ImageMaster 2D Platinum6 User Manual

ImageMaster 2D Platinum6 User Manual
GE Healthcare
ImageMaster 2D Platinum 6.0
User Manual
Contents
1
Introduction
1.1
1.2
1.3
1.3.1
1.3.2
1.3.3
1.3.4
1.4
1.4.1
1.4.2
1.4.3
1.4.4
2
User manual ............................................................................................................ 2
Tutorials..................................................................................................................... 2
Online help ............................................................................................................... 2
Conventions used.................................................................................................. 3
Getting further assistance ........................................................................... 3
How to contact us................................................................................................. 3
Sending e-mail........................................................................................................ 5
System and product information................................................................... 5
GE Healthcare listens........................................................................................... 5
Two-dimensional gel analysis
2.1
2.1.1
2.1.2
2.1.3
2.2
2.2.1
2.2.2
2.3
2.3.1
2.3.2
2.4
2.4.1
2.4.2
2.4.3
2.4.4
2.4.5
2.4.6
2.4.7
2.4.8
2.4.9
2.4.10
2.5
3
About ImageMaster 2D Platinum ............................................................. 1
What’s new in this release? ......................................................................... 1
ImageMaster resources ................................................................................ 2
Two-dimensional electrophoresis ............................................................ 7
Technique ................................................................................................................. 7
Applications ............................................................................................................. 7
Advantages and limitations.............................................................................. 7
Differential gel electrophoresis .................................................................. 8
Technique ................................................................................................................. 8
Internal standard................................................................................................ 10
Understanding gel images .........................................................................11
Anatomy of gel images ................................................................................... 11
Spot and gel relationships.............................................................................. 12
Image analysis workflow ............................................................................13
Acquiring data ..................................................................................................... 13
Setting up a workspace................................................................................... 13
Visualizing and calibrating gels ................................................................... 14
Detecting and quantifying spots................................................................. 14
Annotating spots and pixels.......................................................................... 14
Matching gels....................................................................................................... 14
Analyzing data .................................................................................................... 14
Integrating data.................................................................................................. 14
Reporting results ................................................................................................ 15
Controlling and automating gel analyses............................................... 15
ImageMaster flavors .....................................................................................15
Getting started
3.1
3.1.1
3.1.2
3.2
Installing ImageMaster 2D Platinum .....................................................17
System requirements ....................................................................................... 17
Software installation......................................................................................... 17
eLicensing ..........................................................................................................18
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Contents
3.2.1
3.2.2
3.2.3
3.2.4
3.2.5
3.3
3.4
3.4.1
3.4.2
3.4.3
3.4.4
3.5
4
Starting and exiting ImageMaster ......................................................... 21
ImageMaster window .................................................................................. 22
Menu bar ................................................................................................................ 23
Toolbar .................................................................................................................... 24
Display zone ......................................................................................................... 24
Dockable windows............................................................................................. 28
ImageMaster options ................................................................................... 30
Workspace
4.1
4.2
4.2.1
4.2.2
4.2.3
4.2.4
4.3
4.3.1
4.3.2
4.3.3
4.4
4.4.1
4.4.2
4.4.3
4.4.4
4.5
4.5.1
4.5.2
4.5.3
4.5.4
4.5.5
4.5.6
4.5.7
4.5.8
4.5.9
4.5.10
4.6
4.6.1
4.6.2
4.6.3
4.6.4
4.6.5
4.6.6
4.6.7
iv
Access code........................................................................................................... 18
Finding the physical address of the computer...................................... 18
Installing the GE Healthcare eLicense server ......................................... 19
Collecting and placing an eLicense file..................................................... 20
Test the eLicense................................................................................................. 21
Introduction ...................................................................................................... 33
Workspace window ...................................................................................... 35
Workspace toolbar............................................................................................. 36
Active project........................................................................................................ 36
Navigator ............................................................................................................... 37
File details .............................................................................................................. 37
Setting up a workspace .............................................................................. 37
Creating a workspace ...................................................................................... 38
Opening an existing workspace................................................................... 38
Workspace properties ...................................................................................... 38
Working with projects ................................................................................. 39
Creating a project............................................................................................... 39
Inserting an existing project .......................................................................... 40
Exporting a project............................................................................................. 40
Project properties ............................................................................................... 40
Working with gels .......................................................................................... 41
Creating a subfolder.......................................................................................... 41
Importing a new gel .......................................................................................... 42
Importing a new DIGE gel............................................................................... 43
Adding an existing gel ...................................................................................... 43
Adding an existing DIGE gel........................................................................... 44
Moving gels or folders ...................................................................................... 44
Displaying gel images ...................................................................................... 44
Spot detection ...................................................................................................... 45
Folder properties................................................................................................. 45
Gel properties....................................................................................................... 45
Working with match sets ........................................................................... 46
Creating a match set......................................................................................... 49
Creating a match set from a DIGE gel....................................................... 50
Adding gels to a match set............................................................................. 51
Copying match sets........................................................................................... 51
Setting the match set reference ................................................................... 52
Moving gels or match sets.............................................................................. 52
Displaying match sets...................................................................................... 52
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Contents
4.6.8
4.6.9
4.7
4.7.1
4.7.2
4.7.3
4.7.4
4.7.5
4.7.6
4.8
4.8.1
4.8.2
4.8.3
4.8.4
4.9
Creating a class................................................................................................... 56
Adding gels to a class....................................................................................... 57
Moving gels or classes ..................................................................................... 58
Displaying classes ............................................................................................. 58
Statistical analysis ............................................................................................. 58
Class properties .................................................................................................. 58
Working with reports ....................................................................................59
Creating a subfolder ......................................................................................... 59
Adding reports..................................................................................................... 59
Opening reports.................................................................................................. 60
Moving reports or folders ............................................................................... 60
Working with documents ...........................................................................60
Creating a subfolder ......................................................................................... 60
Adding files............................................................................................................ 61
Opening documents.......................................................................................... 61
Moving documents or folders ....................................................................... 61
4.10
4.11
Gel and report identifiers ............................................................................61
Archiving a workspace ................................................................................62
Backup / Restore workspace ........................................................................ 62
Backup / Restore project ................................................................................ 63
Reports
5.1
5.1.1
5.1.2
5.2
5.2.1
5.2.2
5.2.3
5.2.4
5.3
5.4
5.4.1
5.4.2
5.4.3
5.4.4
5.5
6
Working with classes ....................................................................................54
4.9.1
4.9.2
4.9.3
4.9.4
4.11.1
4.11.2
5
Matching ................................................................................................................ 53
Match set properties......................................................................................... 53
Introduction ......................................................................................................65
Reports menu ...................................................................................................... 65
Analyze menu...................................................................................................... 66
Report toolbars ................................................................................................67
Standard tools ..................................................................................................... 67
Adding comments.............................................................................................. 68
Selecting and navigating ................................................................................ 68
Displaying related reports.............................................................................. 70
Editing report content ..................................................................................72
Customizing reports ......................................................................................74
Settings ................................................................................................................... 74
Column order ....................................................................................................... 75
Column and row size........................................................................................ 75
Sorting data .......................................................................................................... 76
Mouse selection reports ..............................................................................76
Gels
6.1
6.1.1
6.1.2
6.1.3
6.2
6.3
Introduction ......................................................................................................79
Image format ....................................................................................................... 79
Image resolution................................................................................................. 79
Image depth ......................................................................................................... 80
Selecting gels and gel regions .................................................................80
Displaying gels ................................................................................................82
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Contents
6.3.1
6.3.2
6.3.3
6.3.4
6.3.5
6.3.6
6.4
6.4.1
6.4.2
6.4.3
6.4.4
6.4.5
6.4.6
6.5
6.5.1
6.5.2
6.5.3
6.6
6.6.1
6.6.2
6.6.3
6.6.4
6.6.5
6.6.6
6.6.7
6.7
6.7.1
6.7.2
6.7.3
6.8
6.8.1
6.8.2
6.8.3
7
Viewing signal intensity .............................................................................. 94
Loading / unloading gels ................................................................................ 94
Cursor information............................................................................................. 95
Displaying gray levels ...................................................................................... 96
Contrast mapping .............................................................................................. 96
Profile.................................................................................................................... 101
3D view ................................................................................................................ 102
Calibrating and normalizing gels .........................................................106
Intensity calibration........................................................................................ 106
Spot normalization ......................................................................................... 111
Intensity normalization using a scatter plot........................................ 112
Processing gels .............................................................................................114
Delete from disk ............................................................................................... 114
Renaming gels .................................................................................................. 114
Rotating gels...................................................................................................... 115
Flipping gels....................................................................................................... 116
Scaling gels ........................................................................................................ 117
Inverting gray levels....................................................................................... 117
Cropping gels .................................................................................................... 118
Reporting on gels .........................................................................................119
Gel report ............................................................................................................ 119
Gel description report .................................................................................... 120
