ImageMaster 2D Platinum 7.0 - GE Healthcare Life Sciences

ImageMaster 2D Platinum 7.0 - GE Healthcare Life Sciences
GE Healthcare
ImageMaster 2D Platinum 7.0
User Manual
Contents
1
Getting started
1.1
1.2
1.3
1.4
1.5
About this User Manual ................................................................ 9
About the software ........................................................................ 9
System requirements .................................................................... 9
Install the software ...................................................................... 10
eLicensing ...................................................................................... 10
1.5.1
1.5.2
1.5.3
1.5.4
1.5.5
1.6
1.7
1.8
2
Access code................................................................................................. 11
Find the physical address ..................................................................... 11
Install the GE Healthcare eLicense server...................................... 11
Collect and place an eLicense file...................................................... 12
Test the eLicense....................................................................................... 13
Launch the software .................................................................... 13
Customer support ........................................................................ 14
What’s new? .................................................................................. 14
Graphical user interface
2.1
2.2
About the interface ...................................................................... 17
Menu bar ........................................................................................ 17
2.3
Toolbars ......................................................................................... 20
2.2.1 Keyboard shortcuts.................................................................................. 18
2.3.1 Default toolbars......................................................................................... 20
2.3.2 Customize toolbars.................................................................................. 21
2.4
2.5
Status bar ...................................................................................... 22
Display zone .................................................................................. 22
2.5.1
2.5.2
2.5.3
2.5.4
2.6
Sheets ............................................................................................................ 22
Panes.............................................................................................................. 23
Images........................................................................................................... 24
Switch order................................................................................................ 25
Workspace ..................................................................................... 25
2.6.1 Toolbar .......................................................................................................... 26
2.6.2 Navigator ..................................................................................................... 26
2.7
Reports ........................................................................................... 28
2.7.1
2.7.2
2.7.3
2.7.4
2.7.5
2.8
2.9
3
Dynamic content....................................................................................... 29
Content based on enabled spots ....................................................... 29
Toolbars ........................................................................................................ 29
Customize reports .................................................................................... 30
Edit table cells ............................................................................................ 32
Dockable windows ....................................................................... 33
Options ........................................................................................... 34
Image Pool
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3.1
3.2
Introduction ...................................................................................37
Start with good images ...............................................................37
3.2.1
3.2.2
3.2.3
3.2.4
3.2.5
3.2.6
3.3
Resolution .................................................................................................... 37
Image depth................................................................................................ 37
Calibration ................................................................................................... 38
Image editing.............................................................................................. 38
File format.................................................................................................... 38
DIGE file naming convention ............................................................... 38
Preview images .............................................................................38
3.3.1 Open images............................................................................................... 38
3.3.2 Image pool sheet ...................................................................................... 39
3.4
Process images .............................................................................41
3.4.1
3.4.2
3.4.3
3.4.4
3.5
Calibrate images ..........................................................................44
3.5.1
3.5.2
3.5.3
3.5.4
3.5.5
3.5.6
3.6
Display calibration information about images............................ 44
Prepare for a calibration of the image capture device ............ 45
Create calibration of the capture device ........................................ 45
Apply a calibration to an image......................................................... 48
Remove a calibration.............................................................................. 48
Control a calibration................................................................................ 48
Describe images ...........................................................................49
3.6.1
3.6.2
3.6.3
3.6.4
4
Rotate ........................................................................................................... 41
Flip.................................................................................................................. 42
Crop............................................................................................................... 42
Invert gray levels...................................................................................... 44
Gel descriptions......................................................................................... 49
Display gel descriptions......................................................................... 50
Add or edit gel descriptions.................................................................. 50
Permanent gel description categories ............................................ 51
Projects
4.1
Introduction ...................................................................................53
4.1.1 Match hierarchies..................................................................................... 53
4.1.2 Classes........................................................................................................... 55
4.2
Create a project ............................................................................56
4.2.1 The first time the software is launched........................................... 56
4.2.2 At any time .................................................................................................. 57
4.2.3 Add files to project ................................................................................... 57
4.3
Create match hierarchy ..............................................................58
4.3.1
4.3.2
4.3.3
4.3.4
4.3.5
4.4
Create a match set................................................................................... 58
Merge a match set ................................................................................... 59
Set reference............................................................................................... 60
Use existing match sets......................................................................... 61
Export / import a match set................................................................. 62
Create classes ...............................................................................63
4.4.1 Create a class............................................................................................. 63
4.5
4
Handle project items ....................................................................63
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4.5.1
4.5.2
4.5.3
4.5.4
4.6
Display........................................................................................................... 63
Remove ......................................................................................................... 63
Properties..................................................................................................... 64
Move............................................................................................................... 64
Save projects ................................................................................. 64
4.6.1 Project folder............................................................................................... 64
4.6.2 Save ................................................................................................................ 64
4.6.3 Share project .............................................................................................. 64
4.7
Manage projects ........................................................................... 65
4.7.1
4.7.2
4.7.3
4.7.4
4.7.5
5
Add / remove projects ............................................................................ 65
Add files to project.................................................................................... 65
Backup / restore project ........................................................................ 65
Project visibility .......................................................................................... 66
Project properties ..................................................................................... 66
Gels
5.1
5.2
Introduction .................................................................................. 67
Manipulate images ...................................................................... 67
5.2.1
5.2.2
5.2.3
5.2.4
5.2.5
5.2.6
5.3
View signal intensity .................................................................... 71
5.3.1
5.3.2
5.3.3
5.3.4
5.4
Adjust contrast........................................................................................... 71
3D view ......................................................................................................... 75
Profile ............................................................................................................. 78
Cursor information................................................................................... 79
Visually compare images ............................................................ 80
5.4.1
5.4.2
5.4.3
5.4.4
5.4.5
5.5
Move............................................................................................................... 67
Zoom .............................................................................................................. 67
Same position and zoom factor......................................................... 69
Region............................................................................................................ 69
Measure ........................................................................................................ 70
Bookmarks................................................................................................... 71
Sheet reference.......................................................................................... 80
Purpose of aligning images ................................................................. 80
Align images ............................................................................................... 80
Show dual color......................................................................................... 81
Spots overlapped...................................................................................... 82
Grid lines ........................................................................................ 83
5.5.1 Display grid lines....................................................................................... 83
5.5.2 Edit grid lines .............................................................................................. 83
5.6
Gel reports ..................................................................................... 84
5.7
Save, export and print images ................................................... 85
5.6.1 Gel table........................................................................................................ 84
5.7.1 Save ................................................................................................................ 85
5.7.2 Export............................................................................................................. 85
5.7.3 Print images and sheets ........................................................................ 86
6
Spots
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6.1
6.2
Introduction ...................................................................................87
Detect spots in non-DIGE gels ....................................................87
6.2.1 Procedure..................................................................................................... 87
6.2.2 Spot detection parameters................................................................... 88
6.2.3 Spot quantification................................................................................... 89
6.3
Co-detect spots in DIGE gels ......................................................91
6.3.1
6.3.2
6.3.3
6.3.4
6.4
Procedure..................................................................................................... 92
Spot detection parameter..................................................................... 92
Spot quantification................................................................................... 92
Exclude spots.............................................................................................. 93
Select spots ....................................................................................94
6.4.1 Select.............................................................................................................. 94
6.4.2 Spot sets ....................................................................................................... 94
6.4.3 Enabled spots............................................................................................. 96
6.5
Display spots .................................................................................97
6.5.1 Spot shape................................................................................................... 97
6.5.2 Spot color ..................................................................................................... 97
6.6
Edit spots ........................................................................................97
6.6.1 Manual editing........................................................................................... 97
6.6.2 Composite spots ....................................................................................... 98
6.6.3 Propagate spots........................................................................................ 99
6.7
6.8
MW and pI calibration ...............................................................100
Spot reports .................................................................................102
6.8.1 Spot Table ..................................................................................................102
6.8.2 Cursor information.................................................................................102
7
Matches
7.1
7.2
7.3
Introduction .................................................................................105
Display a match hierarchy ........................................................106
Define landmarks .......................................................................107
7.3.1 Rules for defining landmarks.............................................................108
7.3.2 Define Landmark ....................................................................................108
7.4
7.5
Automatic matching ..................................................................110
Select matches ............................................................................110
7.5.1 Select............................................................................................................110
7.5.2 Match count ..............................................................................................111
7.6
Display matches .........................................................................111
7.6.1 Show vectors ............................................................................................111
7.6.2 Show ID.......................................................................................................112
7.7
7.8
Edit matches ................................................................................113
Match reports ..............................................................................114
7.8.1 Match statistics table ............................................................................114
8
Data analysis
8.1
6
Introduction .................................................................................117
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8.2
General settings ......................................................................... 117
8.2.1 Quantification value ..............................................................................117
8.2.2 Statistics......................................................................................................117
8.3
Analyze gels ................................................................................ 120
8.3.1
8.3.2
8.3.3
8.3.4
8.3.5
8.4
Analyze classes ........................................................................... 135
8.4.1
8.4.2
8.4.3
8.4.4
8.4.5
9
Scatter plots ..............................................................................................120
Gel analysis table....................................................................................123
Gel analysis histograms.......................................................................124
Factor analysis.........................................................................................127
DIGE histogram .......................................................................................133
Specify classes .........................................................................................135
Overlapping measures .........................................................................135
Statistical tests.........................................................................................137
Class analysis table................................................................................141
Class analysis histograms ..................................................................143
Annotations
9.1
9.2
Introduction ................................................................................ 147
Create annotations and labels ................................................ 148
9.2.1 Create annotations ................................................................................148
9.2.2 Add labels to existing annotations..................................................148
9.2.3 Link annotations to spots....................................................................148
9.3
Create label categories ............................................................. 149
9.3.1 Predefined label categories................................................................149
9.3.2 Create new categories .........................................................................149
9.4
Connect to protein databases ................................................. 151
9.4.1
9.4.2
9.4.3
9.4.4
9.5
Introduction...............................................................................................151
Set the database .....................................................................................152
Query the database ...............................................................................153
Connext to an executable ...................................................................154
Create specific links ................................................................... 154
9.5.1 Http link:......................................................................................................154
9.5.2 File link:........................................................................................................155
9.5.3 Text link: ......................................................................................................155
9.6
Select annotations and labels ................................................. 156
9.6.1 Select............................................................................................................156
9.6.2 Select menu...............................................................................................157
9.6.3 Reports ........................................................................................................160
9.7
Display annotations and labels ............................................... 160
9.7.1 Annotation flag position ......................................................................160
9.7.2 Visibility of annotations and labels .................................................160
9.8
9.9
Annotation table ........................................................................ 161
Edit annotations and labels ..................................................... 162
9.9.1 Select............................................................................................................162
9.9.2 Edit menu ...................................................................................................162
9.9.3 Annotation table .....................................................................................163
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9.9.4
Import annotations...............................................................................164
10 Data integration
10.1 Introduction .................................................................................165
10.2 Convert projects from earlier software versions .................165
10.3 Acquire images from Twain compatible scanners ...............165
10.3.1 Select source.............................................................................................166
10.3.2 Acquire ........................................................................................................166
10.4 Export data ..................................................................................166
10.4.1 Reports ........................................................................................................166
10.4.2 XML ...............................................................................................................166
10.4.3 Gel and report identifiers.....................................................................167
10.5 Export to spot excision robots .................................................167
10.5.1 GE Healthcare Ettan spot picker......................................................167
10.5.2 Spot pickers from other manufacturers .......................................169
11 Undo, redo and history
11.1 Undo, redo ....................................................................................171
11.2 History ..........................................................................................172
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Getting started 1
1
1.1
Getting started
About this User Manual
All users must read this entire manual to fully understand the safe use of
ImageMaster™ 2D Platinum 7.0. You will find detailed explanations of all the
features and functionalities. The chapters in this manual are generally
organized according to the logical sequence of a 2-DE gel analysis, although
expert users will agree that some steps can be inverted or repeated at some
point.
The user manual contains notes, as defined below.
Note: A Note is used to indicate information that is important for trouble-free and optimal
use of the product.
1.2
About the software
ImageMaster offers a flexible solution for the comprehensive visualization,
exploration and analysis of 2-D gel data.
This version of ImageMaster was developed by a team from the Swiss Institute
of Bioinformatics (SIB) in collaboration with Geneva Bioinformatics (GeneBio) SA
and GE Healthcare.
There are two modules of ImageMaster 2D Platinum 7.0 available for purchase:
1.3
•
ImageMaster 2D Platinum 7.0 DIGE: To be used with conventional 2-DE
and DIGE gels. It is fully functional, enabling you to add and import DIGE
gels directly in the workspace. You can also co-detect DIGE gels using the
algorithm created by the GE Healthcare DeCyder™ software development
team as well as match, report, plot histograms and perform statistical
analyses on DIGE gels.
•
ImageMaster 2D Platinum 7.0: To be used with conventional 2-DE gels. All
menu commands related to DIGE are not functional and are grayed out in
the graphical user interface.
System requirements
In order to install and run ImageMaster, your computer must satisfy the
following requirements:
•
Microsoft Windows XP or Vista operating systems.
•
Administrative permission to install ImageMaster.
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1 Getting started
1.4 Install the software
1.4
•
At least 500 MB of RAM for ImageMaster (recommended: Intel dual-core
processor with 1GB of RAM) and 768 MB RAM for ImageMaster DIGE
(recommended: Intel dual-core processor with 2GB of RAM). The amount of
memory required is determined by the number and size of image files to
be processed simultaneously. Increased memory therefore enhances the
performance when many and/or large images are analyzed.
•
A high-quality display. To take full advantage of the software including the
3D View feature, the color resolution should be set to 24 bit (16.7 million
colors). However, a color resolution of 8 bit (256 colors) is generally
sufficient. It is recommended to use a screen resolution of at least 1024 x
768 pixels.
•
At least 60 MB of available disk space for program files.
•
Internet Explorer 6.0 (Microsoft Corporation), Netscape Navigator 7.0
(Netscape Communications Corporation), Mozilla 1.4 (Mozilla Foundation)
or higher versions. A browser allows you to print reports and to access
scientific databases on the Web.
Install the software
You can install ImageMaster from a CD-ROM or by downloading the installation
package over the Internet. When the CD-ROM is inserted into the appropriate
drive on your computer, the Setup Wizard starts automatically and gives a series
of on-screen instructions. Alternatively, you can double-click on the icon of the
installer file (.msi or .exe file) to launch the Setup Wizard.
The ImageMaster installer creates a default directory on your hard disk called
Program Files\GE Healthcare\ImageMaster 2D Platinum 7.0, in which the
program files are placed. If you want to save the default directory in a different
folder, then browse and select the location before continuing the installation.
Once installation is complete, it is recommended to restart your computer.
1.5
eLicensing
An electronic license (eLicense) file is required to enable ImageMaster to run
once installation is completed.
There are two types of eLicenses:
•
10
Node-locked license (Machine license): To be used on a single computer.
It is practical when only a few computers are used for working with
ImageMaster. A node-locked license file must be placed on the computer
running ImageMaster.
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Getting started 1
•
Floating license (Concurrent license): To be used by all computers
networked to a license server. It is useful when many users, but not all at
the same time, need access to the software. The number of computers
that can simultaneously work with ImageMaster depends on the license,
and is administered via the license server. A floating license file must be
placed on the computer running the eLicense server. This server can either
be installed on a computer running ImageMasteror on any other network
computer (recommended: install on a network computer that is
continually running).
ImageMaster checks that a valid license file is available each time the software
is launched.
1.5.1
Access code
After ordering ImageMaster, a letter including an access code will be sent to the
order’s shipment address. The access code is necessary for collecting the
eLicense files. Store this access code in a safe place.
1.5.2
Find the physical address
You must know the physical address of your computer when collecting the
eLicense file. The address identifies the computer and is used by the licensing
system. If purchasing a node-locked license, you need the physical address of
the computer where ImageMaster is to be installed. If purchasing a floating
license is used, you need the physical address of the computer where the
eLicense server is to be installed.
To find the physical address:
1
On the computer where the eLicense file is to be placed, choose (All)
Programs : Accessories :: Command Prompt in the Windows Start menu.
2
In the Command Prompt window, enter the ipconfig /all. There must be a
space between ipconfig and /all.
3
Note down the Physical Address among the displayed information but
ignore any dashes or colons. If several physical addresses are listed
(dependent on the computer’s network connections), any of them can be
used for identification purposes. The physical address must be correctly
noted for the license file to work.
1.5.3
Install the GE Healthcare eLicense server
This section only applies to floating licenses. If you have a node-locked license,
then skip to the next section.
To install the GE Healthcare eLicense server:
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1 Getting started
1.5 eLicensing
1
On the computer where the eLicense server is to be installed, insert the
ImageMaster 2D Platinum 7.0 installation CD-ROM.
2
Click the Install GE Healthcare eLicense Server button.
3
In the installation window, click Next.
4
Accept the default installation path and start the installation. By changing
the default installation path, the file paths in LMTOOLS will need to be
updated accordingly.
5
Answer Yes to any question about Windows Firewall (this may or may not
appear) in order for the eLicense server to work properly.
6
Verify that the Launch GE Healthcare Software Licensing Server box is
checked and click Finish. LMTOOLS automatically opens once the
installation is successfully completed.
7
Click the Config Services tab and verify that all options are set as in Figure
1-1. The file paths shown (C:\Program Files\GE Healthcare\eLicense server\)
are valid if the default installation was done.
Figure 1-1. The Config Services tab in LMTOOLS.
8
Click the Save Service button, even if no changes were made.
1.5.4
Collect and place an eLicense file
To collect and place a license file:
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Getting started 1
1
Go to the web site http://www.elicensing.amershambiosciences.com/
gtlweb/login.
2
Enter the access code and click Continue.
3
Click Collect Licenses.
4
Select the product (the box is checked).
5
Register your product.
6
Enter the Physical Address (or Host-ID) of the computer where the license
file is to be placed (by default the type of Host-ID is Ethernet). Verify that the
Physical Address is correct. Click Continue.
7
If the data shown is correct, then select Collect License. Otherwise
navigate back in your Web browser to edit.
8
Save the license to a file, or email the license. It is highly recommended to
save the license file. If emailing, the license is delivered as raw text in the
body of the message. Copy and paste the text into Notepad and save as an
ASCII file with the extension .lic.
9
Place the license file. For a node-locked license, the eLicense file must be
placed in the ImageMaster installation directory (C:\Program Files\GE
Healthcare\ImageMaster 2D Platinum, by default ). For a floating license,
the file must be placed in the C:\Program Files\GE Healthcare\eLicense
server\Licenses on the computer running the eLicense server.
10 Restart the computer.
1.5.5
Test the eLicense
In order to test that the license file is correctly placed, start ImageMaster on all
computers where the software is installed.
For a floating license, a FLEXlm License Finder window will display asking to
specify the License Server System or License File. Choose the first option and
then enter the name of the computer on where the license server is installed.
Click OK.
1.6
Launch the software
Double-click the ImageMaster icon (Figure 1-2) found on your computer’s
desktop to start the software. Alternatively, go to (All) Programs : ImageMaster
2D Platinum 7.0 in the Windows Start menu. A splash page appears while the
software is loading. It will disappear automatically.
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1 Getting started
1.7 Customer support
Figure 1-2. ImageMaster icon.
1.7
Customer support
GE Healthcare provides technical and scientific support for ImageMaster.
Please contact us if any problems arise during the installation or use of the
software. Our support team is happy to help you.
How to contact us
By email: [email protected]
You can also find a local office at www.gelifesciences.com/contact.
Product, system and license information
Launch the software and choose Help : About ImageMaster to obtain product,
system and license information. The product name, version number and version
date that are displayed will be requested in technical support issues. You can
view system and license information by clicking on the corresponding buttons.
The Copy to clipboard button in the License Information window can be used
to paste license data into an email. In the System Information window, find facts
about your computer or choose File : Export to save all the information in a text
file.
1.8
What’s new?
The graphical interface and user interaction modes have been entirely updated.
The streamlined analysis in ImageMaster now offers enhanced usability and
speed.
Features have been redesigned to help minimize manual spot editing and
repetitive match editing. Ultimately, tasks are simplified and the reliability of the
results are increased.
The most important new features in ImageMaster, version 7.0 are listed below:
14
•
Fully dynamic tables, histograms, plots and 3-D views in which both
content and selection are continuously updated to stay synchronized with
the corresponding sheet that contains the gel images.
•
Simplified import and visualization of images
•
Improved population matching
•
Management of multiple matches and composite spots
•
Reorganized menu structure with icons
•
Custom and context-related toolbars
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Getting started 1
•
One-click to choose desired layout of sheets and panes
•
Option to surround spot selections by boxes, for easier identification
•
Single tool to select/edit spots and annotations
•
Dedicated landmark tool
•
Measure tool to compute pixel, pI, MW, or real-world (centimeter or inch)
distances between spots
•
Reviewed contrast adjustment feature
•
New 3D View
•
Customized report templates
•
Adaptive display of histograms
•
And much more…
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1 Getting started
1.8 What’s new?
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Graphical user interface 2
2
Graphical user interface
2.1
About the interface
The graphical user interface is divided into four main parts, shown in Figure 2-1.
They are the Menu Bar, the Toolbars, the Status Bar, and the Display Zone.
The Display Zone is the center of the interface. This is where gel images are
arranged in sheets and panes. The Workspace and any reports are dockable
windows found along the edges of the Display Zone.
a
b
e
f
g
d
h
c
Figure 2-1. The ImageMaster window. (a) Menu Bar, (b) Toolbar, (c) Status Bar, (d) Display
Zone, (e) Sheet, (f) Pane, (g) Workspace, and (h) Reports.
2.2
Menu bar
You can choose actions to be performed during your analysis from the Menu
Bar. The menus are context related. This means some of the commands may not
be available all of the time and either go away or are grayed out. For example,
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2 Graphical user interface
2.2 Menu bar
the Select menu allows you to select spots, matches and annotations for
detection and matching. Therefore, this menu is neither available nor displayed
while viewing and editing images in the Image Pool.
Menu
Description
File
Close, save, import, export, print and other basic operations. You
can also exit ImageMaster.
Edit
Undo/redo the last operations, show a history of operations, or edit
(add, modify, delete) specific gels, spots, annotations or matches.
You can also edit spot sets and enable spots.
View
Modify the settings for grid lines, profile or overview in the display,
align images, show dual color or spot overlap in the current sheet,
or change the way gels, spots, annotations or matches are
visualized.
Select
Select specific spots, spot sets, annotations or matches.
Reports
Display tabular or graphical information about selected gels, spots,
annotations or matches, and compute differences and similarities
between gel images. The data analysis is based on robust statistics,
factor analysis, and statistical tests.
Tools
Change display, quantification, and other options at the software
level and customize the user interface (custom toolbars, keyboard
shortcuts). Create and control a calibration tablet while working in
the Image Pool .
Help
Access documentation and obtain license, product, version, or
system information.
Most menu commands also have toolbar icons and/or keyboard shortcuts.
2.2.1
Keyboard shortcuts
Keyboard shortcuts, when available, are designated on the right-hand side of
the corresponding menu command. A list of all shortcuts is given in the
Appendix. Please note the logic behind the key combinations:
Ctrl is used to maneuver gels.
Shift is used to maneuver spots.
Alt is used to maneuver annotations.
Ctrl + Shift is used to maneuver matches.
You can create your own keyboard shortcuts for menu commands.
Note: A new keyboard shortcut must be unique. Be careful not to duplicate a
keyboard shortcut that is already assigned to another menu command.
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Graphical user interface 2
You risk destroying an existing shortcut. However, a given command can
have several, different keyboard shortcuts.
To add your own keyboard shortcut:
1
Choose Tools : Customize.
2
Click the Keyboard tab.
3
Select the menu in the Category drop-down list and then select the
command for which you want to create a shortcut (Figure 2-2).
4
Read through the Key assignments list to see if the shortcut is currently
assigned.
5
Click in the Press new shortcut key box and then press the new
combination on your keyboard.
6
Click Assign. The software warns you when the shortcut is already assigned
to another command and asks if you want to re-assign it. Click No and
come up with a different shortcut.
7
Click Close.
Figure 2-2. Defining keyboard shortcuts in the Customize window.
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2 Graphical user interface
2.3 Toolbars
2.3
Toolbars
2.3.1
Default toolbars
The standard toolbars provided are designed so that you can quickly
manipulate your gel data and apply the most frequently used features. Each of
the tools are described in detail elsewhere in the User Manual.
Tools
This toolbar contains the basic tools of the software. More importantly, their
functions (Move, Zoom, Region, Selection, Measure and Landmark) are not
available in any menu. Although they do have corresponding keyboard
shortcuts.
Display
This toolbar contains the options Undo Zoom/Move, Redo Zoom/Move, Adjust
Contrast and Cursor Information Window.
Image
The image toolbar (Rotate, Flip, Invert Gray Levels, Crop and Add Files to Project)
is contextual; it is only available when working with images in the Image Pool.
Detect And Match Spots
This toolbar is only displayed when working with gels in a Match or Class sheet
(that is, once gels have been imported into a project). You can Detect, Enable
Edit, Match Gels, Add Match, Delete Match and Show Vectors.
Edit Spots
This toolbar is only available when spot edition is enabled. Choose Edit : Spots :
Edit Enabled to get into this mode.
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2.3.2
Customize toolbars
Toolbars can be configured according to your individual specifications.
Toolbar position
To change the position of a toolbar, click the left edge and drag the toolbar to
the position you want. You can drag a toolbar to any of the edges of the user
interface. When a toolbar is dragged outside of the frame of the ImageMaster
window, it becomes a floating window.
Toolbar format
By default, the toolbar icons in the software are small (16x16 pixels). You can
choose to display large icons (24x24 pixels) for better visibility. In addition, when
you move the mouse over an icon, a screentip appears. Perhaps you prefer to
hide all the screentips, or remove the keyboard shortcuts for the tools from the
screentips.
To modify the toolbar format:
1
Choose Tools : Customize.
2
Click the Options tab in the Customize window.
3
Select the desired options in the Other section.
4
Click Close.
Custom toolbars
You can create your own toolbars with icons for the functions you use most
commonly.
To create a custom toolbar:
1
Choose Tools : Customize.
2
Click the Toolbars tab in the Customize window.
3
Click New to create a toolbar. Enter a name for the toolbar and click OK. An
empty toolbar is created below the existing ones.
4
Click the Commands tab in the Customize window.
5
Select the menu from the Category list. Then select the command for which
you want to create an icon and drag it to the empty toolbar.
6
Repeat step 5 to add icons to the toolbar.
7
Click Close in the Customize window.
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2.4 Status bar
2.4
Status bar
The Status Bar at the bottom of the Display Zone is an important resource. It
indicates the total number of gels, spots, matches and annotations that are
selected in the current sheet.
If you move your mouse over a gel image, the Status Bar also indicates the X and
Y coordinates at the cursor position, as well as the image intensity. The unit of
the coordinates can be changed in Tools : Options, under the Display tab.
2.5
Display zone
Gel images are opened in the Workspace and viewed in sheets and panes in the
Display Zone (Figure 2-3). Their layouts can be arranged according to your
requirements.
Figure 2-3. The Display Zone. There are two sheets open here, including the Image Pool.
The current sheet AB is in front and contains four panes (AT1, AT2, BT1 and BT2). Only the
pane AT1 is selected (green tab). By clicking the Layout icon for the pane, different options
(Stacked, Tiled, One Row, One Column, Free) are displayed. Similarly, click the Layout icon
in the upper right corner to re-arrange a sheet.
