time stack or a Z-stack. A multi-
times stack or Z-stack
channel time stack, for instance, then incorporates several color channels. Every
color channel incorporated in the image is reproduced with its own time stack.

How do I recognize a multi-channel image?

You can immediately recognize a multi-channel image by this icon which

appears in front of the image name in the document group or in the Documents
tool window.
In the Properties tool window, you can use the Channels entry, to find out how
many channels are contained in any given image.
A multi-channel image will automatically have its own navigation bar, directly in
the image window. Use this navigation bar to set how a multi-channel image is to
be displayed in the image window, or to change this.
Further information on the navigation bar for multi-channel images is available in
the online help.

28
 Acquiring fluorescence images

Creating multi-channel images

Your software offers you several ways of acquiring a multi-channel image.
Use the Multi Channel automatic acquisition process to acquire a multi-channel
image.
Use the Image > Combine Channels… command, to have several separate
images combined into a multi-channel image.

Displaying multi-channel images

A multi-channel image contains much more data than can be displayed on your
monitor.
Using the navigation
bar

When you load a multi-channel image into your software, you'll see a navigation
bar in the image window, that provides you with access to all of the fluorescence
channels.
 You can have the individual fluorescence images displayed separately or
also as a superimposition of all of the fluorescence images (1).
 Should you have acquired a brightfield of the sample together with the
fluorescence images, you can make this brightfield appear or disappear
(2).
 The individual fluorescence images are monochrome. For this reason,
you can change the color mapping however you like. You can display the
fluorescence channels in the fluorescence colors, use a pseudo color
table of your choice, or also display the original images (3).
Further information on the navigation bar for multi-channel images is available in
the online help.
Hiding the navigation You can also hide the navigation bar. To do this, use the Tools > Options...
bar
command. Select the Images > General entry in the tree view. Clear the Show
image navigation toolbar check box.
Using the "Dimension Alternatively, you can also use the Dimension Selector tool window, to set how a
Selector" tool window
multi-channel image is to be displayed on your monitor, or to change this. There
you can, for example, change the fluorescence colors for individual color
channels.

Saving multi-channel images

Please note: Multi-channel images can only be saved in the TIF or VSI file
format. Otherwise they loose a great deal of their image information during
saving.

29
 Acquiring fluorescence images

Converting multi-channel images

Breaking down a multi- Use the Separate command, to have a multi-channel image broken down into
channel image into its
chosen color channels. The resulting images are still of the "multi-channel" type,
color channels
contain though, only one color channel.
There are several ways of accessing this command:
 Click the Separate Channels button in the Dimension Selector tool
window.
 Use the Separate command from the Dimension Selector tool window's
context menu.
 Use the Image > Separate > Channels menu command.
Reducing the color Use the Extract command, to create a new multi-channel image that is made up
channels in a multi-
of fewer color channels than the source image.
channel image
Select all of the color channels you wish to retain, in the Dimension Selector tool
window. Then use the Extract command in the tool window's context menu.
Converting multi- When you save a multi-channel image in another file format as TIF or VSI, the
channel images while
multi-channel image will also be converted. The multi-channel image then
saving them
becomes a standard 24-bit true-color image. This image will always show exactly
what is currently displayed on your monitor, that is to say, e.g., the
superimposition of all of the channels or possibly only one channel.

Processing multi-channel images

Image processing operations, e.g., a sharpen filter, affect either the whole image
or only a selection of individual images. You can find most of the image
processing operations in the Process menu. Click here for more information on
working with image processing operations.
Select the individual images that you want to process in the Dimension Selector
tool window. The frames you have selected will be highlighted in color in the tool
window.
The dialog box that is opened when you use an image processing operation is
made up in the same way for every operation. In this dialog box, select the Apply
on > Selected frames and channels option, to determine that the function only
affects the selected frames.
Select the Apply on > All frames and channels option, to process all of the
individual images.
An image processing operation does not change the source image's dimensions.
The resulting image is, therefore, comprised of the same number of separate
images as the source image.
00010

30
 Acquiring fluorescence images

6.2. Before and after you've acquired a

fluorescence image
Before you acquire a fluorescence image
Defining observation 1. Define observation methods for your color channels.
methods

Setting up the 2. Use the View > Tool Windows > Microscope Control command, to make the
microscope for the
Microscope Control tool window appear.
acquisition of a
fluorescence image  In the Objectives group, you will find the buttons you use to change
objectives.

 In the Observation method group, you can find a button for every
observation method that has been defined. Observation methods should
have been defined at least for brightfield and for every color channel.
3. Click the required objective's button.
4. Click the button for the observation method with the excitation that has the
longest excitation wavelength (e.g., "Red") .

Setting up the camera for the acquisition of a

fluorescence image
5. Use the View > Tool Windows > Camera Control command to make the
Camera Control tool window appear.
6. Set the image resolution for the acquisition. With a high objective
magnification, you require a lower resolution.
For this purpose, select the required resolution from the Snap/Process list,
located in the Resolution group.
7. Reduce the image resolution in the live mode. When you use a higher
binning in the live mode, the frame rate will be reduced, which enables you to
focus better.
For this purpose, select an entry, e.g., with the supplement "(Binning 2x2)",
from the Live/movie list, located in the Resolution group.
8. Should you work with a color camera: Switch on your camera's grayscale
mode. The Toggle RGB/Grayscale mode button should now look like this
. You can find this button on the Camera Control tool window's toolbar.
9. If it's possible to set different bit depths with your camera, click the Toggle
Bitdepth button to set the maximum bit depth.
.
Switching off the 10. Use the View > Toolbars > Calibrations command, to have the Calibrations
corrections for
toolbar displayed.
brightfield acquisitions
11. Switch off the white balance and the shading correction.

To do that, release these buttons , if they are there and available.

Focusing a 12. Select the automatic exposure time.
fluorescence sample

31
 Acquiring fluorescence images

13. In the Camera Control tool window, click the Live button.
 Should the live-image be too dark, select a higher value in the Camera
Control > Exposure > Exposure compensation list.
 Should the exposure time become longer than 300 ms, reduce it by
increasing the sensitivity or gain.
14. Bring the sample into focus.
 In the camera's black & white mode, you can reduce the diffused light.
Click the Online-Deblur button, located in the Camera Control tool
window's toolbar. You can then decide whether or not you want to apply
the deconvolution filter. It's possible that you might have to lengthen the
exposure time via the exposure time correction.

15. Finish the live mode. To do so, click the Live button in the Camera
Control tool window, once more.
Setting the storage Multi-channel images will be saved by default, as soon as the acquisition has
location
been completed. As file format, the VSI file format will be used.
16. Before you start the acquisition, specify where the file is to be saved.

17. To do this, click the Acquisition Settings button, located in the Process
Manager tool window's toolbar. Select the Saving > Process Manager entry
in the tree view.
 You can find the current directory in the Directory > Base field.
18. Click the [...] button next to the Base field, to change the directory into which
the image is to be saved after its acquisition.

After you've acquired a fluorescence image

Viewing a multi-channel A multi-channel image is made up of the individual fluorescence images. You can
image
set which color channels, resp. combination of color channels, will be displayed
on your monitor. To do this, use the navigation bar in the image window.
Click a color field to make the channel appear or disappear. All of the color
channels that are at the moment displayed on your monitor will be identified by
an eye icon.
The navigation bar also offers you additional possibilities for changing the
appearance of the multi-channel image. Below, is an illustration of the navigation
bar, click one of its buttons and you'll see a description of its functions.

"Properties" tool Numerous acquisition parameters will be saved together with the image.
window
Use the View > Tool Windows > Properties command, to make the Properties
tool window appear. In the Properties tool window, you can find that every color
channel has its own Channel information group. This contains the channel name,
the emission wavelength, the name of the observation method and the exposure
time.
Saving multi-channel The multi-channel image will be automatically saved. You can set the storage
images
directory in the Acquisition Settings > Saving > Process Manager dialog box. The
file format used is VSI.
For the VSI format, a JPEG compression of 90% is preset. You can change the
compression in the Acquisition Settings dialog box under Saving > Process
Manager > Automatic save > Options… .

32
 Acquiring fluorescence images

00363

6.3. Defining observation methods for the

fluorescence acquisition
Before you acquire a fluorescence image, you have to define observation
methods for your color channels. As a rule, observation methods that you can
adapt for your microscope configuration, have already been predefined.
Prerequisite The system has already been configured and calibrated. For this purpose, you
have to enter your hardware components in the Acquire > Devices > Device List
dialog box, and configure them in the Device Settings dialog box. To finalize this
action, calibrate your system by using the Acquire > Calibrations... command.
The tables that follow, contain example configurations for both motorized, and
not motorized, microscopes. Only the hardware components that are relevant for
the acquisition of multi-channel fluorescence images are listed.

Device List Example entries

Non-motorized Motorized microscope

microscope
Microscope

Frame <Name of your BX51 BX61

microscope>

Nosepiece <Name of your Manual Nosepiece Motorized (UCB)

nosepiece>

Mirror Turret <Name of your mirror Manual Mirror BX-RFAA

turret> Turret
Stage

Z-axis <Name of your Not Motorized Motorized (UCB)

controller for the
stage's Z-drive>
Fluorescence/reflected lightpath

Shutter <Name of the Manual Shutter Motorized (UCB)

reflected light
shutter>
Transmission lightpath

Lamp <Name of your Not used UCB Halogen-Lamp

transmission lamp>

Condenser <Name of your Manual Condenser U-UCD8A

condenser>

33
 Acquiring fluorescence images

Device Settings Entries

Nosepiece <Your objectives>

Mirror Turret U-MNU

U-MWB

U-MWG

For a position where there is no mirror cube, select the Free entry.

Condenser With a motorized condenser:

The hardware components Aperture Stop and Top Lens are
additionally listed under the device settings.

Defining the observation method for transmission

brightfield
Hardware components in transmission brightfield: In transmission brightfield,
there is no fluorescence mirror cube in the light path. The reflected light shutter is
closed.
1. Use the Acquire > Devices > Device Customization... command. Activate the
Observation Methods tab.