Gel calibration report..................................................................................... 122
Saving, exporting or printing images .................................................123
Saving gels ......................................................................................................... 123
Exporting gels and windows ...................................................................... 124
Printing gels ....................................................................................................... 125
Spots
7.1
7.2
7.3
7.3.1
7.3.2
7.3.3
7.4
7.4.1
7.4.2
7.4.3
7.5
7.5.1
7.5.2
vi
Moving gels ........................................................................................................... 82
Zoom gels............................................................................................................... 83
Scrollbars................................................................................................................ 87
Transparency ....................................................................................................... 88
Grid lines................................................................................................................. 90
Aligning gels.......................................................................................................... 92
Introduction ....................................................................................................127
Spot ID ...............................................................................................................127
Detecting spots in non-DIGE gels ........................................................128
Procedure............................................................................................................ 128
Spot detection parameters.......................................................................... 130
Spot quantification.......................................................................................... 131
Co-detecting spots in DIGE gels ...........................................................133
Procedure............................................................................................................ 133
Spot detection parameter............................................................................ 134
Spot quantification.......................................................................................... 134
Selecting spots ..............................................................................................135
Spot tool............................................................................................................... 135
Select menu........................................................................................................ 135
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7.5.3
7.6
7.6.1
7.6.2
7.6.3
7.6.4
7.7
Adding / modifying spots ........................................................................ 138
Editing spots .......................................................................................................139
7.8
7.9
7.10
MW and pI calibration ............................................................................... 142
Cursor information ..................................................................................... 143
Reporting on spots ...................................................................................... 145
Spot Report .........................................................................................................145
DIGE histogram .................................................................................................148
DIGE report..........................................................................................................150
Annotations
8.1
8.2
8.3
8.3.1
8.3.2
8.3.3
8.4
8.4.1
8.4.2
8.4.3
8.5
8.5.1
8.5.2
8.5.3
8.5.4
8.5.5
8.6
8.6.1
8.6.2
8.6.3
8.6.4
8.7
8.7.1
8.7.2
8.7.3
8.7.4
8.7.5
9
Spot shape ..........................................................................................................136
Spot color.............................................................................................................137
Show / hide spots.............................................................................................137
Show / hide spot IDs.......................................................................................138
7.7.1
7.10.1
7.10.2
7.10.3
8
Reports..................................................................................................................136
Displaying spots .......................................................................................... 136
Introduction ................................................................................................... 151
Predefined label categories .................................................................... 151
Creating annotations and labels .......................................................... 152
Creating label categories..............................................................................154
Creating sets.......................................................................................................158
Creating specific links.....................................................................................158
Selecting annotations and labels ........................................................ 161
Annotation tool .................................................................................................161
Select menu ........................................................................................................162
Reports..................................................................................................................167
Displaying annotations and labels ..................................................... 167
Annotation flag position................................................................................167
Annotation flag color......................................................................................167
Annotation flagpole color .............................................................................168
Show / hide annotations and labels ........................................................168
Pack / unpack categories .............................................................................170
Adding / modifying annotations and labels ................................... 171
Annotation tool .................................................................................................171
Edit menu.............................................................................................................171
Reports..................................................................................................................174
Importing labels and annotations ............................................................175
Annotations and labels in reports ....................................................... 176
Label report.........................................................................................................176
Annotation report.............................................................................................177
Category report ................................................................................................177
Intra-Class Report ............................................................................................177
Inter-Class Report ............................................................................................178
Matches
9.1
9.2
9.2.1
Introduction ................................................................................................... 179
Matching .......................................................................................................... 181
Automatic matching .......................................................................................181
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Contents
9.2.2
9.3
9.3.1
9.3.2
9.4
9.4.1
9.4.2
9.5
9.5.1
9.6
9.6.1
9.6.2
9.6.3
9.7
9.7.1
9.7.2
Specifying starting matches....................................................................... 182
Selecting matches .......................................................................................183
Menu options..................................................................................................... 183
Reports ................................................................................................................. 186
Displaying matches ....................................................................................186
Show / hide matches ..................................................................................... 187
Show / hide match IDs .................................................................................. 187
Adding / deleting matches ......................................................................188
Adding / deleting matches .......................................................................... 188
Reports on matches ...................................................................................189
Match report ...................................................................................................... 189
Match statistics report................................................................................... 190
Intra-class report ............................................................................................. 190
Synthetic gels ................................................................................................191
Spot filtering....................................................................................................... 192
Creating synthetic gels ................................................................................. 193
10 Data analysis
10.1
10.2
10.2.1
10.2.2
10.2.3
10.2.4
10.2.5
10.2.6
10.3
10.3.1
10.3.2
10.3.3
10.3.4
10.3.5
Introduction ....................................................................................................195
Intra-class statistics ...................................................................................196
Scatter plots....................................................................................................... 196
Descriptive statistics....................................................................................... 198
Intra-Class Histograms ................................................................................. 201
Intra-Class Report ........................................................................................... 205
Factor analysis ................................................................................................. 209
Heuristic clustering ......................................................................................... 214
Inter-class statistics ....................................................................................216
Specifying classes ........................................................................................... 216
Overlapping measures.................................................................................. 216
Inter-Class Histograms.................................................................................. 220
Inter-Class Report............................................................................................ 224
Statistical tests.................................................................................................. 225
11 Data integration
11.1
11.1.1
11.1.2
11.1.3
11.2
11.2.1
11.2.2
11.2.3
11.2.4
11.3
11.3.1
11.3.2
11.3.3
11.3.4
viii
Converting into ImageMaster 2D Platinum format .....................233
From earlier versions of ImageMaster and Melanie......................... 233
From ImageMaster 2D Elite ........................................................................ 233
From Twain compatible scanners............................................................ 236
Exporting and importing ImageMaster 2D Platinum data .......237
XML format......................................................................................................... 237
ImageMaster reports ..................................................................................... 238
Export gel data ................................................................................................. 238
Import gel data................................................................................................. 240
Exporting to spot excision robots ........................................................241
Bruker Proteineer SP spot picker .............................................................. 241
GE Healthcare Ettan spot picker............................................................... 241
Genetix GelPix spot picker ........................................................................... 244
Genomic Solutions ProPic spot picker.................................................... 244
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11.4
11.4.1
11.4.2
11.4.3
Connecting to external protein databases ..................................... 245
Setting the database.......................................................................................245
Querying the database..................................................................................246
SWISS-2DPAGE master gels ........................................................................246
12 Safety, control and automation
12.1
12.1.1
12.1.2
12.1.3
12.1.4
12.2
12.3
12.4
A
A.2.1
A.2.2
A.2.3
A.2.4
A.2.5
A.2.6
A.2.7
A.2.8
A.3
A.3.1
A.3.2
Undo / redo .................................................................................................... 251
History .............................................................................................................. 253
Script ................................................................................................................. 255
Contextual menus ....................................................................................... 259
Workspace contextual menus .............................................................. 259
Workspace name .............................................................................................259
Project name ......................................................................................................259
Gels folder............................................................................................................259
DIGE Gels folder ................................................................................................261
MatchSets folder...............................................................................................262
Classes folder .....................................................................................................263
Reports folder ....................................................................................................264
Documents folder.............................................................................................265
Display zone contextual menus ........................................................... 266
Worksheet, pane or legend..........................................................................266
Image.....................................................................................................................266
Appendix
B.1
B.2
B.3
B.4
B.5
C
Action descriptors ............................................................................................249
Displayed actions.............................................................................................249
History and script navigators .....................................................................250
Gel selection .......................................................................................................251
Appendix
A.1
A.2
B
Introduction ................................................................................................... 249
Shortcut keys ................................................................................................ 269
Gel shortcuts ................................................................................................. 270
Spot shortcuts ............................................................................................... 271
Annotation shortcuts ................................................................................. 272
Match shortcuts ........................................................................................... 273
Appendix
C.1
C.2
C.3
Software references ................................................................................... 275
Statistical methods ..................................................................................... 276
Further reading ............................................................................................ 276
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Contents
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ImageMaster 2D Platinum User Manual 11-0034-38 Edition AA
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Introduction
1
1.1
Introduction
About ImageMaster 2D Platinum
ImageMaster 2D Platinum is a state-of-the-art software application designed to
analyze two-dimensional electrophoresis (2-DE) gels. This version of ImageMaster
has been developed by a team of top researchers from the Swiss Institute of
Bioinformatics (SIB) in collaboration with Geneva Bioinformatics (GeneBio) SA and
GE Healthcare, and integrates the Melanie software. It was conceived in close
collaboration with biologists and proteomics scientists from Professor
Hochstrasser's group in Geneva. The application has advanced to its current level
primarily due to the input of users worldwide.