2.5.1
Sheets
When no dockable windows (Workspace, reports) are open, sheets occupy the
entire Display Zone. Each sheet has a tab, with its name and an icon
representing its type:
•
22
Image Pool: This sheet contains images for viewing and basic
processing.
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•
MatchSet: This sheet is opened by right-clicking on the name of a
match set in the Workspace. Spot detection and matching must be carried
out on this type of sheet.
•
Class: This sheet is opened by right-clicking on the name of one or
several classes in the Workspace. To carry out advanced expression
analysis, you must work in this type of sheet.
When you move the mouse cursor over a sheet tab, the screentip specifies the
type.
Selection
The current sheet is always in front and its name is in bold. Select a sheet by
clicking on its tab.
Close
Click the Close icon in the upper right corner of the current sheet when you are
finished working with it.
Layout
The Layout icon to the left of the Close icon can be used to choose the
arrangement of the panes in a sheet.
Option
Description
Stacked
Panes in a sheet are one on top of another.
Tiled
Panes in a sheet are side by side.
One Row
Panes in a sheet are in a single horizontal line.
One Column
Panes in a sheet are in a single vertical line.
Free
You specify the number of panes laid out horizontally and
vertically in a sheet.
2.5.2
Panes
There can be one or more panes in a sheet (see Figure 2-3). Each pane has a tab
with its name. On the left side of the tab, one or more icons describe the match
or class hierarchy.
Selection
Select a pane by clicking on its tab. Use the Shift or Ctrl keys to make multiple
selections. Click the sheet tab to select all panes in a sheet. Selected panes have
green tabs.
By default, panes are laid out in Tiled mode. When working in a different mode
like Stacked, bring a hidden pane to the front by clicking on its tab.
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2.5 Display zone
Layout
Click the Layout icon on the right side of the tab to change the arrangement of
the images in a pane. The options are the same as for panes in a sheet.
2.5.3
Images
By default, the gel name is displayed in the upper left corner of an image. The
color of the name indicates if the image is selected (green) or not (gray). If an
image name has a red corner, this means that it is the reference image for the
matching. If an image name is a darker green or darker gray than the other
images, then it is the current sheet reference. All other images in the sheet are
compared to this sheet reference when using the menu commands in View :
Sheet.
To hide the image names, choose View : Global : Show Gel Names. The image
name is replaced by three dots.
Move the mouse over an image name to view a screentip specifying the match
hierarchy. In a Class sheet, the class and path to which it belongs are also given.
If the image is the sheet reference, then this fact is included in the screentip.
Selection
Select an image by clicking on its name. Use the Shift or Ctrl keys to make
multiple selections. Click the pane tab to select all images in a pane.
When images are hidden like in Stacked mode, bring an image to the front by
clicking on its tab at the bottom of the pane. Quickly sift through images using
the Page Up and Page Down keys on your keyboard or click on the navigation
triangles in the lower right corner of a pane.
Scrollbars
The scrollbars on the right and bottom edges of each image can be used to
change the zoom factor of an image by dragging one of the ends of the
scrollbar. You can also move the visible area of the gel horizontally or vertically
by clicking in the middle of the scrollbar and dragging left/right or up/down.
Click on the gray square at the intersection of two scrollbars to reset the image
to its full image size.
If you want to view a specific area of your gel image, choose View : Global :
Scrollbars : Adjust. In the Adjust Visible Area window, set the exact horizontal
and vertical start and end coordinates for the area to be displayed. You can do
this in terms of different units: Pixel coordinate, Percentage and Real coordinate
(pI and MW). Please note that pI_MW annotations must be defined in order to
use this function.
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To hide the scrollbars, choose View : Global : Scrollbars : Show. In that case, You
can still move and zoom images by using the Move and Zoom tools in the
toolbar.
2.5.4
Switch order
You can change the order in which images are displayed by dragging the gel
name onto another image. It is then inserted before this image. Similarly, you
can re-order panes by dragging their tabs to a new position.
Swap panes or images by choosing View : Sheet : (Navigation :) Switch or the
Ctrl+F shortcut. This reverses the last re-ordering operation applied to a pane or
image.
2.6
Workspace
The Workspace plays an important role in the software. It allows you to organize
your gels into projects, to specify how the gels are to be matched together, and
to define your classes (or groups) for statistical analysis. A brief description of
the Workspace window is presented here. The utility of the Workspace is
discussed in another chapter.
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2.6 Workspace
The Workspace window is a dockable window and has two main parts: the
Workspace toolbar and the Navigator (Figure 2-4).
Figure 2-4. The expanded view of the Workspace window. The Workspace toolbar and
Navigator (at the left) are always visible. The file details in the expanded view (at the right)
are only displayed when desired.
2.6.1
Toolbar
Commands to create new projects, add files to projects, remove, backup and
restore projects are found under the
Project icon in the Workspace toolbar. .
Click the
Expanded View icon to enlarge the Workspace window to include
details of all files selected in the Navigator. The files can be sorted in ascending
or descending order by clicking a column header. Click the Expanded View icon
again to hide the file information.
2.6.2
Navigator
The Navigator displays all files and folders you can view (Image Pool) and
analyze (projects). The look and feel is similar to Windows Explorer. There is a
hierarchical structure of folders, subfolders and files that can be expanded or
collapsed, dragged and dropped in a new location, copied and pasted, etc.
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When launching the software for the first time, there is an empty Image Pool
folder. Images are opened through the Image Pool. To further analyze images
(detect, match, carry out statistical analysis, etc.), they must be transferred to a
project. You can work on many projects in the Workspace, each of which will
contain one or more root structures with Match and Classes subfolders.
Current sheet
Any item in bold in the Navigator corresponds to items displayed in the current
sheet.
Contextual menus
A contextual menu containing relevant options appears when you right-click an
item in the Navigator.
Navigator icons
Each folder type in the Navigator has a specific icon (Image Pool, Project, Match,
Class). In addition, images and match set folders have icons that indicate their
status. You can distinguish DIGE gels from non-DIGE gels, know which gels have
been detected and matched, and recognize gels used as references for
matching.
Icon
Meaning
Image Pool folder.
Project.
Match folder.
Classes folder
Table 2-1. The folder icons in the Navigator.
Icon
Meaning
Undetected image /
reference image.
Detected image /
reference image.
Matched image /
reference image.
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2.7 Reports
DIGE image (in class).
DIGE gel / reference gel.
Matched DIGE gel /
reference gel.
Match set / reference
match set.
Matched match set /
reference match set.
Class.
Table 2-2. The Navigator icons inform you about the type and status of the image, gel and
match set.
2.7
Reports
Reports are highly practical for organizing and describing your gel data. They
make it much easier to process all of the information.
Reports are not necessarily in table format. Graphical representations of data
such as histograms, scatter plots and 3D views are treated as reports as well. All
reports are dockable windows.
Report
Description
3D View
A three-dimensional view of selected gel regions or areas
around selected spots.
Analyze Gels
Information about each selected spot match such as its
Match ID, value for each spot in the match, and chosen
statistical measures calculated on all spots in the match.
Scatter Plots also provide information (slope, offset,
correlation coefficient, fitting error) that compare the spot
values for two gels.
The Match Statistics Table displays the number of matches
and percentage of matches for each gel at the selected level
of the match hierarchy.
The DIGE Histogram displays the frequency distribution of
the DIGE volume ratios.
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Report
Description
Analyze
Classes
Central tendency, dispersion, and overlapping measures for
classes of gels computed for all selected matches.
Differences between the spot values in several classes can
also be quantified with Statistical Tests such as ANOVA,
Mann-Whitney U test and Kolmogorov-Smirnov test.
Gel Table
Summarized information about the selected gels, such as Gel
ID, file name and path, gel calibration data, gel resolution
and size, number of detected spots, user defined properties
such as sample type, staining or date of the experiment, and
much more. Predefined templates (Properties, Files,
Descriptions, Calibration) allow you to quickly display a
specific subset of fields. You can also create custom
templates.
Spot Table
Specific information about selected spots such as Spot ID
and coordinates, quantification values, attached labels, etc.
Annotation
Table
Information about annotations, including the label content
for each category, the annotation coordinates and Spot ID (if
the annotation is linked to a spot).
2.7.1
Dynamic content
Report content is continuously updated. A report selection is synchronized to
reflect the most current data from the corresponding sheet that contains the gel
images.
Note: Reports are attached to their corresponding sheet. If the sheet is closed,
the reports will be closed as well.
2.7.2
Content based on enabled spots
By default, all spots are enabled and therefore represented in the reports. But
the content of reports can be limited to a subset of spots by deactivating spots
that are not of interest.
2.7.3
Toolbars
Most reports have a toolbar with the following icons in addition to some reportspecific icons and functionalities that are described in later chapters of the User
Manual:
Suspend synchronization
By default, the selection in the report is continuously synchronized with the
corresponding sheet that contains the images and with other reports. Click the
Suspend Synchronization icon to stop the synchronization and render the
selection in the report independent of the selection on the gel, and vice versa.
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2.7 Reports
Save
Enter a name and select the desired format for the file to be saved. Tables can
be saved in tab-delimited text format (.txt), as a Microsoft Excel Workbook (.xls),
or in XML format (.xml). Graphics can be saved in PNG, TIFF or BMP formats.
Print
Normal print options are available when printing graphical reports. When
printing tabular reports, the table is first displayed in your default Web browser.
This is because the XSL stylesheet located in the Template\Reports folder of the
ImageMaster installation directory is used to transform the XML report into an
attractive table. You can then use the print option in your browser to get a
printout.
Copy to clipboard
Export your data directly into another application. First select the desired lines
in a table, or the desired graphics in a window, using the Shift or Ctrl keys. Then
copy the selection to the clipboard by choosing the Copy to Clipboard icon.
Paste directly into the preferred software.
Previous in selection
Click the Previous in Selection icon to skip to the first selected item encountered
when scrolling towards the top of your table.
Next in selection
Click the Next in Selection icon to skip to the first selected item encountered
when scrolling towards the bottom of your table.
Settings
Click this icon to change the display settings for your report. In most cases, this
means setting the visibility (hidden or shown) of the columns. In some cases
(Histograms and Match Statistics Table), you can define other display options.
Select by value
This feature allows you to select items in the report based on a numerical search
criterion. Click the Select by Value icon, then select the measure (that is, column)
you want to use for refinement, and finally set the lower and/or upper limits of
your search interval.
2.7.4
Customize reports
Sorting data in columns
Data in tabular reports can be sorted by the column content. If you click once
on the header of a specific column, a triangle is displayed indicating that the
column's numerical or textual data is sorted in ascending order. When you click
once more on the header, the triangle inverts indicating that the data is sorted
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in the opposite order. Note that ascending order means that numbers are sorted
from 0 to 9 and text is sorted from A to Z.
Column visibility
Load a predefined template or save your own template indicating what
columns should be hidden or shown using the Settings icon in a report toolbar.
This is particularly useful when you only want the essential information to
appear for clarity or for printing purposes.
To apply an existing report template:
1
Click the Settings icon in a report toolbar.
2
In the template window (Figure 2-5), select a template name with the Load
icon. By default, only the Gel Table has more than one predefined template
(Properties, Files, Decriptions, Calibration).
3
Click OK.
To create and save your own report template:
1
Click the Settings icon in a report toolbar.
2
In the template window, select (box is checked) the attributes that you want
to show in the report and deselect (box is unchecked) the attributes to hide
columns.
3
To save the template for later use, click the Save icon. Choose a name from
the list or select <New...: to create one. Click OK.
4
Click OK in the template window.
To delete an existing report template:
1
Click the Settings icon in a report toolbar.
2
In the template window, click the Remove icon and choose the template to
be deleted. Click OK.
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2.7 Reports
3
Click Yes when asked for confirmation.
Figure 2-5. Report template window.
Column order
You can reorganize table columns directly in a report window. To do so, drag the
column header to its new position. You will see red arrows at the insertion point.
Column size
You can enlarge or reduce the column size. Drag the right edge of the column
header until the column is the width you want. To resize columns so that their
whole content is displayed, double-click on their separator. The column to the
left of the cursor is resized.
2.7.5
Edit table cells
In the Spot table, cells containing annotation labels are editable. Double-click in
a cell to start typing or editing your label. When finished, a single click in any cell
quits the editing mode.
Please note that annotation categories of the data type Set are displayed as
check boxes. A checked box means that the item belongs to the Set. An empty
box means that it does not belong to the Set.
The same is true for spot sets represented in any of the tables: a checked box
means that the spot belongs to the set. An empty box means that the spot does
not belong to the set.
To check several boxes simultaneously:
32
1
Use the Shift or Ctrl keys to select the rows in which you want to check or
uncheck the box.
2
Click in the box of one of the selected rows. All rows are now either checked
or unchecked.
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2.8
Dockable windows
The Workspace, reports, and Adjust Contrast window are dockable windows.
Dockable windows make it easier for you to work with numerous windows at the
same time. These windows can be docked (that is, fixed in place) against the left,
right, top or bottom edges of the ImageMaster window and always lie on top
when visible. A visible window can be in one of three modes:
Pinned
The pinned mode enables dockable windows to be locked into position
around the edges of the ImageMaster window. Once a window is pinned, you
can move it to a different location by dragging the title bar (Figure 2-6). Guides
indicate where the window may be docked. By moving the cursor over the
guides, a shaded blue box appears showing where the window will reside if the
left mouse button is released. If you move the dockable window to a nonpredefined location, it becomes a floating window. Moving a docked window
may affect the location and size of other docked windows.
Un-pinned
A visible window in un-pinned mode automatically collapses when not in use
to become a tab at the edge of the ImageMaster window (in Figure 1-7, the
Workspace is in this mode). When you click on a docked tab, the window slides
back into view and is ready for use. You can also click on Minimize to minimize
a window in un-pinned mode.
Floating
A dockable window in floating mode will always appear on top. It can be
dragged to any position within the software or even outside the ImageMaster
window. You can switch in and out of floating mode by double-clicking on the
title bar of a dockable window.
Tabbed groups
Dockable windows can be organized into tabbed groups. This feature extends
your ability to maximize the use of limited screen space by combining multiple
dockable windows into one window. In order to form a tabbed group, drag the
title bar of a dockable window into the center of another. You will see the nested
tabs at the bottom of the docked window. In order to separate a tabbed group,
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2.9 Options
drag a tab away from the docked window or double-click on the tab. Please note
that tabular reports and graphical reports cannot be grouped together.
Figure 2-6. The Class Analysis Histograms window previously docked at the right edge will
now be docked to the right of the Class Analysis Table window, in the lower left corner of
the ImageMaster window.
2.9
Options
You can set various parameters that influence your work in ImageMaster. These
settings are accessed by choosing Tools : Options in the menu. More detailed
information about the options are provided in the related chapters of the User
manual. However, an overview of the settings, per tab, is given below.
Display
• Indicate the default spot colors (for enabled, disabled, selected and
overlapped spots) and the color for the match vectors.
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•
Choose the units to express coordinates in the Status Bar and Measure
tool.
•
Indicate if spot selections should be surrounded by boxes, for easier
localization.
Annotation
• Define the annotation categories that should always be available in the
software, and set their attributes and display properties.
Gel descriptions
• Define the gel descriptions that should always be available in the software.
Quantification
• Specify the spot quantification value to be used for non-DIGE (Value) and
DIGE (DIGE Value) experiments. This spot quantification value is used in Gel
and Class Analysis Reports.
•
2
Compute the spot areas in mm , based on gel resolution (default), or pixels.
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Image Pool 3
3
Image Pool
3.1
Introduction
This chapter is about opening and processing images in the Image Pool. You
must be familiar with the concepts described in the last chapter (the Workspace,
sheets, panes and images) in order to get the most out of it.
3.2
Start with good images
Your gels must first be converted into an image file by an appropriate imaging
device. During digitization, a gel is resolved into a two-dimensional matrix of
squares, or pixels. Each pixel in the generated image file is characterized by its
X and Y coordinates, and its signal intensity, or gray value.
To make any analysis meaningful, it is important to start with good quality
image files. The following paragraphs give some helpful tips on what resolution,
depth and image formats should be used to obtain the best possible results.
3.2.1
Resolution
The scanning resolution of a gel is critical as it influences the amount of visible
detail in the image. A low resolution corresponds to a large pixel size or a small
number of pixels (or dpi, dots per inch). When the image resolution is too low,
individual spots cannot be distinguished. On the other hand, when the scan
resolution is too high, the image file becomes very large. This slows down the gel
analysis significantly. A resolution between 150 and 300 dpi is generally
sufficient for gel analysis.
3.2.2
Image depth
The range of potential gray levels in an image varies according to the image
8
depth. In the case of an 8-bit image, one pixel has 256 (2 ) possible gray values
(0 to 255). Images scanned with a higher image depth contain more information.
16
A 16-bit image (2 = 65536 gray levels) will reveal more subtleties. We strongly
recommend an image depth of at least 12 bits for gel analysis. 16 bits is
preferred.
Please make sure to use gray scale images for your analysis, and not color
images. The extra color information (one intensity value for each color channel)
is of no value.
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3.3 Preview images
3.2.3
Calibration
Some imaging devices can measure more gray levels (for example, 100000 or
more) than can be stored in the availabe image formats. These instruments
encode the gray values using a nonlinear calibration curve (generally encoding
low intensity values with higher accuracy than the high intensity values), to
conserve as much information as possible in the saved files. ImageMaster uses
the calibration curve contained in these image files to recalculate the measured
gray values and use them for display and quantification.
Other devices convert raw pixel values into real-world units (generally optical
density or OD). This means that the range of gray values is the same no matter
what the original image depth. There are image capture devices, such as the GE
Healthcare ImageScanner™ in conjunction with the LabScan™ software, that
even allow you to perform this type of calibration. When this is the case,
ImageMaster takes into account the conversion tables or calibration formulas
stored in the files. You can also calibrate such an image capture device within
ImageMaster using calibration step wedges or calibration strips.
3.2.4
Image editing
General purpose graphics software such as Adobe Photoshop ignore or even
remove calibration information. Therefore, you should not use them to flip,
rotate, crop or invert your images. Instead, use the software that came with your
scanner or the dedicated tools in ImageMaster.
3.2.5
File format
The supported input formats are GEL (Molecular Dynamics), MEL (ImageMaster),
TIFF (Tag Image File Format), IMG (Fuji), GSC and 1SC (Bio-Rad). Please note that
the TIFF format does not include calibration information.
3.2.6
DIGE file naming convention
To facilitate the import of DIGE images, it is recommended that the file names
for the group of two or three images contain a common string and their
respective dye names (Cy2, Cy3, Cy5).
3.3
Preview images
3.3.1
Open images
You can open gel images that are in any of the above-mentioned input formats
(.mel, .gel, .tif, .img, .gsc, .1sc).
To open gel images:
1
38
Do one of the following:
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Image Pool 3
•
Choose File : Open.
•
In the Workspace window, click the Project icon. In the Add Files
window, search for Files of Type “All Image Files (*.mel; *.tif; *.gel;
*.img)”.
•
In the Navigator of the Workspace window, right-click on the Image
Pool folder and select Add.
2
In the Open Files or Add Files window, browse the directory where the
image file is located, select its name and click Open. Use the Shift or Ctrl
keys to make multiple selections. Note that you can select files from
multiple DIGE gels.
3
For the different types of images, do the following:
•
For non-DIGE images: Specify the staining. If the staining cannot be
found in the list, you can type a new one. Click OK.
•
For DIGE images: Select the images that are part of the same DIGE gel
(Figure 3-1). By default, the software proposes image combinations
based on the file names. Click Add to confirm one DIGE gel at a time, or
Add All if the proposed combinations are all correct. The suggested
names for the created DIGE gels must be edited and confirmed
individually.
Figure 3-1. Create DIGE Gel window. The first list contains the Cy2 images to be opened,
the second the Cy3 images and the third the Cy5 images. The software proposes the
combination of images based on the file names.
Note: The software checks the image resolution before opening a file. If the
resolution is too low, you get a warning message. If it is higher than 300
dpi, you are able to scale the image in order to reduce its size.
3.3.2
Image pool sheet
The gel images appear in the Image Pool folder in the Workspace and are
automatically opened in the Image Pool sheet (Figure 3-2). Non-DIGE images all
appear in a single pane with the name Files. A separate pane is displayed for
each DIGE gel, containing the images belonging to that gel.
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3.3 Preview images
Remove
Gels remain in the Image Pool until they are added to a Project. When you are
finished working with an image, right-click on it and select Remove. To remove
all images in the Image Pool, right-click on the Image Pool folder and select
Remove All. Please note that this action never affects the original image files
(which always remain unchanged in their original location).
Hide / display
If you do not want to display all gels in the Image Pool sheet, you can right-click
on certain items in the Image Pool folder and select Hide. To show a hidden
image, right-click on it and select Display.
To open the Image Pool sheet at any time, right-click on the Image Pool folder in
the Workspace and select Display.
Properties
To rename a gel or image, or to see the file path, right-click on a gel and select
Properties.
Figure 3-2. Image Pool folder in the Workspace and Image Pool sheet.
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3.4
Process images
The Image toolbar provides the basic tools to process images: Rotate, Flip, Crop
and Invert Gray Levels, which can also be found in the Edit : Gels menu. These
tools are only available when the Image Pool is the current sheet. They are also
optional and should only be applied when needed.
Processing your images in ImageMaster does not affect your original image
files. Copies are saved in a temporary folder until the images are added to a
project and saved in the corresponding project folder.
Please note that these operations are simultaneously carried out on the two or
three images in a DIGE gel.
3.4.1
Rotate
Selected images can be rotated by 90° CCW (counterclockwise) or 90° CW
(clockwise). Free rotation (the third icon) can also be applied, although it should
be avoided since it modifies the original data.
To freely rotate an image:
1
Select the gel image to be rotated in the Image Pool sheet.
2
Click the Free Rotate icon.
3
A red grid appears on the image. The bold horizontal grid line plays the role
of landmark to help you visualize the rotation. It becomes the new
horizontal in your rotated image.
4
Click anywhere in the image and rotate the grid while holding the left
mouse button. Release the button when the bold line is parallel with what
should be the new horizontal in your image (Figure 3-3). You can also
manually enter a rotation angle in the Rotation Tool window.
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Figure 3-3. Rotation Tool window. The grid is rotated until its bold line is parallel with what
should be the new horizontal reference. When the mouse button is released, the gel image
is rotated.
3.4.2
Flip
When gel images are scanned in the wrong direction, you can Flip Horizontally
or Flip Vertically to produce their correct mirror image.
3.4.3
Crop
You can crop your gel images with the Crop tool. This creates new gels that only
contain the selected area and removes the outer area.
When you crop a gel, you get the choice to create a new image (a number is
appended to the existing name) or to overwrite the image in the Image Pool.
Note: Gels in the Image Pool are copies of the original image file.
Crop area
The crop area is a region that can have an anchor attached to it. You can
position the anchor on an easily recognizable protein spot. As the region moves
with the anchor, and vice versa, you can easily crop a similar part of each gel by
correctly positioning the crop areas (of the same size) in the gels (Figure 3-4).
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To define a crop area:
1
Click the Region tool and place the cursor at the top left position of the area
you want to crop. Hold down the left mouse button and move the cursor to
the bottom right position (a dashed box is displayed). Release the mouse
button at the end point.
2
Move a crop area by clicking inside the box and dragging it. Change the size
of the area by dragging a corner or edge. If the box is reduced to its
minimum size, its appearance changes to a blue circle. To remove an area,
double-click on the image.
3
If you want to attach an anchor to the crop area, hold the Alt key while
clicking on an easily recognizable protein spot. A dark blue circle will be
centered on the spot. Note that this anchor may be located inside or
outside the crop area.
4
To change the position of the anchor, hold the Alt key and click another
spot. To remove the anchor, hold the Alt key and click on the anchor.
5
Propagate the crop area to the other images in your sheet by holding the
Shift key while clicking in the crop area.
6
Adjust the position of the crop area in each gel by moving it so that the
anchor is centered on the appropriate spot.
Figure 3-4. Identical crop areas in three gel images.
7
Click the Crop icon to crop the image according to your area.
Crop area export / import
Crop areas can be exported and imported to ensure that the final size of all your
cropped gels is identical, even between work sessions.
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To export a crop area:
1
With the Region tool, define a crop area as described above.
2
Select the gel in which you defined the crop area.
3
Choose Edit : Gels : Crop Area : Export to save the crop area to a file with
the extension .cpt (Crop Tool).
4
Browse to locate a folder and enter a file name. Click Save.
To import a crop area:
1
Select the gels to be cropped to a previously defined crop area.
2
Choose Edit : Gels : Crop Area : Import and select the previously saved file.
Click Open.
3
The crop area appears on the selected images.
4
By clicking inside the area with the Region tool and dragging it, you can
move the crop area to superimpose the anchor on a characteristic spot.
3.4.4
Invert gray levels
You can invert the gray levels of selected gels. This means that if your image
shows white spots on a black background, the inversion displays black spots on
a white background (the required mode for analysis in ImageMaster).
Note: When your images open in the software with white spots on a black
background this may indicate that you used incorrect scanner settings or
that your files are not properly imported. Please verify your image
acquisition parameters. If you think the software does not correctly
support your files (that is, saved in one of the recommended input
formats), please contact our technical support service.
3.5
Calibrate images
3.5.1
Display calibration information about images
It is recommended to use calibrated images for your gel analysis. To verify if
your images were calibrated, and possibly view the calibration information:
•
44
Choose Reports : Gel Table, click the Settings icon and select the
Calibration template from the Load drop-down list. The Calibration Unit,
Formula, Name, Creator and Date are displayed in the report. If nothing
appears in these columns, the gels were not calibrated. Create a
calibration according to the following sections.
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•
If your image was calibrated with LabScan 5.0 or 6.0, you can select the
image and choose Edit : Gels : Show Calibration Plot to view the
calibration curve. See below for details about the calibration curve.
If you are digitizing your images using a flatbed document scanner and do not
have calibrated images yet, you can prepare and apply a calibration at this
point, before adding your images to a project. It is generally recommended to
perform this type of intensity calibration on the image capture device once
every month.
3.5.2
Prepare for a calibration of the image capture device
Scan a calibration step tablet
To calibrate the image capture device, you need to scan a calibration step tablet
or calibration strip along with your gels. These step tablets have known intensity
values (expressed in optical density, OD, or diffuse density, DD) published by the
manufacturer of the step tablet.
Please note that for the purpose of 2D gel analysis, it is only useful to calibrate
the image capture device when working in transparent mode. No calibration
needs to be done when you do reflective scanning. When both a transparent
and a reflective calibration strip are provided, be sure to use the appropriate
calibration step tablet.
Calibration tablet file
The OD values for the step tablet have to be specified in a Calibration Tablet File,
together with other information such as the height and width of the tablet, and
the number of steps.
An example of such a Calibration Tablet File (Kodak2.tab) can be found in the
Template\Tablet folder of the ImageMaster installation directory. This
Calibration Tablet File is made for use with the Kodak Step Tablet no. 2. If you do
not use this specific step tablet, you can copy the file and edit the data to make
your own Calibration Tablet File. You can edit the file with tools such as Windows
Notepad.
As the intensity values supplied with your step tablet are generally expressed in
diffuse density (DD), you have to convert them to OD values. For this purpose, the
manufacturer of the step tablet should provide the appropriate relationship. For
the Kodak Step Tablets no. 2 and 3, for example, this is OD = 1.4 DD.