2. Click the New Observation Method button.

 A dialog box, in which you can enter a name, will be opened.
3. Enter a name for the new observation method, then close the dialog box with
OK. You could, e.g., name your observation method, "MyBF".
Setting up the 4. Select the mirror turret in the Available components list.
mirror turret
 In the dialog box's central area, the settings for the hardware
components that have been selected, will be displayed.
5. In transmission brightfield, the mirror turret isn't allowed to contain a mirror
cube. Therefore, select the Adjust entry in the Status list, located in the
middle of the dialog box, and in the list that's below that one select the Free
entry.
 For manual microscopes: Where a manual microscope is concerned, the
mirror turret can't be automatically moved to the position you want. For
this reason, when you use a manual microscope, you'll receive a
message that asks the user to make this setting manually.
This message will also appear when you are defining the observation
method. Confirm the message with OK.
6. Select the condenser.
For motorized
microscopes: Setting Select the Adjust entry in the Status list, located in the middle of the dialog
up the condenser box, and in the list that's below that one, the Free entry.
7. Select the hardware component Aperture Stop.
Select the Adjust entry in the Status list, located in the middle of the dialog
box. Enter "75%" in the field below that.
8. Select the hardware component Top Lens.
Select the Adjust per objective entry in the Status list.

34
 Acquiring fluorescence images

 In the middle of the Device Customization dialog box, you can now set
the top lens for each objective separately.
 The top lens is only used for objectives with higher magnifications
(upwards of 10x) and is swung out for lower magnifications.
9. Specify for which objectives the top lens should be brought into the light path
and for which objectives it should be removed from the light path.
To do so, select the Use with this objective check box for all objectives.
In the Selected components list, each objective with a lower magnification
than 10x needs to show the Out status. If that isn't the case, click in the
middle of the dialog box on the Out button.
In the Selected components list, each objective with a higher magnification
than 10x or exactly 10x needs to show the In status. If that isn't the case,
click in the middle of the dialog box on the In button.
For motorized 10. Select the transmission lamp. You will find this lamp in the Available
microscopes: Setting components list, under the Transmission entry.
up the transmission
lamp 11. Select the Adjust entry in the Status list, located in the middle of the dialog
box. Set "9 V" for the lamp.
Use the Switch on/off the lamp button, to make sure that the lamp is switched
on.
 The button in the central area of the dialog box looks like this when the
lamp has been switched on.

Saving the observation 12. Click the OK button, to save the new observation method.
method
 The Microscope Control tool window will then contain a new button with
this observation method's name.
 You can now use the observation method in the Process Manager tool
window, for the acquisition of a multi-channel fluorescence image.

Defining observation methods for fluorescence

channels
Task Define an observation method for the acquisition of a fluorescence image. It also
makes sense to do this when you don't work with a motorized microscope, since
then the acquired image will be automatically colored with the correct
fluorescence color.
Summary The following hardware components belong to an observation method for
fluorescence channels. How you integrate these hardware components with the
observation method, is described in detail in these step-by-step instructions.

Hardware component Settings

Fluorochromes Assign the fluorescence colors.

Mirror Turret Choose the mirror cube to be used.

Fluorescence shutter Open the fluorescence shutter for the image acquisition.

Transmitted light lamp Switch off the transmitted light lamp.

35
 Acquiring fluorescence images

1. Use the Acquire > Devices > Device Customization... command. Activate the
Observation Methods tab.

2. Click the New Observation Method button.

3. Enter a name for the new observation method, then close the dialog box with
OK. Name the observation method, e.g., "Blue".
Defining the 4. Assign the fluorescence channel a fluorochrome (e.g., "Blue" or "DAPI") and
fluorochrome a color (e.g., Blue at 470 nm). To do so, select the Fluorochromes entry in
the Available components list.
Select the Use entry in the Status list.
In the Fluorochrome list located below that one, select the fluorochrome to be
used, e.g., the entry Blue or DAPI.
You can change the fluorescence color in the Color list, should that be
necessary.
 It is important in all cases to define the fluorochrome for a fluorescence
observation method, even if you don't automatically change any device
settings. When the fluorescence color is linked to the observation
method, every image you acquire with this observation method will be
automatically colored in the corresponding color. This is valid irrespective
of whether you work with a manual or a motorized microscope.
For motorized It can make sense to use this setting and the additional settings for motorized
microscopes: Setting microscopes also for manual microscopes. When you use a manual microscope,
up the mirror turret you'll receive a message that prompts you, to make this setting manually. As well
as this, the device settings are saved together with the acquired image.
5. Select the mirror turret in the Available components list.
 In the dialog box's central area, the settings for the hardware
components that have been selected, will be displayed.
6. For the acquisition of a fluorescence image a specific mirror cube has to be
used. Therefore, select the Adjust entry in the Status list, located in the
middle of the dialog box, and in the list that's below that one, select the mirror
cube you want.
For motorized 7. In the Available components list, select the shutter that is located below the
microscopes: Setting Fluorescence/reflected entry.
the shutter for the
fluorescence light path 8. When the fluorescence acquisition is made, this shutter must be open.
Therefore, select the Use for acquisition entry in the Status list, located in the
middle of the dialog box.
 Then the shutter will be opened before the image acquisition is made,
and closed when this has been done, to avoid bleaching of the sample.
For motorized 9. Select the transmission lamp. You can find this lamp in the Available
microscopes: Setting components list, under the Transmission entry.
up the transmission
lamp 10. For the acquisition of a fluorescence image, the lamp must be switched off.
Select the Adjust entry in the Status list, located in the middle of the dialog
box.
Use the Switch on/off the lamp button, to make sure that the lamp has been
switched off.
 The button in the central area of the dialog box looks like this when the
lamp has been switched on.

Including camera It's usually better to use a black & white camera for acquiring fluorescence
settings images. Should you use a camera that can be toggled between a color mode and

36
 Acquiring fluorescence images

a grayscale mode, you can integrate the grayscale mode into the observation
mode.
This setting is not necessary when you acquire fluorescence images with the
Multi Channel acquisition process. Before the Multi Channel acquisition process
starts, your software checks whether or not your camera is working in the gray
scale mode. You will then receive a corresponding message, and can reset the
camera before the image acquisition is made.
11. Select your camera in the Available components list.
12. Select the Use entry in the Status list.
13. Some cameras offer gray scale modes in different bit depths. Select the gray
scale mode with the highest bit depth from the Image type list.
Saving the observation 14. Click the OK button, to save the new observation method.
method
 The Microscope Control tool window will then contain a new button with
this observation method's name.
 You can now use this color channel for the Multi Channel acquisition
process.

Using predefined observation methods

As a rule you don't have to completely redefine the observation method required.
Use one of the predefined observation methods, and customize it for your
microscope.
1. Use the Acquire > Devices > Device Customization... command. Activate the
Observation Methods tab.
 In the Name list, you can find all of the observation methods that have
been predefined.
Should no observation methods be available, click the Select Predefined
Observation Methods button. Click the Select All button. Click OK.
 As soon as filter cubes have been entered for the mirror turret, in your
device settings, the appropriate observation methods will be
automatically set up. You will always find the observation method
beneath the filter cube's name.
2. Select a fluorescence channel (e.g., "U-MNU"), in the Name list.

3. Click the Rename Observation Method button.

 The Enter a New Observation Method Name dialog box opens.
4. Enter a more general name (e.g., "Blue" or "DAP"), then click OK.
 The Selected components list contains the following hardware
components. There can be more or fewer components, depending on
what you have chosen in the way of hardware components in the device
list.
 In the mirror turret, the corresponding mirror cube (e.g., "U-MNU") will be
selected.
 The transmission lamp will be switched off.
 The transmission light path's shutter will have the Use for acquisition
status. This means that it will be open when the image is acquired.
5. Assign a fluorochrome (such as "Blue" or "DAPI") to the fluorescence
channel.
6. Click the OK button, to save the new observation method.

37
 Acquiring fluorescence images

 The Microscope Control tool window will then contain a new button with
this observation method's name.
00370 26072011

6.4. Acquiring and combining fluorescence

images
You software supports several image types. The multi-channel image usually
shows a sample that has been stained with several different fluorochromes.
However, it is also possible to acquire a multi-channel image that consists only of
one single channel.

The illustration shows three fluorescence images of the same sample position.
Each image shows another fluorochrome.

Acquiring a fluorescence image

Selecting the 1. Use the View > Toolbars > Observations Methods command, to have the
fluorescence
Observation Methods toolbar displayed.
observation method
2. To load an observation method, click the button with the name of the
fluorescence observation method you want.
 For manual microscopes: Loading a fluorescence observation method
defines that a fluorescence image is to be acquired. For your software,
all observation methods using the Fluorochromes component are
automatically identified as fluorescence observation methods.
 For motorized microscopes: If you load an observation method, this
leads to the microscope being brought into a defined condition. To do so,
all of the microscope's motorized components will be brought into exactly
the position that has been defined for these components in the
observation method.
Selecting the exposure 3. Use the View > Tool Windows > Camera Control command to make the
time for the acquisition
Camera Control tool window appear.

4. Switch to live mode. To do so, click the Live button in the Camera
Control tool window.
 For motorized microscopes: The reflected light shutter will be
automatically opened.
This behavior will be specified when the observation method is defined.
For the shutter, the Use for acquisition status should have been selected.
5. In the Camera Control tool window, select the Exposure > Manual option.
6. Some cameras offer the SFL mode for fluorescence acquisitions (e.g., the
DP72). Switch this mode on.
7. Optimize the exposure time.
Should the exposure time become longer than 500 ms, reduce it by
increasing the sensitivity or gain.
To do this, change the value in the Exposure > Sensitivity field, or use the
Gain slide control.

38
 Acquiring fluorescence images

Focusing a 8. Bring the image into focus.

fluorescence sample

9. Finish the live mode. To do so, click the Live button in the Camera
Control tool window, once more.
 For motorized microscopes: The reflected light shutter will be closed.
 In the image window, you will now see the fluorescence image that has
been acquired. The fluorescence image has the image type "Multi-
channel image" even if it consists only of one single channel.
You can immediately recognize a multi-channel image by this icon
which appears in front of the image name in the document group or in
the Documents tool window.
 The fluorescence image will be displayed using the fluorescence color
that you have defined together with the observation method.
Changing the 10. You can use the Dimension Selector tool window, to define how the
fluorescence image's
display
fluorescence image is to be displayed on your monitor, or to change this.
There you can, for example, change the fluorescence color.
Saving the 11. Use the File > Save As... command afterwards, to save the new multi-
fluorescence image
channel image. Use the TIF file format.
00395 05012011

Combining channels
The Image > Combine Channels... command creates a new multi-channel image
from several separate images. A description of the dialog box can be found in the
online help.