ImageMaster 2D Platinum ensures fast and reliable image comparisons. It easily
manages multiple image analyses and offers the possibility to automate
detection and matching steps. However, it is the versatility of the software that
empowers you, the scientist. There are numerous interactive tools for optimizing
and manipulating data. Furthermore, attain a higher level of quantitative and
qualitative analysis using the robust and sophisticated techniques provided in the
application. ImageMaster integrates filtering, querying, reporting, statistical and
graphing options so that you can easily view, compare, analyze and present your
results. From the raw experimental images to the preparation of a publication,
ImageMaster takes you successfully through each stage of an image study.
1.2
What’s new in this release?
The most important new features in ImageMaster 2D Platinum, version 6.0 are
listed below:
•
Population matching
•
Support of DIGE technology (co-detection, DIGE histograms)
•
Intelligent multi-worksheet display
•
Dockable report windows
•
Workspace with management of gels, match sets and classes
•
Scrollbars for moving and zooming gels
•
Improved cropping option
•
Adjust contrast in 3D View
•
Measure histogram in spot report
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1
Introduction
1.3
ImageMaster resources
1.3.1
User manual
The ImageMaster 2D Platinum software, incorporating Melanie, is intended to be
intuitive and comprehensive. We encourage you to read through the User Manual
in order to exploit its full potential. This guide explains in detail all the functions
and advantages of ImageMaster 2D Platinum. If new to the software, you will
quickly master the fundamental concepts and features. If already familiar with
the software, the manual will inform you of new functionalities and help to
optimize your image analyses.
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 many of
the steps can be inverted or repeated at some point. The last chapters are more
specialized and have no defined place in the general procedure of an image
analysis study.
1.3.2
Tutorials
You can familiarize yourself with ImageMaster 2D Platinum by working through
the online tutorials. These tutorials offer a step-by-step guide on how to analyze
your gel images and report results. They are supplied with gel images and related
files, enabling you to carry out an entire image analysis using the various tools
available in the software.
You can access the different tutorials from the Help menu in the main program
window. For each of the tutorials, the option Help > Tutorials > Tutorial X > Open
will prepare your environment, restore the necessary files, and display the
tutorial text. The option Help > Tutorials > Tutorial X > Result shows the expected
outcome after running the corresponding tutorial.
1.3.3
Online help
The user manual is also available online from within the ImageMaster software.
To consult the online manual:
2
1
Choose Help > User Manual from the menu.
2
ImageMaster opens the online document.
3
Find the desired section from the Contents, Index or through a Search.
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Introduction
1.3.4
Conventions used
Before getting started with ImageMaster, you should have a working knowledge
of your computer's operating system and its conventions, including how to use a
mouse and standard menus and commands. You need to be able to adeptly
open, save and close files. If you wish to review any of these skills, please refer to
the documentation that came with your computer.
Convention
Use
Bold
Highlights different options or parameters that are
inherent to ImageMaster 2D Platinum. It also draws
attention to newly discussed terms.
Italic
Highlights menu options, window names, icon names,
file paths and folder names.
Italic > Italic
The arrow indicates a menu choice. For example, you
would choose the italicized menu title and then click on
the italicized option.
NOTE!
Highlights important comments, issues or tips.
Shift
Designates holding down the Shift key.
Ctrl
Designates holding down the Control key.
Left click
Means clicking the left mouse button.
Right click
Means clicking the right mouse button.
Double click
Means clicking the left mouse button twice.
Drag
Signifies positioning the cursor on an object and
holding down the left mouse button while moving the
mouse.
Table 1-1. Conventions used in the ImageMaster documentation.
1.4
Getting further assistance
GE Healthcare provides technical and scientific support for the ImageMaster
software. Please contact us if any problems arise with the installation or use of
the program. Our support team is happy to help you.
1.4.1
How to contact us
E-mail us at [email protected]
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1
Introduction
Support numbers:
4
Country/Region
Tel
Fax
Asia Pacific
+852 2811 8693
+852 2811 5251
Australasia
+ 61 2 9899 0999
+61 2 9899 7511
Austria
01/57606-1619
01/57606-1627
Belgium
0800 73 888
03 272 1637
Canada
1 800 463 5800
1 800 567 1008
Central, East, & South
East Europe
+43 1 982 3826
+43 1 985 8327
Denmark
45 16 2400
45 16 2424
Finland & Baltics
+358-(0)9-512 39 40
+358 (0)9 512 39 439
France
01 6935 6700
01 6941 9677
Germany
0761/4903-490
0761/4903-405
Italy
02 27322 1
02 27302 212
Japan
+81 3 5331 9336
+81 3 5331 9370
Latin America
+55 11 3933 7300
+55 11 3933 7306
Middle East & Africa
+30 210 9600 687
+30 210 9600 693
Netherlands
0165 580 410
0165 580 401
Norway
815 65 555
815 65 666
Portugal
21 417 7035
21 417 3184
Russia & other C.I.S. &
N.I.S
+7 (095) 232 0250
+7 (095) 230 6377
South East Asia
60 3 8024 2080
60 3 8024 2090
Spain
93 594 49 50
93 594 49 55
Sweden
018 612 1900
018 612 1910
Switzerland
0848 8028 12
0848 8028 13
UK
0800 616928
0800 616927
USA
+1 800 526 3593
+1 877 295 8102
+7 (095) 956 1137
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Introduction
1.4.2
Sending e-mail
To provide us with the essential technical and customer information when
mailing to [email protected], please proceed as follows:
To send an e-mail to technical support:
1
Choose Help > Online Support from the menu.
2
Enter your personal details in the Customer Information box. Please note
that this data is used to update our customer database. In this way, we
can contact you with certainty when it is time to send new patches or
installation files.
3
In the Online Support box, specify whether you want to ask a question,
make a suggestion, or report a bug. Depending on your choice, the
software asks you subsequent questions that help us to supply you with
an appropriate answer.
4
Click on the E-mail button. A new message is created with your default
e-mail software, automatically directed to [email protected] Highlight the <Paste clipboard> text with your mouse
and then Edit > Paste the contents of your clipboard into the mail
message. The e-mail now contains your questions and contact
information, as well as useful data about your ImageMaster version,
work session and computer system. If the problem is file specific, then
attach any relevant files. Send us this message. Alternatively, you can
print the clipboard contents and send it to us by fax.
1.4.3
System and product information
To quickly obtain product and system information, for your own benefit or during
a technical support call, choose Help > System & Product Information from the
menu. A window shows the useful data about your ImageMaster version and
computer system.