3.5.3
Create calibration of the capture device
You can create a calibration once you have scanned the step tablet and have a
correct Calibration Tablet File.
To create a calibration:
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1
Open the step tablet image file. If necessary, rotate the image so that the
light steps are displayed at the top.
2
Choose Tools : Calibration Tablet : Create.
3
In the Load Step Tablet Definition window, browse to the folder where you
saved the Calibration Tablet File (.tab) specifically tailored to your step
tablet (see above), select the file and click Open.
4
A red calibration step overlay appears on the image and the Create
Calibration window is displayed (Figure 3-5).
5
Adjust the position of the steps by dragging the overlay while holding the
left mouse button. The size of a step on the red overlay (the distance
between two short horizontal lines) should correspond exactly to the size of
a step on the image. If this is not the case, you must adjust the height of the
tablet in the Calibration Tablet File.
Figure 3-5. Image of the step tablet with the red calibration step overlay. The Create
Calibration window shows the calibration curve and the OD values for the different steps
(on the left). When selecting a step in the list, the corresponding step becomes
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automatically highlighted in green on the step tablet overlay and in the calibration graph.
6
At the left of the Create Calibration window, you see the theoretical optical
density (OD) values of the different steps in the tablet (the values you
entered in the Calibration Tablet File). Some steps are automatically
deselected (grayed out) because of their unreliable values. You can deselect
additional ones if you estimate that they should be excluded from the
calibration process. At the right of the Create Calibration window you see
the calibration curve between the logarithmic transmittance values on the
X-axis, and the OD intensities on the Y-axis. Note that the measured
intensity values for each step are calculated as median intensities over all
the pixels in the small rectangle area for each step (on the step overlay). The
horizontal dispersion intervals in blue (or gray for deselected spots)
represent the intensity ranges when 10% of the less intense and 10% of the
most intense pixel values are removed. The calibration formula and error
are given below the graph.
7
You can display some reports to judge the quality of your scanner
calibration (see below for more details).
8
Once you are satisfied with the calibration, close the Create Calibration
window. The software asks if you want to apply this new calibration. If you
answer Yes, ImageMaster applies the calibration to the image.
The icons in the toolbar of the Create Calibration window and their use are:
Open another step tablet definition.
Save the calibration (with the extension .cal).
Print the calibration graph.
Copy the calibration graph to the clipboard.
Display related reports:
•
The Fitting Table displays the calibration formula.
•
The Calibration Table displays for each step: the step number, the
measured average gray level, the theoretical intensity value, and the
fitting error (difference between the curve and the point).
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3.5.4
Apply a calibration to an image
Once you have created a calibration using a step tablet, and saved this
calibration in the step tablet image file or a calibration file (.cal), you can apply
the calibration to newly-scanned image files.
To apply a calibration to a gel:
1
Display and select the gels the calibration should be applied to.
2
Choose Edit : Gels : Apply Calibration.
3
Select the source of the calibration information. This can be an open gel (or
step tablet image) that was already calibrated or you can select a file (.cal
or .mel) from the hard disk.
All pixel and spot values subsequently calculated and displayed in the software
correspond to the calibrated values.
3.5.5
Remove a calibration
You can remove a calibration from a gel. Note that this can also be done for gels
that were already calibrated when you imported them into the software.
To remove a calibration from a gel:
1
Display and select the gels from which you want to remove the calibration.
2
Choose Edit : Gels : Reset Calibration.
3
Confirm your choice.
3.5.6
Control a calibration
The Control calibration mode allows you to verify if you are using the correct
calibration. It requires a different, specially calibrated step tablet (for example,
Kodak Step Tablet no. 3), which you compare to your previous calibration results.
In other words, you have a calibration step tablet for everyday use and a
specially calibrated control step tablet to verify your calibration periodically.
To control a calibration:
48
1
Scan your control step tablet (for example, Kodak Step Tablet no. 3) and
open the image in ImageMaster. If necessary, rotate the image such that
the light steps are displayed at the top.
2
Choose Tools : Calibration Tablet : Control.
3
You are asked to load the calibration to be controlled. This calibration could
have previously been saved using the Save icon in the Create Calibration
window (.cal) or can simply come from a calibrated image file (.mel) such as
the calibrated step tablet image obtained in the section above.
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3.6
4
Next load the definition of the control step tablet. This Control Tablet File
must be specifically adapted to this new step tablet. That is, it should have
been edited with a tool such as Windows Notepad so that it contains the
appropriate OD values, height, width, and number of steps corresponding
to the control step tablet.
5
The red calibration step overlay appears on the image of the control step
tablet and the Control Calibration window is displayed.
6
Select the Spot or Annotation tool in the ImageMaster toolbar and adjust
the position of the steps.
7
You should now verify that the calibration curve is passing through the data
points correctly and with minimum dispersion intervals. If this is not the
case, try to find out why your current calibration does not seem to work
properly.
Describe images
3.6.1
Gel descriptions
A gel description is information you can enter about the gel image, which can
be used for later reference by yourself or any colleagues. This information can
include sample type, gel running protocol, date of the experiment, operator
name, pH range, SDS gel percentage. All of this data is entered as Gel
Descriptions.
When opening images in the software, you are asked to specify the staining.
Staining is a standard Gel Description. This information is simply informative,
that is, it is not used by the software except when you specify the Cy™ dye for
DIGE images that do not have this information in their file name.
Each image can only have one description of a given category. For example, a
gel description category Treatment could contain the description “Drug 1” for
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3.6 Describe images
certain images and “Drug 2” for other images (Figure 3-6). You can define any
number of categories for an image or a given set of images.
Figure 3-6. Gel descriptions in a Gel Table (using the Descriptions template).
3.6.2
Display gel descriptions
Gel descriptions can be displayed in a Gel Table. Select the predefined
Descriptions template in this table to display only the columns for the gel
description categories.
To display gel descriptions:
1
Choose Reports : Gel Table.
2
Click the Settings icon.
3
In the Gel Table window, select the predefined Descriptions template from
the Load icon drop-down list. The boxes for the FileName and all gel
description categories (by default, only Staining and Comment) are
checked.
4
Click OK.
3.6.3
Add or edit gel descriptions
You can edit a gel description for one or more selected gels.
To add or edit gel descriptions:
50
1
Select the images for which you want to add or edit descriptions.
2
Choose Edit : Gels : Edit Description.
3
In the Add Gel Description window, select an existing gel description
category or create one. Click OK.
4
Enter the gel description that applies to the selected images. Click OK.
5
The new gel descriptions are displayed in the Gel Table.
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Alternatively, you can define gel descriptions by clicking on the Add Description
icon in the Gel Table toolbar. It’s behaviour is identical to that of the Edit : Gels :
Edit Description menu.
You can delete all gel descriptions of a certain category for the selected gels by
choosing Edit : Gels : Delete Description.
3.6.4
Permanent gel description categories
When gel description categories are created as described above, they are only
used for the gels that were selected during the creation of the categories. To
make gel description categories permanent in the software (always availabe
from the category list), you must define them in the Options.
To create permanent gel description categories:.
1
Choose Tools : Options.
2
Click the Add in the Gel Descriptions tab.
3
Enter a category name in the Add Category box and click OK.
4
The category is displayed in the permanent category list.
To remove a permanent category, select it from the list and click Delete.
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4.1
Projects
Introduction
A project includes all gels, spots, matches, annotations, spot sets, and other
information produced and analyzed during the course of a specific gel study.
You can create or add many projects in the Workspace.
A project in the Workspace can include one or more match hierarchies (for
example, AB in Figure 4-1), each of which contains a Match folder and a Classes
folder. The Match folder describes how gels or populations of gels, called match
sets, should be matched together. The Classes folder is where the biological
question is stated, through the definintion of classes of gels to be compared.
Figure 4-1. Project structure.
The next sections further explain the principles of match hierarchies and
classes, and how they can be used.
4.1.1
Match hierarchies
All images in an experiment are not equally easy to compare, even when the
gels are run in a highly controlled way. Typically, gels belonging to the same
biological group are easier to match (that is, corresponding spots are easier to
find) than images from different biological populations.
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4.1 Introduction
It is therefore recommended to use hierarchical match structures to create
more efficient match designs. Figure 4-2 and Figure 4-3 show an example of a
match hierarchy, or root match set, AB and submatch sets A, B, AT1, AT2, BT1
and BT2. Gels or match sets with a red marker are used as the reference in the
matching and always appear first in the list.
Figure 4-2. The Match folder. In this example experiment, a set of samples from bacteria
were cultivated with either substrate A or with substrate B. Under both growing
conditions, two treatments were tested. Therefore, 4 different populations exist. A gel was
run for each of the 3 samples in a population, giving a total of 12 gel images.
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BacteriaAB
1
A
A_T1
B
A_T2
A_T2_Gel3
A_T2_Gel2
A_T2_Gel1
A_T1_Gel3
A_T1_Gel2
A_T1_Gel1
B_T1
B_T1_Gel3
B_T1_Gel2
B_T1_Gel1
B_T2
B_T2_Gel3
B_T2_Gel2
B_T2_Gel1
Figure 4-3. A tree view of the example match hierarchy described above.
An important advantage of this type of match hierarchy is that it minimizes the
number of difficult match combinations. Instead of having to match 9 images
(those belonging to AT2, BT1 and BT2) to a global reference (for example,
A_T1_Gel1) that is not belonging to the same population, only 3 slightly more
complex match combinations must be performed (AT1 versus AT2, BT1 versus
BT2, and A versus B). This significantly reduces time spent on match editing.
There is another important reason why it is useful to adopt hierarchical
population matching instead of matching all images against a unique arbitrary
reference image. Only spots matched with a spot in another gel are included in
Gel and Class Analysis Tables (all spots are of course presented in the Spot
Table). The likelihood of a spot being matched is much higher when matching
with a gel from the same biological population. Spots that are represented in a
single population (submatch set) are therefore included in the analysis, even if
they are not in the global match reference. This considerably reduces the
number of spots missed in the analysis.
Note: DIGE gels are inherent match sets. A DIGE gel (the entity with its
composing images) is treated as any other non-DIGE gel when setting up
a match hierarchy.
Once created in the Workspace, you can display a complete match hierarchy
(for example, AB in Figure 4-2) in a sheet and carry out spot detection on all the
included gels. After defining one or two landmarks, the entire experiment is
matched in a matter of seconds, and matches are automatically propagated at
each level of the match hierarchy.
4.1.2
Classes
In the Classes folder, you state your biological questions. This means that you
define a class for each set of gels that you want to compare with other such
entities. Your goal, by comparing classes and therefore the gels within those
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4.2 Create a project
classes, is to find the protein expression variations between different biological
states.
The classes created in the Classes folder of a given match hierarchy can contain
any of the images in that hierarchy. One image can be in different classes. In
Figure 4-4, for example, the same 12 images can be compared as part of the 4
classes AT1, AT2, BT1 and BT2, or as part of the two classes T1 and T2.
You can define classes at any time, even in the very beginning of your gel
analysis study when no spots are detected. To carry out statistical analysis,
however, your gels must be detected and matched.
Figure 4-4. The Classes folder.
4.2
Create a project
4.2.1
The first time the software is launched
As long as no projects have been created or added to the Workspace (for
example, the first time you open the software), you are prompted to create a
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project (see Figure 4-5). Enter a project name, browse to where the project folder
should be saved, and possibly add a comment.
Figure 4-5. New Project window.
4.2.2
At any time
You can create a new project at any time by selecting New from the Project icon
drop-down list in the Workspace toolbar (Figure 4-6). Again, enter the Project
Name, Location and Comment (Figure 4-5).
Figure 4-6. Project icon drop down menu in the workspace.
4.2.3
Add files to project
Use the Add Files to Project icon in the Image toolbar to add gels from the
Image Pool to a project (Figure 4-7). Add an existing project or create a new in
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4.3 Create match hierarchy
the Add Files to Project window. An existing or new match set name must be
entered as well.
Figure 4-7. Add Files to Project window. A project can be created by choosing <New...: from
the Project list.
4.3
Create match hierarchy
4.3.1
Create a match set
The easiest way to create a match hierarchy is by adding gels from the Image
Pool to a project. The idea is to select only the gels that should be added to a
particular match set (for example, gels from the same population or same
experimental batch) and then to create this match set. Different options exist:
•
Select gels in the Image Pool folder and drag them onto a project name.
Enter a name for the new match set.
•
Select gels in the Image Pool sheet and drag them onto a project name in
the Workspace. Enter a name for the new match set.
•
Select gels in the Image Pool sheet and click Add Files to Project in the
Image toolbar. Set or create the destination project, and then click <New...:
in the MatchSets field (Figure 4-8). Enter a name for the new match set.
Alternatively, create an empty match set by right-clicking on the project name
and selecting Create MatchSet in the contextual menu. Drag images from the
Image Pool (folder or sheet) into the new match set. Gels selected in the Image
Pool sheet can also be added to an existing match set by clicking the Add Files
to Project icon.
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Figure 4-8. Add Files to Project. The three images of population A_T1 are selected in the
Image Pool sheet. After having clicked the Add FIles to Project icon, the destination project
and new name for the MatchSet can be set.
4.3.2
Merge a match set
Once match sets have been created, they can be merged into higher level
match structures for further matching. Select the match sets, right-click on one
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4.3 Create match hierarchy
of them, choose Merge MatchSet from the contextual window, and give a name
for the new match set (Figure 4-9). A hierarchy (Figure 4-2) can be created.
Figure 4-9. Match sets BT1 and BT2 are selected for merging. Match sets AT1 and AT2 were
already merged this way into a match set A.
4.3.3
Set reference
The reference for each match set must be carefully chosen. This is because
automatic matching compares the spots in the reference to those in the other
images. If a spot is absent from the reference, it cannot be matched
automatically (although it can be matched manually with spots in other gels).
The rule of thumb is to choose the gel or match set with the most and the best
quality spots as the reference.
Within each match set, the gel or match set that has a red marker and appears
first in the list is used as the reference in the matching process. To change the
match reference, drag the desired gel or match set onto the name of its parent
match set so that it moves into the first position.
Note: You can change the match reference as long as the images in your match
set have not been matched. Once they are, the reference image can no
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longer be changed.
4.3.4
Use existing match sets
You may wish to carry out several analyses, using different matching schemes.
You can copy match sets to use them in another configuration. In Figure 4-10,
for example, the match sets A_T1, A_T2, B_T1 and B_T2, were copied to be used
in populations per treatment (T1, T2) rather than growing substrate (A, B).
Because existing matches are conserved when copying match sets, this can
save a lot of work.
Figure 4-10. Match sets AT1, AT2, BT1 and BT2 were copied to be used in the match
hierarchy T1T2.
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T1T2
Bacteria
3
T1
A_T1
A_T1_Gel3
A_T1_Gel2
A_T1_Gel1
T2
B_T1
B_T1_Gel3
B_T1_Gel2
B_T1_Gel1
A_T2
A_T2_Gel3
A_T2_Gel2
A_T2_Gel1
B_T2
B_T2_Gel3
B_T2_Gel2
B_T2_Gel1
Figure 4-11. A tree view of the match hierarchy in Figure 4-10. The match sets A_T1, A_T2,
B_T1, and B_T2 have been reused in a different configuration compared to the example
in Figure 4-3.
You can simply drag one or more match sets to a new destination, or copy (rightclick on the match set) and paste them in the new destination (right-click on the
destination project or match set). The destination can be a match set or open
project. Copies of the match sets, including the existing matches, are created. A
number is appended to the orginal name. You can then rename the copied
match sets (see below).
To copy a match set in the same project, hold down the Ctrl key while dragging
the match set onto the project name.
4.3.5
Export / import a match set
The easiest way to provide others with access to the data is to export a match
hierarchy (the match set that is the parent of the project, containing the folders
Match and Classes). Simply right-click on the parent match set and choose
Export MatchSet. The entire match set (including images, matches, spots,
annotations, spot sets) is compressed into a single .exp file.
A .exp file can be imported into a project by right-clicking on the project name
and choosing Import MatchSet.
If the project data are saved in a folder on a shared network, colleagues having
access to this folder can open and work with the project, and therefore the
match sets.
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4.4
Create classes
4.4.1
Create a class
In the Match folder of a hierarchy, select all images that should be added to a
single class (group of biologically-related images). You can select match sets as
well. However, only one type of item (images or match sets) should be selected
at a time.
•
Option 1: Drag the selection onto the Classes folder of the same hierarchy.
Enter a name for the new class and click OK. You can also use the Copy
and Paste options in the contextual menus.
•
Option 2: Right-click on one of the selected items and choose Add In
Class. Enter a name for the new class and click OK.
Alternatively, create an empty class by right-clicking on the Classes folder and
selecting Create Class in the contextual menu. Drag images or match sets into
the new class.
4.5
Handle project items
Several operations are available for most items in a project. These are often
found in a contextual menu by right-clicking an item.
4.5.1
Display
To display one or more images, match sets or classes in a new sheet, right-click
on a selected item and choose Display. Please note that only complete match
sets or classes are displayed. This means that if you select one image in a match
set, all images in the match set are shown.
In the resulting sheet, only the highest level and lowest level items (match sets
or classes) are specifically visualized, in the sheet and panes, respectively. To
select an intermediary level, click its icon in the pane tab of the match reference
for that level.
Once the images are displayed in a sheet, you can start working with them. You
can change the layout settings to focus on certain images and hide others.
4.5.2
Remove
To permanently remove an item from the project, select Remove in its
contextual menu. This will delete the item from your hard disk.
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4.6 Save projects
4.5.3
Properties
When choosing Properties for an item, some of its attributes are shown such as
its name, creator, file path, etc. Here is where you can change the name of an
item or enter a comment to describe the item.
4.5.4
Move
You can rearrange your images, match sets and classes to change their position
in the list or move them into another match set or class. Drag your image, match
set or class to the desired position. It is generally inserted after the item you drop
it on. Whenever there is a possibility to insert it inside or after an entity, you are
asked to specify.
4.6
Save projects
4.6.1
Project folder
When you create a project, you are asked to specify a name and a location on
your hard disk. All the data related to the project (images, spots, matches,
annotations, spot sets) are saved in this location, in a folder with the name of the
project. All users that have access to this folder are able to open, view and work
with the project.
The project folder contains the following files and folders:
•
Projectname.prj: This project file is the link between all the other data files
in the project folder. If you want to add an existing project to your
workspace, you must search for, and open, its .prj file.
•
Raw Images: This folder contains the raw image files in .mel format.
•
MatchSet: This folder contains the gel (.gda) and match (.mda) data in
subfolders. The .gda files are used to store the spot information. The .mda
files contain the match information.
4.6.2
Save
The software automatically saves your work when you close a sheet or exit the
software. You can also save your work by choosing File : Save. This saves all your
data in the corresponding project folder on the hard disk.
4.6.3
Share project
If the project data are saved in a folder on a shared network, colleagues having
access to this folder can open and work with the project. The project must be
added to the colleague’s Workspace using the procedure described below.
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4.7
Manage projects
4.7.1
Add / remove projects
Remove
You can remove a project from your workspace by selecting Remove in the
Project icon drop-down list in the Workspace toolbar. Select the project(s) to be
removed and confirm. This does not delete the project folder from your hard
disk.
To permanently remove a project folder, you must delete it from your hard disk.
Make sure that this folder is not accessed by colleagues in your network before
you delete it.
Add
A previously removed project can be re-inserted to your workspace at any time.
Another user can also add it to his/her workspace.
To insert an existing project:
1
Select Add in the Project icon drop-down list in the Workspace toolbar.
2
In the Add Files window, browse the directory where the project file (.prj) is
located, select its name and click Open.
4.7.2
Add files to project
Add in the Project icon drop-down list in the Workspace toolbar also enables
you to add project elements instead of entire projects. You can open the
following file types:
•
Project (*.prj)
•
Project Backup (*.bkp)
•
Export File (*.exp)
•
All Image Files (*.mel; *.tif; *.gel; *.img)
•
MatchSet Data (*.mda)
•
Gel Data (*.gda)
4.7.3
Backup / restore project
It is good practice to do regular backups so that you can recover your work at
any time. With the Backup function in the Workspace, one or more project(s) can
be archived by writing all project-related data (images, spots, matches,
annotations, spot sets, enabled spots) into a single compressed file with the
extension .bkp. This file can then be restored when needed.
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4.7 Manage projects
To backup a project:
1
In the Workspace, select Backup in the Project icon drop-down list.
2
Select one or more projects to backup, and confirm when prompted.
3
In the Backup Project window, browse to the directory you want, enter a file
name and click Save.
4
The backup file (with the extension .bkp) is archived.
To restore a project:
1
In the Workspace, select Restore in the Project icon drop-down list.
2
In the Restore Project window, browse the directory where the backup file
is located, select its name and click Open.
3
Select the project(s) to restore from the list. Use the Ctrl or Shift keys to
make multiple selections. Click Restore.
4
If the project already exists in the Workspace, there is the possibility to
replace it with the archived project. If the project is not present in your
workspace, you are given two options for restoring a project. By restoring
to the original file path, the files are put back in the original project folder.
By restoring to a new file path, the project is replicated in a new location,
which must be specified. Click Restore.
4.7.4
Project visibility
When there are several projects in the Workspace, it can be useful to
temporarily hide some of them. Select Display in the Project icon drop-down list
and check (or uncheck) the Visibility box in front of the projects that should be
hidden (or shown).
4.7.5
Project properties
To quickly view details (creator, comments, etc.) about a project, right-click on
the project and select Properties in the contextual menu.
You can also modify the Project Name and Comment.
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5
5.1
Gels
Introduction
This chapter presents operations that specifically relate to gel images. You will
learn how to manipulate images, use different tools to view signal intensities,
and discover ways to visually compare images. The last section explains how to
save, export and print gel images.
5.2
Manipulate images
The main toolbar provides the following tools to deal with images :
5.2.1
Move
Select this tool, click in the image and hold down the left mouse button while
moving the cursor. The image changes position. Release the button at the
position you want.
Alternatives
There are other ways to change positions in gels:
•
Use the View : Gels : Navigation : Move menu.
•
Use the shortcut keys for the above-mentioned menu commands.
•
Use the scrollbars.
Move all gels
To move all gels in the current sheet in the same way, hold down the Shift key
while changing the position in one of the gels.
5.2.2
Zoom
Select this tool, click repeatedly in the area of the gel where you want to zoom
in. Right-click repeatedly on the gel to zoom out.
You can also define a zoom area: place the cursor at the top left corner of the
area, hold down the left mouse button, and move to the bottom right position (a
red box is displayed). Release the mouse button at the end point.
Apply to all gels
To move all gels in the current sheet to the same position with the same zoom
factor, hold down the Shift key while zooming in or out on one of the gels.
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5.2 Manipulate images
Alternatives
There are other ways to zoom gels:
•
Use the mouse scroll wheel to zoom in or out.
•
Use the View : Gels : Navigation : Zoom menu.
•
Use the shortcut keys for the above-mentioned menu commands.
•
Use the scrollbars.
•
To temporarily enlarge an area in a gel image, hold down the Ctrl key while
the Zoom tool is activated. The area under the cursor is enlarged as if you
were looking through a magnifying glass.
Overview option
When you zoom in on a gel, it can be helpful to have an overview of where the
region is localized on the full image (Figure 5-1). This overview enables you to
easily locate and move to any region you want on your gel.
Choose View : Global : Show Overview to show or hide the overview of each
image in its lower right corner. The green rectangle in the overview corresponds
to the current view of the gel. You can drag the green rectangle to another
position to display a new region.
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Figure 5-1. Overview option activated. In the lower right corner of all images in the Display
Zone is a small overview of the entire gel with a green rectangle corresponding to the
visible gel area.
5.2.3
Same position and zoom factor
To move all gels in the current sheet to the same position with the same zoom
factor, double-click on one of the gels. The corresponding position in the
different gels is estimated by interpolating between the surrounding matches,
or if no matches exist, between the two nearest landmarks. Finally, when no
landmarks exist, the gels are aligned at the same location using the X and Y
coordinates.
Note: For more information about landmarks, see 7.3.
5.2.4
Region
A region is a rectangular area in an image that is of interest for displaying a 3D
View, for previewing spot detection parameters or adjust contrast settings, for
cropping, etc.
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5.2 Manipulate images
Select this tool, place the cursor at the top left corner of the area you want to
define, hold down the left mouse button, and move to the bottom right position
(a dashed box is displayed). Release the mouse button at the end point (Figure
5-2).
Figure 5-2. Region tool. The left gel shows a region after selection, the middle gel during
selection, and the right gel shows when the region has been reduced to its minimum size.
Edit region
You can move a region by clicking inside the box and dragging it. You can also
change the size of the box by dragging a corner or edge. To remove a region,
double-click on the gel. If the box is reduced to its minimum size, then its
appearance changes to a blue circle.
Apply to all gels
To define the same region on all gels in the current sheet, hold down the Shift
key while drawing the box on one of the gels or while clicking in an existing
region.
5.2.5
Measure
Select this tool to measure pixel, pI, MW, or real world (centimeter or inch)
distances between two pixels in an image. Click on a pixel (for example, center
of first spot), hold down the left mouse button and move to the next pixel (for
example, center of second spot). The horizontal and vertical distances between
the start and end points are displayed (Figure 5-3).
The units of the displayed coordinates can be changed by choosing Tools :
Options and looking under the Display tab.
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Figure 5-3. Measured distance between pixels on an image. In the example, the horizontal
and vertical distances between the starting (left) and end (right) points are 2.51 cm and
1.11 cm, respectively.
5.2.6
Bookmarks
You can bookmark a view of your images in a sheet. This saves the currently
visible areas. Bookmarks allow you to return to the same area on your gels at a
later time or to show a similar area on the same images opened in a different
sheet.
Create
Choose View : Sheet : Bookmarks : Create and enter a name.
Load
To go back to previously visible areas, choose View : Sheet : Bookmarks : Load
and select the name of the corresponding bookmark. Select the name of
another sheet in the list to reproduce the visible areas of that sheet in the
current sheet.
Delete
To remove a bookmark from the list, choose View : Sheet : Bookmarks : Delete.
5.3
View signal intensity
The digitized image is composed of individual pixels, each of which is
characterized by its X and Y coordinates, and its signal intensity (raw pixel value).
This section describes different approaches to explore the signal intensity and
adapt the way it is visualized.
5.3.1
Adjust contrast
Sometimes the gray levels displayed by default are so low that small spots are
hardly visible. To emphasize these very faint spots, you can adjust the contrast
of the image and/or display images using pseudo colors.
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5.3 View signal intensity
Choose View : Gels : Adjust Contrast and select a region of interest on one or
more images using the Region tool. These regions will allow you to preview the
contrast and pseudo color settings before applying them to the selected
images. The size and position of the preview regions can be adjusted at any
time. After having adjusted the settings as described below, click Apply. The new
contrast settings are saved with the image file.
Note: Any Adjust Contrast changes only influence how the image is displayed
on your screen and do not affect the underlying data, spot detection, and
quantitation.
Gray level mapping
Some scanners are able to scan 2-DE images with 100000 gray levels or more.
Because common computer screens are only able to display 256 gray levels,
mapping must be undertaken between the image gray levels and the 256
screen gray levels. By default, the software uses a linear mapping function
where the lightest point in the image is mapped to 0 (white) and the darkest
point is mapped to 255 (black).