Which images can you combine?

Gray-value images You can combine a series of gray-value images into a multi-channel image.
These can be either 8-bit gray-value images or 16-bit gray-value images. The
prerequisite therefore, is that all of the separate images have the same bit depth,
image size, and image calibration.
Multi-channel Multi-channel images don't necessarily have to be made up of several color
images channels. There can also be multi-channel images that only contain one
fluorescence channel. You can also combine these images into a new multi-
channel image that then contains several fluorescence channels. The
prerequisite therefore, is that all of the separate images have the same bit depth,
image size, and image calibration.

Multi-
You can combine several multi-dimensional images into one image. Prerequisite
dimensional images for such an operation is that all of the individual images only differ in one
dimension (color channel, focus position, or time point), and are of the same
image size.
One example of this are two single color time stacks, that are each made up of
50 frames. Each time stack was acquired with a different color channel. In this
case you can create one multi-channel time stack.
Transmission image Sometimes another image that shows the same position on the sample in the
transmitted light mode, belongs to a series of fluorescence images. You can
combine such a transmission image with the multi-channel image.
The transmission image doesn't have to be of the same image type as the
separate images. However, the image size, image calibration, and the bit depth
have to be the same as the fluorescence images.

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 Acquiring fluorescence images

Example: You can use a true-color image as a transmission image. When the
individual fluorescence images have a bit depth of 16 bit, you can only use a 48-
bit true-color image as a transmission image.

Combining fluorescence images

1. Load the gray-value images that you want to combine into a multi channel
fluorescence image. The sample was, for example, marked with the
fluorochromes, DAPI, and Texas Red.
2. Activate the first image in your software's document group.
Combining channels 3. Use the Image > Combine Channels... command.
 The Combine Channels dialog box opens.
 In the Available Images table, the active image will be automatically
entered as the first color channel.
 When you have acquired the individual fluorescence images with a
suitable observation method, the name of the color channel and the
fluorescence color will be read out of the image and automatically used
in the Combine Channels dialog box.
4. In case you have to change the channel name or want to do so: Click once in
the Name cell. Enter a description of the color channel, e.g., the name of the
fluorochrome used "Texas Red".
You can increase the width of the row so that the description will fit into it.
5. When the fluorescence color can't be read out of the image, the first channel
will, by default, be assigned the color 'Red". To change the active color, click
this color field. Select one of the colors from the palette on the Standard tab,
or activate the Custom tab, to define a color of your choice.
Click the OK button, to close the color palette and return to the Combine
Channels dialog box.
6. In the next free row, click the Images cell. You open a picklist containing all
of the images that you can combine with the active image. Select your next
image. As soon as you click in another row, the new image will be included in
the table.
7. You can now change the characteristics of the new channel. Give the new
channel a name and assign it a color.
8. It's possible to shift the frames in relation to one another. To do this, if
necessary, use the arrow buttons in the Pixel Shift group.
9. You can set the weighting of the individual color channels. Increase, e.g., the
value in the Intensity field, to weight a channel more strongly.
10. Click the OK, button to create your multi-channel image.
A new image document with the default name "Image_<serial No.>" is
created.

With the Combine Channels command, you combine fluorescence images (1)
and (2) into a multi-channel image (3), this can be done with more than two
images also.

40
 Acquiring fluorescence images

Saving a multi-channel 11. Use the File > Save as... command, to save your new multi-channel image.
image
Always use the TIF or VSI file format when saving an image.
Viewing a multi-channel 12. Use the Dimension Selector tool window, to change the fluorescence color,
image
to choose another color mapping, or to switch individual color channels off
and back on.
13. Use the Adjust Display tool window, to change the display of a multi-channel
image on your monitor. You can e.g., change the weighting of individual color
channels in relation to one another.

Combining fluorescence images with a transmission

image
1. Load one or more fluorescence images and the transmission image that you
want to lay over the color channels.
2. Activate the transmission image in your software's document group.
3. Use the Image > Combine Channels... command.
 The Combine Channels dialog box opens.
 If you want to use a true-color image as a transmission image, that
image will be automatically selected in the Transmission list.
When the transmission image is a gray-value image, it will be
automatically entered in the Available Images table.
4. If necessary, choose the transmission image in the Transmission list.
5. Click once in the Images cell. You get a picklist containing all of the loaded
images that you can combine with the selected transmission image. Select
the fluorescence image you want.
6. If necessary, change the new fluorescence channel's properties. Give the
channel a name and assign it a color.
7. Click the OK button, to create the resulting image.
A new image document with the default name "Image_<serial No.>" is
created.
 In the document group, you can then see a superimposition of all of the
images you've combined.
 The resulting image is a multi-layer image with two image layers.
Normally, the two image layers are not of the same image type. For this
reason, the image has this icon in the document group.

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 Acquiring fluorescence images

With the Combine

Channels command,
you combine several
fluorescence images
into a multi-channel
image (1). If you've
acquired the
transmission image at
the same place on the
sample (2), you can
combine it with the
multi-channel image to
make a multi-layer
image (3).

Viewing a multi-layer
image 
The navigation bar will be displayed at the top of the image window. You
can find a button for showing and hiding the transmission image next to
the button for the individual color channels. The eye icon indicates that
the transmission image is currently visible.

8. Click this button in the navigation bar, to hide the transmission image.
 Now, you will only see the multi-channel fluorescence image.

9. Click this button and show the transmission image again.

 Now, you see a superimposition of the transmission image and the multi-
channel fluorescence image.
Moving the 10. Use the View > Tool Windows > Layers command to make the Layers tool
transmission image
window appear.
with respect to the
multi-channel image 11. In the Layers tool window, click the [+] sign (1) and open the image's layers.

You can now see the image's individual layers: transmission mage (2)
and multi-channel image (3 ). The height map can't be seen because it's
absolutely transparent at the moment. The icon at the left side of the
multi-channel image means that it is not possible to move the multi-
channel image.
12. Select the transmission image in the Layers tool window.
13. Activate the Toolbox toolbar. To do this, use, e.g., the View > Toolbars >
Toolbar command.

42
 Acquiring fluorescence images

14. Click the Move button on the Toolbox toolbar.

 On the image window, the mouse pointer will change its shape.
15. Move the whole image with the left mouse button depressed.

16. Click, e.g., the Selection Tool button on the Toolbox toolbar, to leave the
move mode.
Changing the weighting When you display the transmission image and the multi-channel image
between a transmission
simultaneously in the image window, the transmission image will cover the multi-
image and a multi-
channel image channel image, and for this reason, you can't see the multi-channel image. You
can display both images transparently, and in that way be able to see parts of
both images.
17. To start with, select the image layer in the Layers tool window. To do so,
simply click the layer's name.
18. The layer you have selected will then be shown with a colored background in
the tool window.

19. Then, click the Set Layer Opacity button. You will find this button in the
tool window's toolbar.
 In the tool window, a slide control will then appear, with which you can
set the degree of transparency.

20. Use the slide control to set the degree of transparency you want. At a value
of 100% the image layer is opaque. At a value of 0% the image layer will be
completely faded out.
21. When you're satisfied with the transparency setting, click once on any place
on the user interface.
Saving a multi-layer 22. Use the File > Save as... command, to save your new multi-layer image.
image
Always use the TIF or VSI file format when saving an image.
00067 25072011

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 Acquiring fluorescence images

6.5. Acquiring multi-channel fluorescence

images
Use the Multi Channel automatic acquisition process to acquire a multi-channel
fluorescence image.
Example: Define a process for the acquisition of a multi-channel fluorescence
image (e.g., "Blue", "Green", and "Red"). When you set up the fluorescence
sample, illuminate it as little as possible to minimize the bleaching effect.

At the top of the illustration you can see the individual fluorescence images (1).
Below, you can see the superimposition (2) of the separate fluorescence images.

Defining the "Multi Channel" acquisition process

Choosing acquisition 1. Use the View > Tool Windows > Process Manager command to make the
process
Process Manager tool window appear.
2. Select the Automatic Processes option.

3. Click the Multi Channel button.

 The button will appear clicked. You can recognize this status by the
button's colored background.
 The [ C ] group will be automatically displayed in the tool window.
4. Should another acquisition process be active, e.g., Multicolor, click the button
to switch off the acquisition process.
 The group with the various acquisition processes should, for example,
now look like this:

Adding color channels 5. Click the Add Channel button (1).

44
 Acquiring fluorescence images

 A context menu will open.

The context menu will list all of the observation methods that are
currently defined.
6. Select the color channel that is to be acquired first, e.g., "Red".
7. Select the other channels (e.g., "Green" and "Blue") in the same manner.
Note that the fluorescence images are later on acquired in the same order as
they are listed there.
Selecting the exposure 8. Use the View > Tool Windows > Camera Control command to make the
times for the individual
Camera Control tool window appear.
color channels
9. Click the small plus sign (2) next to the first color channel.

 The channel has now been activated (3). The active color channel will be
shown highlighted in color in the tool window.
 The color channel entries in the Process Manager tool window are
organized like a tree view. Expand an entry to open a list with additional
information about the selected color channel.
 When you activate the color channel, you also automatically select the
corresponding observation method. You can recognize which
observation method is active, by the microscope icon . At the same
time, this means that the microscope is now set up correctly for the
acquisition of the fluorescence image for the first color channel.

10. Switch to live mode. To do so, click the Live button in the Camera
Control tool window.
 The reflected light shutter will be automatically opened.
This behavior will be specified when the observation method is defined.
For the shutter, the Use for acquisition status should have been selected.
11. In the Camera Control tool window, select the Exposure > Manual option.
12. Some cameras offer the SFL mode for fluorescence acquisitions (e.g., the
DP72). Switch this mode on.
13. Optimize the exposure time.
Should the exposure time become longer than 500 ms, reduce it by
increasing the sensitivity or gain.
To do this, change the value in the Exposure > Sensitivity field or use the
Gain slide control.

14. In the Process Manager tool window, click the Read settings button.

 This exposure time will be automatically adopted for the active channel,
and will be shown in the Process Manager tool window (4).

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 Acquiring fluorescence images

15. Then, activate the other channels and set the exposure time for each of
them.

16. Finish the live mode. To do so, click the Live button in the Camera
Control tool window, once more.
 The reflected light shutter will be closed.
Focusing a 17. Activate the first channel.
fluorescence sample
18. Switch to live mode.
19. Bring the image into focus.
If your microscope stage is equipped with a motorized Z-
drive, a focus regulator will be at your disposal in the
Microscope Control tool window.