You can also get information about your ImageMaster version by looking at the
release number given under About ImageMaster in the Help menu. Choosing Help
> About ImageMaster displays the program splash page.
1.4.4
GE Healthcare listens
The GE Healthcare staff is attentive to your suggestions. Many of the new
features and enhancements in this software are a direct result of conversations
with our customers. We truly appreciate any comments, criticisms or ideas that
would help us to improve the software. Please do not hesitate to contact us by email at [email protected]
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1
Introduction
6
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2.1
Two-dimensional electrophoresis
2.1.1
Technique
Two-dimensional electrophoresis (2-DE) can effectively resolve thousands of
proteins in a complex biological mixture by separating the proteins based on two
independent properties: isoelectric point (pI) and molecular weight (MW).
Separation in the first dimension is achieved by re-hydrating an immobilized pH
gradient (IPG) strip with the protein sample and subsequently applying an electric
field. The charged proteins migrate within the pH gradient until they reach their
isoelectric point, where they lose their net charge and therefore their mobility.
Non-equilibrium pH gradient electrophoresis (NEPHGE) can also be used to
accomplish this separation.
After the isoelectric focusing (IEF) step, the strip is placed at the cathodic end of a
homogeneous or gradient sodium dodecyl sulfate polyacrylamide gel (SDSPAGE), possibly along with molecular weight marker proteins flanking one or both
ends of the IEF strip. In an electric field, the negatively-charged SDS-protein
complexes then migrate towards the anode with differing velocities depending on
their size. Small molecules move faster and further. Larger ones move more
slowly and cover less distance.
The proteins are finally visualized by radiolabeling or detected with a variety of
staining methods such as silver, Coomassie blue or fluorescent stains. Adapted
image capture devices are used to generate digital images that can be analyzed
with a 2-DE image analysis software such as ImageMaster.
2.1.2
Applications
Two-dimensional (2-D) gel electrophoresis is a leading research tool in
proteomics. This technique enables the identification of protein expression
changes such as those associated with the exposure to pathogens, drugs or
other stimuli, or alterations linked to different stages of development. Thus, gel
electrophoresis is invaluable in gene expression studies for the discovery of
protein targets, disease markers, and drug candidates, or to evaluate drugs for
toxicity. And this is just to name a few examples.
2.1.3
Advantages and limitations
2-DE is currently the most powerful protein separation technique, allowing the
parallel investigation of thousands of proteins in a single step. The technology is
compatible with almost all protein types. Gels with narrow pH ranges permit very
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high resolution and large protein loads so that even poorly expressed proteins
can be observed.
However, scientists wanting to compare a set of gel images may notice that the
quality and reproducibility of 2-D gels are sometimes mediocre. Poor resolution,
high noise levels, and large distortions in the protein patterns often hamper the
analysis process. The reasons for this are extensive: unskilled sample preparation
leads to unintended biological variability, poor sample solubilization produces
insufficient resolution, vertical and horizontal streaking caused by salt in the IPG
strip makes proper detection or quantification difficult, dust particles or droplets
lead to many spot artifacts, etc.
The crucial algorithms in 2-D gel analysis software have been significantly
improved over the years in order to account for such difficulties. In addition,
program interfaces have gained a lot of ground in user-friendliness. In spite of
these advances, no image analysis software can solve all experimental problems.
The desire to draw valuable information from low quality gels remains an illusive
goal. Before starting a 2-D gel image analysis, it is therefore essential to optimize
all steps in the gel production process in order to assure the best possible quality
and reproducibility. Careful experimentation remains the only way to guarantee
significant end results. This includes refining protocols for the sample preparation
of each starting material, pre-fractionating the sample, using precise and correct
experimental conditions during 2-D separation and protein staining or labeling,
employing larger gel formats or narrower pH ranges, and so on. Image
acquisition parameters also play a very important role.
2.2
Differential gel electrophoresis
2.2.1
Technique
Conventional 2-DE techniques require the separation of each sample on an
individual gel in order to compare protein abundance in different samples. This
one-sample-per-gel approach introduces a high level of system variation to the
data such as the variation that arises from differences in protein uptake into the
first dimension strip, second dimension gel running, etc. This high level of system
variation can outweigh the often subtle, induced biological changes that the
experiment is intended to detect, for example, differences that are caused by a
disease state, drug treatment or life-cycle stage.
To further complicate this problem, it is also necessary to separate the induced
biological changes within an experiment from the inherent biological variation. In
other words, the differences between two individual animals, cultures or plants,
that are present irrespective of the applied experimental conditions. To achieve
this, multiple sample replicates must be incorporated within each experimental
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design. This requires the separation and analysis of a large number of samples
and slows the process if each sample must be separated on a different gel.
Differential gel electrophoresis (DIGE) is a method for pre-labeling protein samples
prior to two-dimensional electrophoresis. By allowing different samples to be run
on a single gel, one of which can be an internal standard, it can significantly
simplify the above mentioned problems.
The system is based upon the use of up to three cyanine dyes possessing unique
fluorescent properties. The dyes (Cy2, Cy3 and Cy5) are mass- and chargematched N-hydroxy succinimidyl ester derivatives that differentially attach to
lysine residues in a protein. Therefore the labeled proteins migrate
simultaneously on the 2-DE gel but still produce distinct excitation and emission
spectra.
The design of a DIGE experiment is straightforward (Figure 2-1). Protein samples
are labeled with the different DIGE dyes and subsequently mixed on the same 2DE gel. Electrophoresis is applied to separate the proteins according to molecular
weight and isoelectric point. The gel is then scanned using instrumentation such
as the Typhoon Variable Mode Imager (GE Healthcare) to generate overlaid, multichannel images for each gel.
A
Pool: A+B
B
Cy3
Cy2
Cy5
+
Figure 2-1. Simple scheme for DIGE. Three protein extracts are labeled, combined on one 2DE gel and then separated according to the fluorescence spectra.
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Depending on the amount of sample available, different dye to protein ratios can
be used. Minimal labeling with Cy2, Cy3 and Cy5 is most straightforward to use.
When only a small amount of sample is available, saturation dyes might be the
best choice. The Cy3 and Cy5 saturation dyes are also mass and charge
matched, except that they are maleimide fluors that react with thiols found on
cysteine residues to form a thioether.
Keep in mind that if no lysine or cysteine amino acid (which are targeted by the
minimal and saturation dyes, respectively) is present in a protein, the protein is
undetectable.
2.2.2
Internal standard
This type of methodology allows an internal standard to be included. Ideally, the
internal standard consists of a subset taken from all of the samples within the
experiment. The internal standard is labeled with one of the cyanine dyes
(Typically Cy2 for minimal dyes and Cy3 for saturation dyes) and run on each gel
in the experiment. This creates an image that is the average of all experimental
samples, with all proteins in the experiment represented. The presence of the
internal standard in every DIGE gel provides an inherent link between samples.
It is the use of an internal standard in 2-DE experiments that permits quantitative
and statistical analyses. First, each protein spot in a sample can be compared to
its representative spot within the internal standard on the same gel, to generate
a ratio of relative protein levels. Quantitative comparisons of samples between
gels are made based on the relative change of sample to its in-gel internal
standard. This process effectively removes the system (gel-to-gel) variation
enabling accurate quantitation of induced biological change between samples.
The need to run gel replicates is also eliminated, thus reducing the number of gels
required per experiment.
An additional benefit of using an internal standard in DIGE technology is that
matching between gels is more apparent. The internal standard image is
common between all gels in an experiment, therefore matching can be
performed between internal standard images which have similar spot patterns.
Conventional 2-DE requires matching between different samples on different
gels, which introduces differences in spot patterns from sample-to-sample and
gel-to-gel variation. Matching between internal standards allows matching
between identical samples, so variations in spot patterns are due only to
electrophoretic differences.