This is illustrated in Figure 5-4. The histogram displays the frequency with which
each gray level (from 3524 to 28530) occurs in the first image of the sheet. The
low gray levels (on the left of the histogram) corresponding to the background
occur very often, whereas the high gray levels (on the right of the histogram)
corresponding to the darkest spots are much less frequent. The red line
indicates that the minimum and maximum gray levels (3524 and 28530,
respectively) are remapped by the default linear mapping. The vertical axis for
the red remapping graph corresponds to the 256 screen gray levels.
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Figure 5-4. Adjust contrast function.
Change the default mapping
The software offers two complementary ways to change the default mapping
function in order to improve the visual display of the gels (Figure 5-5):
•
You can define the minimum and maximum gray levels (that is, look at only
the light or the dark regions in the images). Do this by decreasing the size
of the gray level range that is to be remapped linearly to the screen gray
levels. Move the left or right borders of the slider that is found below the
histogram function. Once the size (interval) of the slider is decreased, you
can also move the interval to the left or right. Alternatively, you can type
valid numbers in the boxes at the lower left and right corners of the
histogram.
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5.3 View signal intensity
Figure 5-5. Adjustments to the gray level mapping for the selected image are immediately
reflected in the preview region. In this example, the maximum gray level was set to 13300
by moving the right side of the slider to the left. All pixels with a gray level higher than
13300 appear as black. By setting the Bending slider to -3, the image becomes darker.
•
You can additionally use a non-linear mapping function. The Bending
parameter, that is, slider to the left of the histogram, expands or
compresses the contrast at the dark or light ends of the range. When the
bending parameter is positive, the image is lighter. When the bending
parameter is negative, the image is darker.
Contrast
Select the image for which you want to see the current minimum and maximum
gray level settings. When you make changes to these settings, the list will
display Modified to reflect this fact.
Unit
Two different units can be used for displaying the gray level minimum and
maximum:
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•
Intensity uses the raw pixel values as displayed in all the reports. When a
calibration is done, these correspond to the calibrated pixel values.
•
%: Chooses the scale as a percentage of the total gray level range in the
histogram.
Region
You can display the histogram for the gray levels in the:
•
Whole Image. This is the default option.
•
Selected Region. The software only considers the gray levels that are
present in the selected region. This option is practical in combination with
the % unit and a relatively small region. In this case, you enter an adaptive
mode that allows you to adjust to the local gray levels. The effect is a
regional increase in contrast that is useful for viewing very faint spots.
Colors
The software offers various color palettes to visualize the intensities in your
image. The Gray+Saturation palette is helpful when you want to visualize
saturated spots or the background. It corresponds to the default Gray option,
except for the maximum gray value (saturation) that is displayed in red, and the
minimum value (background) that appears in blue. To decrease the stringency
on what is considered saturation or background, you can modify the minimum
and maximum gray levels.
Invert
You can inverse the gray levels by checking the Invert box.
5.3.2
3D view
Another way to examine the intensity variations in gel images is by looking at
the three-dimensional view of gel regions (Figure 5-6). In this type of view, the X
and Y axes represent the pI and MW values, whereas the pixel intensity is plotted
along the third dimension (Z axis). The resulting image shows a peak for each
protein spot, with a peak height that is proportional to the spot intensity. 3D
views can be rotated in any direction to look at the interesting spots from all
sides, thus facilitating spot editing or matching decisions.
The 3D View window can be displayed by choosing Reports : 3D View. If spots
are selected (see 6.4.1), the area around these spots is visualized for all images
in the sheet (Figure 5-7). If only regions were defined on images in a sheet, these
regions are shown in the 3D View (Figure 5-6). If both spots are selected and
regions are defined, the area around the spots is shown by default but you can
choose to display the regions.
The layout of the images in the 3D View reflects as much as possible the layout
of the images in the sheet. Bold black lines separate images that are in different
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5.3 View signal intensity
panes. The pixel gray levels are overlaid on 3D views; the top of the peaks are
therefore always darker. You can restrict the number of images displayed in the
3D View by setting their Visibility in the Display Options (see below).
By default, 3D views can be rotated by dragging the mouse while holding down
the left button. If your mouse has a scroll wheel, it can be used to zoom in or out
on the view. You can change to one of the other image manipulation tools
avaible in the Tools icon drop-down list (see below for more details).
Note: All views in the 3D View window are manipulated simultaneously.
Right-clicking is reserved for the contextual menu. It allows you to select the
spot where your cursor is positioned and to quickly access one of the image
manipulation tools described below.
Figure 5-6. 3D views of the regions defined in A_T1_Gel1 and A_T2_Gel2.
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Figure 5-7. 3D views for the selected spots.
The following tools are available in the 3D View toolbar:
Tools
To manipulate 3D views with your left mouse button, you can switch to one of
the following options:
•
Rotation: Rotate the images using your mouse.
•
Zoom/Contrast: Zoom in or out by dragging the mouse horizontally.
Adjust the contrast, or height of the spots, by dragging the mouse
vertically on the view.
•
Translation: Choose one of the suboptions (Z, Y, X, XY) to move the
views along the desired axis.
•
Auto Rotate: Let the images rotate automatically around the Z axis so that
you can view the spots from all sides.
•
Show Default View: Return to the original view.
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5.3 View signal intensity
•
Stack
Stack: Display the images one on top of the other, instead of using the
default tiled view.
•
Next/Previous: Move to the next or previous image in the stack.
•
Animate: Switch automatically between the different images in the stack.
This option is useful for visualizing the expression variations in a set of
images.
•
Transparent Mode: Display one of the images (Reference) with a
transparent surface. This option is useful for visualizing expression
variations in a single, static view. To move one view with respect to the
other, choose Translation in the Tools icon. Hold down the Ctrl key (gray)
or the Shift key (green for the Reference) while dragging the view.
•
Set Reference: Select the image to be used as the Reference for the
Transparent Mode.
•
Transparency Settings: Choose the color for the display of the Reference,
and increase or decrease the transparency.
•
Display options
Visibility: Select the images to be displayed in the 3D View window.
•
Spot Shape: Set the way the spots are displayed in the 3D View (Crossed,
Outlined, Filled, None).
•
Display Options: Display the X (pink), Y (purple) and Z (blue) axes, the
coordinates of the point on which these axes are centered, and change
the way the surfaces of the 3D Views are visualized (Grid, Wireframe,
Smooth).
•
Color Palette: Combine the 3D View with one of the color palettes
available.
5.3.3
Profile
It is sometimes difficult to judge if spots should be split, are saturated or not, or
have other problems such as so-called donut structures (low intensities in the
center compared to the borders). It is important to identify such problems as
they will lead to incorrect spot quantitation.
The Profile function, activated or deactivated by choosing View : Global : Show
Profile can help in such cases. Red curves represent the intensity variations of
the gel in the vertical and horizontal directions at the position of the mouse
cursor (Figure 5-8). The Profile can clarify the intensity changes in a gel and
assist in making editing decisions, without the need to open an additional
window, as is the case with the 3D View tool.
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Figure 5-8. Profile feature. Red curves represent the intensity variations of the gel in the
vertical (right) and horizontal (bottom) directions at the position of the mouse cursor. Green
lines indicate the exact position of the cursor, whereas the numbers indicate the minimum
and maximum gray levels in a specific profile view.
5.3.4
Cursor information
At any given time, the Cursor Information window can be used to display
information on pixels such as the pixel intensity and the X and Y coordinates
expressed in pixels, in pI and MW units (if available), and in cm or inches. Note
that if spots are detected, the Cursor Information window also displays spot
information.
The Cursor Information window is availabe by choosing View : Global : Cursor
Information, or by clicking the corresponding icon in the Display toolbar. Place
the cursor over the pixel for which you want to display information.
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5.4 Visually compare images
5.4
Visually compare images
When working in MatchSet or Classes sheets, the software provides different
tools to visually compare images to the sheet reference.
5.4.1
Sheet reference
You can set the sheet reference by choosing View : Sheet : Set Reference. All
other images in the sheet are compared to this reference image for the options
in the View : Sheet menu (Align Images, Show Dual Color,Spot Overlap) .
5.4.2
Purpose of aligning images
The Align Images feature facilitates the visual comparison of images that
demonstrate large variations in protein migration. It is especially helpful when
the images are in Stacked mode. It modifies (Figure 5-9) the images in the sheet
so that they are better superimposed with the sheet reference and therefore
with each other. This allows easy identification of corresponding spots.
The Align Images feature is a purely visual tool. It is not used in, and will not
improve, the matching process. The only reason to align your images is to ease
their visual comparison (possibly in combination with the Dual Color mode).
5.4.3
Align images
To align images, the software needs to know which positions in the different
images correspond to each other, that is, represent the same protein form. This
is done by defining landmarks as described in 7.3. Landmarks should be added
gradually so that you can monitor their individual influence on the alignment
(automatically updated after each landmark addition). Immediately remove
landmarks that decrease the alignment quality. The alignment algorithm then
deforms the images to superimpose the landmarks.
Note: As opposed to matching, landmarks do not need to be linked with spots
to carry out image alignment. Therefore, no spots need to be detected.
To align images in a sheet:
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1
Set the desired image as the sheet reference, with View : Sheet : Set
Reference.
2
Select the Landmark tool and define a few landmarks, bearing in mind the
rules listed in 7.3.
3
Choose View : Sheet : Align Images.
4
If parts of your images are still not sufficiently aligned, you can add extra
landmarks. The alignment is automatically updated.
5
To view the original images, choose View : Sheet : Align Images. The
original images replace the aligned ones.
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(a)
(b)
Figure 5-9. Image (a) before and (b) after alignment. The match vectors and landmarks
are displayed.
Alternatively, double-click in an image (with the Move tool selected) to locally
superimpose the different images:
•
If the images are matched, the image positions are synchronized based on
the surrounding matches.
•
If the images are not matched but have landmarks, the image positions
are synchronized based on a simple interpolation between the two nearest
landmarks.
•
If no matches or landmarks are present, the image positions are
synchronized based on the same image location (X,Y coordinates).
5.4.4
Show dual color
Choose View : Sheet : Show Dual Color to display each of the images (the sheet
reference and the current image) in one of two colors: red and cyan (Figure
5-10). When the pixel colors of the two superimposed gels are added:
•
Overlapping spots appear as shades of gray.
•
Cyan spots are present only in the current gel.
•
Red spots are present only in the sheet reference.
•
Halos of cyan or red around dark spots indicate that the protein is over or
under expressed, respectively, compared to the sheet reference.
The less color you see in the dual color mode, the more similar the gels. Of
course, this is only true if the gels are correctly aligned (see above) and
superimposed.
Note: It is recommended to perform any operations related to gel alignment
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5.4 Visually compare images
(especially the definition of landmarks), before entering the Dual Color
mode to avoid a slow down due to the recalculation of the overlaid
images.
Figure 5-10. Dual color view.
5.4.5
Spots overlapped
Chooose View : Sheet : Spot Overlap once spots have been detected and the
visible spots on the sheet reference will be shown in blue on the image (Figure
5-11). Thus, you can easily compare the position and size of the red spots in the
current gels with the blue spots on the sheet reference (shown in Crossed or
Outlined mode).
You can change the default color to be used for overlapped spots by selecting
the Display tab in Tools : Options, and clicking the Overlapped box.
(a)
(b)
Figure 5-11. Spots overlapped with (a) outlined and (b) crossed spot shapes.
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5.5
Grid lines
5.5.1
Display grid lines
Choose View : Global : Grid Lines : Show to display grid lines over your images
(Figure 5-12). Grid lines can be used to evaluate distances between spots, in
terms of pixel coordinates, pI/MW units, or length units (cm or inch). Grid lines
are also a helpful way to visualize deformations in aligned gels because the grid
lines are warped in the same way as the image.
5.5.2
Edit grid lines
Choose View : Global : Grid Lines : Edit to change the grid properties. The
software partitions the visible area into the Number of subdivisions entered by
the user (Figure 5-12). The graduations can be attached to the Gel or the Screen.
This means that the software divides the gel width/height or the visible screen
area by the number of subdivisions. The Coordinate Units can be Centimeters,
Inches, Pixels, or pI/MW units, provided data is available in the annotation
category pI_MW. The pI/MW grid can also be displayed over gels that do not
contain this information, but that were matched to a gel having pI and MW
values.
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5.6 Gel reports
.
Figure 5-12. Grid lines in cm attached to the gel image.
5.6
Gel reports
5.6.1
Gel table
Choose Reports : Gel Table to display a table (Figure 5-13) with summarized
information about the images in the current sheet. The available columns are:
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•
File name, ID, path, size, modification and creation dates.
•
Image height and width expressed in pixels (Rows and Columns), pixel size
(PixSize).
•
Minimum and maximum gray levels before (MinGray and MaxGray) and
after calibration (MinValue and MaxValue).
•
Calibration information (Calibration Formula, Unit, Name, Creator and
Date).
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•
Number of spots and annotations.
•
Staining method and user-defined gel descriptions.
•
MatchSet to which the image belongs and the Class to which it was
assigned.
Figure 5-13. Gel Table.
You can customize the Gel Table to display only the columns of interest. Four
predefined report templates are already available from the Load icon in the
Settings of the Gel Table toolbar:
5.7
•
Properties: Shows various properties of the gel images: File Name, image
height and width in pixels, maximum gray levels before and after
calibration, Modification Date, as well as the MatchSet and Class to which
it belongs.
•
Files: Shows information on the image files: File Name, ID, Path, Size,
Creation and Modification Dates, as well as the MatchSet and Class to
which they belong.
•
Descriptions: Shows the user-defined gel descriptions.
•
Calibration: Provides calibration-related information: File Name, ID and
Path, as well as Calibration Formula, Name, Creator and Date.
Save, export and print images
5.7.1
Save
The software automatically saves your images and all associated data as part
of the project when you close a sheet or exit the software. You can also save
your work by choosing File : Save.
5.7.2
Export
You can either export images to a file or to the clipboard for direct pasting into
another software. The procedure is very similar to exporting images to files.
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5.7 Save, export and print images
However, you can only export one image at a time to the clipboard. Finally, you
can also export a view of the current sheet.
The export options are available in the File : Export menu:
•
Image to Clipboard
•
Image to File
•
Sheet to Clipboard
•
Sheet to File
Rather than saving your gel images in the ImageMaster file format, you may
want to export them to a different file format (TIFF, BMP or PNG). The gel images
are exported as 8-bit, flat, rasterized images without any structure. This means
that gel components such as spots and annotations are saved exactly as they
appear on the screen, but are no longer recognizable as ImageMaster objects
and therefore become part of the image. Consequently, exported gel images
should only be used for presentation purposes and not for further analysis with
any software package.
Adapt the size of the exported image by zooming in or out. If a region is selected
on the image, you are given the choice to export the entire image or the selected
region only.
5.7.3
Print images and sheets
Print options
The software provides various printing options in the File : Print menu:
•
•
Images: Print selected images or image areas (if defined with the
Region tool). This option prints one image per page.
Sheet: Print the current sheet.
Whatever your choice, the image is printed as it is displayed on the screen
retaining objects and properties such as spots, annotations, contrast mapping
and pseudo colors, alignment, zoom, grid, etc.
Note: With a zoom factor of 1, the printed gel image takes the full paper width.
You can adapt the zoom factor to decrease the size of the printed image.
Page Setup
You can change print parameters such as printer name, paper size, paper
orientation, etc. Choose File : Page Setup. This command opens the standard
print window where printer-related settings can be modified.
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6
6.1
Spots
Introduction
Once gels have been added to a project and you have taken a good look at
them, you are ready to detect spots. A spot delineates a small region in the gel
where protein is present. This shape is automatically differentiated by a spot
detection algorithm and quantified; its intensity, area and volume are
computed.
There are two different spot detection algorithms implemented:
6.2
•
Non-DIGE images are detected with the ImageMaster algorithm.
•
DIGE images are co-detected with GE Healthcare’s DeCyder 2D algorithm.
Detect spots in non-DIGE gels
6.2.1
Procedure
The ImageMaster spot detection algorithm is optimized to give relevant
biological results with minimum user interaction. You can preview spot
detection and fine-tune a few parameters before automatically locating all the
spots in your image.
To detect spots automatically:
1
Display the gels to be detected by selecting a match set in the Workspace.
Right-click and select Display in the contextual menu.
2
Choose View : Spots : Outlined to visualize the spot borders.
3
Click the Region tool. Draw preview regions on one or more gels. Note that
you can still draw, resize or move regions while setting the detection
parameters.
4
Select the gel images for spot detection.
5
Choose Edit : Spots : Detect.
6
The Detect Spots window appears on-screen and the spots in the preview
regions of the selected images are detected with the default parameters. If
you do not want to recalculate the spots in the preview regions for each
parameter change, turn the Auto Preview option off. To manually refresh
the preview regions, simply click the Preview button.
7
Adjust the detection parameters (Figure 6-1). In particular optimize Smooth
to detect all real spots and split the overlapping ones. Subsequently, filter
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6.2 Detect spots in non-DIGE gels
out the noise by changing the Saliency and Min Area values. See below for
more details on spot detection parameters.
8
When you are satisfied with the preview, click OK to detect all spots in the
selected gels using the specified parameter values. Note that you can still
change your gel selection at this point.
9
The spot shapes are displayed on the images.
Figure 6-1. Adjusting spot detection parameters in real time.
6.2.2
Spot detection parameters
Spot detection parameters are best adjusted in the following order:
88
•
Smooth: Set this parameter first. It fixes the number of times ImageMaster
smooths the image before detecting spots, using a smooth-by-diffusion
algorithm. The Smooth parameter should be optimized to detect all real
spots and split as many overlapping spots as possible without being
concerned about noise spots (these can be filtered out with the Saliency
and Min Area parameters).
•
Saliency: This parameter is a measure based on the spot curvature. It
indicates how far a spot stands out with respect to its environment. Real
spots generally have high saliency values whereas artifacts and
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background noise have small saliencies. Although the Saliency is an
efficient quantity for filtering spots, it is also highly dependent on the
images (for example, image resolution and depth). Some gels need a
saliency value of 10 for correct filtering. Others may necessitate a value of
5000. To estimate the saliency range to use with your images, you can
display the Cursor Information window or Spot Table and look at the
saliency value given for a spot that you would like to suppress. Enter this
value in the Saliency field in the Detect Spots window. The spot detection
algorithm then discards all spots with saliencies smaller than the specified
threshold.
•
Min Area: After setting an appropriate Saliency to filter out all noise spots,
there may still be noise in your gel that cannot be eliminated without
suppressing real spots. This often happens with dust particles that consist
of a few very dark pixels. Get rid of these artifacts by using the Min Area
parameter. It eliminates spots that have an area smaller than the specified
threshold (expressed in number of pixels).
6.2.3
Spot quantification
The software automatically computes the amount of protein present in each
spot. Figure 6-2 illustrates the principles of spot quantification in the
ImageMaster algorithm. Measuring the protein quantification values in this way
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6.2 Detect spots in non-DIGE gels
has the advantage of being more robust and reproducible when calculating
protein expression variations (relative quantification).
0.75 * Intensity
Intensity
Intensity
Figure 6-2. Spot quantification. The 3D View in ImageMaster reflects the spot shape and
volume of what will effectively be quantified. The spot outline corresponds to the area at
75% of the spot height when measured from the peak.
90
•
Intensity: The software first calculates the intensity of a spot. The intensity
is based on the highest calibrated pixel intensities in the spot from which
the background has been withdrawn. The background is defined as the
minimum pixel value in the spot neighborhood.
•
Area: The area of a spot is not determined at the spot base because the
base is often arbitrary and difficult to determine. ImageMaster computes
the area at 75% of the spot intensity, as measured from the peak of the
spot. The spot outlines displayed in ImageMaster exactly encircle this
2
computed spot area (expressed in mm ).
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•
Vol: The volume of a spot is calculated as the volume above the spot
outline, which is situated at 75% of the spot height (as measured from the
peak of the spot). In Figure 6-2, the measured volume of the spot is striped.
Please note that the volume values, like the intensities, depend on pixel
intensity calibration.
•
%Vol: The relative volume of a spot is calculated as indicated below. It is a
normalized value that remains relatively independent of variations due to
protein loading and staining by considering the total volume over all the
spots in the image. This means that in an image where, globally, the spots
are darker than in another image, the majority of spot volumes is higher.
However, the bulk of %Vol should be similar to those in the compared
image, at least for gels with similar spot patterns.
Vol
%Vol = ----------------------- × 100
n
∑ VolS
S=1
where VolS is the volume of spot S in a gel containing n spots.
Note: When you look at a detected spot in the 3D View, you will notice that the
borders are not localized at the base of the spot, especially for intense
spots. This is normal because the spot contours displayed effectively
reflect the quantified parts of the spot, and they do not correspond to the
'whole' spot, which is difficult to define.
6.3
Co-detect spots in DIGE gels
The co-detection algorithm is designed to simultaneously process one, two or
three images derived from a single gel.
•
Single detection: one image.
•
Double detection: two images.
•
Triple detection: three images.
Single detection is performed on images of fluorescently post-stained gels used
for picking, a case where there is a single image associated with the gel.
Double and triple detection takes advantage of the inherent co-migration
benefits of the CyDye™ DIGE Fluor dyes. A set of co-run images are merged
together to include all spot features in a single image. Spot detection and spot
boundary definition is then performed using pixel data from all the individual
raw images and the merged image. The resultant spot map is overlaid back
onto the original image files. Since the spot boundaries and the detection areas
are identical for all images, the spots are effectively already matched. This
process results in highly accurate volume ratio calculations.
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6.3 Co-detect spots in DIGE gels
6.3.1
Procedure
To perform spot detection on DIGE images:
1
Display the gels to be detected by selecting a match set in the Workspace.
Right-click and select Display in the contextual menu.
2
If desired, select a subset of gels to be detected.
3
Choose Edit : Spots : Detect.
4
In the DIGE Spot Detection window, enter an estimation (see below) of the
Number of Spots present in the images. Click OK.
6.3.2
Spot detection parameter
When detecting DIGE images, you must enter an estimation of the Number of
Spots present in the images. It is recommended that this value be overestimated
to compensate for the detection of non-protein spots on the image, for example,
dust particles which can subsequently be excluded from the analysis using spot
filtering.
If all the spots are not identified, the spot detection process can be repeated
with a higher number of estimated spots.
For example, for a mammalian lysate run on an 24 cm pH 4-7 Immobiline™
DryStrip and a large format gel, such as the Ettan™ DALT Gel (20 cm x 26 cm), a
value of 2500 for the estimated Number of Spots should be satisfactory.
6.3.3
Spot quantification
Volume, area, intensity, slope, and volume ratio for individual spots are
automatically calculated and included in the Spot Table and Cursor Information
window.
•
Vol: Spot volumes (sum of pixel intensities within the spot boundary) are
always expressed with background subtracted. Background is subtracted
on a spot specific basis by excluding the lowest 10th percentile pixel value
on the spot boundary from all other pixel values within the spot boundary.
The spot volume is the sum of these corrected values.
•
Vol ratio: Volume ratios (volume of current image spot/volume of DIGE
reference image spot) indicate the change in spot volume between two
images.
Note: When using single detection the volume ratio value is 1.0 for all spots
since there is no second image.
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6.3.4
Exclude spots
To exclude small, non-protein spots on DIGE images, it is recommended to filter
the spots based on the Volume. More precisely, spots that have a maximum
volume in the two or three images that is lower than a given threshold can be
deleted.
To exclude DIGE spots based on maximum volume:
1
Choose Reports : Spot Table.
2
Click the Select by Value icon in the toolbar of the table.
3
Select Vol in the displayed list. Make sure the >= box is checked and enter
a value to be used as the cutoff for the volume. Deselect the <= box and
click OK (Figure 6-3).
4
All spots with a volume higher than the given threshold are selected,
together with their matched spots (which may have volumes that are
smaller than the threshold). These are the spots that are kept.
5
Choose Select : Spots : Inverse Selection.
6
The current selection includes all spots for which the maximum volume in
any of the two or three DIGE images is lower than the given threshold.
7
Choose Edit : Spots : Delete to permanently delete the selected spots from
your DIGE gels.
Figure 6-3. Select by Value can be used to filter spots based on their volume.
Note: Instead of deleting spots, you can exclude them from the analysis by
disabling them, as described in 6.4.3.
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6.4 Select spots
6.4
Select spots
6.4.1
Select
Spots can be selected with the Select tool. Once selected, they are highlighted
in green, unless the default spot colors were modified. All matched spots are
selected as well.
If an annotation is attached to a spot, the annotation is also selected. Similarly,
if you select an annotation or label with the Select tool, the linked spot is also
selected.
To select more than one spot, select the first one and then hold down the Shift
key while clicking on additional spots.
To select all spots in a region, place the cursor at the top left position of the
desired region, hold down the left mouse button, and then drag the cursor to the
bottom right position. All spots in the designated region are selected.
To deselect all spots, click in the gel (not on a spot).
Surrounding box
By default, spot selections are surrounded by green boxes. This makes it is
easier to localize selected spots, especially when working at low zoom factors.
To deactivate this option, choose Tools : Options, and uncheck the
corresponding box in the Display tab.
Alternatives
There are other ways to select spots:
•
Choose a command in the Select menu.
•
Select spots in reports.
6.4.2
Spot sets
It is possible to focus your analysis on particular spots by creating and saving
spot sets for later selection or combination.
Create
Select spots you want to include in a spot set, either manually or by selection in
a report. Then choose Edit : Spot Sets : Create, enter a name for the new spot
set and click OK.
Spot sets can be visualized as columns in various reports (Figure 6-4). If they are
not displayed by default, you can add them by checking the corresponding box
in the Settings of the report window (only for tabular reports). Once the column
is displayed, you will see a checked box for spots that belong to the set, or an
empty box for spots that do not belong to the set. Click in a box to change its
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state. If several spots are selected, you can change the state by clicking in one
box while holding down the Shift key.
Figure 6-4. Spot sets Anova p<0.001 and Ratio : 2 in a Class Analysis Table. New spot sets
can be created by clicking on the Create Spot Set icon.
Spot sets can also be created by clicking the Create Spot Set icon in a report
toolbar. All currently selected spots are automatically included in the newly
created spot set.
Delete
Delete a spot set by choosing Edit : Spot Sets : Delete and selecting the spot
set(s) to be deleted.
Combine
You can combine two spot sets using logical operators, in order to create a new
spot set or change the current selection (Figure 6-5). Four operators are
available:
•
And: Keeps spots that belong to both spot sets.
•
Or: Keeps spots that belong to either one or both spot sets.
•
Not equal: Keeps spots that belong to only one of the two spot sets.
•
Exclude: Keeps spots that belong to the first spot set and do not belong to
the second spot set.
Figure 6-5. Combine Spot Sets window.
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6.4 Select spots
To combine two spot sets:
1
Choose Edit : Spot Sets : Combine.
2
From the Inputs drop-down lists, select the spot sets to be combined. The
current selection can also be used as input.
3
Specify the operator (And, Or, Not Equal or Exclude).
4
Enter a new name for the resulting spot set. Alternatively, the output can be
immediately reflected in the current selection. Click OK.
Select
To select spot sets, choose Select : Spot Sets.
6.4.3
Enabled spots
By default, all spots are enabled and therefore represented in the reports. To
exclude a specific subset of spots, you can disable protein spots that are not of
interest, or specifically define a set of spots to be enabled. Only enabled spots
appear in reports.
The options related to the creation, saving or loading of sets of enabled spots
can be chosen from the Edit : Enabled Spots menu:
Set
Select the spots you want to focus your analysis on and choose this option to
enable the selected spots. After deselection, excluded spots are disabled and
appear in yellow.
Add
Use this option to add selected spots to the current set of enabled spots.