20. Click the Read Z-offset button in the Process Manager tool window to
adopt the current Z-position of your microscope stage (5).

21. Activate the other channels, focus the sample and transfer the current Z-
position of the microscope stage to the acquisition process.
 The first color channel is always used as a reference for the Z-offset.
Below the other color channels you can find the Z-offset value. It shows
how the focus positions of the individual color channels differ from each
other.
22. Select the Use Z-offset check box (6).

Finishing the process 23. Finish the live mode.

definition
 The reflected light shutter will be closed.

24. Click the Save Process Definition button in the toolbar at the top of the
Process Manager tool window to save the acquisition parameters for the
process that has just been defined.
 A channel contains an observation method, an exposure time, a
sensitivity or gain value, and where necessary, a focus position. All of
these settings will be saved together with the process definition.
 You can now use the acquisition parameter for this acquisition process
again, at any time.

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 Acquiring fluorescence images

Acquiring and viewing a multi-channel fluorescence

image
Defining the acquisition 1. Define a process for the acquisition of a multi-channel fluorescence image, or
process
load acquisition parameters that have already been saved. To do this, click
the Load Process Definition button, located in the Process Manager tool
window's toolbar.
Starting the acquisition
process 2. In the Process Manager tool window, click the Start button.
 If you use a manual microscope, you'll receive several different
messages about switching the mirror cube and opening and closing the
shutter.
3. For microscopes that aren't motorized: Follow the instructions and make the
necessary settings on your microscope.
 The acquisition of the multi-channel fluorescence image starts
immediately. The order of the color channels in the Process Manager
tool window corresponds to the order in which the color channels were
acquired.
 The acquisition has been completed when you can again see the Start
button in the Process Manager tool window.
 The multi-channel fluorescence image will be automatically saved. You
can set the storage directory in the Acquisition Settings > Saving >
Process Manager dialog box. The preset file format is VSI.
 In the image window, the superimposed fluorescence image of all
channels is displayed.

Viewing a multi-channel
image 
The navigation bar will be displayed at the top of the image window. It
contains a button for each channel to enable you to display or hide that
channel. The eye icon indicates that the channel is currently visible.
4. Click the color channel button in the navigation bar to have a color channel
displayed or hidden. Take a look at all of the color channels one by one.
5. When you've finished, superimpose all of the channels again.

6. Click the Tile View button located in the navigation bar to change the
image window view.

47
 Acquiring fluorescence images

 You will then see all of the color channels that have been acquired.

7. Compare the color channels.

8. Click the Single Frame View button on the navigation bar.

 You will then once more see the superimposition of all of the color
channels in the image window.
Viewing information on 9. Use the View > Tool Windows > Properties command to make the Properties
the individual color
tool window appear.
channels
 In the Properties tool window, you will find that every color channel has
its own Channel information group.
10. Should an information group has been reduced: Click the plus sign to
have all of the information displayed again.
 The color channel's name, the corresponding wavelength, the
observation method, and the exposure time will all be shown for each
color channel.

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 Acquiring fluorescence images

Acquiring a multi-channel fluorescence image

together with a transmitted light image
Simultaneously with the multi-channel fluorescence image, acquire a transmitted
light image, e.g., a phase contrast image.
Defining the acquisition 1. Define an acquisition process for a multi-channel fluorescence image. To do
process
this, follow the step-by-step instructions described above.
Adding the acquisition 2. Click the Add Channel button. Choose an observation method for the
of a transmitted light
acquisition of a transmitted light image, e.g., phase contrast, differential
image to the acquisition
process interference contrast (DIC), or brightfield.
3. Click on the transmitted light channel in the Process Manager tool window.
 The channel has now been activated. Your microscope will be set in the
transmitted light mode.
4. Click the small plus sign next to the transmitted light channel.
 You'll now see a table with additional information about the transmitted
light channel.
5. Make sure that the Transmission overlay check box has been selected. Only
then is the transmitted light image assigned its own layer that lies over the
fluorescence channels.
Defining the transmitted 6. Switch to the live mode.
light image acquisition
7. Select manual exposure time in the Camera Control tool window. Set the
sensitivity to the lowest ISO value or the gain to the value of "0". Optimize the
exposure time.

8. In the Process Manager tool window, click the Read settings button.
 The exposure time will be adopted for the channel.
9. Finish the live mode.
Starting the acquisition
process 10. In the Process Manager tool window, click the Start button.
 Then, together with your fluorescence images, a transmitted light image
will also be acquired and saved together with the multi-channel
fluorescence image. The result of this acquisition process is a multi-layer
image that you can view with the Layers tool window.
11. Take a look at the multi-channel fluorescence image with the superimposed
transmitted light image in the image window.
00369 05012011

49
 Creating stitched images

7. Creating stitched images

Acquiring a stitched image without a motorized XY-
stage (Manual MIA)
Example: You want to acquire an image of a large sample area. Use the Manual
MIA acquisition process, to acquire several individual images of adjoining
positions on the sample, and to have them combined into a stitched image.
Prerequisite The camera is aligned parallel to the XY-stage. The angle between camera and
stage should be smaller than 1°.
1. Switch to the "Acquisition" layout. To do this, use, e.g., the View > Layout >
Acquisition command.
Selecting the objective 2. On the Microscope Control toolbar, click the button with the objective that
you want to use for the acquisition of the stitched image.
Setting the image 3. Switch to the live mode, and select the optimal settings for your acquisition,
quality
in the Camera Control tool window. Pay special attention to setting the
correct exposure time. This exposure time will be used for all of the stitched
image's individual images.
4. Find the position on the sample at which you want to start acquiring the
stitched image.
5. Finish the live mode.
Selecting the 6. Activate the Process Manager tool window.
acquisition process
7. Select the Manual Processes option.

8. Click the Manual MIA button.

 The button will appear clicked. You can recognize this status by the
button's colored background.
 The Manual MIA group will be automatically displayed in the tool window.
 Should the Instant EFI acquisition process have been active, it will be
automatically switched off. You can, however, use images with extended
depth of focus for the stitched image. To do this, before you acquire each

of the individual images, click the Instant EFI button, located in the
Manual MIA group.
Selecting the 9. Make quite certain that the Auto Align button appears clicked. It should then
acquisition parameters

look like this: .

 Then, your software will search for the same image structures in
neighboring individual images. The stitched image will be put together in
such a way that image areas that are the same will be superimposed.
Acquiring a stitched
image 10. Click the Start button.
 Your software switches into the live mode.
11. Bring the sample into focus.
12. Click on one of the arrow buttons to set the side of the current image at
which the next image is to be arranged. For example, click this button if
the next image is to be laid to the right of the current image.

50
 Creating stitched images

 Your system now acquires an image at the current position on the

sample. In the image window you now see, on the left (1) the acquired
image, and on the right (2) the live-image is displayed.

Since you haven't moved the sample, the live-image still shows the
current sample position, too, which means that you now see the current
image twice.
The two images overlap. Since the live-image is shown transparent, you
see both images in the overlap area simultaneously.
13. Make a note of a significant structure on the live-image's right border. You
will find the same sample structure in the overlap area. On the illustration, a
significant structure has been indicated by a circle.
14. Now move the stage very slowly to make the structure on the live-image
move to the left. Keep moving the stage until the image structures in the
overlap area lie as exactly over each other as possible. The image structures
need not lie precisely over each other, since your software will match the
individual images with each other.
 In the overlap area (3), the same image segments are shown now. This
enables your software to seamlessly combine the two images.

 You can reverse the direction in which your stage moves, in the Device
Settings > Stage dialog box. Depending on how you can best orient
yourself, the live-image will then move to the left or to the right, when you
move your stage to the right.
15. Check whether both images have been correctly combined. Otherwise you
can undo the last step by using the Undo last frame button. You can then
move the stage again, and match the structures better.
 During the acquisition, you can change the current stitched image's
zoom factor, e.g., to see certain parts in the overlap area better. You will
find an overview on the possibilities of changing an image's zoom factor
in the online help.
16. Define your way through the sample, with the arrow buttons, and follow that
with the stage.
In this manner, you can display a sample in any form you like in the stitched
image. The illustration shows a stitched image that is made up of 9 individual
images, and the stage path.

51
 Creating stitched images

17. Click the Stop button when you want to end the acquisition of the
stitched image.
 You see the completed stitched image (4) in the image window.
Since the individual images can lie a little askew of each other, the
stitched image isn't as a rule, rectangular, but contains empty areas on
its borders (5). These areas will, as a rule, be cut off in the stitched
image.

 The stitched image will, by default, be automatically saved. The storage

directory is shown in the Acquisition Settings > Saving > Process
Manager dialog box. The preset file format is VSI.
Properties of the  By default, in the overlap area, the intensity values of two adjoining
stitched image
individual images will be matched with each other, to make the image's
overall impression homogeneous.
 Stitched images are calibrated. This means that you can measure
distances and objects on a stitched image.

52
 Creating stitched images

Acquiring a stitched image with a motorized XY-stage

(XY-Positions / MIA)

Example: You want to acquire an image of a large sample area. Use the
automatic MIA acquisition process, to scan a rectangular area of the sample and
to have adjoining images combined into one stitched image.
Prerequisites  The stage has been set up and initialized, i.e. its stage limits have been
defined.
 The camera is aligned parallel to the XY-stage. The angle between
camera and stage should be smaller than 1°.
 The shading correction has been set up.
1. Switch to the "Acquisition" layout. To do this, use, e.g., the View > Layout >
Acquisition command.
Selecting the objective 2. On the Microscope Control toolbar, click the button with the objective that
you want to use for the acquisition of the stitched image.
Selecting the 3. Activate the Process Manager tool window.
acquisition process
4. Select the Automatic Processes option.

5. Click the XY-Positions / MIA button.

 Should another acquisition process be active, e.g., Multicolor, click the
button to switch off the acquisition process.
 The group with the various acquisition processes could now look like
this, for example:

 The XY group will be automatically displayed in the tool window.

Using the software 6. If your microscope is equipped with a motorized Z-drive, you can switch on a
autofocus
software autofocus.
In the Process Manager tool window, click the Software Autofocus button.