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2.3
Understanding gel images
2.3.1
Anatomy of gel images
Gels
A gel is a 2-DE image that has been digitized and stored on a drive. It contains the
raw input data from which proteins can be detected and quantified. The proteins
represent the gray values for all the pixels in the image. Every pixel is
characterized by its X and Y coordinates, which represent the horizontal and
vertical positions of the pixel on the image. The pixel's raw value, or gray value, is
the signal intensity of the pixel (Z axis in a three-dimensional view).
The software recognizes gels from several image formats, but the gels must be
saved in the ImageMaster file format (.mel) in order to store data about spots,
matches, annotations, etc. If a gel is exported with its objects into a foreign
format, then the data simply becomes part of the saved image and loses any
structure or value.
Gels can be duplicated, deleted, cropped, filtered, flipped or scaled. Gels can also
be aligned, meaning that they are distorted in order to superimpose their image
with another gel.
Various items can be defined on a gel for detailed examination. These are spots,
regions, annotations and labels (Figure 2-2). Matches, match sets and classes are
entities that give structure to the relationships between gels (Figure 2-3) and will
be discussed in the following section.
Annotations
Region
Labels
Spots
Figure 2-2. Items on a gel: region, spots, annotations and labels.
Regions
A region is a rectangular area of a gel that is defined using the Region tool. When
a region is set, certain actions may be limited to it, such as previewing spot
detection parameters, adjusting gray levels, viewing three-dimensional plots of
spot intensities, cropping, etc.
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Spots
Spots represent the proteins on the gel. They can be automatically detected by
ImageMaster or manually edited by the user. Spots can be quantified. Their
intensity, area and volume are computed.
Annotations and labels
Spots or individual points (pixels) can be marked by annotations. An annotation is
defined by its position and its set of labels. It contains relevant information that is
separated into label categories (predefined and/or user-defined categories).
The predefined categories in ImageMaster include pI_MW (holds standard pI and
MW values for a pI/MW calibration), Ac (contains database accession numbers),
Landmark (defines tie points for gel warping and matching), Set (marks spots that
share the same properties) and Comment (keeps user information). Moreover, an
unlimited number of user-defined categories can be added. Labels can also
contain direct links to other files, Web sites and query engines.
2.3.2
Spot and gel relationships
Matches
A match represents the relationship between corresponding spots in different
gels. It connects the same protein in the different gels. It is composed of a spot ntuple (S1, S2, ..., Sn) where S1 is a spot in the first gel, and Sn a spot in the last gel.
Matches can be manually defined by the user or automatically determined by
ImageMaster's powerful gel matching algorithm. The match is the basic element
for searching and investigating protein expression changes across gels, through
the use of reports, histograms, and statistical methods.
Match sets
To be able to initiate the matching process in ImageMaster 2D Platinum 6.0, gels
must be part of a Match set. A match set includes gels or populations of gels (in
sub match sets) that should be compared and therefore matched together. Every
match set is represented by a Master image. This master image is created by
ImageMaster based on the Reference for the match set, chosen by the user.
Classes
A class is a set of gels or gel populations that you can compare with other such
entities. In the Classes folder, you state your biological questions. For example, a
class of gels obtained from the infected tissue samples of different patients can
be compared to a class of gels representing healthy tissue from other patients.
A class is the basic element for searching and investigating protein expression
changes across gels, through the use of intra-class reports, histograms, and
scatter plots. You can also compare different classes through inter-class
analyses. To compare gels in a class or within different classes, the gels must be
matched together. This means that they must belong to the same match set.
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Class A
Class B
A2
A1
S1
B1
S2
B2
S3
S4
Master AB
S1
Match set AB
Figure 2-3. Definition of matches, match sets and classes. Spots S1, S2, S3, and S4 together
form a match. Note that the spot in Master AB has the same number as the corresponding
spot in A1. This is because A1 is the reference image for Match set AB, on which the Master
AB is based. The Match set AB contains all the gels to be matched (A1, A2, B1 and B2), and
is represented by a master image (Master AB). A class regroups gels from a particular
biological state that has to be compared with another such state. The spots in the selected
match (green) are under-expressed in Class B with respect to Class A.
2.4
Image analysis workflow
A typical image analysis, both for non-DIGE and DIGE gels, would consist of the
following steps. Please note that the specificities of DIGE gel analysis are
explained in the corresponding chapters.
2.4.1
Acquiring data
Your gel images must first be digitized using an image capture device. This will
generally be done with a separate software. You can open gels from TWAIN
compatible scanners from within ImageMaster. Please consult Chapter 11 for
more details about this . In Section 6.1 you will also find recommendations on
image scanning (format, resolution, depth).
2.4.2
Setting up a workspace
You must set up a workspace to open and work on gel images. The workspace
allows you to organize your gels into projects, to define match sets and classes
and to keep accompanying data, such as reports and image documents in
project related folders. Your preferred ImageMaster settings are also saved in the
workspace file. Detailed information about the workspace is found in Chapter 4.
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2.4.3
Visualizing and calibrating gels
The next step is to handle the gel files (open, save, print, close), manipulate the
gels images (select, move, zoom, stack, align), possibly transform the gels (rotate,
crop, scale) and view the signal intensity (adjust contrast, profile, 3-D view). At this
stage, it may also be necessary to calibrate your gels. Information about all these
features is given in Chapter 6.
2.4.4
Detecting and quantifying spots
Once you are familiar with your gel images, you can perform automatic spot
detection. In Chapter 7 you will learn everything you need to know about spots,
including how to select, display and edit spots, as well as how to view their
properties and quantification values.
2.4.5
Annotating spots and pixels
Individual pixels and spots in a gel image may be labeled with annotations. These
annotations can be used in functionalities such as calibration, alignment and
matching, or be utilized to mark spots with their particular characteristics.
Chapter 8 explains how to create, use, select and display labels, categories and
annotations. It further describes how to create links to external databases or data
sources of any format (text, file, html, etc.).
2.4.6
Matching gels
After spots were detected and match sets defined, you can match your gel
images. In other words you find the corresponding protein spots in different gels.
Chapter 9 introduces you to all the necessary concepts and functionalities for gel
matching and the creation of synthetic gels.
2.4.7
Analyzing data
Chapter 10 offers an overview of the powerful data analysis and classifications
tools that are used to study the variations in protein expression among gels or
classes of gels. The data analysis step may be carried out at two different levels.
The intra-class statistics tools include scatter plots, descriptive statistics, and
factor analysis. For inter-class analyses, the so-called overlapping measures and
various statistical tests can be used. Heuristic clustering can help finding classes.
2.4.8
Integrating data
You may want to export spot coordinates to a spot excision robot, export gel data
to a database (for example, via XML format), or import experimental information
to be included in annotations. The necessary tools are described in Chapter 11 of
this manual.
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2.4.9
Reporting results
You can display information on specific gels and gel components (spots, matches,
classes, annotations) at any moment during your analysis. You will learn the
details about displaying, using, saving, customizing and editing reports in Chapter
5. Additionally, you will find information on specific report types in the various
chapters of this document.
2.4.10
Controlling and automating gel analyses
As with reporting results, you can check the operations that were carried out on
your gels at any time. Do this with the History function that is described in
Chapter 12. The latter also instructs you on how to create Scripts for automating
parts of your analysis and explains the multiple undo/redo feature.
2.5
ImageMaster flavors
The powerful suite of features that normally apply to 2-DE gels in ImageMaster
can now be used in conjunction with DIGE gels. There are two modules of
ImageMaster 2D Platinum 6.0 available for purchase:
•
ImageMaster 2D Platinum 6.0: The software version to be used with
conventional 2-DE gels. All menu options related to DIGE are not functional
and are grayed out in the graphical user interface.
•
ImageMaster 2D Platinum 6.0 DIGE: The fully functional version to be used
with conventional 2-DE and DIGE gels. You can 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, match, report, plot histograms and perform statistical analyses on
DIGE gels.
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Getting started
3.1
Installing ImageMaster 2D Platinum
3.1.1
System requirements
In order to install and run ImageMaster, your computer must satisfy the following
requirements:
•
Microsoft Windows 2000 or XP operating systems.
•
Administrative permission to install ImageMaster.