Remove
Use this option to remove selected spots from the current set of enabled spots.
Save
Use this option to save the currently enabled spots as a new spot set. Enter a
name and click OK.
Load
Use this option to enable spots belonging to an existing spot set.
Select
To select the enabled spots, choose Select : All Enabled Spots.
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6.5
Display spots
6.5.1
Spot shape
Once spots are detected, you can choose how to display their shapes (outlined,
crossed, filled, outline/filled) on the gels from the View : Spots menu.
6.5.2
Spot color
By default, enabled spots are displayed in red, disabled spots in yellow, selected
spots in green and overlapped spots in blue. You can change these default
colors in the Display tab in the Options window (accessible by choosing Tools :
Options). Click the colored box you want to change and the Color window opens.
Choose the preferred color from the spectrum and click OK.
6.6
Edit spots
Note: Except for deleting spots, spot editing is not allowed on DIGE gels.
Quantitative protein data, and in particular the spot volume, are highly
dependent on an optimal and reproducible definition of the spot borders and a
correct splitting of partially overlapped spots. To guarantee reproducibility of
quantitative work it is therefore recommended to create spots by using the
automatic spot detection algorithm in the software and to avoid manual editing
as much as possible.
However, spot detection differences can still occur. In particular, some spots are
differently split in gels to be compared. The software offers the following
solutions to deal with detection variations between gels without calling for spot
editing:
•
Create multiple matches. In practice, this means that you can match
“composite spots” that are treated as unique entities in the quantitation.
•
Propagate all or selected spots from one image to the other images.
Note: Both solutions require prior matching. Therefore, before doing any spot
editing, first match your images and only then consider to use one of the
options described below
6.6.1
Manual editing
You must enter the special spot-editing mode to manually edit spots. Choose
Edit : Spots : Edit Enabled, or click the corresponding icon in the Detect And
Match Spots toolbar. The Edit Spots toolbar displays. Selecting the Edit Enabled
option again disables spot editing.
Note: For edited spots, the Saliency value becomes zero. This can be used to
quickly check which spots have been edited.
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6.6 Edit spots
The following spot editing tools are available:
Create spots
Click this icon and draw the outline of the new spot. Alternatively, double-click
the desired location in your image, and set the disc radius for the circular spot
to be drawn in the Create Spot window.
Delete spots
Click this icon, select the spot to be deleted, and confirm.
Split spots
Click this icon, select a spot to be split, and draw a line through the spot at the
position where the separation should occur. Make sure you start and finish
outside the spot.
Merge spots
Click this icon, select two or more spots to be merged, and draw a trajectory
through the selected spots. Make sure to start and finish in the same spot.
Grow spots
Click this icon, select a spot to be grown, and outline the area you would like to
add. Make sure to start and finish within the selected spot.
Shrink spots
Click this icon, select a spot to be shrunk, and outline the area you would like to
suppress. Make sure to start and finish outside the selected spot.
6.6.2
Composite spots
As shown in Figure 6-6, the software allows you to match several spots in one
gel with multiple spots in other gels. In the figure, the three selected spots in gel
A_T1_Gel1 are matched to the single green spot in A_T2_Gel1 and the two
selected spots in B_T1_Gel1, and so on.
Once the match has been effectively created, the three spots in A_T1_Gel1, for
example, are treated as a single entity in the quantification. The quantification
value for A_T1_Gel1 displayed in the different reports is the one obtained after
combination of the values from the three individual spots.
This is an efficient solution for dealing with spot detection differences without
subjective and time-consuming spot editing. When selecting a spot on a gel, any
matched spots on the other images are automatically selected as well. Select all
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spots to be matched and click the Add Match icon in the Detect And Match
Spots toolbar.
Figure 6-6. Composite spots can be defined through multiple matches
6.6.3
Propagate spots
You can propagate all or selected spots from one image to the other matched
images (Figure 6-7). This allows you to quantify identical areas on all gels.
•
For matched spots: the spot in the destination image is replaced with the
shape of the spot in the source image.
•
For non-matched spots: the spot from the source image is copied to the
equivalent location in the destination image. This position is extrapolated
from the surrounding match vectors.
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6.7 MW and pI calibration
Matches are automatically created between the original and the propagated
spots.
Figure 6-7. All spots from gel A_T1_Gel1 have been propagated to the other images.
To propagate spots from one matched image to another:
6.7
1
Select spots on one image.
2
Choose Edit : Spots : Propagate.
3
Select one or more images you want to copy the spots to.
4
The new spots are added to the selected images.
MW and pI calibration
If you have a gel with pI/MW standards, the software can compute approximate
pI and MW values for all the spots/pixels in this image, as well as any other
images matched to it.
Define pI_MW annotations for a certain number of spots/pixels in the gel. The
calculated pI and MW values for all spots in this gel and any matched gels are
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automatically available in the Spot Report, DIGE Report or Cursor Information
window. In addition, pI and MW grid lines can be displayed over the images.
To define pI_MW annotations on an image:
1
Select the image for which you know the pI and MW values for several
protein spots. Spots may or may not have already been detected in this
image.
2
Click Select in the toolbar.
3
Double-click on a spot (pixel) for which you know the pI and/or MW values.
4
Select the pI_MW category in the Create Annotation by Click window.
5
Enter the known pI and MW values, respectively, separated by a space.
Replacing one of the values with -1 means that no value is set.
6
Do this for a sufficient number of protein spots that are distributed over the
whole image area. Obviously, the more spots and annotations, the better
the approximated pI and MW values will be.
ImageMaster does the following to calculate approximate pI and MW values. In
the case of pI, it looks up the two closest annotations to the left and to the right
of the spot for which the pI will be determined and then interpolates between
these two points. Since ImageMaster does not have any information about the
experimental (possibly non-linear) pI scale, the calculated values are only
approximate. In the case of MW of the spots, the procedure is similar, except that
ImageMaster searches for the closest spots above and below the spot for which
the MW will be determined and it makes a logarithmic interpolation.
Extrapolating pI and MW values is more complicated. For example, if the pI of a
spot on the extreme right side of your gel is to be determined, the software looks
for the two closest spots to the left of the spot in question. If these two spots are
sufficiently distant from each other (in order to decrease the error), the value for
the spot in question can then be extrapolated.
Normally, the pI and MW values in the Spot Report or Cursor Information
window should be the same as in the defined pI_MW annotations. However, this
is only the case if the annotations are attached to actual spots and not to pixel
positions in the image. If an annotation is attached to a pixel, the pI and MW
values for the spot that lies closest to it will be slightly different from that of the
pixel (to which the annotation is attached). You can solve this ambiguity by
linking the annotation to the spot.
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6.8 Spot reports
6.8
Spot reports
6.8.1
Spot Table
The Spot Table (Figure 6-8), obtained by choosing Reports : Spot Table, displays
summarized information about enabled spots:
•
Name of the image on which they were detected.
•
Spot ID (see below), and Match ID if the spot was matched.
•
Coordinates of the spot’s center of gravity (X and Y).
•
Quantification values: Intensity, Area, Vol, and %Vol. Depending on the
spot detection algorithm used, the Saliency, Vol Ratio, and Slope will also
be given.
•
Calculated pI and MW values, if pI_MW annotations were defined on the
image, or a matched image.
•
All linked labels and spot sets.
Figure 6-8. Spot Table.
Spot ID
Each spot in a gel has a unique identifier, called the Spot ID. Spot IDs of deleted
spots are not reused. ImageMaster attributes a new ID to each new spot. When
a spot is split, the child spot for which the coordinates are closest to the parent
spot keeps the existing spot ID, the other child spot gets a new ID. When two
spots are merged, the resulting spot is attributed the ID of the initial spot that
was closest to the new center of gravity.
6.8.2
Cursor information
The Cursor Information window is available from the menu View : Global :
Cursor Information Window or by clicking the corresponding icon in the
Display toolbar. It can be used at any time to display information on pixels and
spots located at the position of your mouse cursor (Figure 6-9).
Information on the pixel under the cursor:
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•
Name of the image.
•
Calibrated pixel intensity.
•
X and Y coordinates, expressed in pixels, in pI and MW units (if available),
and in cm or inches.
Information on the spot under the cursor:
•
Spot ID.
•
Quantification values: Intensity, Area, Vol, and %Vol. Depending on the
spot detection algorithm used, the Saliency, Vol Ratio, and Slope are also
provided.
Figure 6-9. Cursor Information window.
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6.8 Spot reports
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Matches 7
7
7.1
Matches
Introduction
Matching is a key operation in 2-DE image analysis. Basically, the image
matching algorithm compares gel images to find matches between related
spots, that is, spots representing the same protein in the gels. A match is
therefore composed of spot n-tuples (S1, S2, ..., Sn) where S1 is a Spot ID in the
first gel, ..., and Sn a Spot ID in the last gel.
The matching algorithm always starts from the reference image or match set,
and looks for each spot in this reference, if corresponding spots in the other
images are found. If a spot is absent from the reference, it cannot be matched
automatically. However, if you have several match sets in your hierarchy, there
is a good chance that the spot is on the reference of at least one of them. If so,
the spot will turn up in the analysis. Subsequently, additional spots can be
matched to it manually (Figure 7-1).
Matches are propagated at each level of the hierarchy. This means that once all
match sets are effectively matched, spots from one gel can be directly
compared with those in any of the other gels.
Note: A DIGE gel is an inherent match set for which the co-run images are
automatically matched. To subsequently match different DIGE gels,
proceed like any other match sets.
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7.2 Display a match hierarchy
Figure 7-1. Reference and matching. The selected spot is absent from groups AT1 and AT2,
including the sheet reference A_T1_Gel1. Since the spot is present in the references
B_T1_Gel1 and B_T2_Gel1, it is matched to corresponding spots and turns up in the
analysis (Gel and Class Analysis Tables).
7.2
Display a match hierarchy
In the example below, the match hierarchy AB was displayed by right-clicking on
its name in the Workspace and selecting Display. In the resulting sheet, only the
lowest level (AT1, AT2, BT1, BT2) and highest level (AB) items are specifically
visualized, in the panes and sheet, respectively. To select intermediary levels (A,
B), you can use the corresponding icon in the pane tab of the reference (for
example, AT1) for that level (A).
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At the same time, the red markers in these icons indicate what item in a given
match level is used as the reference in the matching.
Figure 7-2. Match hierarchy AB displayed in a sheet. The match level A was selected by
clicking on the corresponding icon in the pane tab of match set AT1.
7.3
Define landmarks
ImageMaster is designed to match gels with minimum user intervention.
Nevertheless, when the gels are very distorted or different, you may need to
help the matching process by specifying a few landmarks. Landmarks are points
that relate corresponding spots in each of the gels to be matched.
In some cases, no landmarks are required. More often, a single landmark is
sufficient for quick and efficient matching. If the matching results are not
satisfactory, you can repeat the automatic matching procedure using
additional landmarks.
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7.3 Define landmarks
7.3.1
Rules for defining landmarks
The addition of suspect or badly positioned landmarks can worsen the
alignment results. The following rules must be considered when defining
landmarks:
•
The number of landmarks should be kept to a minimum. There is no point
in putting a landmark on each spot.
•
Landmarks should be well distributed over the whole images (covering
both the X and Y directions). To correct for local distortions, it is
recommended to define landmarks around the distorted regions rather
than within those regions.
•
Landmarks should only be defined on spots that clearly represent the
same protein form. Protein variants definitely should not be used as
landmarks.
•
Landmarks should be placed on small, sharp spots (of similar area), rather
than on large diffuse ones (which may differ considerably in size) to reduce
the error in the position.
•
When a spot is missing on a gel (sometimes happens to border spots), you
should not put a landmark (that is, validate a landmark) in a hypothetical
spot position. Missing landmarks are not an issue.
7.3.2
Define
Landmark
Landmarks can be defined using the dedicated tool as described below.
To define landmarks:
1
Click the Landmark icon in the toolbar.
2
Place the mouse cursor over a known, well-defined spot in the first
reference gel and click. A validated landmark symbol (bold orange circle)
appears on the spot (Figure 7-3).
3
In the other images, drag the non-validated symbols (green circle with
orange plus sign) onto the corresponding spots. If the symbol is already on
a good spot, double-click to validate it.
4
Repeat steps 2 and 3 to add more landmarks.
In certain gels, the symbols only become visible once the landmark has been
validated in the reference. In the example, the landmark must be validated in
the image B_T2_Gel1 before any symbols appear in the images B_T2_Gel2 and
B_T2_Gel3.
Sometimes, you may want to move or zoom your images during the
landmarking process. When you click the Move or Zoom tools, the orange
landmark symbols disappear, and only labels with the landmark numbers are
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left. When you click the Landmark tool again, the symbols are reactivated and
you can continue defining landmarks.
To delete landmarks, you must delete the corresponding annotations. Choose
Select : Annotations : By Category and select Landmarks in the list. Then
choose Edit : Annotations : Labels : Delete.
Figure 7-3. Placing of landmarks. Note the difference between the validated and nonvalidated landmark symbols.
In general, it is good to validate one or two landmarks on all the images in the
hierarchy, so that they are used for matching at all levels. If it turns out after the
matching that the gels within the lowest hierarchies (for example, AT1, AT2, BT1
and BT2) have been properly matched, but that the higher level matches (for
example, A and B) are not satisfactory, you can add additional landmarks to the
higher levels only. Do this by validating the landmark only on the reference
images.
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7.4 Automatic matching
7.4
Automatic matching
An entire hierarchy can be matched automatically.
To match the gels in a match hierarchy:
1
Select the gels to be matched in the sheet. All the gels, even in a multilevel
hierarchy, can be selected.
2
Choose Edit : Matches : Match Gels. Or click
3
If several match sets are selected, you are asked to choose the ones to be
matched in the Match Gels window (Figure 7-4). Items preceded by a ~ sign
still require matching. Use the Ctrl or Shift keys to make multiple selections.
All match sets can be selected at the same time. Click OK.
Match Gels in the toolbar.
Figure 7-4. Match Gels window. In this example, all seven match sets were selected for
matching. The items preceded by a ~ sign are not yet matched.
Match vectors are displayed in blue. The vector pattern is proof of consistency.
If there is a mismatch, the vector has a different length and/or orientation.
If the matching did not work properly, you can rematch a particular match level
after having added additional landmarks to the appropriate images.
7.5
Select matches
7.5.1
Select
When you select a spot with the Select tool, the matched spots are
automatically selected.
Alternatives
There are other ways to select matches:
•
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Choose an option in the Select : Matches menu (All, Inverse Selection,
Multiple Matches).
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•
Select matches in reports.
7.5.2
Match count
The software allows you to select spots present:
•
In a certain number of gels: the Match Count in a Gel Analysis Table gives
the number of gels in which the spot is present (detected and matched).
•
In a certain number of classes: The Match Count in a Class Analysis Table
gives the number of classes in which the spot is present (detected and
matched).
Click the Select By Value icon in these tables to refine your selection. For
example, select only spots that are present in at least X out of Y gels, or X out of
Y classes.
7.6
Display matches
You can visualize matches by selecting them. The software also provides the
following tools to display matches.
7.6.1
Show vectors
Right after matching, the software automatically displays the match vectors in
blue. Vectors link the spots in a gel with the corresponding spots in the sheet
reference. This sheet reference has a darker green gel name and should not be
mistaken for the match reference, which has a red component. The sheet
reference can be changed by choosing View : Sheet : Set Reference.
A blue upside down triangle on a spot indicates that the spot was matched to
one or more spots in other gels, but not to a spot in the sheet reference (Figure
7-5).
A spot with a triangle means that the corresponding position in the sheet
reference lies outside the visible area (Figure 7-5).
To hide the match vectors, or on the contrary, to display them when they are not
visible, choose View : Matches : Show Vectors.
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7.6 Display matches
Figure 7-5. Different representations of matches. The selected spots in match set AT2 with
a blue upside down triangle are matched, but not with a spot in the sheet reference. Spots
with blue triangles, as the one indicated with a green arrow, are matched, but the
corresponding position in the sheet reference lies outside the visible area.
To minimize the match vectors (after having moved or zoomed an image), select
the Move tool and double-click in the image so that it is synchronized with the
other images and the sheet reference.
You can change the default color for the match vectors by choosing Tools :
Options and going to the Display tab.
7.6.2
Show ID
Choose View : Matches : Show ID to display Match IDs for selected spots on
selected gels. Previously, the gels must be matched. To hide the Match IDs again,
possibly in selected gels only, choose View : Matches : Hide All ID.
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7.7
Edit matches
Sometimes it may be an advantage to manually add or delete matches after the
automatic matching procedure. Use the options in the Edit : Matches menu, or
the corresponding icons in the Detect and Match Spots toolbar.
Note that matches were created in a hierarchical manner. This means that spots
may be properly matched in one match set level, but not in another. To delete
your matches, it is therefore important to select the appropriate images/panes.
Delete match
Select spots that were incorrectly matched, make sure the appropriate match
level (gels/panes) is selected, and click the Delete Match icon to remove the
spots from the match.
Add match
Select all spots to be matched and click the Add Match icon to add the spots to
the match.
While selecting spots, you may see that some spots were already matched.
When selecting one of them, the others are automatically selected. Make sure
that the existing matches are correct. If this is not the case, delete them before
proceeding.
Multiple matches
ImageMaster enables the creation of multiple matches (Figure 7-6). In contrast
to a single match, where only a single spot is selected per gel, a multiple match
implies that one or more spots in one image can be matched to several spots in
other images.
All the spots from such a multiple match on a given gel image are considered as
a single spot in the subsequent data analysis. The calculated quantification
values for this composite spot reflect the size, intensity, and abundance of the
combined spots. Therefore, this is a solution to avoid spot editing.
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7.8 Match reports
Figure 7-6. Multiple matches. The selected spot was correctly matched throughout match
set A and within BT2. But due to spot splitting in B_T1_Gel1, it was not matched within
match sets BT1 and B. Rather than merging the two spots in B_T1_Gel1, the spots can
both be included in the match and treated as a single entity in the spot quantification.
Review matches
To review matching, choose Select : Matches : All and specify the hierarchical
level at which you want to select the matches. Matched spots are highlighted in
green. The matching of any red spots should be examined. However, this can
also be done during data analysis.
7.8
Match reports
7.8.1
Match statistics table
Choose Reports : Analyze Gels : Match Statistics Table to display the number
and percent of matches found for each of the images. By default, the numbers
are calculated based on All MatchSets in the hierarchy. But you can select a
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particular match set by clicking the Choose MatchSet icon in the toolbar of the
Match Statistics Table (Figure 7-7).
Figure 7-7. Match Statistics Table.
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8
8.1
Data analysis
Introduction
To study the variations in protein expression among a series of gels, the gels
should be matched together (be part of the same match hierarchy).
Data analysis can be carried out at two different levels:
8.2
•
Analyze Gels: Study protein expression changes within a set of gels,
without taking populations into consideration. This type of analysis can be
carried out on both MatchSet and Classes sheets. The analytical methods
used include scatter plots, descriptive statistics, histograms, and factor
analysis.
•
Analyze Classes: Find significant protein expression changes between
classes of gels. For this type of analysis, images must be placed in classes
and opened in a Classes sheet. The analytical methods used include
descriptive statistics per class, histograms, overlapping measures, and
statistical tests.
General settings
The following concepts and settings are used in the different analytical methods
described later in this chapter.
8.2.1
Quantification value
The quantification value found in various tables, graphs and plots is a software
option. Choose Tools : Options and select the Quantification tab to set:
•
Value: To be used for the analysis of conventional 2-D gels. You can
choose between Intensity, Area, Vol, %Vol, or Saliency. The default unit is
%Vol.
•
DIGE Value: To be used for the analysis of DIGE gels. You can choose
between Intensity, Area, Vol, %Vol, Vol Ratio, or Slope. The default unit is Vol
Ratio.
8.2.2
Statistics
Central tendency and dispersion are the most frequently-used descriptive
statistics. They are calculated in Gel or Class Analysis Tables and Histograms.
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These statistics summarize spot values from a match. The central tendency
allows you to localize a center for the data, whereas the dispersion indicates
how closely the data points fall around the center.
Note: Absent spots, with zero values, are also considered in the calculation of
statistics (for both central tendencies and dispersion).
Specify and display the statistics by clicking the Statistics icon in a report
toolbar (Figure 8-1):
Figure 8-1. Statistics common to Gel or Class Analysis Tables and Histograms. The third
section is specific to different report types and is described in the corresponding sections
of the User Manual.
Central tendency
The central tendency gives the general location of a variable. This is commonly
calculated by the arithmetic mean (also known as the average or center of
gravity of a distribution), the median (the middle value which divides the sample
in two equal parts) or the midrange (middle location between the two sample
extremes).
Mean and midrange values are very sensitive to extreme values (outliers) and
can be seriously affected by a single observation. On the other hand, the median
is highly resistant to outliers. A compromise is given by the trimmed mean (or
trimmed midrange) where a predefined number of outliers are removed from
the sample. The trimmed measures are more robust than the mean (or
midrange) but are more sensitive than the median.
The percentage slider in the Statistics window allows you to remove outliers and
obtain the different central tendencies. A 100% value means that all the spot
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values available in a match are used to calculate the statistics (no outliers are
suppressed). With a value of 80%, for example, 10% of the minimum values and
10% of the maximum values are discarded from the sample and the trimmed
measure is calculated with the remaining values.
•
Select Mean and the arithmetic mean is calculated, that is, the sum of all
the sample values divided by the sample size.
•
Select Midrange and the midpoint of the sample value is calculated, that
is, the middle location between the two sample extremes.
•
Obtain a Trimmed Mean (or Trimmed Midrange) by selecting Mean (or
Midrange) and discarding the desired percentage of outliers with the
percentage slider.
•
Obtain the Median by selecting Mean and discarding 50% of the values at
each of the extremities, that is, select 0% in the percentage slider.
•
Select Reference... and the value from the spot in a specified reference gel
is taken as the central tendency. This option is only available for Gel
Analysis Reports.
Dispersion
The dispersion measures the variability of the sample data as indicated by how
clustered or scattered the data points are around their center value. There are
numerous measures of variability: standard deviation, range, interquartile
range, and so on.
Like the statistics for central tendency, these measures make use of all the
available sample data and can be heavily influenced by outliers. Therefore, you
can also restrict the sample to the central values by trimming out the extreme
values with the percentage slider.
•
Select Mean Squared Deviation (M.S.D.) and the square root of the
average squared difference of each sample value to the center location is
calculated.
•
Select Mean Absolute Deviation (M.A.D.) and the mean of the absolute
difference between each value and the central value is calculated. It is not
affected as much by outliers as the Mean Squared Deviation because the
differences are not squared.
•
Select Half-range Size and the difference between the largest and the
smallest values divided by 2 is calculated.
Examples
• The Mean 100% and the Mean Squared Deviation 100% are the most
commonly-used statistics (Figure 8-2, a). Note that the standard deviation
is the Mean Squared Deviation multiplied by N, where N is the sample size.
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This difference comes from the fact that the standard deviation should be
an unbiased estimator.
•
The Median (Mean 0%) and Mean Absolute Deviation 100% are much more
robust to outliers than the statistics above (Figure 8-2, b).
•
The Midrange 100% and Half-range 100% define an interval that includes
all sample values (Figure 8-2, c).
•
The Midrange 50% and Half-range 50% are known as order statistics and
interquartile ranges (Figure 8-3).
(a)
(b)
(c)
Figure 8-2. Histograms showing the sensitivities of central and dispersion values. (a) Mean
100% and M.S.D. 100%, (b) Median and M.A.D. (c) Midrange 100% and Half-range 100%.
(a)
(b)
(c)
(d)
Figure 8-3. Histograms showing the effect of suppressing outliers. Midrange and halfrange values are given for (a) 100%, (b) 80%, (c) 50% and (d) 33%.
Note: Histograms are described further in 8.3.3.
8.3
Analyze gels
8.3.1
Scatter plots
To analyze gel similarities or experimental variations such as disparities in stain
intensity or sample loading, you can produce Scatter Plots for matched spots
(Figure 8-4) by choosing Reports : Analyse Gels : Scatter Plots.
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Figure 8-4. Scatter plot for matched spots.
Scatter plots give an idea of the relationship between the spot values from two
gels by searching for the linear dependence between the spot values of one gel
(variable X) and the corresponding values in the sheet reference (variable Y).
Remember that you can change the sheet reference (darker gel name) by
choosing View : Sheet : Set Reference.
The linear dependence is defined as the best-fit line through the data points.
The best-fit line is described by a slope and its offset from the equation y = slope
× x + offset.
The goodness-of-fit for this approximation is given by the correlation coefficient
Corr. This coefficient can vary between -1 and 1, where an absolute value near
1 indicates a good fit. The spot values of one gel can be predicted, to some
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extent, by the values of the other gel. On the other hand, a low value indicates
that the data could not be approximated by a straight line.
The types of conclusions that can be drawn from the regression line equations
and the correlation coefficients are:
1.0 × x + 0
and Corr = 1
indicates that the spot values for all matched
spots are the same in the two gels.
1.2 × x + 0
and Corr = 0.95
indicates that almost all spot values are
approximately 20% higher in the sheet
reference.
1.0 × x + 0.2
and Corr = 0.95
indicates that almost all spot values are
shifted by +0.2 with respect to the sheet
reference.
In general, when the data are highly correlated (Corr close to 1) but the best-fit
line is far from identity (1.0 × x + 0), you should search for possible reasons to
explain why your values are systematically biased. Stain intensity variations,
differences in protein loading, or image acquisition problems are some typical
causes.
In the Scatter Plots window you can visualize a scatter plot for each gel in the
sheet versus the sheet reference, together with the best-fit line, correlation
coefficient and the number of matches displayed.
Scatter plots are interactive. You can click on the points representing the
matched spots. This in turn selects the spots in the gels and other reports.
Slider
Move the slider in the toolbar of the Scatter Plots window to view the scatter
plots for the other images in the sheet.
Scatter table
Click the Scatter Table icon in the toolbar to show or hide the table below the
plot. The Scatter Table displays, for each pair of matched spots in the scatter
plot, the corresponding spot values in the gels and the error in relation to the
regression line.
Copy formula to clipboard
Select the Copy Formula to Clipboard option from the Save icon in the dropdown list to copy the regression formula and correlation coefficient to the
clipboard for use in other applications.
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8.3.2
Gel analysis table
The Gel Analysis Table and Histograms provide valuable tools for looking at
unusual matches within a set of gel images. Analyzing protein expression
changes, checking spot detection or verifying matching operations are a few of
the potential uses. Choose Reports : Analyse Gels : Table to display the Gel
Analysis Table (Figure 8-5). This report describes, for each match:
•
The spot values from each gel.
•
The Max value, that is, the highest of all these spot values.
•
The Central Tendency and Dispersion over all the gels in the sheet,
regardless of if they belong to different populations or not.
•
The Coefficient of Variation (Coef. Variation), which is the dispersion
divided by the central tendency. It measures the relative variability of the
spots in a match by correcting for the magnitude of the data values, thus
giving a measure that has no units. When you choose the Median and
Mean Absolute Deviation statistics, this measure is also known as the
Coefficient of Dispersion.
•
The Range Ratio, which is the maximum value divided by the smallest
value in the sample specified. To specify the sample, click the Statistics
icon in the Gel Analysis Table toolbar, and suppress outliers by setting the
percentage slider in the Range Ratio section to the desired value.
•
The Separability, which is the highest difference between two
consecutively sorted values in the whole sample. It measures the greatest
gap that you can have if you want to split the spot values in a match into
two separate classes.