 The Software Autofocus group will be automatically displayed in the tool

window.
7. In the Software Autofocus group, select the Multiposition / MIA check box.
8. If the sample surface is not plane or if it is inclined to the objective, choose
the Every MIA frame option. Now, the software autofocus will be performed
before every image acquisition.

53
 Creating stitched images

Putting the stage

navigator on display 9. In the Process Manager tool window, click this button .
 The Stage Navigator tool window will be shown. When you have
acquired an overview image of your sample, you will see this area of the
image in the stage navigator's image segment.
10. Set the magnification for the image segment in the Stage Navigator tool
window. To do this, use the zoom buttons at the bottom left of the tool
window (2).
The current stage position will be shown by a yellow rectangle in the image
segment (1). You should choose a magnification that enables you to see this
rectangle clearly.

 Further information on the Stage Navigator tool window is available in

the online help.
Defining the MIA scan
area 11. In the Process Manager tool window, click this button .
 The system will automatically switch into the live mode.
 The Define MIA Scanning Area dialog box will open.
12. Move the XY-stage to the top left-hand corner of the MIA scan area you want
(3).
13. Focus, then select the optimal settings for your acquisition in the Camera
Control tool window. Pay special attention to setting the correct exposure
time. This exposure time will be used for all of the stitched image's individual
images.
14. Confirm the starting position in the Define MIA Scanning Area dialog box,
with OK.
15. Move the XY-stage to the bottom right-hand corner of the MIA scan area (4).
Confirm this position in the Define MIA Scanning Area dialog box, with OK.

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 Creating stitched images

 In the Stage Navigator tool window, the MIA scan areas that have been
defined are displayed. Here, you can immediately see how many
individual images are required for the acquisition of the stitched image,
when the current magnification is used.

Acquiring a stitched
image 16. Click the Start button.
 The acquisition has been completed when you can once more see the

Start button in the Process Manager tool window, and the progress
bar has been faded out.
 You see the completed stitched image, in the image window. The
individual images won't be saved separately.

Combining individual images into a stitched image

Loading and selecting 1. Load the images you want to combine, resp. acquire a suitable set of
images
images.
2. Open the Gallery tool window. To do this, use, e.g., the View > Tool
Windows > Gallery command.
3. Select all of the images you want to combine, in the Gallery tool window.
Assembling images 4. Use the Process > Multiple Image Alignment... command. This command is
only active when more than one image of the same image type has been
selected.
 The Multiple Image Alignment dialog box will open. A description of the
dialog box can be found in the online help.
 The dialog box's stitching area will display a preview of the individual
images.
5. If necessary, while keeping your left mouse button depressed, drag on the
bottom left-hand corner of the dialog window to enlarge it. Alternatively,
doubleclick the header of the dialog box to enlarge the dialog box to full-
screen size.
6. Check whether the images' positions are correct. You can change the
arrangement of the individual images, e.g., by exchanging two images in the
stitching area by Drag&Drop.

55
 Creating stitched images


The illustration shows the stitching area with four individual images. On
the left, the images 1 and 2 are not in the correct position. Image 1
(green frame) will therefore be dragged onto image 2 (red frame). On the
right, you see the stitching area after the two images have been
interchanged.
7. When the individual images overlap, select the Correlation option in the
Output > Alignment list.
Then, your software will search for the same image structures in neighboring
individual images. The stitched image will be put together in such a way that
image areas that are the same will be superimposed.
8. Click the OK button to carry out the automatic image alignment.
 The Multiple Image Alignment - Manual Align dialog box opens.
 The stitched image will be displayed.
Checking a stitched 9. Check the stitched image on display.
image
Use the zoom buttons in the dialog box to zoom in the stitched image in the
dialog box.

10. Should individual images have been incorrectly assembled, you can
manually shift one or more of them, in respect to one another.
To do this, click in the image you want to shift, then drag it with your left
mouse button depressed, in the required direction.
 The currently selected image will be displayed semi-transparently, to
make it easier for you to find the point of contact with the neighboring
image.


Two images were not correctly aligned with each other. There is a
misalignment. When the manual alignment has been made, the two
images fit together seamlessly.
11. Select the Cut Edges check box, to clip the image in such a way that there
are no longer any empty areas visible on its borders.
 In the preview, the image edges that are to be clipped will be displayed
semi-transparently.
12. Select the Equalize check box, if the images aren't homogeneously
illuminated. Then the intensity values of the individual images will be
matched with one another, which will make the background appear more
homogeneous.
13. Click OK.
 A new image with the name "Image_<consecutive No.>" will be created.

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 Processing images

8. Processing images
The Process menu offers numerous image processing functions, with which you
can change an acquired image (e.g., increase the image contrast or the image
sharpness).

Processing images
1. Load the image that you want to process, or activate the image in the
document group.
 Please note that the Process menu will only be visible when an image
window is active in the document group.
2. Use one of the commands in the Process menu, e.g., Process >
Enhancement > Adjust Intensity... .
 The image processing dialog box opens. The image processing
operation that is active is shown in the dialog boxes header.

3. Click the small arrow next to the Preview button to open a list of all of
the preview functions. Select the Original and Preview entry.
 This preview function displays the same image segment twice in the
dialog box. The first one shown is the source image. The second is the
image that results when the current parameters are used.
 Most of the image processing operations need one or two of the
parameters that are shown in the Settings group.
4. Change the image processing operation's parameters. After every change
that is made in a parameter, the operation will be immediately applied to the
source image, and the resulting image will be shown in the preview window.
Click the Default button, to readopt the preset parameters in the Settings
group, when the current parameter doesn't make sense to you.
5. When you have found the optimal parameters, click the OK button to have
the active image processing operation applied to the image with the active
parameters.
 The image processing dialog box is closed.
 Please note that the image processing operation changes the source
image. No new image document will be created. You can, however use
the Edit > Undo command to restore the source image.
00175 22022011

57
 Processing images

8.1. Fluorescence Unmixing

Multi-channel fluorescence microscopy
In the multi-channel fluorescence microscopy, different cell structures will be
visually separated by acquiring them separately, then displaying them in different
colors. To achieve this, one stains the sample with several suitably chosen
fluorochromes. Each of these labels a special cell structure. The fluorescence
images will then be acquired. The fluorescence image 1, created with
fluorochrome 1 shows cell structure 1, the fluorescence image 2 created with
fluorochrome 2 shows cell structure 2, etc.. The individual images will be
combined into a multi-channel fluorescence image that shows the different cell
structures in different colors. When, for example, three fluorochromes are used, a
three channel fluorescence image will be created.

Problem with the visual separation of the structures

Filter sets in the Your microscope has an appropriate filter set for each fluorochrome, this set
microscope
comprises an excitation filter, a dichromatic mirror, and an emission filter. When
the fluorochrome 1 is excited by light from the wavelength range 1a, it emits light
in the wavelength range 1b. When fluorescence image 1 is acquired, the
excitation filter 1 takes care that only light from a narrow range within the
wavelength range 1a reaches the sample from the microscope's illumination
source. At the same time, the dichromatic mirror 1 and the emission filter 1 take
care that, from the sample, only light from a narrow range within the emission
wavelength range 1b, reaches the camera.
Overlapping of the The problem with this procedure is, that the wavelength ranges of the different
wavelength ranges
fluorochromes overlap. If this overlapping didn't occur, the aspired visual
separation of the different cell structures in the resulting multi-channel
fluorescence image would be perfect.
Neither the excitation wavelength ranges nor the emission wavelength ranges
have sharp limits, and they lie very close to one another, where numerous
fluorochromes are concerned. Therefore, the excitation wavelength ranges 1a,
2a, 3a, ... of the fluorochromes 1, 2, 3, .... normally overlap. The same applies to

58
 Processing images

the emission wavelength ranges 1b, 2b, 3b, ... As well as that, there are also
overlappings between excitation wavelength ranges and emission wavelength
ranges.
Excitation and In the spectra that follow, you can see a graphical demonstration of the way in
Emissions spectra
which the excitation intensities and the emission intensities of several
fluorochromes that are often used, depend on the wavelength. The way in which
the different wavelength ranges overlap, can clearly be seen in these spectra.

Spectral unmixing Owing to the spectral overlapping, the aspired visual separation of the different
cell structures only succeeds partially. When, for example, the light that excitation
filter 1 lets through, also excites fluorochrome 2 a little, and part of the light that
fluorochrome 2 then emits can pass the emission filter 1; cell structure 2 will also
be dimly visible in fluorescence image 1. One can then speak of an unwanted
"spectral mixing" of the individual fluorescence images.

Spectral unmixing
The spectral mixing can be subsequently removed from a digitally recorded multi-
channel fluorescence image, by recalculation. That's to say, the image will be
"spectrally unmixed". When you do this, it improves the visual separation of the
different cell structures in the image, and improves the image quality. To do that,
use the Process > Enhancement > Fluorescence Unmixing... command.
00360

59
 Processing images

Carrying out a fluorescence unmixing

The spectral unmixing of a multi-channel fluorescence image takes place in two
steps. The first step is the calibration of the color channels with the help of
reference images. When the experimental conditions don't change, you will only
need to carry out this step once. In the second step, the actual spectral unmixing
takes place.
You require precisely one reference image for each color channel that is to be
calibrated. Each reference image must have exactly the same number of color
channels as the image that is to be unmixed. In the instructions that follow, it will
be assumed that you have acquired a three channel fluorescence image and
want to carry out a spectral unmixing with it. For a two channel fluorescence
image the procedure is analogical.

Calibrating color channels

When the experimental conditions don't change, you will only need to carry out
the calibration once. When you've done that, you can spectrally unmix all of the
three channel fluorescence images that are acquired later, on the basis of this
calibration.
Acquiring reference 1. Set up three samples that in each case have only been stained with one of
images
the three fluorochromes.
Alternatively, you can use a single sample that has been stained with all
three fluorochromes. In this case, there must be three areas on the sample
that have each been stained with only one fluorochrome.
2. Acquire a three channel fluorescence image of each of the three samples
(alternatively, of each of the three areas on your sample).
When you do this, use either the excitation filter appropriate for each of them,
and a multiband emission filter or a multiband excitation filter and the
emissions filter appropriate for each of them.
The experimental conditions must be the same as they were when the image
that is to be spectrally unmixed was acquired.
 Differences in the exposure times of the individual color channels will be
automatically linearly corrected when a spectral unmixing is carried out.
Nevertheless, as a rule it makes sense not to change the exposure times
when the reference images are acquired.
 The result will be three multi-channel fluorescence images. Each of them
contains three channels. These will, in what follows, be designated as
"reference image 1", "reference image 2" and "reference image 3".
Reference image 1 is to be used to calibrate color channel 1, that
belongs to fluorochrome 1. With the reference images 2 and 3 the
method is analogical.
 Each of the reference images will be displayed in its own window, in the
document group. The reference image that was last acquired will be the
currently displayed, active image.
 Should you have already acquired the three reference images at an
earlier point in time, you can load them into the document group by using
the File > Open > Image... command.