•
At least 256 MB RAM for ImageMaster (512 MB is recommended) and 768
MB RAM for ImageMaster DIGE (1024 MB is recommended). The amount of
memory required is mainly 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 and approximately
400 MB for the full installation of tutorials.
•
Internet Explorer 6 (Microsoft Corporation), Netscape Navigator 6 (Netscape
Communications Corporation), Mozilla 1.4 (Mozilla Foundation) or higher
versions. A browser enables you to print reports and to access scientific
databases on the Web.
3.1.2
Software installation
You can install ImageMaster from a CD-ROM or by downloading the installation
package over the Internet. When you insert the CD-ROM into the appropriate
drive on your computer, the Setup Wizard starts automatically and guides you
through a series of screens. Alternatively, you can double click on the icon of the
installer file (for example SetupIMPv600.msi) to launch the install program.
The ImageMaster installer creates a default directory on your hard disk called
Program Files\GE Healthcare\ImageMaster 2D Platinum, in which the program
files are placed. If you want to save the default directory in a different folder, then
browse and open the folder before continuing the installation.
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Once installation is complete, it is recommended to restart your computer.
3.2
eLicensing
An electronic license (eLicense) file is required to enable ImageMaster to run once
installation is completed.
There are two types of eLicenses:
•
Node-locked license (Machine license): This type of license is for a single
computer. It is practical when only one or a few computers are used for
working with ImageMaster. A node-locked license file must be placed on the
computer running ImageMaster.
•
Floating license (Concurrent license): This type of license can be used by all
computers connected via network to a computer with a license server
installed. It is useful when many users, but not all at the same time, need
access to the ImageMaster program. The number of computers that can
simultaneously use 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 the ImageMaster program or on any other network
computer (It is recommended to install it on a network computer that is
always running).
To be able to start ImageMaster, a valid license must be found. ImageMaster will
check that the license file is available at every subsequent login. To obtain and
correctly place a license file, follow the activation steps below.
3.2.1
Access code
After ordering ImageMaster, a letter including an access code will be send to the
order’s shipment address. The access code is necessary for collecting the
eLicense files (see Section 3.2.4). Store this access code in a safe place.
3.2.2
Finding the physical address of the computer
The physical address is necessary when collecting the appropriate eLicense file.
The address identifies the computer and is used by the licensing system. If a
node-locked license is used, it is the physical address of the computer where
ImageMaster is installed. If a floating license is used, it is the physical address of
the computer where the license server is installed.
To find the physical address:
1
18
On the computer where the eLicense file should be placed, select Start >
(All) Programs > Accessories > Command Prompt.
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2
When the command-prompt appears, click on it and enter the
command ipconfig /all (making sure to leave a space between the
ipconfig and the /all).
3
Among the displayed information, make a note of the Physical Address
but without any dashes or colons. If the computer has several Physical
Addresses (there can be one for each network, i.e. Ethernet, card in the
computer), any of them can be used for identification. Please verify that
the Physical Address is correctly noted. Otherwise the license file will not
work.
4
Close the command-prompt window.
3.2.3
Installing the GE Healthcare eLicense server
The installation in this section should only be performed when using a floating
license. If only node-locked licenses are used, continue from Section 3.2.4.
To install the GE Healthcare eLicense server
1
On the computer where the eLicense server will be installed, insert the
ImageMaster 2D Platinum installation CD.
2
Click on the Install GE Healthcare eLicense Server button.
3
The installation window is shown. Click Next.
4
Accept the default installation path and start the installation (if the
default installation path is changed, the paths below need to be updated
accordingly).
5
A question concerning the Windows Firewall may appear. To allow the
license server to work properly, you must answer Yes.
6
At the end of the procedure, ensure that the Launch GE Healthcare
Software Licensing Server box is checked and click Finish. This
automatically launches the license server dialog (LMTOOLS).
7
Click the Config Services tab and ensure that all options are set as in
Figure 3-1. Note that the paths shown (C:\Program Files\GE
Healthcare\eLicense server\) are valid if the default installation is made.
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Figure 3-1. Configuration in the Config Services tab in LMTOOLS.
8
Click the Save Service button, even if you made no changes in the
previous steps.
9
Close LMTOOLS.
3.2.4
Collecting and placing an eLicense file
Next, an eLicense file should be collected and placed in the appropriate folder on
your computer. In the case of a node-locked license, the license file should be
placed in the ImageMaster installation folder (which is by default C:\Program
Files\GE Healthcare\ImageMaster 2D Platinum). For a floating license, the file
should be in the folder C:\Program Files\GE Healthcare\eLicense server\Licenses
on the computer running the license server (see Section 3.2.3).
To collect and place a license file:
20
1
Go to the web site http://www.elicensing.amershambiosciences.com/
gtlweb/login.
2
Enter the access code and press Continue.
3
Click on Collect Licenses.
4
Step 1 - Select the product to collect (mark the check box).
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5
Step 2 - Register your product.
6
Step 3 - Enter the Physical Address (Host-ID) of the computer where you
will install the license file (The expected type of Host-ID shall by default
be ETHERNET). Ensure that the Physical Address is correct. Click on
Continue.
7
If the data shown in the next screen is correct, select to Collect License.
Otherwise use Back to edit.
8
Save the license to a file, or e-mail the license. It is strongly
recommended to save the license file. Close the Download Complete
window after saving the file. If the license is e-mailed, the content is
delivered as raw text in the e-mail body. The text must then be copied to
an ASCII-text file given the extension .lic.
9
Place the license file in the appropriate folder if this was not already
done when saving the license file, or if the license file was e-mailed.
10 Restart the computer.
3.2.5
Test the eLicense
After installation of ImageMaster, downloading and placing the license file, start
ImageMaster on all computers where the program is installed to test that the
license file can be found (see below).
When using a floating license, a FLEXlm License Finder window will be displayed
asking to specify the License Server System or License File. Choose the first
option and then enter the name of the computer on which the license server is
installed. Click OK.
Note that depending on the license acquired (ImageMaster 2D Platinum DIGE or
ImageMaster 2D Platinum), you will or will not be able to use the DIGE
functionalities in the software.
3.3
Starting and exiting ImageMaster
ImageMaster starts like any other software by selecting Start > Programs > GE
Healthcare > ImageMaster 2D Platinum from the Windows menu. You can also
double click the ImageMaster icon (Figure 3-2) on the desktop. The ImageMaster
splash page appears while the software is loading. You can click on the logo to
make it disappear.
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Figure 3-2. ImageMaster icon.
Select File > Exit in the menu to close the program. You can also click on the Close
button in the upper right corner of the program window. ImageMaster prompts
you to save any modifications made to gel images and matching data before
exiting. The open workspace will be saved automatically.
3.4
ImageMaster window
By reading through the following pages, you will become acquainted with the
graphical user interface. It is divided into four main parts, shown in Figure 3-3,
which are the Menu Bar, the Toolbar, the Display Zone, and Dockable Windows.
Each of them will be discussed in more detail below.
a
b
d
d
c
d
Figure 3-3. The ImageMaster window. (a) Menu Bar, (b) Toolbar, (c) Display Zone and (d)
Dockable Windows.
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3.4.1
Menu bar
If gels, spots, annotations or matches are selected, then you can choose actions
from the menu bar to be performed on them. This is done by choosing the
appropriate menu and moving the cursor to the desired option while holding
down the left mouse button. Certain menu options have sub-options. You can
choose one of these by moving the mouse over the small arrow on the right-hand
side of the menu item.
Menu
Usage
File
Perform basic functions such as close, save, import, export and
print. You can also exit ImageMaster.
View
Modify the worksheet layout or name. Change the arrangement
of images in a pane.
Edit
Undo/redo the last operations, show a history of operations, or
edit (add, modify or delete) specific gels, spots, annotations or
matches.
Show
Change the way gels are displayed, show/hide specific spots,
annotations or matches, or modify their physical properties
(shape, color or visibility of IDs).
Select
Select specific gels, spots, annotations or matches based on their
ID, value, content or other properties.