•
The Match Count, which is the number of gels in which the spot is present
and matched.
Figure 8-5. Gel Analysis Table.
In addition to the standard functionalities for saving, printing, copying content
to the clipboard and navigating in the report, the following tools in the Gel
Analysis Report are particularly useful:
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Select by value
Select items in the table based on a numerical search criterion. Choose the
measure (that is, column) to be used for refinement, and set the lower and/or
upper limits of your search interval.
Create spot set
Create a spot set to annotate spots of interest for use at a later stage. All
currently selected spots are automatically included in the newly created spot
set, which appears as a column in the table. You see a checked box for spots
that belong to the set, or an empty box for spots that do not belong to the set.
Factor analysis table
Carry out a factor analysis. For more information, see 8.3.4.
Statistics
Set the statistics to be used in the report. These settings are common to the Gel
Analysis Table and Gel Analysis Histograms.
Settings
Some of the above-mentioned columns may not be displayed by default. Click
the Settings icon to show or hide columns in the table.
Normalization
In order to simplify comparisons across matches, the spot values in the Gel
Analysis Table can be normalized relative to their gel analysis statistics. Select
the desired type of normalization in the drop-down list in the toolbar. The
following options are available:
Value
Relative
Ratio
Normalized
8.3.3
Raw spot value.
Spot value – Central tendency
Spot value
---------------------------------------Central tendency
Spot value – Central tendency
-----------------------------------------------------------------------Dispersion
Gel analysis histograms
Histograms are a way to look at matched spots. The Gel Analysis Histograms
window (Figure 8-6) is displayed by choosing Reports : Analyze Gels :
Histograms.
In the histograms, the vertical orange bars correspond to the spot values, the
blue horizontal line represents the chosen central tendency and the red lines
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delimit the range defined by [Central value - Dispersion, Central value +
Dispersion]. The Match ID is displayed at the bottom of each histogram.
Figure 8-6. Gel Analysis Histograms.
The following tools in the Gel Analysis Histograms are very useful:
Slider
To see the matches displayed on additional pages, use the slider in the toolbar.
When a match is selected on an image or in another report, it is automatically
highlighted in a histograms window.
Statistics
Set the statistics to be used in the histograms. These settings are common to the
Gel Analysis Table and Gel Analysis Histograms.
Settings
Click the Settings icon to select one of the following options:
•
Sorted Values, to sort the spot values in ascending order (Figure 8-7).
(a)
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Figure 8-7. Histograms on matches with (a) unsorted values and (b) values sorted in
ascending order.
•
Adaptive Gradations, to adjust the histogram gradations according to the
spot values in each match. Deselect this option to display an identical
gradation in all histograms (Figure 8-8).
(a)
(b)
Figure 8-8. Histograms on matches with adaptive gradations set (a) individually for each
histogram and (b) according to the minimum and maximum values in all histograms.
•
Show Labels, to display a table with the gel names. Alternatively, when
you place the mouse over a letter in the histograms, a screentip displays
the full image name and the spot value in the image.
Normalization
You can display normalized spot values to simplify the comparisons across
matches. These normalized values are particularly useful in combination with
the histograms:
Value
Raw spot value.
Relative
Spot value – Central tendency
This normalization sets the central tendency values to 0, and if
the Adaptive Gradations option (see above) is deactivated, you
have a good overview of the dispersion and therefore of the
homogeneity of the matches. This normalization is sensitive to
high spot values.
Ratio
Spot value
---------------------------------------Central tendency
This normalization divides all values by the central tendency and
thus gives a ratio for all data. If you deactivate the Adaptive
Gradations option (see above), all histograms have the same
scale and thus it becomes easier to detect matches that do not
have homogenous values. This normalization is more sensitive
to low spot values.
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Normalized
Spot value – Central tendency
-----------------------------------------------------------------------Dispersion
This normalization is a compromise between the two
normalizations described above and is sensitive to all values.
8.3.4
Factor analysis
The visual task of comparing gels is rather difficult when dealing with a large
number of gels that consist of thousands of spots. It can be hard to determine if
different sample populations exist and to characterize their different protein
expression profiles. Factor analysis helps here by condensing the information
contained in such huge data sets into a smaller number of factors, or
dimensions, that explain most of the variance observed. The factor analysis tool
is used to examine the interrelationships between large numbers of variables
(that is,, spot values for a series of gels) and to explain these relationships (for
example, gel populations) in terms of common underlying factors (or
associations with specific spot patterns).
Factor analysis is a complex statistical technique, whose comprehensive
description is beyond the scope of this manual. For more information,
references are given in the Appendix.
Perform a factor analysis
A factor analysis is carried out on all or selected matches in the Gel Analysis
Table. You must judge which of the options described below is most applicable
to your analysis.
To carry out a factor analysis:
1
Click the Factor Analysis Table icon in the Gel Analysis Table toolbar.
2
If any matches are selected, the software asks if you want to use all or only
the selected rows.
3
The Factor Analysis Table (Figure 8-9) is displayed with the lines
corresponding to the axes that can be drawn in the Factor Projection Plot.
Select two axes to be displayed in this plot (generally the first two ones).
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4
Click the Factor Projection Report icon in the Factor Analysis Table toolbar.
Figure 8-9. Factor Analysis Table.
The Factor Projection Plot (Figure 8-10) displays the projection of each match
(cross) and each gel (blue vector) on the two factorial axes. The blue curve
represents a part of the correlation circle; its form is linked to the scale of the
axes. When matches are selected on the plot, they are automatically
highlighted on the gels and/or any open reports.
The Factor Projection Table (Figure 8-10) displays the contribution of each
match to the two axes displayed in the Factor Projection Plot. The Quality
measures if the projection of the match is well represented on the factorial
subspace.
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Figure 8-10. Factor Projection Report includes the Factor Projection Plot (top) and the
Factor Projection Table (bottom).
You can use the following tools in the Factor Projection Report:
Factor analysis table
Display the Factor Analysis Table from which the two axes for the current Factor
Projection Report were selected.
Factor projection table
Show or hide the Factor Projection Table corresponding to the displayed plot.
Factor projection plot
Show or hide the Factor Projection Plot corresponding to the displayed table.
Displayed items
Choose the items to be displayed from the drop-down list in the toolbar of the
plot. You can choose matches (crosses), gels (blue vectors), or both.
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Matches displayed
Enter the number of matches to be displayed in the plot.
Interpret a factor analysis
How to interpret a factor analysis is explained with an example of six gels run to
compare the effect of two treatments T1 (A_T1_Gel1, A_T1_Gel2, A_T1_Gel3)
and T2 (A_T2_Gel1, A_T2_Gel2, A_T2_Gel3) on bacteria cultivated on substrate
A. The gels were detected and matched, and a Gel Analysis Table displayed.
The Factor Analysis Table (Figure 8-9) summarizes the variance accounted for
by successive axes (or factors), expressed as a percent of the total variance.
Thus, factor 1 accounts for 91.8% of the variance, factor 2 for 5.4%, and so on.
The coordinates for each gel projected on these axes is also listed.
The number of factors equals the number of gels being analyzed. Factor
analysis cannot be performed with less than two gels. The more gels you use,
the more reliable the results are likely to be, and the more factors are calculated.
Since the first factors are generally the best ones for characterizing gels and
matches that behave similarly, the factors are ranked in order of importance.
Figure 8-11 shows the Factor Projection Plot obtained when the first two axes in
Figure 8-9 were selected. In the example, only the 20 most significant matches
are displayed on the projection plot. The further away a spot is from the origin,
the more important it is likely to be in terms of characterizing the gels. If all
matches were shown, one would find that many of them cluster around the
origin of the graph. This illustrates that the majority of matches, that is, protein
spots, are not meaningful in classifying the gels.
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Figure 8-11. Factor Projection Report. The ten matches with highest contribution to the
second axis are selected.
To find a possible meaning for a given factor, one should first identify the
matches that largely contribute to this factor. The Factor Projection Table is used
for this purpose. When sorting the matches according to their contribution to
the first axis, one discovers that the matches at the top of the table are those
with the highest relative volumes, as found in the Gel Analysis Table. In fact, the
first axis is generally correlated with protein abundance.
The Factor Projection Table also contains the Quality for each match. This
number tells you how close the distance of the projection is to reality. Matches
with very similar behavior (similar expression profiles across gels) are close in
space. However, when projected onto a two-dimensional subspace, matches
that are actually far apart may appear close together. It is therefore important
to look at the Quality to judge if matches are effectively close. If the values are
high for both matches, the chance is great that they are indeed nearby and have
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a similar behavior. If one of the matches has a low value, any interpretation
becomes tentative.
Gels that are adjacent on the graph are likely to be similar to each other. They
may correspond to the same population. This is clearly the case in the example
above. The gels from T1 cluster together below the horizontal axis, whereas the
gels corresponding to T2 lie above.
The closer a match is to a given set of gels, the more characteristic it is likely to
be of those gels. That is, the more important the match is in determining why
those gels are different from other gels. In Figure 8-11, one can observe that the
matches 48, 123, 180, 181 and 248 are close to the gels belonging to T1. The
histograms in Figure 8-12 show that these matches have high spot values for
the T1 gels and low values for the T2 gels. Matches 237 and 338, on the other
hand, are characteristic of the T2 population, with higher spot values in the T2
gels. In our example, the second axis appears very important for separating the
gels into two classes. It is related to the ratio between the mean spot values in
each population of gels. The matches in the upper part of the graph have ratios
that favor the T2 population, while those below the horizontal axis have ratios
that favor the T1 gels.
Comments on factor analysis
Factor analysis is used to examine the protein expression pattern within each
match. The quality of the output depends on the quality of spot matching.
Hence, it may be useful to exclude spots that are not well matched across all
gels (using Match Count in the Gel Analysis Table). Nevertheless, in cases where
a majority of spots were properly matched, the inclusion of all matches in the
factor analysis can yield good results with no preliminary match filtering
necessary.
This statistical method, based on data variation and their standard deviations,
highlights the natural formation of populations among the gels and allows
identification of matches (that is, matched spots) that are characteristic of these
classes. However, one should be very critical when analyzing factor analysis
plots since the results can be greatly influenced by outliers, bad matches, and
so on. Factor analysis can provide valuable indications in some cases, but not in
others.
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Figure 8-12. Gel Analysis Histograms for the ten matches ranked highest for their
contribution to the second axis in the factor analysis example. Matches 48, 123, 180, 181
and 248 are characteristic of population T1, matches 237 and 338 are characteristic of
population T2.
8.3.5
DIGE histogram
A DIGE Histogram can be displayed when detected DIGE gels are selected. It
shows, for each non-reference image, data associated with detected spots in
the image, plotted against Log Volume Ratio on the X-axis (Figure 8-13). It has
two different Y-axes:
•
The left Y-axis displays the spot frequency. The blue curve represents the
frequency distribution of the log volume ratios.
•
The right Y-axis represents the Measure parameter (see below) selected
from the Measure tool in the DIGE Histogram window. A plotted single
data point on the histogram represents an individual protein spot.
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The name of the DIGE reference for the current image, used for the calculation
of the Log Volume Ratio, appears at the lower right corner of the DIGE
Histogram window.
The DIGE Histogram is dynamic. You can click on the data points representing
spots to select them on the gels and any other open reports.
Figure 8-13. DIGE Histogram.
Two report-specific tools are available:
Slider
Move the slider in the toolbar of the DIGE Histogram window to view the
histograms for the other images in the sheet.
Measure
Select one of the following options from the Measure icon:
134
•
Max Slope: Largest gradient associated with the co-detected spots.
•
Area: Number of pixels within the spot boundary.
•
Max Intensity: Largest pixel value associated with the co-detected spots.
•
Max Volume: Volume of the largest co-detected spot.
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8.4
Analyze classes
8.4.1
Specify classes
To identify protein expression variations between populations of gels, you need
to specify what gels belong to which population by creating classes. Classes are
created in the Workspace.
Often, you already know the populations for your set of gels. This is the case
when you are comparing gels from healthy tissue extracts with those from
disease-associated samples. When you have no preliminary knowledge of the
populations in the set of gels, you can draw possible conclusions from factor
analysis.
8.4.2
Overlapping measures
Spot values for a given match within each class can be summarized by the
central tendency and dispersion. In addition to these descriptors, the software
computes overlapping measures between the class intervals, where class
intervals are defined by the ranges [Central value - Dispersion, Central value +
Dispersion].
The overlapping measures quantify the overlap between these intervals, and
thus evaluate how different the protein expression changes between the
classes really are. They take into account both the difference between the
central tendency in each population and the dispersion. The following
overlapping measures are available:
•
Gap: Maximum difference between the range of the current class and the
range of one of the other classes (Figure 8-14, c-b in the case of Class A).
Negative values indicate overlap whereas positive values are nonoverlapping class ranges.
•
Ratio: Maximum ratio between the lower limit of one of the other classes
and the upper limit of the current class (Figure 8-14, c/b in the case of
Class A). Absolute values smaller than 1 indicate overlap, whereas
absolute values higher than 1 show that there is no overlap. In order to
easily distinguish matched spots that are over or under expressed in one
of the classes, the ratio value is preceded by a minus sign when the protein
spot is under expressed for the class in question, compared to the other
class. Positive values are attributed to the Ratio value in the overexpressed class.
•
Normalized: Maximum percentage of the current class range not
overlapping with the range of one of the other classes (Figure 8-14, (c-a)/
(b-a) in the case of Class A). A value smaller than 1 indicates overlap. For
example, 0.25 implies that 25% of the current class range is not recovered
by one of the other classes. In the same way, a value of 1.5 indicates that
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NON-OVERLAPPING CLASSES
there is a gap equivalent to 50% of the current class range to the furthest
other class. The normalized overlapping is not symmetrical, the value from
Class A compared to Class B is not the same as the value from Class B
compared to Class A.
0
2
6
8
1.5
Class A
a
b
c
Class B
d
2
Gap:
Ratio:
Normalized:
0
OVERLAPPING CLASSES
4
2
Class A
Class B
2.0
1.4
1.5
2.0
1.4
2.0
[c-b]
[c/b]
[(c-a)/(b-a)]
4
6
8
0.75
Class A
a
b
c
Class B
d
0.5
Gap:
Ratio:
Normalized:
Class A
Class B
-1.0 [c-b]
0.8 [c/b]
0.75 [(c-a)/(b-a)]
-1.0
0.8
0.5
Figure 8-14. Scheme demonstrating how the Gap, Ratio, and Normalized values are
calculated for two non-overlapping classes (upper part) and two overlapping classes
(lower part). Arrows above or below each class range illustrate how the Normalized
measure relates to this class range.
Note: The formulas mentioned above only apply to Class A in the current
example. Their purpose here is to illustrate the principles of overlapping
measures. Many different cases (and therefore formulas) exist.
A value of 1e6 (1000000) in the Class Analysis Histograms characterizes the
case where the protein is completely absent from a class. The software cannot
compute ranges. A value of 0 for the Ratio or Normalized measures indicates
that the particular class is entirely covered by another one.
Note: As indicated in their definition, the Gap, Ratio, and Normalized values
always calculate the MAXIMUM difference, ratio or percentage with
respect to ANY of the other classes. This means that when the Ratio
values for three populations (for example, A, B, C) are compared, the
software calculates the ratio of A with respect to B and of A with respect
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to C, but only displays the highest value in the column for class A. The
number shown does not indicate with respect to which class the value
was obtained. The idea is to quickly enable you to find a match (that is,
protein marker) that distinguishes the current class from any of the other
classes. Once such protein markers are found, the Class Analysis
Histograms can be used to study the match in more detail.
8.4.3
Statistical tests
The software provides three statistical tests: ANOVA, Mann-Whitney U test, and
the Kolmogorov-Smirnov test. These tests are used to analyze differences in
protein expression between classes of gels. The idea is to draw conclusions
about the significance of the protein expression changes by extrapolating
information from the data you collected. For example, when you have two
samples (classes) with different means (that is, different means for the spot
values of a particular match), you might want to know if the data were sampled
from populations with different means or if the populations have the same
mean with the observed difference being a coincidence of random sampling.
You can calculate the probability of observing a certain difference (or larger)
between sample means in an experiment of this size, for populations that in
reality have the same mean. If the probability is small, you can conclude that the
difference is not likely to be caused by random sampling and assume instead
that the populations have different means.
Note: You can display the desired statistical values for each match in the Class
Analysis Table. These values should be considered as qualitative
indications of the variations in protein expression between two
populations. To draw quantitative conclusions, you must verify that the
restrictive assumptions of the various tests are met. In addition, one
should always check the results by visual inspection of the spots, since the
conclusions may be erroneous due to inaccuracies in detection or
matching.
Note: The given statistical values are useless when the samples (classes) do not
consist of more than two gels. Your objective should always be to work
with the largest possible sample sizes.
One-way ANOVA
Analysis of Variance (ANOVA) is one of the most important statistical tests
available for biologists. It is essentially an extension of the logic of Student's ttests to those situations where the comparison of the means of several groups
is required. Thus, when comparing two means, ANOVA gives the same results as
the t-test for independent samples.
One-way ANOVA tests the null hypothesis that all populations have identical
means. It generates a P value that answers this question: If the null hypothesis
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is true, what is the probability that randomly selected samples vary as much (or
more) than actually occurred?
It is based on the same assumptions as the t-test:
•
The samples are randomly selected from, or at least representative of, the
larger populations.
•
The two samples are independent. There is no relationship between the
individuals in one sample as compared to the other.
•
The data are sampled from populations that approximate a Gaussian
distribution.
If you are not able to assume that your data are sampled from Gaussian
populations, then non-parametric tests like the Mann-Whitney or KolmogorovSmirnov tests can provide a better analysis. However, these test only allow you
to compare two samples at the same time.
Mann-Whitney or Wilcoxon test
The Mann-Whitney U test or rank sum test is the non-parametric substitute for
the two-sample t-test when the assumption of normality (Gaussian bell-shaped
distribution) is not valid. It is equivalent to the Wilcoxon rank sum test. It should
only be used for comparing two unpaired samples. The assumptions of the
Mann-Whitney U test are:
•
The variable of interest is continuous (not discrete) and the measurement
scale is at least ordinal. This means that repeated values (ties) are not
acceptable. When ties are present in your data, there is an approximation
provided in the calculations, but the exact results no longer hold.
•
The distributions of the two samples are identical (although not necessarily
normal) and differ only in location (that is, central tendency).
•
The two samples are independent.
To perform the Mann-Whitney test, the software first ranks all the spot values
from low to high, paying no attention to which of the two classes (for example,
X and Y) each value belongs. Then each value is given a rank number. The
smallest number gets a rank of 1. The largest number gets a rank of N, where N
is the total number of spot values in the two classes. If two values are the same,
then they both get the average of the two ranks for which they tie. Finally, the
ranks in each class are summed, thus giving WX and WY, which are used to
calculate the Mann-Whitney test statistic, U. The formula for UX is as follows (the
formula for UY is obtained by replacing X by Y):
nX ( nX + 1 )
U X = W X – ------------------------2
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The smaller of the two calculated U values corresponds to the number of shifts
needed in order that the spot values from the two populations do not overlap.
For the first example in Figure 8-15, no shifts are necessary since the spot values
of classes X and Y are already separated. On the contrary, for the second
example the Mann-Whitney test indicates that the first two values from Y (or the
three last values from X) have to be swapped four times in order to separate the
samples.
The software displays the smaller of the two calculated U values in the Class
Analysis Table. The lower the number, the higher the probability is that the
means of the two samples are different. Knowing this value, and the sample
size, you can easily look up the probabilities in a Mann-Whitney table.
Attention should be payed when analyzing the results of a Mann-Whitney test.
First, the assumptions are often violated. This is the case when spots are
completely absent in one of the classes (in this case you have repeated values
of 0). Moreover, if you have small samples, the Mann-Whitney test is
meaningless. If the total sample size is seven or less, the test always gives a
probability (of finding different means, in the case of identical populations)
greater than 0.05, no matter how little the samples differ.
Y
Spot values
X
X
Ranks
X
Y
1.5
1.5
3
4
5
Spot values
0.000
0.000
0.044
0.059
0.068
0.144
0.237
0.240
0.249
0.255
0.308
6
7
8
9
10
11
0.034
0.045
0.056
0.058
0.064
0.069
0.075
0.078
0.104
0.106
0.126
WX
nX
UX
51 15
6 5
30 0
WX
nX
UX
Y
Ranks
X
1
2
3
Y
4
5
6
7
8
9
10
11
25 41
6 5
4 26
Figure 8-15. Two examples to illustrate the Mann-Whitney and Kolmogorov-Smirnov
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tests. In (a) the two classes do not overlap at all, whereas in (b) 4 shifts are needed to
completely separate the spot values of the two classes. The bold values correspond to the
spot values and ranks from class Y, the others belong to class X.
Kolmogorov-Smirnov test
The Kolmogorov-Smirnov test tries to determine if two data sets differ
significantly. It is used to test if two samples may reasonably be assumed to
come from the same distribution. It has the advantage of making no
assumption about the distribution of the data and is frequently preferred over
the Mann-Whitney rank sum test where there are many ties (repeated values).
Other tests (for example, the t-test) may be more sensitive if the data meet the
requirements needed for that test. The assumptions of the KolmogorovSmirnov test are:
•
The probability distributions are continuous.
•
The measurement scale is at least ordinal.
•
The two samples are mutually independent.
In the Kolmogorov-Smirnov test, the data points in each sample (spot values for
a particular match in a class) are sorted in ascending order and converted into
an empirical distribution function (EDF). This function gives the fraction of data
points to the left of a given value z. For the second example in Figure 8-15, the
ordered data points from class X are: 0.034, 0.045, 0.056, 0.064, 0.069, and 0.078.
The fraction of data points to the left of each of these z values can easily be
calculated and plotted (full line) in an Empirical Distribution Plot (Figure 8-16):
It is clear that no data lie strictly below 0.034, 1/6 = 17% of the data is strictly
smaller than 0.045, 2/6 = 33% of the data is strictly smaller than 0.056, 3/6 =
50% of the data is strictly smaller than 0.064, and so on.
The same procedure can be followed for the second sample (class Y in our
example, the dashed line in Figure 8-16). The Kolmogorov-Smirnov test statistic
D is then defined as the maximum distance between the empirical distribution
functions (EDF) for the two samples. In the example, D is 0.63 (0.83-0.20). If D is
greater than a particular decision limit (critical value found in a Kolmogorov-
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Smirnov table), there is a statistically significant difference between the two
samples. However, the test provides no insight as to what causes the difference.
Fraction of data points to the left
of Spot value z
Empirical Distribution Plot
1.00
0.80
0.60
D
0.40
0.20
Class X
Class Y
0.00
0.02
0.04
0.06
0.08
0.1
0.12
0.14
Spot value z
Figure 8-16. The Empirical Distribution Plot for the spot values of match 613 (Figure 8-15),
in Classes X and Y. The Kolmogorov-Smirnov statistic D corresponds to the maximum
distance between the two empirical distribution functions.
8.4.4
Class analysis table
The Class Analysis Table provides valuable data for finding significant protein
expression changes between populations of gels. Using this data, it is possible
to differentiate one class from the others based on a few matches/spots.
Choose Reports : Analyse Classes : Table to display the Class Analysis Table
(Figure 8-17). For each match, there is a description of:
•
The Center (central tendency), Dispersion, Gap, Ratio or Normalized
values for each class, depending on the selection made in the drop-down
list (see below). By default, the Center values are displayed.
•
The Max value, that is, the highest from all these class values.
•
The Match Count, which is the number of classes in which the spot is
present and matched.
•
The results from the statistical tests: the ANOVA probability P, the Wilcoxon
U statistic, and the Kolmogorov D statistic.
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•
The columns corresponding to spot sets.
Figure 8-17. Class Analysis Table.
In addition to the standard functionalities for saving, printing, copying content
to the clipboard and navigating in the report, the following tools in the Class
Analysis Table are particularly useful:
Select by value
Select items in the table based on a numerical search criterion. Choose the
measure (that is, column) to be used for refinement, and set the lower and/or
upper limits of your search interval.
Create spot set
Create a spot set to annotate spots of interest for use at a later stage. All
currently selected spots are automatically included in the newly created spot
set, which appears as a column in the table. You see a checked box for spots
that belong to the set, or an empty box for spots that do not belong to the set.
Statistics
Set the statistics to be used for calculating the Center and Dispersion value of
each class, and consequently the Gap, Ratio and Normalized values. These
settings are common to the Class Analysis Table and Class Analysis Histograms.
Note: The Center and Dispersion values define the interval that characterizes
the protein sample of each class in a match. To characterize a class only
by the central value, set the Dispersion percentage slider to 0%. This is
useful if you want to calculate the difference or ratio between the central
values of your classes.
Settings
Some of the above-mentioned columns may not be displayed by default. Click
the Settings icon to show or hide columns from the table.
Displayed value
The displayed statistical descriptor or overlapping measure can be selected
from the drop-down list in the toolbar. You can display the Center (central
tendency), Dispersion, Gap, Ratio or Normalized values for the different classes.
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8.4.5
Class analysis histograms
You can visually investigate the statistical and overlapping descriptors of
populations by displaying Class Analysis Histograms. Choose Reports : Analyse
Classes : Histograms.
When the Center (central tendency) value is selected for display in the dropdown list, the Class Analysis Histograms display all the individual spot values in
each match, separated for each class by vertical gray lines (Figure 8-18). The
classes are characterized by their central tendency (blue horizontal line) and
dispersion interval (bounded by the outer red lines). The Match IDs appear below
each histogram.
When displaying the Dispersion, Gap, Ratio or Normalized values (Figure 8-19),
each class is represented by a single value (red bar).
Figure 8-18. Class Analysis Histograms with Center values displayed.
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Figure 8-19. Class Analysis Histograms with Ratio displayed.
The histograms can be selected to highlight the corresponding spots on the gels
and in any open reports.
The following tools in the Class Analysis Histograms are very useful:
Slider
To see the matches displayed on additional pages, use the slider in the toolbar.
When a match is selected on an image or in another report, it is automatically
highlighted in a histograms window.
Statistics
Set the statistics to be used for calculating the Center and Dispersion value of
each class, and consequently the Gap, Ratio and Normalized values. These
settings are common to the Class Analysis Table and Class Analysis Histograms.
Settings
Click the Settings icon to activate one of the following options:
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•
Sorted Values, to sort the classes according to their central value and sort
the spot values inside each class (Figure 8-20). This can simplify the search
for non-overlapping intervals.
(a)
(b)
Figure 8-20. Class Analysis Histograms with (a) unsorted values and (b) values sorted in
ascending order.
•
Adaptive Gradations, to adjust the histogram gradations according to the
values in each histogram. Deselect this option to display an identical
gradation in all histograms (Figure 8-21).
(a)
(b)
Figure 8-21. Class Analysis Histograms with adaptive gradations set (a) individually for
each histogram and (b) according to the minimum and maximum values in all histograms.
•
Show Labels, to display a table with the gel or class names. Alternatively,
when you place the mouse over a letter in the histograms, a screentip
displays the full image or class name and the value in that image or class.
Displayed value
The displayed statistical descriptor or overlapping measure needs to be
selected in the drop-down list in the toolbar. You can display the Center (central
tendency), Dispersion, Gap, Ratio or Normalized values for the different classes.
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9
Annotations
9.1
Introduction
Individual pixels and spots in a gel image can be labeled with annotations.