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 Processing images

Defining ROIs 1. Activate reference image 1 in the document group.

2. In the Life Science Applications toolbar, click the New ROI - 3 Points Circle
button.
 Should the toolbar not be visible, put it on display by using the View >
Toolbars > Life Science Applications command.
3. Search out an area in reference image 1, in which fluorochrome 1 is
especially bright and glows as evenly as possible.
4. Define a circular ROI within this area with three mouse clicks.
 This ROI was defined for the fluorochrome 1. It will be automatically
assigned the name "ROI 1".
 You can still subsequently change the size and position of this ROI.
 You can change this automatically created name. To do so, use the
Measurement and ROI tool window. In it, click the ROI's name to change
it. Should the tool window not be visible, put it on display by using the
View > Tool Windows > Measurement and ROI command.

5. Click the New ROI - 3 Points Circle button once more.

6. Search out a dark area in the background of reference image 1, in which, as
far as possible, no fluorochrome can be seen.
7. Define a circular ROI within this area with three mouse clicks.
 This ROI was defined for the image background. It will be automatically
assigned the name "ROI 2".
8. Using the same procedure, define in reference image 2 an ROI for the
fluorochrome 2, and an ROI for the image background.
9. Using the same procedure, define in reference image 3 an ROI for the
fluorochrome 3, and an ROI for the image background.
Finishing the calibration 1. Activate reference image 1 in the document group.

2. In the Life Science Applications toolbar, click the Fluorescence Unmixing

button, to open the Fluorescene Unmixing dialog box.
3. Activate the Calibration tab.
4. Enter the label for fluorochrome 1 in the Name field. This is, at the same
time, the name for the calibration for color channel 1.
5. Select reference image 1 in the Image list.
6. In the ROI list, located immediately below the Image list, select "ROI 1" which
was defined for the fluorochrome 1.
7. In the Background subtraction group, select the ROI option for the
background correction of reference image 1.
8. In the neighboring list to the right, select "ROI 2" that was defined for the
image background in reference image 1.
9. Click the Save button.
 The calibration of color channel 1, has now been completed.
 The Fluorochrome 2 >> button will become available.
10. Click the Fluorochrome 2 >> button to skip to the calibration of color channel
2.
 The name of the Fluorochrome 1 group will then change to
Fluorochrome 2.

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 Processing images

11. Then, using the same procedure, calibrate color channel 2 with reference
image 2 and fluorochrome 2.
12. Then, using the same procedure, calibrate color channel 3 with reference
image 3 and fluorochrome 3.
13. Click the Cancel button, to close the Fluorescence Unmixing dialog box.

Spectrally unmixing a three channel fluorescence

image
1. In the document group, activate the three channel fluorescence image you
want to spectrally unmix.
 Should you have already acquired the image at an earlier point in time,
you can load it into the document group by using the File > Open >
Image... command.
2. In the Life Science Applications toolbar, click the New ROI - 3 Points Circle
button.
3. Search out a dark area in the background of your image, in which, as far as
possible, no fluorochrome can be seen.
4. Define a circular ROI within this area with three mouse clicks.
5. This ROI was defined for the image background. It will be automatically
assigned the name "ROI 1".

5. In the Life Science Applications toolbar, click the Fluorescence Unmixing

button, to open the Fluorescene Unmixing dialog box.
6. Activate the Linear Unmixing tab.
7. In the Fluorochrome 1 list, select the calibration with which the fluorescence
image in your multi-channel fluorescence image's color channel 1 is to be
corrected.
 The name of this calibration is identical with the label you gave the
fluorochrome 1 while you were performing the calibration.
8. In the Fluorochrome 2 list, select the calibration with which the fluorescence
image in your multi-channel fluorescence image's color channel 2 is to be
corrected.
9. In the Fluorochrome 3 list, select the calibration with which the fluorescence
image in your multi-channel fluorescence image's color channel 3 is to be
corrected.
10. In the Background subtraction group, select the ROI option for the
background correction of your image .
11. In the neighboring list to the right, select "ROI 1" which was defined in your
image for the image background.
12. Click the OK button to carry out the spectral unmixing and to close the dialog
box.
 A new image document will be created for the spectrally unmixed image.
The source image will not be changed.
 It can occur that, immediately after the spectral unmixing, the image will
not be optimally displayed on your monitor. In this case, click the Apply
button in the Adjust Display tool window. When you do this, the image
contrast on your monitor will be automatically optimized. The actual
image data will not be changed.
13. Save the spectrally unmixed image if you need it.

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 Measuring images

9. Measuring images
Your software offers a wide range of measurement functions. They enable you to
quickly count objects and measure segments and areas. All the results will be
saved together with the image and can also be issued as a sheet.
Prerequisites For making measurements, correctly calibrated images are an essential
prerequisite. Images that you have acquired with your software will have been
automatically correctly calibrated when you have specified the objective you
used.
Should the image not yet have been calibrated, use the Image > Calibrate
Image... command to carry out a calibration.

Selecting the measurement environment

Measuring with help of Switch to the Count and Measure layout when you want to measure images. You
the tool window
will find the Measurement and ROI tool window in the bottom section of this
layout. In this tool window you have fast access to all measurement functions
and settings which relate to the measurement. This tool window is at the same
time the measurement display and contains all of the values that have been
measured on the active image.
Note: Should, right at the bottom of the user interface, several tool windows lie
one over the other, activate the Measurement and ROI tool window, by clicking
on the header of the Measurement and ROI tab. The tabs can be found
under the tool windows.
Measuring with help of Should you need more room for displaying the image, you can also use the
the toolbar
Measurement and ROI toolbar instead of the tool window. All the measurement
functions can also be found on the Measurement and ROI toolbar. To have this
toolbar displayed use the View > Toolbars > Measurement and ROI command.
Begin a measurement by simply clicking the appropriate button. In this case you
will only see the measurements that you have carried out in the image.
Measuring with help of The Measure menu also contains all of the measurement functions. To start a
menu commands
measurement, simply use the corresponding menu command. In this case you
will only see the measurements that you have carried out in the image.

Starting a measurement
Begin a measurement by selecting the measurement function you want. You will
find the measurement function in the Measurement and ROI tool window, on the
Measurement and ROI toolbar, or in the Measure menu.
Working in the As soon as you have clicked a measurement function, your software will
measurement mode
automatically switch to a measurement mode. In the measurement mode, your
mouse pointer will take on the shape of a cross on the image. You can make as
many measurements as you like with the measurement function that has been
selected. The continuous measurement mode is valid for all loaded images. You
can, therefore, easily measure numerous images one after the other.
The selected measurement function's button will keep its clicked appearance and
in this way show you the current measurement function. You can recognize this
status by the button's background color.
Finishing the You will remain in this measurement mode until you explicitly switch it off. To do
measurement mode
this, click the Select Measurement Objects button. You can find the button
either in the Measurement and ROI tool window or on the toolbar.

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 Measuring images

Changing the default The continuous measurement mode described above is preset by default. You
measurement mode
can change this default setting. To do this, use the Tools > Options... command.
Select the Measurement and ROI > General entry in the tree view. Select the
Switch to 'Select' mode after creation check box. Then, when you have
completed a measurement, you will automatically leave the measurement mode
again. This means you have to select the measurement function again before
you start each interactive measurement.

Displaying and saving measurement results

The measurement results will be displayed directly on the image and in the
Measurement and ROI tool window. Use the View > Tool Windows >
Measurement and ROI command to have the tool window displayed.
Saving the The measurements will be saved along with the image, if you save the image in
measurement results
the TIF or VSI file format.
You can, however, also export the measurement results in a results sheet, and
save this as a file. To do this, use the Export to Excel or Export to Workbook
command.
Showing and hiding The measurement results will be shown on the image in a special data layer, the
measurement results in
measurement layer. On your monitor, image and measurement layer are shown
an image
together. The data of each, however, is individually stored if you use the TIF or
VSI image file format. Try and picture the measurement layer as a transparency
which is placed over the image. When you measure an image, the image data
will not be changed by having the measurement results displayed on it.
You can, at any time, hide or show the measurement layers.
To do so, use the Layers tool window. There you have access to all of an image's
layers. The eye icon identifies all of the layers that are currently on display on
your monitor.
Click the eye icon in front of the measurement layer to hide the measurements.
Click an empty cell without an eye icon, to make the corresponding layer
reappear.
You can export the measurement results from the Measurement and ROI tool
window as a sheet, for example, to be able to save the measurement results in
their own file, independently of the image. You will find the functions, e.g., as a
button next to the measurement functions on the Measurement and ROI tool
window's toolbar.

Click the Export to Workbook button to export measurement results from the
Measurement and ROI tool window's results sheet to a workbook. Use this export
possibility to save the measurement results in a file format that you can at any
time load and edit with your software.

Click the Export to Excel button to export the results to an MS-Excel sheet.
Use this export possibility, for example, when you want to evaluate the
measurement results still further. This will also enable you to supply the results to
other users who don't have your software.

Measuring in the live mode

All of the measurement functions are also available in the live-image. You can
therefore, e.g., quickly measure a segment in the live-image.

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 Measuring images

Measurement precision
How precise the measurement is, depends on the X/Y-calibration and the
image's current zoom factor.
Influence of the X/Y- The X/Y-calibration defines the width and height of the sample area that is
calibration
represented by one pixel. For example, it could be that one pixel displays a
sample area of 10 µm x 10 µm. A pixel is the smallest image structure that can
be measured. For this reason, the maximum measurement precision where this
example is concerned, is 10 µm.
Influence of the zoom The zoom factor tells you how large the image will be displayed on your monitor.
factor
With a zoom factor of 100%, one pixel on the monitor equals exactly one pixel in
the image. With a zoom factor of 50%, one pixel on the monitor equals 2 x 2
pixels in the image. When you make a measurement, you should use the zoom
factor 100% whenever possible. Then you will achieve a maximum of
measurement precision. Should the zoom factor 100% not be possible, because
the image area you want to measure can't then be completely seen, choose the
largest possible zoom factor under 100%.
Click here if you wish to change an image's zoom factor.
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9.1. Measuring images

Your software offers a wide range of measurement functions. They enable you to
quickly count objects and measure segments and areas.
The following step-by-step instructions present the measurement functions to you
by way of several examples.