Analyze
Compute differences and similarities between gel images (data
analysis based on robust statistics, factor analysis, statistical
tests and clustering techniques). All reports containing calculated
properties are found in this menu.
Reports
Display information on selected gels, spots, labels, annotations,
categories or matches, and use the navigation tools in the reports
to view the data on your gels. The three-dimensional (3D) view is
treated as a report and is therefore located in this menu.
Tools
Remove or rename gels on the hard disk, create new gel images
from selected ones (flip, rotate and scale gels, or create synthetic
images), run scripts, manage calibrations and change
ImageMaster settings.
Window
Display pixel and/or spot information, open zoom windows or
mouse selection reports and get a list of all open report windows.
Help
Obtain support online, access the official ImageMaster Web site
or obtain information about the current version of ImageMaster.
Keyboard shortcuts
Several menu options can be activated by keyboard shortcuts. These are
indicated at the right-hand side of the corresponding menu option. A list of all
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shortcuts is given in Appendix B. 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.
3.4.2
Toolbar
Objects on the screen can be displayed, selected or processed using the various
options in the ImageMaster menus. However, they can also be manipulated with
the tools provided in the toolbar. The fine points of these tools are explained in
other sections of the User Manual but the main features are as follows:
When the Hand tool is activated, you can move your image in
order to show other parts of it.
The Magnify tool lets you repeatedly zoom in (left click) or out
(right click) on the whole image each time by a factor of two.
The Region tool is useful for selecting a rectangular area within
an image. This region can be used for cropping gels, selecting
objects within a region, or getting a preview zone for spot
detection or contrast adjustment.
The Spot tool lets you select or edit individual spots. The tool
changes when in the Edit Enabled mode.
The Annotation tool is used to add, edit, move or select
annotations and labels.
3.4.3
Display zone
Gel images are opened from the Workspace (see Chapter 4 for details) and
subsequently displayed in the Display Zone (Figure 3-4). The Display Zone is laid
out in three levels: Worksheets, Panes and Images. Their layouts can be
customized according to your requirements. A Status Bar is located at the
bottom of the Display Zone.
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c
b
a
d
Figure 3-4. The Display Zone. (a) Worksheet, (b) Pane, (c) Image and (d) Status Bar.
Worksheets
Images are always displayed in worksheets. Each worksheet has a banner, which
is the horizontal bar located at the top containing the worksheet name and data
set type (enclosed in brackets []). The color of the worksheet banner indicates
whether it is selected (green) or not (gray). The selected worksheet is always at the
front of the Display Zone. In order to select an inactive worksheet, click on its
banner. The worksheet is brought to the front of the Display Zone.
To rename a worksheet:
1
Select the worksheet.
2
Choose View > Rename Worksheet in the menu.
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3
In the Rename Active Worksheet box, input the name you want. Click OK.
4
The new worksheet name appears in the banner.
To close a worksheet:
1
Select the worksheet. If all of its contents are not already selected press
Ctrl+A.
2
Choose File > Close Images in the menu.
3
The worksheet is removed from the Display Zone.
4
In order to close all worksheets at one time, choose File > Close All in the
menu.
Panes
When an image or folder of images is opened in a worksheet, it is automatically
placed in a pane. The pane banner is labeled with the same name that was given
to the folder of images in the Workspace. Random images are opened in a pane
with the generic label Gels. The color of the pane banner indicates whether it is
selected (green) or not (gray). In order to select a pane, click on its banner.
To close a pane:
1
Select the pane. If all of its contents are not already selected, then click
on its banner.
2
Choose File > Close Images in the menu.
3
The pane is removed from the worksheet.
A worksheet can contain one or more panes. Panes can be laid out so that they
do not overlap (tiled panes) or so they do overlap (stacked panes). Stacked panes
resemble a pile of papers lying one on top of another; only the topmost pane is
displayed in full. You can move a pane to the top of the stack by clicking on its
banner.
By default, ImageMaster displays panes in tiled mode.
To stack panes:
26
1
Select the worksheet.
2
Choose View > Worksheet Layout > Stacked in the menu.
3
The panes are stacked.
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You can also fix the number of panes displayed horizontally and vertically in a
worksheet. Choose View > Worksheet Layout > Free in the menu. In the Worksheet
Layout box, input the following values:
•
Number of Columns: Constrains the panes positioned one next to the other
in the worksheet.
•
Number of Rows: Constrains the panes positioned one above the other in
the worksheet.
Images
A pane can contain one or more gel images. Each image has a legend with its
name in the upper left corner. The color of the legend indicates whether the image
is selected (green, or red for a master image) or not (gray, or pink for a master
image). In order to select an image, click on its legend.
To close a gel image:
1
Select the gel image. Use the Shift or Ctrl keys to make multiple
selections.
2
Choose File > Close Images in the menu.
3
The image is removed from the pane.
Images can be laid out so that they do not overlap (tiled images) or so they do
overlap (stacked images). By default, ImageMaster displays images in tiled mode.
To stack images:
1
Select the pane.
2
Choose View > Pane Layout > Stacked in the menu.
3
The images are stacked.
You can also fix the number of images displayed horizontally and vertically in a
pane. Choose View > Pane Layout > Free in the menu. In the Pane Layout box,
input the following values:
•
Number of Columns: Constrains the images positioned one next to the
other in the pane.
•
Number of Rows: Constrains the images positioned one above the other in
the pane.
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When the number of open images in a pane exceeds the number of displayed
images (for example, in stacked mode) you can make an image visible by clicking
on its tab at the bottom of the pane.
In order to sift through images (for example, in stacked mode), use the Page Up
and Page Down keys on your keyboard. Alternatively, you can choose View >
Pane Layout in the menu and select Previous or Next.
Switch order
You can change the order by which the gel images are displayed by clicking on
the gel legend and dragging the image onto another gel. It is inserted before this
gel. Similarly, you can re-order panes by clicking on their banners and dragging
them to their new position.
You can swap worksheets, panes and images. To do so, use View > Switch, or the
Ctrl+F shortcut. This reverses the previous re-ordering operation carried out on a
worksheet, pane or image.
Contextual menus
When you click the right mouse button on a worksheet banner, pane banner or
gel legend in the Display Zone, a contextual menu appears containing the main
options used to work with images, panes or worksheets. If you right click directly
on a visible gel image, then another contextual menu offers the possibility to edit,
show or select objects like gels, spots, annotations and matches. These short
menus duplicate some of the commands found in the menu bar but are ordered
differently and are quicker to access at any time. A list of all contextual menus is
given in Appendix A.
Status bar
The status bar is an important resource because it indicates the total number of
gels, spots and annotations that are currently selected in the active worksheet.
3.4.4
Dockable windows
The workspace and reports are displayed in dockable windows that can be set
according to your preferred style of working with numerous windows at a time.
Dockable windows can be docked (i.e. 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 Dockable (pinned), Auto Hide (un-pinned) or Floating
mode.
Dockable (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 3-5). ImageMaster
displays blue arrow guides to indicate where the window may be docked. By
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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 non-predefined location, it becomes a floating window. Moving a
docked window may affect the location and size of other docked windows.
Figure 3-5. The Gel Report window previously docked at the bottom edge will now be
docked at the right edge of the ImageMaster window.
Auto hide (un-pinned)
A visible window in Auto Hide (un-pinned) mode
automatically collapses when
not in use to become a tab on the edge of the ImageMaster window. When you
click on a docked tab, the window slides back into view and is ready for use.
You can also click on Collapse
to minimize a window in Auto Hide mode.
Floating
A dockable window in Floating mode will always appear on top. It can be dragged
to any position within the program or even outside the main program window.
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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, drag a
tab away from the docked window or double click on the tab.
The workspace cannot be added to a tabbed group. In addition, tabular reports
and graphical reports cannot be grouped together.
3.5
ImageMaster 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 this manual.
However, an overview of the settings, per tab, is given below.
General
• Raw Image: Indicates whether the raw image data of newly opened gels
should be kept in memory. If the available RAM on your computer is low,
then it is better not to use this option. See Section 6.4.1 for details on loading
raw image data.