Annotations are used to flag items with their specific characteristics (protein
name, database accession number and so on) or to mark spots with common
characteristics. They also offer the possibility of linking and associating gel
objects to external query engines or data sources of any format (text, html,
spreadsheet, multimedia, database) located locally or on the Internet.
Figure 9-1. Annotations are composed of an annotation basis (square or cross), an
annotation flagpole and a set of labels (flag).
An annotation is defined by its position on the gel (X and Y coordinates) and its
set of labels. Each label belongs to a predefined or user-defined category. As
shown in Figure 9-1, each annotation is composed of a basis, a flagpole, and a
flag that consists of a set of colored labels.
Spot and pixel annotations
You can create two types of annotations (Figure 9-1):
•
Annotations linked to pixels are visualized with a cross basis. They are
simply connected to a pixel and have the same coordinates as that pixel.
•
Annotations linked to spots are visualized with a square basis. They are
linked to a spot and have the same coordinates as that spot, that is, as the
spot's center of gravity. These annotations are automatically selected
when the linked spot is selected and vice versa.
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9.2 Create annotations and labels
9.2
Create annotations and labels
9.2.1
Create annotations
The creation of annotations essentially consists of taking three steps.
1
Select the spots or pixel on which you would like to create an annotation:
•
•
Choose the Select tool and double-click on a spot or a pixel.
Select several spots, make sure to select only the images on which
you want to create new annotations, and choose Edit : Annotations :
Add.
2
Choose one of the existing categories or create a new category. The
creation of new categories is described in Section 9.3.
3
Enter the desired label content.
9.2.2
Add labels to existing annotations
Labels can be added to an existing annotation.
1
Select the annotation to which you would like to add a label:
•
Choose the Select tool and double-click at the basis of the
annotation to which your label should be added.
•
Select several annotations, make sure to select only the images on
which you want to create new labels, and choose Edit : Annotations :
Labels : Add.
2
Choose one of the existing categories or create a new category. The
creation of new categories is described in Section 9.3.
3
Enter the desired label content.
Note: One annotation may have many labels, but it can only contain one label
from each category. If one spot contains several proteins, it may need to
carry different labels from the same category. You can do this by linking
additional annotations to the spot.
9.2.3
Link annotations to spots
The software allows you to link an annotation to a spot. This is helpful when you
want to link an additional annotation to a spot (to attach multiple labels of the
same category), or when you missed a spot to which you wanted to link a newly
created annotation.
To link an annotation with a spot, click on the annotation basis and drag it to a
spot. If, for some reason, an annotation already exists within a spot but is not yet
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linked, you can also select the annotation and choose Edit : Annotations : Link
with Spot.
To unlink an annotation from a spot, select the annotation and drag it outside
the spot or choose Edit : Annotations : Unlink from Spot.
9.3
Create label categories
9.3.1
Predefined label categories
The software offers some predefined label categories:
•
Ac: This category is provided to hold the protein's accession number (AC)
taken from a user-defined database and can be the linked to
ImageMaster's remote database query engine. When such a link is
defined, double-clicking on a label of this category displays the
corresponding protein entry in the selected database with the default
Internet browser.
•
pI_MW: This category contains the known isoelectric point (pI) and
molecular weight (MW) information, which is subsequently used to
compute approximate pI and MW values for any point in a gel. You should
enter the pI value first and MW value second, separated by a space. By
replacing one of the values by -1, you indicate to the program that no
value is set.
•
Comment: This category is an example of a general category where users
may store their comments.
9.3.2
Create new categories
When you create a new category during one of the procedures described in
Section 9.2, you must enter the constraints and attributes for the new category
in the Create Category window (Figure 9-2). This section explains the different
options.
Note: User-defined categories are only available from the category list as long
as there is at least one label of this category in the open gels (in any of the
open sheets). To create categories that are permanently available, you
must define them in the Annotations tab of the Options window
(available by choosing Tools : Options). The same category constraints
must be defined.
At any moment, you can change the category constraints by choosing Edit :
Annotations : Categories : Edit Attibutes, or rename a category by choosing
Edit : Annotations : Categories : Rename a Category.
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9.3 Create label categories
Figure 9-2. Create Category window.
Data type
By default, the labels can contain any character. However, to ensure coherent
annotation data, the label contents can be constrained to one of the following
Data Types:
•
Text. Can contain any character.
•
Number: Can only contain numerical values.
•
Auto-Numbering. An incremental number (per gel) is entered
automatically as the new label.
•
Set. Use this data type to mark spots with common properties. The labels
in such a category do not contain specific information. They only display
the name of the category to which they belong. Note that labels of this
category are displayed in the form of check boxes in tables. A checked box
indicates that the spot belongs to the category.
Is unique
When you check the Is Unique box, you indicate that each label on a gel within
the new category should be unique. The software will not accept a new label
when an identical one already exists. You are asked to enter a new label.
External engine
ImageMaster offers the possibility to link spots on gel images to protein data in
2-DE or other databases. All you have to do is input the appropriate query
format (database address and query engine) in the External Engine field of the
new label category and enter valid database accession numbers (AC) as labels.
This functionality is further described in Section 9.4.
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When you subsequently double-click on a label of such a category, the software
opens your default Internet browser and launches an HTTP query that takes the
form of a Web page address.
Note: You can define a different query engine for each label category and
therefore you can link one protein spot to different database entries.
Display properties
User-defined categories use a gray background color by default. You can
change the default color by clicking on the Color box. The new background color
is used for all labels of that specific category.
You can choose to hide the category by default, by clicking the By default
hidden box. In this case, you must choose View : Annotations : Visible
Categories to make the category visible by checking its box.
9.4
Connect to protein databases
9.4.1
Introduction
ImageMaster can link spots on gel images to protein data in 2-DE or other
databases. Such databases contain information on proteins identified on 2-DE
images, such as pI and MW values, bibliographical references to protein related
literature, information on protein functions, etc. If your computer has Internet
access, you can remotely query and retrieve protein data related to spots on
your gels.
Note: The ImageMaster software provides access to several databases on the
Internet. It is the responsibility of the user to acquire the database
licenses, if needed. In particular, the PROSITE and SWISS-2DPAGE
databases are copyrighted, and all commercial users of these databases
are required to purchase a database license from GeneBio. No license fee
is charged to academic users for non-commercial use. For questions
about obtaining a license subscription for the PROSITE and SWISS2DPAGE databases, please contact GeneBio (www.genebio.com).
ImageMaster takes advantage of the fact that virtually all databases on the
Web, and in particular those containing 2-DE and other protein data, use CGI
scripts to enable data queries. A CGI (Common Gateway Interface) script is a
program or script file executed on a Web server in response to a user request.
The CGI script transmits information (such as a database accession number or
object identifier) from the client to a database engine, receives back the results,
and displays them to the client.
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9.4 Connect to protein databases
9.4.2
Set the database
To query a remote database through the Internet, you must send HTTP queries
(for instance, http://www.expasy.org/swiss-2dpage/P02649) to a CGI script on a
server. The HTTP query must be composed of:
•
The database HTTP address (http://www.expasy.org/).
•
The database query engine (swiss-2dpage/).
•
The database accession number (P02649).
In ImageMaster, the database HTTP address and query engine are entered as
constraints to the annotation category (see Figure 9-3). Type them in as one
string in the External Engine field (for example, http://www.expasy.org/swiss2dpage/).
Figure 9-3. Setting the External Engine as a category constraint.
Federated 2-D PAGE databases
A list of federated 2-D PAGE databases, with the required database query
formats, can be found at http://www.expasy.org/ch2d/2d-access.html, or by
clicking on the Url button to the right of the External Engine field. Copy the
desired database address and query engine from this site.
Other databases
It is possible to find the required query format for databases that are not
federated or that do not contain 2-DE data. Directly query the database until
you find a specific protein (or other) entry. Then copy the address of the
corresponding Web page in your browser to the External Engine field of the
Create Category window, without including the accession or identification
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number of the current entry. Generally, this address consists of the database
HTTP address and query engine followed by a question mark (?) or equal sign (=).
For example, you might display the entry for the protein structure 1BMT in the
Protein Data Bank (PDB): http://www.rcsb.org/pdb/explore/
explore.do?structureId=1BMT. The required query format for ImageMaster is:
http://www.rcsb.org/pdb/explore/explore.do?structureId=
9.4.3
Query the database
The database accession number (for example P02649) is entered as a label of
the particular category linked to a spot.
When you subsequently double-click on the label while the Selection tool is
selected, ImageMaster opens your default Internet browser and launches an
HTTP query that takes the form of a Web page address.
As a result, the entry for the protein with the given accession number opens in
your browser (Figure 9-4). In the case of the above example, this would be the
entry for Human Apo E (Gels) in the SWISS-2DPAGE database.
Figure 9-4. The SWISS-2DPAGE entry for Human Apo E. Entries from this database contain
full protein names, bibliographic references, annotations (such as protein function or
pathological variations), and the pI and MW of the related spots on the 2-DE maps. It also
includes cross-references to numerous other databases.
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9.5 Create specific links
9.4.4
Connext to an executable
Analogous to an HTTP query, where the content of a label is transmitted to a CGI
script on a server, ImageMaster allows you to pass on the content of a label as
the first parameter to any executable. When you double-click on a label that has
an executable defined in the External Engine field of the label category, the
executable runs with the label content as a parameter.
To define an executable in the External Engine field, click on the Exe button and
locate the .exe file.
9.5
Create specific links
As seen above, it is possible to link labels to remote database entries by defining
an external search engine for a particular category. By using specific keywords
in the labels of any category, you can also create links to Web pages, files, and
text (Figure 9-5).
To create a specific link, you should add an annotation to a gel and include the
following items in the label field:
•
A short descriptor that will be the visible part of the label.
•
A keyword indicating the type of link (http:, file:, text:).
•
A link content, which contains the information necessary to establish the
link or the content of the link (in the case of a text link).
To indicate that a label is linked, its short descriptor is followed by three dots.
When you double-click on such a linked label, you do not enter the typical
editing mode, but the link (Web page, file or text) is automatically opened with
the appropriate software. Alternatively, to open any linked label, choose View :
Annotations : Linked Data in the menu.
Note: You can define links in any label category but you can only have one link
per label.
9.5.1
Http link:
You can link spots or pixels to specific Web pages. A double-click on an httplinked label will launch your Internet browser and automatically call the
corresponding Web page.
You can, for example, create a direct link to the ExPASy Proteomics Server
(Figure 9-5). In this case, the label content should contain the string “http:”
followed by the address of the Web page.
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9.5.2
File link:
You can link spots or pixels to software files. Double-clicking on a file-linked
label launches the specified file with the default system application associated
to the file extension.
The linked files can be placed in a specific directory, which is defined by
choosing Tools : Options in the menu and by setting the Annotations folder in
the Annotations tab. In this case, you only need to give the name and file
extension when creating the link. You can create subfolders in the Annotation
folder to arrange your files. The file names indicated in the labels must then
contain the name of the subfolder (for example, AA composition\AA P10413.xls).
Alternatively, you can link labels with files located anywhere on your system.
You should then include the complete file path when referencing the file.
For example, you can link a protein spot to an Excel file containing the amino
acid composition of a protein (Figure 9-5). The label should then consist of the
string “file:” followed by the file name (with its extension).
9.5.3
Text link:
In some cases, you might want to associate a long text comment with a specific
protein spot, but without overloading the display. The solution is to create a text
link, rather than a very long annotation label. Double-clicking on the linked label
is sufficient to display a window containing the entire text (Figure 9-5).
Text links are particularly useful for attaching bibliographic references to a spot,
for instance, or any other comment such as the one in Figure 9-5. Please note
that the string "text:" must first be inserted, followed by the text you would like
to associate with the spot.
To connect general information about the gel, other tools are better adapted.
Comments can be attached to projects, match sets, and classes in the
workspace. Additionally, you can specify gel descriptions.
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9.6 Select annotations and labels
Figure 9-5. The annotation on the spot containing protein P10413 includes linked labels
such as a link to a Web site (top), a link to a file (bottom right) and a text link (bottom left).
9.6
Select annotations and labels
9.6.1
Select
Annotations and labels can be selected with the Select tool. The selected labels
or annotations are highlighted in green and displayed in front of the other
annotations (Figure 9-6).
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(a)
(b)
Figure 9-6. (a) Annotation hidden by some other annotations. (b) Same annotation
displayed in front of all other annotations, after selection.
If the annotation is attached to a spot, the spot is also selected. Similarly, if you
select a spot, the attached annotation is selected.
To select a label, click on the label. You can select more than one label by using
the Shift key.
To select an annotation, click on the annotation basis. To select more than one
annotation, select the first one, then hold down the Shift key and select
additional annotations.
To select all annotations in a region, position the cursor at the top left position
of the desired region, hold down the left mouse button, and then drag the cursor
to the bottom right position. All annotations in the defined region are selected.
To select annotations in more than one region, hold down the Shift key while
selecting additional regions.
To deselect all annotations, click on a gel (not on an annotation).
9.6.2
Select menu
You can select specific labels and annotations with the options in the Select :
Annotations menu. Please note that this also selects hidden annotations.
•
By Content: This feature enables the selection of labels (belonging to one
or several categories that must be selected) based on their content. When
the Regular Expression box in the window is not checked, the entered
string of characters is taken literally, and the program selects all labels
containing this string. By activating the Regular Expression option, regular
expressions can be used in the search field (see below for details).
•
By Category: This feature enables the selection of all labels belonging to
one or several categories. Use the Shift and Ctrl keys to pick several
category names at a time.
•
All: This highlights all annotations in the selected gels.
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9.6 Select annotations and labels
•
Common Labels: This option allows the retrieval of all sets of identical
labels within a gel or among a series of gels, for all the categories chosen
in the window.
Regular expressions
Regular expressions provide a mechanism to select specific strings from a set
of character strings. Regular expressions use symbols and syntax elements to
describe a generalized pattern. ImageMaster invokes the standard Extended
Regular Expressions to search patterns in labels, the essentials of which are
summarized in Table 9-1 .
158
Syntax
Description
Example
.
Matches any one character.
e.oli matches:eaoli, eboli,
ecoli...
[…]
Matches any character listed
between the brackets. [a-z] indicates
the range of characters between A
and Z and [0-9] is any numeral from 0
to 9.
P[a-d] matches:Pa, Pb, Pc,
Pd
[^…]
Matches any character except those
listed between the brackets.
P[^bd] matches:Pa, Pc, Pe
but not Pb or Pd
?
Matches the preceding element zero
or one times.
P0?1 matches:P1 and P01.
+
Matches the preceding element one
or more times.
P0+1 matches:P01, P001,
P0001, ...
*
Matches the preceding element zero
or more times.
P0*1 matches:P1, P01,
P001, P0001, ...
{n}
Matches the preceding element
exactly n times.
P0{3}1 matches:P0001 but
not P01 or P001
{n,}
Matches the preceding element at
least n times.
P0{2,}1 matches:P001,
P0001, … but not P01
{n,m}
Matches the preceding element at
least n times, but not more than m
times.
P0{1,3}1 matches:P01
P001, and P0001, but not
P1 or P00001
()
The characters between parentheses
form a subexpression.
P(24)+ matches:P24,
P2424, P242424, ...
|
Matches the expression before or
after the vertical line. Mostly used
within a subexpression.
P(ab|cd)1 matches:Pab1
and Pcd1
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Syntax
Description
Example
^
A circumflex outside a bracket
expression anchors the element it
starts with to the beginning of a
string; such an element can match
only a sequence starting at the first
character of a string.
^(ec).* matches:ecoli,
ecoli_eftu but not
eftu_ecoli
$
A dollar sign outside a bracket
expression anchors the element it
terminates with to the end of a string;
such an element can match only a
sequence ending at the last
character of a string.
.*(ecoli)$ matches:ecoli,
eftu_ecoli but not
ecoli_eftu
Table 9-1. Regular expressions available to search patterns in labels. Please note that the
term element used in the description indicates a character or a subexpression.
The characters ^.[$()|*+?{\ have a special meaning in certain contexts. If your
labels contain any of these special characters, you must enter a backslash in
front of them if you want to include them as normal characters in your search
expression. You must also release the backslash character itself from the
expression. For example, the search pattern R\*.* returns the result R*3.24 but
not R/2.87.
Nevertheless, bracketed expressions are an exception to the rule. Inside
bracketed expressions, all special characters, including the backslash, lose their
special meaning (for instance, [*\+?{}.] matches exactly any of the characters
inside the brackets).
The order of rank for the regular expressions described above is as shown in
Table 9-2 . For example, the regular expression abc2|3de matches either the
string abc2 or the string 3de (rather than the string abc2de or abc3de) because
concatenation has a higher ranking order than alternation.
Escaped characters
\<Special character:
Bracket expression
[]
Grouping
()
Single-character
duplication
* + ? {m,n}
Concatenation
Anchoring
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9.7 Display annotations and labels
Alternation
|
Table 9-2. Ranking order (from high to low) for regular expressions.
9.6.3
Reports
You can select annotations by selecting their corresponding lines in the
Annotation Table, available by choosing Reports : Annotation Table.
9.7
Display annotations and labels
You can change the way annotations and labels are displayed.
9.7.1
Annotation flag position
Sometimes you may want to move an annotation flag because you are
preparing an illustration or want to see what lies underneath. To interactively
change an annotation's flag position, click on one of the labels and drag the flag
to the desired position (Figure 9-7).
(a)
(b)
Figure 9-7. (a) Default and (b) modified annotation flag position.
9.7.2
Visibility of annotations and labels
You can quickly cover entire gel images with a considerable number of
annotations and labels, which are not always necessary at any given moment
in the analysis. Therefore, you can hide all annotations or certain label
categories in selected gels. These options are available from the View :
Annotations menu:
•
160
Visible categories: Sets the visibility state of the various categories on the
selected gels. To hide a category, make sure its box is unchecked. On the
other hand, select an empty check box to show the corresponding
category. Click once or twice in a grayed check box (category that takes
different visibility states in the various selected images) to hide or show the
corresponding label category in the selected gels.
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•
Show All: Makes all the annotations on the selected gels visible, including
the labels that were hidden.
•
Hide All: Hides all the annotations on the selected gels, including the cyan
cross or square that remains visible when all labels, but not annotations,
are hidden.
When you click on an annotation that has hidden labels, all of its labels are
displayed on the screen during the time it remains selected (Figure 9-8). The
hidden labels disappear when the annotation is deselected.
(a)
(b)
(c)
(d)
Figure 9-8. (a) Unselected annotation with two labels. (b) Unselected annotation with 1
label hidden. (c) Unselected annotation with all labels hidden. (d) When the annotation is
selected the hidden labels become visible.
9.8
Annotation table
The Annotation Table (Figure 9-9), available by choosing Reports : Annotation
Table provides specific information about annotations, and consequently labels
and categories:
•
Name of the image on which the annotation was created.
•
SpotID, if the annotation is attached to a spot.
•
Coordinates of the annotation.
•
Calculated pI and MW values, if pI_MW annotations were defined on the
image, or a matched image.
•
A column for each label category, with the label content in the
corresponding cells.
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9.9 Edit annotations and labels
Figure 9-9. Annotation Table.
9.9
Edit annotations and labels
You can add and modify annotations and labels in different ways. The Select
tool is helpful if you want to add or modify a single or a few labels. The menu
options are more adapted to the creation of a large number of annotations or
labels simultaneously. Reports are useful for editing existing annotations.
9.9.1
Select
When double-clicking on a label while the Select tool is activated, the Edit Label
window is displayed. Change the text in this box to modify your label content.
The Select tool should also be used to change the position of an annotation. In
this case, simply select an annotation and drag its basis to the new location.
9.9.2
Edit menu
Various options for editing labels or annotations are available from the Edit :
Annotations menu. All of these features can be used to edit several labels or
annotations at a time.
Delete annotations and labels
Two possibilities are available for deleting selected labels or annotations:
•
Edit : Annotations : Delete deletes the selected annotations.
•
Edit : Annotations : Labels : Delete deletes the selected labels.
To delete labels from a specific category only, first select all labels from the
desired category with Select : Annotations : By Category.
Edit labels
You can change the content of selected labels (belonging to a single category)
by choosing Edit : Annotations : Labels : Delete and entering the desired
modifications in the Edit Labels window.
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Copy / paste annotations
You can select annotations in a given gel image and copy them to the
corresponding locations in other gels. This is a means of creating a set of similar
annotations in a series of gels. Subsequently, you may need to adjust the
annotation positions.
Select annotations on one image and choose Edit : Annotations : Labels : Copy.
Then select the gels into which you want to paste the annotations and choose
Edit : Annotations : Labels : Paste. Adjust the annotation positions using the
Select tool.
Copy / paste labels
Instead of copying entire annotations, you can also copy distinct labels from one
gel to selected spots or annotations in a series of gels.
Select the labels you would like to copy and choose Edit : Annotations : Labels
: Copy. Then select spots or existing annotations into which you want to paste
the labels and choose Edit : Annotations : Labels : Paste.
Propagate to matched
When gels have been matched, labels selected in one gel can be copied to their
corresponding spots in other gels by choosing Edit : Annotations : Labels :
Propagate to Matched. This is particularly useful when you have annotated one
image during analysis, and want to propagate the labels to all matched gels.
Duplicate labels
You can copy selected labels to another category by choosing Edit :
Annotations : Labels : Duplicate. Since the selected labels may belong to
different categories, this option can be used to merge several categories into a
new one. However, only one label per annotation can be duplicated at a time.
9.9.3
Annotation table
You can edit labels via the Annotation table, available from Reports :
Annotation Table. Make sure you have displayed the desired categories for
editing, by clicking the Settings icon.
Create labels and label categories
Click on the Add Label icon in the Annotation Table to create new labels for
selected spots. During this process, you can create a new label category, which
will be inserted as an extra column in the table.
Add or modify labels
You can directly add new labels to the appropriate cells of the Annotation Table,
or edit existing ones. Double-click in a cell to start typing your label, or modifying
an existing label. When finished, a single click in any cell quits the editing mode.
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9.9 Edit annotations and labels
Please note that categories using the Data Type Set are displayed in the form of
check boxes in the table. These boxes show if the corresponding item belongs
to the category (checked box) or not.
9.9.4
Import annotations
You can import annotations from a file into open gels. Choose Edit :
Annotations : Import to import annotations from an Annotation Report or a
tab-delimited text file containing the required columns: SpotID, X, Y, and a
column for each category to be imported.
If the Spot ID is not known, use -1 in this field (or remove this column) and the
software will position the label in the corresponding X and Y positions of the gel.
If X and Y positions are not known, use -1 in these fields and the software will
position the label on the spot with the corresponding Spot ID.
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10 Data integration
10.1
Introduction
Projects that were created with earlier versions of ImageMaster 2D Platinum
can be imported to version 7.0. It is also possible to import images from TWAIN
compatible scanners. After analysis, data can be exported as reports or to spot
excision robots.
10.2
Convert projects from earlier software versions
ImageMaster can
Convert Projects created with versions 4, 5 and 6 of
ImageMaster 2D Platinum. Images analyzed with older versions must be added
to a project in order to be imported into this new version and for spots,
annotations and match data to be recovered. You can do this batch conversion
for many projects at a time.
Choose File : Import : ImageMaster 2D Platinum or Melanie Data, and indicate
the folder where some or all of your projects (.prj files) are saved. The software
searches and displays all project files found in this folder, and allows you to
select projects for conversion (Figure 10-1). Then you must indicate the location
where the converted projects should be saved. After conversion, the projects are
automatically inserted in the workspace.
Figure 10-1. Select projects from previous ImageMaster versions for Batch File
Conversion.
10.3
Acquire images from Twain compatible scanners
ImageMaster can also acquire images directly from TWAIN compatible
scanners.
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10.4 Export data
10.3.1
Select source
You must indicate the scanning source by choosing File : Import : Twain : Select
Source. All TWAIN compatible scanners attached to your PC are automatically
recognized by ImageMaster. This selection only needs to be done once (unless
you want to change to a new image capture device).
10.3.2
Acquire
Launch the scan with File : Import : Twain : Acquire. The scanner software
opens, giving you the opportunity to change the necessary settings, and
subsequently initiate the scanning process. Once this is done, the image is
saved in ImageMaster file format and added to the Image Pool.
10.4
Export data
10.4.1
Reports
Data obtained in the analysis can be exported for use in other applications by
saving the various tabular reports. Tables can be saved in text format (.txt), as a
Microsoft Excel Workbook (.xls), or in XML (.xml) format. See below for more
details on XML.
Graphical reports can be saved in PNG, TIFF or BMP formats.
10.4.2
XML
XML stands for eXtensible Markup Language and was created as a crossplatform, software, and hardware independent tool to structure, store, and
exchange information. It allows the creation of customized tags, enabling the
definition, transmission, validation, and interpretation of data between
applications and organizations.
XML files can be viewed in the latest versions of Web browsers such as Internet
Explorer, Netscape and Mozilla Firefox. However, as XML was designed to
describe data and not to display data, it does not look like a Web page. An XML
document contains color-coded root and child elements. A plus (+) or minus (-)
sign to the left of the elements can be clicked to expand or collapse the element
structure. If you want to view the raw XML source, you must select View : Source
from the browser menu.
XML does not use predefined tags, as is the case for HTML. Therefore, the
browser does not understand the meaning of the tags and does not know how
to display the XML document. Therefore, XSL (eXtensible Stylesheet Language)
stylesheets must be used in addition to the XML document to transform the XML
into the sort of document that is recognized by the browser. This is the case
when tabular reports or the history are printed from ImageMaster. The software
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uses the XSL stylesheets located in the Template folder of the ImageMaster
installation directory to print attractive documents. If you are familiar with XML
and XSL, you can even create personalized templates for printing. Note that XSL
stylesheets can also be used to convert an XML file into another XML file.
The main interest in XML format is that external applications can easily extract
necessary data. Moreover, the files can be converted to other user-defined
formats.
Note: Because the XSL stylesheets are specific to the browser you use, you will
find that different versions (both for printing reports and the history) are
installed with the software (in the Template\Report and Template\Script
folders of the installation directory). ImageMaster will therefore ask you to
choose the appropriate XSL template each time you print a report or
history. Look at which template works with your browser and delete the
other one. Next time, the software automatically opens the remaining file
and does not ask you to make a choice
10.4.3
Gel and report identifiers
ImageMaster allocates a unique identifier (ID) to each project, match set, gel
image, etc. This is useful to assure data consistency, allow reliable identification
of objects across a computer network and enable database integration.
The IDs in ImageMaster are UUIDs (Universal Unique IDentifiers), which are 128bit numbers that are guaranteed to be unique through combinations of
hardware addresses, time stamps and random seeds. These IDs allow the
ImageMaster objects to be uniquely recognized. The software can therefore
detect, for example, if you delete a gel and replace it with another one with the
same name.
Note: You must be very careful when manipulating project, match set, gel and
other ImageMaster files outside the software, in order not to corrupt the
data.
10.5
Export to spot excision robots
You can export spots to an excision robot. In addition, it may be useful to save
them as part of a spot set, or annotate them. This allows you to easily select
them later on to add experimental data such as mass spectrometry
information.
10.5.1
GE Healthcare Ettan spot picker
To use the Ettan Spot Picker, two adhesive markers should be placed on the gel
before scanning. These markers are used for the calibration of the coordinates,
that is, for determining the correspondence between the X and Y positions of the
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10.5 Export to spot excision robots
analyzed gel image and the coordinates of the actual gel located on the spot
picker (Figure 10-2).