Measuring image objects interactively

Task You want to measure the diameter of some cells.
To do this, load a suitable image, or acquire one.
Subsequently edit the measurement. Delete some of the measurements you've
made. Enter the results in a MS-Excel sheet.
1. If necessary, use the View > Tool Windows > Measurement and ROI
command to have the Measurement and ROI tool window displayed.
 You'll find the tool window at the lower edge of the user interface. It's
possible that it may be covered by the Count and Measure Results tool
window. Click the Measurement and ROI tab at the bottom of the user
interface, to bring the tool window into the foreground.
Loading an image 2. Acquire an image or load one.

During the installation of your software some sample images have been
installed, too. Regarding the information as to where the example images are
located, please refer to the online help. You can follow these step-by-step
instructions when you use the exemplary image "Neurons.tif".

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Setting the labeling The measurement results will be written into the image according to the default
color
settings, in red font color and without a background. This can't be easily read
against the superconductor's structure. Change the labeling settings.
3. Use the Tools > Options... command.
4. Click the Measurement and ROI > Measurement Display entry in the tree
view.
5. Click in the Background Color field, and choose, e.g., the color "Black".
6. Select the Text color > Fixed colors option, then select the color "White" from
the palette to see the measurements in white and the labeling in black in the
image.
7. Close the dialog box with OK.
Measuring lengths
8. Click the Arbitrary Line button, located on the toolbar at the top of the
tool window.
9. Click with your left mouse button at the starting point and end point of the
reference distance.
10. If you have measured a reference distance, you can immediately proceed
with the next measurement.

11. Click the Arbitrary Line button again to end the length measurement.
12. Take a look at the results in the tool window and in the image.
 The illustration shows the image with three executed measurements.
The measurement 2 has been selected

Deleting measurements 13. Click one of the measurement results in the Measurement and ROI tool
window.
 The corresponding line will be marked in the image.
14. Press the [Del] key.
 The measurement will be deleted both in the image and in the tool
window.
 When a measurement has been deleted, the tool window contains one
measurement less. The IDs of the remaining measurements won't be
changed by the deletion of a measurement.

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 When you've completed the measurements, you should switch off the
measurement mode, since otherwise, you might inadvertently select your
measurements and move them.
15. Check whether one of the buttons on the Measurement and ROI tool
window's toolbar appears clicked. Release this button
Exporting results to
MS-Excel 16. To do this, click the Export to Excel button.
17. In the In/Output dialog box, you set up the directory in which the data is to be
saved, and enter the name of the MS-Excel sheet. Adopt the file type "Excel-
Sheet (*.xls)“.
18. Click the Save button to have the MS-Excel sheet with the measurement
results saved.
Closing the image 19. Click the Close button, located at the top right of the document group.
 You have changed the image because you've added interactive
measurements. For this reason, you'll receive a query whether you wish
to save the image or not.
20. Save the image in the TIF or VSI file format. The measurements will then
also be saved in the image file. They can at any time, be edited deleted or
augmented.

Outputting various measurement parameters

Task You want to measure some cells.
Have a variety of measurement parameters, such as the area, the perimeter and
the diameter, output. Have the diameter shown in the image.
1. Acquire an image or use the example image "BadTissue.tif". Regarding the
information as to where the example images are located, please refer to the
online help.
Measuring areas
2. In the Measurement and ROI tool window, click the 2 Points Circle
button.
3. With your left mouse button, click in the center point of the hexagonal
structure.
4. Move your mouse, and in the process drag out the circle. Match the circular
object as well as possible to the structure. Click the left mouse button.

5. Click the 2 Points Circle button again, and switch off the measurement
mode.
6. Take a look at the result in the Measurement and ROI tool window.
 The illustration shows the image with a circle measured.

Viewing the list of 7. In the Measurement and ROI tool window, click the Select Measurements
measurement
parameters button.

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 Measuring images

 In the dialog box, you'll see a list with all of the available measurement
parameters. At the bottom of the dialog box you'll see a list of the
measurement parameters that are calculated for all objects.
 A detailed description of the dialog box can be found in the online help.
Outputting additional 8. Go to the list of all of the available parameters, then click the "Diameter"
measurement
measurement parameter.
parameters
 On the right, an illustration shows you how the parameter is calculated.

You can see that there are different ways in which the diameter of a 2D
object can be calculated.
9. Click the "Mean" entry in the list under the illustration, to select the "Mean
(Diameter)" measurement parameter. When you do this, the mean value of
all of the possible diameters is determined.
10. Click the Add 'Mean (Diameter)' button.
 This measurement parameter will be adopted in the list. All of these
measurement parameters will be displayed in the tool window.
11. Close the dialog box with OK.
12. Take a look at the result for the circle's diameter in the Measurement and
ROI tool window.
Outputting 13. Open the Select Measurements dialog box.
measurement
parameters in the 14. At the bottom of the list of all of the calculated measurement parameters,
image click the "Mean (Diameter)" measurement parameter.

15. To the right of this list you'll see a button with a blue arrow . Click this
button to move the measurement parameter to the top of the list.
16. Close the dialog box with OK.
17. Take a look at the result for the circle's diameter in the image.
Note: The measurement display in the image has to be updated once, so that the
settings that have been changed are also taken into account. You update the
measurement display, for instance, by adding another measurement, or by once
selecting an existing measurement in the image.

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 Measuring images

Measuring several images

Task You want to measure cells on multiple images. To do so, acquire some image
and measure them one after another. Have the results from all images displayed
simultaneously. Take a look at the mean value for all of the measurements.
Loading an image 1. Acquire an image or load one.

During the installation of your software some sample images have been
installed. You can carry these step-by-step instructions out directly with the
example images "Clematis04.tif" and "Clematis05.tif". Regarding the
information as to where the example images are located, please refer to the
online help.
Measuring cells 1. Activate the first image in the document group.

2. Click the Arbitrary Line button located on the toolbar at the top of the
Measurement and ROI tool window. Measure the diameter of several cells.
3. Activate the next image. Measure the diameter of several cells on this image,
too.

4. Click the Arbitrary Line button again, and switch off the length
measurement.
 Cells have been measured on both images.

Displaying the 5. In the Measurement and ROI tool window, click the Measurement and ROI
measurement results of
all of the images Options button.
6. Select the Measurement and ROI > Results entry in the tree view.

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 Measuring images

7. Clear the Show measurement objects: Only of the active image check box.
8. Close the dialog box with OK.
 Now the results for both images will be shown simultaneously in the tool
window.
Viewing the statistical 9. In the Measurement and ROI tool window, click the Measurement and ROI
parameter
Options button.
10. Select the Measurement and ROI > Results entry in the tree view.
11. Select the Mean check box.
12. Close the dialog box with OK.
 Now, in the Measurement and ROI tool window under the measurement
results, the chosen statistical parameter (1) will by shown. You can see
there the mean value of the layer thickness for all of the measured
images.

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 Measuring images

9.2. Measuring intensity profiles

With the Measure > Intensity Profile... command you can measure the intensity
profile over the time (time stack) or over the different Z-positions (Z-stack). An
image series can be a time stack or a Z-stack.
As a result you will obtain an intensity profile that shows how the intensity within
one, or within several image segments, changes over a period of time or over the
different Z-positions.
Example of use You can use intensity profiles to measure how concentrations change with time.
For example, when you make experiments with triggering the calcium flow with
ATR, and use suitable fluorescence stains.
Before using the To calculate an intensity profile, all of the pixels within a specific image segment
command
will be evaluated. You software will, e.g., determine the mean intensity of all of
the pixels.
Before you can measure an intensity profile, you have to define this image
segment. To do this, define one or more ROIs (Regions Of Interest) on the
image. To define these ROIs, you can, for example, use the buttons on the Life
Science Applications toolbar. Further information on working with ROIs is
available in the online help.
Supported image types With the Measure > Intensity Profile... command you can measure the following
image types:

Time stacks, whose frames are gray-value images.

Z-Stacks, whose frames are gray-value images.
Multi-channel Z-stacks, multi-channel time stacks
The command is only available for gray-value images. Use the Image > Mode >
Grayscale command, to convert the an image into a gray-value image.
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Measuring an intensity profile on a multi-channel Z-

stack
Task You have acquired a focus series for several fluorescences. You want to know
how the intensity develops at a variety of positions on the sample at a variety of
Z-positions.

The illustration shows an overview over the frames in a multi-channel Z-stack.

The multi-channel image contains a red and a blue color channel. For the
acquisition of the Z-stack a through-focus series was taken of the sample. The
sample can only be seen clearly, and sharply focused, in the middle of the Z-
stack.
1. During the installation of your software some sample images have been
installed, too. Regarding the information as to where the example images are
located, please refer to the online help. Load e.g., the multi-channel Z-stack
with the name "PeroxysomOrganelles.tif" from this directory.

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 Measuring images

 When you load a multi-channel Z-stack, it will be automatically displayed

in the Single Frame View in the image window.
Displaying a suitable 2. Use the navigation bar at the top of the image window.
image for the definition
of the image segment

3. Move the slide control slowly, and by doing so display frames acquired at
differing Z-positions in the image window. Search out a Z-position at which
the sample can be clearly recognized.
4. Use the View > Toolbars > Life Science Application command, to have the
Life Science Application toolbar displayed.
Defining ROIs (Region 5. Rotate the mouse wheel to change the zoom factor. Enlarge the image until
Of Interest)
you can see at least one enlarged segment of the sample in the image
window, that is fluorescing in red.

6. Click the New ROI - Polygon button on the Life Science Applications
toolbar.
7. By clicking with your left mouse button, define an area on the image that only
includes red fluorescing sample positions.
8. Rightclick to finish the definition of the ROI.
9. Then define another ROI on an image segment that only includes green
fluorescing sample positions.