Once the gel has been digitized and analyzed with ImageMaster, the software
can generate a pick list. This list contains the location, in pixels, of the center of
each spot you wish to pick, as well as the pixel coordinates for the centers of the
two reference markers.
To export a file with spot coordinates for use by the spot picker, you first have to
open the image files and perform image analysis (spot detection is mandatory).
You should then annotate the reference markers on the marker spot or on the
pixel in the center of the marker. The basis of an annotation on the marker spot
is displayed as a small square, while a pixel annotation has a crossed basis.
Note: The two annotation options can be used in a single gel (one marker with
a spot annotation and the other with a pixel annotation), as long as the
annotations are centered on the markers.
To create a pick list:
1
Identify the two reference markers on your image.
2
Zoom the image to better see the left reference marker.
3
Check if the marker is detected as a nice, round spot. If it is not, select the
spot(s) on the marker and delete by choosing Edit : Spots : Delete.
4
Click on the Select tool.
5
Double-click on the marker spot, or if such a spot is not present, on the pixel
that is in the middle of the marker.
6
Select the Comment category.
7
Enter IR1 as the label text.
8
If an annotation attached to a pixel is not in the center of the reference
marker, you can move it to the appropriate position. Do this by clicking on
its basis (cross) and holding down the left mouse button while dragging the
annotation to its new position.
9
Move your gel to see the second (right) reference marker.
10 Repeat the procedure, but enter the label text IR2 this time.
11 Once the two markers have been annotated, select spots to pick (Figure
10-2).
12 Choose File : Export : Spots to Picker : GE Healthcare Ettan.
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13 For each gel, you will be asked to save a pick list in text or XML format (only
the text file can be read by the Ettan Spot Picker).
Figure 10-2. Reference markers IR1 and IR2. IR1 is attached to a pixel (cross basis). IR2 is
attached to a spot. Both options can be used in a single gel. Spots to be picked are
selected (highlighted in green).
10.5.2
Spot pickers from other manufacturers
The software can also export spots to spot pickers from Bruker Daltonics,
Genetix and Genomic Solutions.
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10 Data integration
10.5 Export to spot excision robots
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Undo, redo and history 11
11 Undo, redo and history
11.1
Undo, redo
The software allows you to cancel any unwanted modifications to the images
or layout of the current sheet by choosing Edit : Undo in the menu (Figure 11-1).
Figure 11-1. Undo window.
Each action in the Undo/Redo list is registered along with the time at which it
was carried out.
By default, all actions are displayed. If you specifically want to track permanent
changes that have been made to the image data (essentially the commands
available under the Edit menu), choose Only Edit from the Filter drop-down list.
All operations performed can be reversed except for modifications to the
Workspace and functionalities linked to opening and saving files. This includes
image rotation, flipping, cropping, and the inversion of gray levels.
To undo specific actions:
1
Choose Edit : Undo.
2
Select a prior action to be undone.
3
Click OK. The selected action and all following actions are undone
automatically.
If you are not satisfied with your latest undo or you canceled too many
operations, you can restore the actions.
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11 Undo, redo and history
11.2 History
To redo specific actions:
11.2
1
Choose Edit : Redo.
2
Select the action to be redone.
3
Click OK. The selected action and any previous actions are redone.
History
You can display a history of the modifications that have been carried out during
the present work session on the images or layout of the current sheet. Choose
Edit : History : Show to open the History window (Figure 11-2).
Figure 11-2. History window.
Please note that the action names displayed in the History window are the same
as those used in Undo/Redo. As for Undo/Redo, you can choose Only Edit from
the Filter drop-down list to restrict the displayed actions to those that bring
about permanent modifications to the image data.
Some actions are preceded by a + or - node allowing the item to be expanded
or collapsed. Once an action is expanded (by clicking on the plus sign), you see
parameters and values that further describe the action.
Modifications to the Workspace and functionalities linked to opening and
saving files (including image rotation, flipping, cropping, and inversion of gray
levels) are not included in the History.
Insert marker
You can place a marker in the History by choosing Edit : History : Insert Marker.
Clear
Clear the list of actions in the History by choosing Edit : History : Clear.
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Undo, redo and history 11
Refresh
The content of the History is not automatically updated when you work on the
images while the History window is still open. Click the Refresh icon to update
the list.
Save, print, copy
By using the options in the Save icon drop-down list in the History window, the
content of the History can be:
•
Saved in an XML type file with the extension .hst.
•
Printed. It will first display in your Web browser using the XSL stylesheet
located in the Template\Script folder of the ImageMaster installation
directory. Use the print option in your browser to get a paper copy.
•
Copied to the clipboard.
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11 Undo, redo and history
11.2 History
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Shortcuts A
Appendix A
Shortcuts
A.1
Shortcut keys
Several menu commands can be activated by keyboard shortcuts. They are
indicated to the right of the corresponding command in the menus. Please note
the logic behind the key combinations:
Ctrl for gels.
Shift for spots.
Alt for annotations.
Ctrl + Shift for matches.
Some exceptions do exist. The most important ones are the following two
shortcuts used for undoing and redoing actions carried out on a gel.
Shortcut
Menu Command
Ctrl+Z
Edit : Undo...
Ctrl +Shift+Z
Edit : Redo...
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A Shortcuts
A.2 Tool shortcuts
A.2
Shortcut
Tool
Ctrl+1
Move
Ctrl+2
Zoom
Ctrl+3
Region
Ctrl+4
Selection
Ctrl+5
Measure
Ctrl+6
Landmark
A.3
176
Tool shortcuts
Gel shortcuts
Shortcut
Menu Command
Ctrl+F
View : Sheet : Navigation : Switch
Ctrl+I
View : Global : Grid Lines : Show
Ctrl+J
View : Global : Grid Lines : Edit…
Ctrl+O
File : Open…
Ctrl+P
View : Global : Show Profile
Ctrl+S
File : Save
Ctrl+W
File : Close : Sheet
Ctrl+<Down:
View : Gels : Navigation : Move : Down
Ctrl+<Left:
View : Gels : Navigation : Move : Left
Ctrl+<Right:
View : Gels : Navigation : Move : Right
Ctrl+<Up:
View : Gels : Navigation : Move : Up
F2
View : Show All
F3
View : Hide All
F5
Select : Unselect All
Page Down
View : Sheet : Navigation : Previous Page
Page Up
View : Sheet : Navigation : Next Page
Shift+<Down:
View : Gels : Navigation : Zoom : Out
Shift+<Up:
View : Gels : Navigation : Zoom : In
ImageMaster User Manual 28-9381-02 Edition AA
Shortcuts A
A.4
Spot shortcuts
Shortcut
Menu Command
Shift+1
View : Spots : Crossed
Shift+2
View : Spots : Outlined
Shift+3
View : Spots : None
Shift+A
Select : Spots : All
Shift+E
Edit : Spots : Edit Enabled
Shift+N
Select : Spots : Inverse Selection
Shift+X
Edit : Spots : Delete
A.5
Annotation shortcuts
Shortcut
Menu Command
Alt+A
Select : Annotations : All
Alt+C
Edit : Annotations : Labels : Copy
Alt+D
Edit : Annotations : Delete
Alt+E
Edit : Annotations : Labels : Edit…
Alt+F
Edit : Annotations : Add…
Alt+H
View : Annotations : Hide All
Alt+J
View : Annotations : Visible Categories…
Alt+L
Edit : Annotations : Link with Spot
Alt+U
Edit : Annotations : Unlink from Spot
Alt+V
Edit : Annotations : Labels : Paste
Alt+X
Edit : Annotations : Labels : Delete
Alt+Y
View : Annotations : Show All
A.6
Match shortcuts
Shortcut
Menu Command
Ctrl+Shift+A
Select : Matches : All
Ctrl+Shift+G
Edit : Matches : Add Match
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A Shortcuts
A.6 Match shortcuts
178
Shortcut
Menu Command
Ctrl+Shift+J
View : Matches : Show ID
Ctrl+Shift+K
View : Matches : Hide All ID
Ctrl+Shift+N
Select : Matches : Inverse Selection
Ctrl+Shift+U
Edit : Matches : Delete Match
Ctrl+Shift+Y
View : Matches : Show Vectors
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References B
Appendix B
References
B.1
Software
Appel RD, Hochstrasser DF, Roch C, Funk M, Muller AF and Pellegrini C (1988)
Automatic classification of two-dimensional gel electrophoresis pictures by
heuristic clustering analysis: A step toward machine learning. Electrophoresis 9:
136-142.
Appel RD, Hochstrasser DF, Funk M, Vargas JR, Pellegrini C, Muller AF and
Scherrer J-R (1991) The MELANIE project - from a biopsy to automatic protein
map interpretation by computer. Electrophoresis 12: 722-735.
Appel RD, Palagi PM, Walther D, Vargas JR, Sanchez J-C, Ravier F, Pasquali C and
Hochstrasser DF (1997) Melanie II - a third generation software package for
analysis of two-dimensional electrophoresis images: I. Features and user
interface. Electrophoresis 18: 2724-2734.
Appel RD, Vargas JR, Palagi PM, Walther D and Hochstrasser DF (1997) Melanie
II - a third generation software package for analysis of two-dimensional
electrophoresis images: II. Algorithms. Electrophoresis 18: 2735-2748.
Appel RD and Hochstrasser DF (1998) Computer analysis of 2-D images. In: Link
AJ (ed) Methods in Molecular Biology, Vol 112: 2-D Protocols for Proteome
Analysis, pp 363-381. Totowa NJ: Humana Press.
Miller MJ, Olson AD and Thorgeirsson SS (1984) Computer analysis of twodimensional gels: automatic matching. Electrophoresis 5: 297-303.
Pun T, Hochstrasser DF, Appel RD, Funk M, Villars-Augsburger V and Pellegrini C
(1988) Computerized classification of two-dimensional gel
electrophoretograms by correspondence analysis and ascendant hierarchical
clustering. Applied and Theoretical Electrophoresis 1: 3-9.
Vargas RJ (1996) Two-dimensional gel electrophoresis computer analysis
systems: from image acquisition to protein identification. Ph.D. thesis, Faculty of
Science, Geneva University.
Wilkins MC, Hochstrasser DF, Sanchez J-C, Bairoch A and Appel RD (1996)
Integrating two-dimensional gel databases using the Melanie II software.
Trends in Biochemical Sciences 21: 496-497.
B.2
Statistical methods
Armitage P and Berry G (1987) Statistical methods and medical research. Oxford,
London: Blackwell Scientific Publications.
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179
B References
B.3 Further reading
Kim JO and Mueller CW (1978) Introduction to factor analysis: What is and how
to do it? Newbury Park: Sage Publications.
Tabachnick B and Fidell LS (1996) Using multivariate statistics (3rd edition). New
York: Harper Collins College Publishers.
B.3
Further reading
Appel RD, Bairoch A, Sanchez J-C, Vargas JR, Golaz O, Pasquali C and
Hochstrasser DH (1996) Federated 2-DE database: a simple means of publishing
2-DE data. Electrophoresis 17: 540-546.
Appel RD, Sanchez J-C, Bairoch A, Golaz O, Ravier F, Pasquali C, Hughes GJ and
Hochstrasser DF (1996) The SWISS-2DPAGE database of two-dimensional
polyacrylamide gel electrophoresis. Nucleic Acids Research 22: 3581-3582.
Binz PA, Mueller M, Walther D, Bienvenut WV, Gras R, Hoogland C, Bouchet G,
Gasteiger E, Fabbretti R, Gay S, Palagi P, Wilkins MR, Rouge V, Tonella L, Paesano
S, Rossellat G, Karmime A, Bairoch A, Sanchez JC, Appel RD and Hochstrasser DF
(1999) A molecular scanner to automate proteomic research and to display
proteome images. Analytical Chemistry 71: 4981-4988.
Binz PA, Wilkins MR, Gasteiger E, Bairoch A, Appel RD and Hochstrasser DF (1999)
Internet resources for protein identification and characterization. In: Kellner R,
Lottspeich F, Meyer HE (eds) Microcharacterization of Proteins, 2nd ed., pp. 277300. Weinheim: Wiley-VCH.
Hoogland C, Baujard V, Sanchez J-C, Hochstrasser DF and Appel RD (1997)
Make2ddb: a simple package to set up a 2-DE database on the WWW.
Electrophoresis 18: 2755-2758.
Hoogland C, Sanchez J-C, Bairoch A, Hochstrasser DF and Appel RD (1999) The
SWISS-2DPAGE database: what has changed during the last year. Nucleic Acids
Research 27: 289-291.
Link AJ (ed) (1998) Methods in molecular biology, Vol 112: 2-D Protocols for
Proteome Analysis. Totowa NJ: Humana Press.
Lopez MF (2000) Better approaches to finding the needle in a haystack:
optimizing proteome analysis through automation. Electrophoresis 21: 10821093.
Sanchez J-C, Wilkins M, Appel RD and Hochstrasser DF (1997) Identifying
proteins for proteome studies. In: Creighton ET (ed) Protein Function: a practical
approach, 2nd ed., pp. 1–27. IRL Press.
Wilkins MR, Williams KL, Appel RD and Hochstrasser DF (eds) (1997) Proteome
research: new frontiers in functional genomics. Berlin Heidelberg: Springer
Verlag.
180
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References B
Unlu M, Morgan ME, and Minden JS (1997) Difference gel electrophoresis: a
single gel method for detecting changes in protein extracts. Electrophoresis 18:
2071–2077.
Tonge R, Shaw J, Middleton B, Rowlinson R, Rayner S, Young J, Pognan F,
Hawkins E, Currie I and Davison M (2001) Validation and development of
fluorescence two-dimensional differential gel electrophoresis proteomics
technology. Proteomics 1: 377–396.
ImageMaster User Manual 28-9381-02 Edition AA
181
B References
B.3 Further reading
182
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Index
Symbols
% 74
%vol 89
.bmp 85
.gda 64
.mda 64
.png 85
.prj 64
.tif 85
Numerics
3D view 75
animate 78
auto rotate 77
color palette 78
display options 78
next/previous 78
rotation 77
set reference 78
show 77
spot shape 78
stack 78
tools 77
translation 77
transparency settings 78
transparent mode 78
visibility 78
zoom/contrast 77
A
access code 11
adaptive gradations 124, 144
add
files to project 57, 65
labels 163
matches 113
spots 96
adjust
contrast 71
region contrast 75
algorithm 89
matching 105
align images 80
analysis
class 117
gel 117
analyze
classes 28, 117, 135
gels 117, 120
animate 78
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183
annotations 147
copy 163
create 147
create categories 163
delete 162
display 160
flag position 160
hide 160
import 164
MW and pI 100
options 34
paste 163
report 160, 161, 163
select 156
shortcuts 177
show 160
table 28
tool 156
apply
calibration 48
region to all gels 70
zoom to all gels 67
area 89, 134
auto rotate 77
automatic matching 110
autonumbering 150
B
backup 65
BMP 85
bookmarks
create 71
delete 71
load 71
Bruker Proteineer SP spot picker 167
C
calibration 37
apply 48
control 48
copy to clipboard 47
create 44
fitting table 47
images 44
MW and pI 100
open 47
print 47
remove 48
save 47
table 47
tablet file 44
categories 163
center 141
central tendency 118, 123, 141
184
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CGI script 151
choose matchset 114
classes
analysis 135
analysis histograms 143
analysis table 141
create 63
folder 55
co-detection 91
coef. variation 123
coefficient variation 123
collect eLicense 12
color palette 78
colors 75
combine spot sets 94
command prompt 11
common labels 157
compare images 80
composite spots 98
concurrent license 11
contact
information 14
us 14
contextual menus 27
contrast 74, 77
control calibration 48
copy
annotations 163
formula to clipboard 120
labels 163
to clipboard 29
Corr 120
correlation coefficient 120
create
annotation categories 163
annotations 147
calibration 44
gel descriptions category 49
label categories 149
labels 147
match hierarchy 58
pick list 168
projects 56
specific link 154
spot set 124, 142
spot sets 94
spots 97
toolbar 21
crop 41
area 41
export area 41
import area 41
current sheet 27
cursor information 79, 102
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185
customize
reports 30
toolbars 21
D
data
analysis 117
type 150
database options 34
DD 44
DeCyder 2D algorithm 87
default mapping 73
define landmarks 107
delete
annotations 162
labels 162
matches 113
spot sets 94
spots 97
describe images 49
descriptions 49
desktop shortcut 13
DIGE 9, 87
co-detect spots 91
exclude spots 93
file naming convention 37
histogram 133
spot detection parameter 92
spot quantification 92
value 117
disable spots 96
dispersion 119, 123, 141
display
annotations 160
calibration information 44
gel descriptions 49
labels 160
match hierarchy 106
matches 111
options 34, 78
projects 63
properties 151
spots 97
zone 17, 22
displayed
items 129
value 142, 144
dockable windows 22
floating 33
minimize 33
pinned 33
tabbed groups 33
un-pinned 33
double detection 91
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dual color 81
duplicate labels 163
E
EDF 140
edit 17
annotations 163
enabled 96
gel descriptions 49
grid lines 83
matches 113
region 70
spots 97
table cells 32
eLicense 10
collect 12
file 12
place 12
server 11
test 13
empirical distribution
function 140
plot 140
enabled spots 96
ExPASy 154
export 85, 166
crop area 41
match set 62
spot coordinate file 167
spot excision robot 167
external engine 151
external protein databases
query 153
set 152
F
factor analysis 127, 135
comments 132
interpret 130
table 124, 129
factor projection
plot 129
table 129
files 17
format 37
link 154
names 37
first time software launched 56
fitting table 47
flag position 160
FLEXlm license finder 13
flip 41
floating 33
floating license 11
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187
free 22, 23
rotate 41
further reading 180
G
gap 135, 141
Gaussian distribution 137
GE Healthcare Ettan spot picker 167
gels 67
analysis histograms 124
analysis table 123
description options 34
descriptions 49
export 85
ID 167
print 85
reports 84
save 85
shortcuts 176
table 28, 49, 84
general settings 117
good images 37
graphical user interface 17
gray level mapping 72
gray+saturation 75
grid lines
display 83
edit 83
show 83
grow spots 97
GUI 17
H
half-range size 119
help 17
hide
annotations 160
ID 112
labels 160
histograms
class analysis 143
DIGE 133
gel analysis 124
history 172
clear 172
copy 172
insert marker 172
print 172
refresh 172
save 172
http
link 154
queries 151
188
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I
image pool 22, 39
display 39
hide 39
properties 39
remove 39
sheet 39
images 67
calibrate 44
calibration 37
compare 80
depth 37
describe 49
editing 37
export 85
file format 37
file names 37
good 37
manipulate 67
open 38
preview 38
print 85, 86
process 41
resolution 37
save 85
selection 24
to clipboard 86
to file 86
import 164, 166
annotations 164
crop area 41
labels 164
match set 62
install
eLicense server 11
software 10
installation cd-rom 10
installer 10
intensity 74, 89
interface 17
interpret a factor analysis 130
invert 75
gray levels 41
ipconfig 11
is unique 150
K
keyboard shortcuts 18
Kolmogorov-Smirnov 140
L
labels 164
add 163
copy 163
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create 147
create categories 149
delete 162
display 160
duplicate 163
hide 160
modify 163
paste 163
report 160, 161
select 156
show 160
landmarks
define 107
tool 107
launch
software 13
software first time 56
license
concurrent 11
floating 11
information 14
machine 10
node-locked 10
LMTOOLS 11
load spots 96
log volume ratio 133
M
machine license 10
MAD 119
manage projects 65
manipulate images 67
Mann-Whitney 138
manual editing 97
mapping
default 73
gray levels 72
match
count 110, 123, 141
ID 112
match sets
existing 61
export 62
import 62
merge 59
matches
add 113
create hierarchy 58
delete 113
display 111
display hierarchy 106
displayed 129
edit 113
gels tool 110
190
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hierarchy 53, 105
multiple 113
reference 105
report 114
review 113
shortcuts 177
statistics table 114
vectors 111
matching 105
automatic 110
reference 59
max 123, 141
intensity 134
slope 134
volume 134
mean 118
absolute deviation 119
squared deviation 119
measure 70, 133
measures overlapping 135
median 118
menu
bar 17
select 157
merge
match set 59
spots 97
midrange 118
min area 88
minimize 33
modify labels 163
move
alternatives 67
double-click 67
projects 63
tool 67
MSD 119
multiple matches 113
MW 100
N
navigator 25
contextual menus 27
icons 27
next in selection 29
next/previous 78
node-locked license 10
non-DIGE 9, 87
detect spots 87
spot detection parameters 88
spot quantification 89
normalization 124
normalized 124, 126, 135, 141
number 150
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191
O
OD 44
one
column 22, 23
row 22, 23
one-way ANOVA 137
open images 38
options 34, 117
print 86
overlapping
measures 135
spots 82
overview option 68
P
page setup 86
panes 22
close 23
free 23
layout 23
one column 23
one row 23
selection 23
stacked 23
tiled 23
paste
annotations 163
labels 163
physical address 11
pI 100
pick list 168
pinned 33
pixel distances 70
place eLicense 12
plots
empirical distribution 140
scatter 120
PNG 85
populations 55
preview images 38
previous in selection 29
print 29, 85
images 86
options 86
sheets 86
process images 41
product information 14
profile 78
projectname.prj 64
projects
add 65
backup 65
convert data to new version 165
create 56
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display 63
folder 64
import older versions 165
manage 65
move 63
properties 63
remove 63, 65
restore 65
save 64
propagate spots 99
properties 63
purchase
software 9
Q
quantification
options 34
value 117
query external protein databases 153
R
range ratio 123
ratio 124, 126, 135, 141
raw images 64
recycle bin 64
redo 171
reference 118
match 59
set 80
sheet 80
references 180
software 179
statistical methods 179
region
adjust contrast 75
apply to all gels 70
edit 70
tool 69
regular expressions 157
relative 124, 126
remove
calibration 48
projects 63
spots 96
rename category 163
reports 17, 163
3D view 28, 75
analyze classes 28
analyze gels 28
annotation 161
annotations 160
columns 31
customize 30
edit labels 163
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193
export 166
factor projection 127
gel 84
gel analysis histograms 124
ID 167
import 166
labels 160, 161
match 114
spot 102
toolbar 29
resolution 37
restore 65
review matches 113
rotate 41
rotation 77
S
saliency 88
save 29, 85
projects 64
spots 96
scatter
plots 120
table 120
scrollbars 24
select 17, 94
all 157
annotations 156
by category 157
by content 157
by value 29, 93, 124, 142
common labels 157
enabled spots 96
labels 156
matches 110
menu 157
spot sets 96
tool 110, 156
selected region 75
separability 123
set 150
external protein databases 152
reference 59, 78, 80
spots 96
settings 29, 124, 142, 144
setup wizard 10
sheets 22
align images 80
close 22
current 27
free 22
image pool 39
layout 22
one column 22
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one row 22
print 86
reference 80
selection 22
stacked 22
tiled 22
to clipboard 86
to file 86
shortcuts 175
annotations 177
gels 176
matches 177
redo 175
spots 177
tools 176
undo 175
show
annotations 160
default 3D view 77
dual color 81
ID 112
labels 124, 160
overview 68
profile 78
vectors 111
shrink spots 97
signal intensity 71
single detection 91
slider 120, 124, 133, 144
smooth 88
software references 179
sorted values 124, 144
specific links
create 154
file 154
http 154
text 154
specify classes 135
spot
coordinate file 167
excision robots 167
ID 102
spot pickers 167
Bruker Proteineer SP 167
GE Healthcare Ettan 167
spots 87
add 96
co-detection parameter 92
color 97
composite 98
create 97
delete 97
detection algorithm 87
DIGE co-detection 91
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195
disable 96
display 97
edit 97
enabled 96
frequency 133
grow 97
load 96
manual editing 97
merge 97
non-DIGE detection 87
overlapped 82
propagate 99
remove 96
report 102
save 96
select 94
sets 94
shape 78, 97
shortcuts 177
shrink 97
table 28, 102
stack 78
stacked 22, 23
statistical methods references 179
statistics 117, 124, 142, 144
tests 137
status bar 17, 22
step tablets 44
student’s t-test 137
suspend synchronization 29
switch order 24
system
information 14
requirements 9
T
tabbed groups 33
tables
annotation 28
calibration 47
class analysis 141
edit cells 32
factor analysis 127
fitting 47
gel 28, 84
gel analysis 123
match statistics 114
scatter 120, 122
spot 28, 102
tests
ANOVA 137
Kolmogorov-Smirnov 137, 140
Mann-Whitney 137, 138
student’s t 137
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Wilcoxon 138
text 150
link 154
TIFF 85
tiled 22, 23
toolbar 17, 20
create 21
format 21
position 21
tools 17
add files to project 57
adjust contrast 71
annotation 156
cursor information 79
landmark 107
match gels 110
measure 70
move 67
profile 78
region 69
select 94, 110, 156
shortcuts 176
show dual color 81
zoom 67
translation 77
transparency settings 78
transparent mode 78
trimmed
mean 118
midrange 118
triple detection 91
t-test 137
Twain compatible scanners 165
U
undo 171
unit 74
un-pinned 33
update gels 164
UUID 167
V
value 117, 124, 126
vectors 111
view 17
signal intensity 71
visibility 78
vol 89, 92
ratio 92
W
whole image 75
Wilcoxon 138
workspace 22
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197
class folder 63
classes 63
match folder 58
match hierarchy 58
navigator 25
projects 56
toolbar 25
X
XML format 166
Z
zoom 67, 77
alternatives 68
apply to all gels 67
overview option 68
198
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For contact information for your local office,
please visit
www.gelifesciences.com/contact
GE Healthcare Bio-Sciences AB
Björkgatan 30
751 84 Uppsala
Sweden
www.gelifesciences.com
GE, imagination at work and GE monogram are trademarks of General
Electric Company.
Cy, CyDye, DeCyder, Ettan, ImageMaster, Immobiline, LabScan and
ImageScanner are trademarks of GE Healthcare companies.
Any use of this software is subject to GE Healthcare Standard Software EndUser License Agreement for Life Sciences Software Products. A copy of this
Standard Software End-User License Agreement is available on request.
This version of ImageMaster has been developed by the Swiss Institute of
Bioinformatics in collaboration with GeneBio and GE Healthcare.
All intellectual property rights on the ImageMaster Platinum Software
belong to the Swiss Instiute of Bioinformatics.
© 2008 Swiss Institute of Bioinformatics - All rights reserved. Reproduced by
permission of the owner.
Swiss Institute of Bioinformatics
CMU-Rue Michel-Servet 1, CH-1211 Geneva, Switzerland
ImageMaster 2D Platinum uses the DeCyder co-detection algorithm.
© 2005 General Electric Company – All rights reserved.
ImageMaster 2D Platinum uses the TIFF library.
© 1988-1999 Sam Leffler and 1991-1999 Silicon Graphics, Inc - All rights
reserved.
ImageMaster 2D Platinum uses software developed by the Apache
Software Foundation (www.apache.org).
© 1999-2003 The Apache Software Foundation - All rights reserved.
All third party trademarks are the property of their respective owners.
All goods and services are sold subject to the terms and conditions of sale
of the company within GE Healthcare which supplies them. A copy of these
terms and conditions is available on request. Contact your local GE
Healthcare representative for the most current information.
GE Healthcare UK Ltd
Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK
GE Healthcare Bio-Sciences Corp
800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA
GE Healthcare Europe GmbH
Munzinger Strasse 5, D-79111 Freiburg, Germany
GE Healthcare Bio-Sciences KK
Sanken Bldg. 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan
imagination at work
28-9381-02 AA 06/2008
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