10. Click the New ROI - Rectangle button.

11. Define a square in a dark image segment that has no fluorescing objects.
This ROI will be used as a reference for the background correction.
Calculating an intensity
profile 12. On the Life Science Applications toolbar, click the Intensity Profile
button.
 The Intensity Profile - <Name of the active image> dialog box opens.
 Your software will recognize the image type, and will select the
appropriate option in the Method group. In this example the Z-stack
option is preset.
13. Select the Results: Average check box.
 In the Intensity profile group, all of the ROIs that have been defined on
the active image will be listed. In this example, you'll find three ROIs
there (two on sample positions showing different fluorescence colors and
one on the background).
14. Each ROI defines a specific image segment. Now, select the image
segments for which intensity profiles are to be calculated.
In this example, select both of the ROIs at fluorescing sample positions.
15. In the Background Subtraction group, select the ROI option.
 In the list next to the ROI option, all of the ROIs that have been defined
on the active image will be listed.
16. In the list, select the ROI that was defined on the image background.
17. Click the Execute button.

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 The intensity profiles will be calculated and displayed in the Intensity

Profile tool window.
Viewing intensity 18. If necessary, use the View > Tool Windows > Intensity Profile command, to
profiles
show the tool window.

You can see two diagrams, each with two curves. Along the X-axis the Z-
position, that's to say, the height, has been plotted. The intensity range has been
plotted along the Y-axis.

For each of the image's color channels an individual diagram will be created. The
name of the corresponding color channel will be displayed in the diagram's
header. On the left you see the results for the green color channel, on the right,
those for the red one.
In each diagram you will see a curve for each ROI that has been defined. The
diagram contains a caption with the name of the ROI concerned. The green
curve was measured on the ROI on the green fluorescing position on the sample,
the red on the red fluorescing position.

Exporting and saving 19. In the Intensity Profile tool window's toolbar, click the Export to Workbook
an intensity profile
button.
 A new workbook will be created in the document window. This workbook
contains results sheets with all of the results.
When you've measured a multi-channel image, you'll find an individual
work sheet for each of the color channels.
20. Use the File > Save as... command, to save a workbook.
 A workbook will be saved in the file format OWB. This format is an
exclusive file format and can only be opened with your software.
Workbooks are, obviously, therefore not suitable for using to exchange
data with other application programs. When you want to use the results
in another application program, use the MS-Excel-Export, for this
purpose.
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9.3. Measuring the colocalization

What is colocalization?
In the fluorescence microscopy, it can occur that the fluorescence signals emitted
by two parts of a sample (e.g., molecules) that have been stained with different
fluorochromes, interfere with each other. In these cases, the different parts of the
sample lie very close to one another, or one over the other. The effect of the
interference of fluorescence signals is termed "colocalization".
In the digital image analysis, the colocalization of fluorescence signals can be
measured. This is done by detecting pixels that have the same intensity in both
color channels. These measurements are carried out on multi-channel images,
and are always valid for one channel pair.

Examples for colocalization

1) Superimposed signals in the green and blue color channels. The colocalized
pixels are displayed in white

2) Superimposed signals in the blue and red color channels. The colocalized
pixels are displayed in white

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 Measuring images

Measuring the colocalization on the whole frame

Use the Colocalization button to start a measurement of colocalization. You will
find this button on the Life Science Applications toolbar. This button isn't
available in all software versions.
1. Load the multi-channel image you want to use for the colocalization
measurement.

2. On the Life Science Applications toolbar, click the Colocalization button.

If this toolbar is not displayed, use the View > Toolbars > Life Science
Applications command.
 The Colocalization dialog box opens.
3. In the Channels field, choose the two color channels for which the
measurement of colocalization is to be carried out.
4. When you work with multi-channel time stacks or multi-channel Z-stacks:
Determine in the Apply on group, whether the colocalization measurement is
to be carried out on all frames or only on selected frames. Should you want
to limit the image selection, select the Selected frames entry, then click the
Dimension Selector button.
 Then you can limit the image selection in the Dimension Selector tool
window. Further information on this tool window is available in the online
help.
5. In the Target area group, select the Channel segmentation entry in the Area
field.
6. Click the Options... button, then select the Colocalization channel (Image),
and Measurement results (Workbook) check boxes.
7. Pay attention to the displayed results in the preview, and in the Results
group.
8. If necessary, change the position and size of the intensity range in the
scatterplot.
 Then only the colocalization of the pixels that lie within the chosen
intensity range are shown in the preview.
9. Click the OK button to finish the measurement of colocalization.
 A new image that contains the colocalization channel will be created.
 At the same time, a workbook that contains the results of the
colocalization measurement, will be displayed.
10. If required, use the File > Save as... menu command, to save the new image
and the workbook.

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 Measuring images

Measuring the colocalization on a part of the image

(ROI)
Frequently, a colocalization of fluorescence signals occurs only in a small image
segment. In this case, it makes sense to define a ROI (Region of Interest) then
determine the colocalization only within this ROI. You can also define several
ROIs. ROIs can have any shape you wish. General information on working with
ROIs is available in the online help.
1. Load the multi-channel image you want to use for the colocalization
measurement.

2. On the Life Science Applications toolbar, click the Colocalization button.

If this toolbar is not displayed, use the View > Toolbars > Life Science
Applications command.
 The Colocalization dialog box opens.
3. In the Channels field, choose the two color channels for which the
measurement of colocalization is to be carried out.
4. When you work with multi-channel time stacks or multi-channel Z-stacks:
Determine in the Apply on group, whether the colocalization measurement is
to be carried out on all frames or only on selected frames. Should you want
to limit the image selection, select the Selected frames entry, then click the
Dimension Selector button.
 Then you can limit the image selection in the Dimension Selector tool
window. Further information on this tool window is available in the online
help.
5. Click the Options... button, then select the Colocalization channel (Image),
and Measurement results (Workbook) check boxes.
6. In the Target area group, click once in the Area field, to open the picklist.
Select the ROI entry.
 Next to the field, to the right, the buttons with the various ROI forms are
displayed.
7. Click the button for the required ROI form that you want to set up. You have
the choice between a rectangle, a circle and a polygon.
 The mouse pointer will appear in the image window. The Colocalization
dialog box is hidden.
8. Define the first ROI with clicks of your left mouse button. When you have
completed the definition of your ROI, click your right mouse button, then
select the Confirm Input command in the context menu.
 You will then once more see the Colocalization dialog box. The ROI you
have defined will now be shown in the preview image.
9. If required, define further ROIs.
10. Select the required ROIs. To do this, click once in the box to the left of the
ROI's name.

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 Measuring images

11. Pay attention to the displayed results in the preview, and in the Results
group.
12. If necessary, change the position and size of the intensity range in the
scatterplot.
 Then only the colocalization of the pixels that lie within the chosen
intensity range are shown in the preview.
13. Click the OK button to finish the measurement of colocalization.
 If you haven't changed the default settings for colocalization, a new
image, that contains the colocalization channel, will be created.
 At the same time, a workbook that contains the results of the
colocalization measurement, will be displayed. The columns in the
workbook contain the supplement "ROI".
14. If required, use the File > Save as... menu command, to save the new image
and the workbook.
15. The multi-channel image will also have been changed when the ROI was
defined. Therefore, if you want to keep the ROI, save it also.

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 Measuring images

Measuring the colocalization on a color channel

You can also measure the colocalization on image structures. Where images are
concerned on which the image structures that are to be analyzed are numerous,
and are spread over the whole image, this procedure is quicker than setting a lot
of ROIs. If, for example, you want to measure the colocalization on image
structures that have been stained with the (blue fluorescent) fluorochrome DAPI,
select the blue channel. Then define the threshold values for this channel.
The colocalization measurement on a color channel only makes sense on a
multi-channel image with at least three color channels.
Example:
On the image, the colocalization of the red and green pixels within the area
marked in blue (cell nucleus) is to be measured. All other positions on the image
where pixels colocalize are to be ignored.

1. Load the multi-channel image for which you want to carry out a colocalization
measurement.

2. On the Life Science Applications toolbar, click the Colocalization button.

 The Colocalization dialog box opens.
3. When you work with multi-channel time stacks or multi-channel Z-stacks:
Determine in the Apply on group, whether the colocalization measurement is
to be carried out on all frames or only on selected frames. Should you want
to limit the image selection, select the Selected frames entry, then click the
Dimension Selector button.
 Then you can limit the image selection in the Dimension Selector tool
window. Further information on this tool window is available in the online
help.
4. Click the Options... button, then select the Colocalization channel (Image)
and Measurement results (Workbook) check boxes.
5. In the Target area group, select the Channel segmentation entry, in the Area
field.
 In the Area field, a picklist with all of the available channels will open.

6. Select the channel of the fluorochrome with which the image structure that is
to be analyzed, has been stained. In the example shown above, this is the
"DAPI" channel.

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7. Click the button located next to the Area field, on its

right-hand side.
 A picklist with the different methods for setting threshold values, will
open.
8. Select the Automatic Threshold... method.
 This method requires the user to make the smallest number of settings.
Therefore, you should only use the other methods for setting threshold
values, when the Automatic Threshold... method doesn't lead to the
result you wanted. An overview on "Thresholds" is available in the online
help.
 The Separation Channel Threshold dialog box opens. Your software will
carry out an automatic setting of threshold values. In the image window,
you will now see the image structures that are detected by the automatic
threshold settings.
9. In the Channel group, select the required channel again (in this case "DAPI").
10. Check in the image window, whether the automatic threshold setting has
correctly found the image structures that are to be analyzed.
 In the Separation Channel Threshold dialog box, select the Dark or
Bright option in the Background group, should the Automatic option not
lead to the results you want.

11. When the image structure that is to be analyzed has been correctly found,
click the OK button.
 You will then once more see the Colocalization dialog box. In the
preview, the image structures found via the Channel segmentation will
now be displayed with a yellow outline. The colocalized pixels shown lie
exclusively within these image structures.

12. If required, change the position of the white rectangle (gate) in the
scatterplot. By doing this you'll change the observed intensity range. You can

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now, e.g., have pixels with a lower colocalization shown. Pay attention to the
display in the Results group.
13. Click the OK button to finish the measurement of colocalization.
 A new image that contains the colocalization channel will be created.
 At the same time, a workbook that contains the results of the
colocalization measurement, will be displayed. The columns in the
workbook contain the supplement "Separation channel".
14. If required, use the File > Save as... menu command, to save the new image
and the workbook.
15. The multi-channel image will also have been changed when the Channel
segmentation was defined. Therefore, if you want to keep these settings,
save it also.
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