Cursor information
• Set the attributes to be shown in the Cursor Information window (see
Section 7.9).
Display
• Indicate the default spot colors (for normal and selected spots) and spot
shapes to be used (Crossed, Outlined, Filled, Outlined/Filled). Choose the
color for overlapped spots and match vectors in an image.
Grid settings
• Type: Specify whether the grid graduations should just be regular
subdivisions of the visible space - in Fixed mode - or a subdivision in terms of
real coordinates at whole multiples - in Adaptive mode (see Section 6.3.5).
•
30
Units: Choose the default grid units to be used for displaying a grid over a
gel. Note that you can still set specific grid properties for each gel (see
Section 6.3.5).
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•
Divisions Number: Specify the default number of grid subdivisions to be
displayed in the horizontal and vertical directions (see Section 6.3.5).
Categories
• Set the constraints for the label categories (see Section 8.3.1) and define the
color for the labels of each category.
Gel descriptions
• Add or delete gel descriptions and change their constraints (see Section
6.7.2).
Quantification
• Volume quantification: Indicate if you wish to compute the spot areas in
mm2, based on gel resolution (default for new workspaces), or pixels (default
for workspaces containing imported ImageMaster 2D Elite experiments).
Database
• Annotations folder: Select the folder where all linked files should be located.
These files are linked to spots or pixels in a gel via file link labels (see Section
8.3.3).
•
Database: ImageMaster offers full support for the Bruker Proteineer suite. In
this context, the Server Name, User Name and Password field are used for
exporting gel data to the Bruker ProteinScape database. If the Bruker tools
are not installed, these parameters are inactive (grayed out).
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4
Workspace
4.1
Introduction
The Workspace is the command center of ImageMaster. It can be seen as the
place where all gel, matching, and analysis data is centralized and from where all
operations carried out in the software are controlled.
It is where you perform the following actions:
•
Manage gels, match sets, classes, reports and documents.
•
Import new gels and directly convert them into ImageMaster format.
•
Open gels, for viewing and processing.
•
Define match sets and reference gels / match sets for matching.
•
Create classes for statistical analysis.
•
Add comments to describe and explain your experiment.
•
Save all this information in the workspace file (with the extension .mws).
The workspace is a systematic and efficient way to clarify your research and
avoid unnecessary work. The workspace ideally should reflect the structure and
design of your research. This encourages you to think, right from the beginning,
about the way the study should be constructed and about the information you
would like to obtain from your analysis.
The workspace also guides you through the gel analysis workflow (Figure 4-1) as
major operations such as spot detection, matching and statistical analysis are to
be prepared in the workspace. To understand how this works, you should note
that every project in the workspace is organized into five default folders: Gels,
MatchSets , Classes, Reports and Documents:
•
First, gels are imported in the Gels
folder where possible relationships
between gel images are specified (e.g. DIGE). The gels are then opened in the
Display Zone and spot detection is carried out.
•
Subsequently, MatchSets
are created to specify how the gels will be
matched together. Each match set is characterized by a reference gel and
can belong to a higher level match set. Once match sets are defined, the
corresponding gels can be opened and effectively matched.
•
Sets of gels that will be compared are grouped in Classes
. This is where
the biological question is stated. Classes must be defined in order to
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proceed with statistical analysis and the discovery of protein variations,
differences and similarities in a series of gels.
•
Saved ImageMaster reports are inserted in the Reports
folder. The
objects of interest in these reports can be sorted, selected, visualized on the
gels and exported in different formats.
•
Documents related to the project can be inserted in the Documents
folder. Graphical files containing exported gel images, histograms, 3D views,
or scatter plots are examples of files that can be added to the Documents
folder. So are spreadsheet files, text files, Microsoft Word, Microsoft
PowerPoint files, and many more.
Workspace
Project folders
Operation
Gels
Detecting
MatchSets
Matching
Classes
Analysing
Reports
Exporting
Documents
Figure 4-1. Image analysis from start to finish in the Workspace.
The gels in the workspace have icons that indicate what stage in the analysis
procedure your image has reached (Table 4-1 , Table 4-2). You can distinguish
DIGE gels from non-DIGE gels, see which gels have been detected and matched,
and tell which gels are used as reference for matching.
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Icon
Meaning
Non-detected gel.
Detected gel.
Matched gel / reference gel.
Table 4-1. The gel icons indicate what stage in the analysis procedure your image has
reached. Non-DIGE gels.
Icon
Meaning
Non-detected DIGE image / DIGE reference image (e.g. standard,
pool).
Detected DIGE image / DIGE reference image (e.g standard, pool).
Matched DIGE image / DIGE reference image (e.g. standard, pool).
Table 4-2. The gel icons indicate what stage in the analysis procedure your image has
reached. DIGE gels.
The beauty of the ImageMaster workspace is that you can customize it according
to your own requirements. Coworkers can look or work with the same data but
organize their workspace differently.
The Workspace is essentially symbolic. Changes to the workspace do not affect
images, reports, matches and master gels that are saved on your hard disk.
However, the information in the workspace project is necessary to reconstruct
the match sets and retrieve the matches. This is the reason why it is important to
regularly save the workspace (saves the projects) and why the workspace is
saved by default when closing the software.
4.2
Workspace window
Click the Workspace
tab below the ImageMaster toolbar to display the
Workspace window. The window (Figure 4-2) contains the Workspace Toolbar, the
Active Project, the Navigator and File Details.
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Workspace
a
b
c
d
Figure 4-2. Workspace window. a) Toolbar, b) Active Project, c) Navigator and d) File Details.
4.2.1
Workspace toolbar
The following icons are available from the Workspace toolbar:
Create a New workspace. In the Create New Workspace box, specify
the Workspace Name, Location and Comment. By default, a
workspace file (with the extension .mws) is saved in the
ImageMaster\Workspaces folder found in the user’s My Documents
directory.
Open an existing workspace. In the Open Workspace box, browse
the directory where the workspace file is located, select its name
and click Open.
Automatically Save the current workspace including changes made
to any projects. When you close ImageMaster, the workspace and
projects are also saved. Keep in mind that modifications done to
images, reports or matches are not saved with the workspace. To
save these changes, choose one of the options under File > Save.
4.2.2
Active project
The Active Project displays the name of the project from which the active
worksheet has been opened. This is particularly useful when different projects are
contained in the workspace and worksheets have been opened from several of
these.
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4.2.3
Navigator
The Navigator displays all files and folders related to a workspace. The look and
feel is similar to the Windows Explorer file manager. 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.
NOTE! The main difference from Windows Explorer is that the Navigator only
gives a symbolic view of your gel images, reports and inserted documents.
This means that you can delete images, reports and documents from a
workspace without losing the data stored on the hard disk. However, keep in
mind that removing gels from a project may affect your match sets.
The fundamental organization of the Workspace is fixed. At the top of the
Navigator is the Workspace Name. A workspace contains any number of
Projects, each of which represents a study of related gels. Every project is
organized into Gels, MatchSets, Classes, Reports and Documents. These default
folders cannot be deleted or renamed. However, you can organize the Workspace
according to your needs by adding subfolders to the hierarchical structure.
Workspace contextual menus
Right click on a folder, subfolder or file to open a contextual menu from which you
choose an action to be carried out. Detailed descriptions of all the contextual
menus in the Workspace are provided in Appendix A.
Moving files or folders
Move folders, subfolders and files in the Workspace by dragging and dropping or
using the Copy and Paste options in the contextual menus.
4.2.4
File details
Detailed information about selected files is given on the right side of the
Workspace window. The details include the Name, MatchSet and Class to which
the image belongs, dates Created and last Modified, Size (in bytes), File Type, File
Path and ID. The files can be listed in ascending or descending order by clicking
on the column header that you want to sort by.
4.3
Setting up a workspace
In ImageMaster 2D Platinum 6.0, gel images can only be opened from the
workspace. It is therefore necessary to set up a workspace as soon as you start
working with the software.
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4.3.1
Creating a workspace
To create a workspace:
1
Click on the New icon in the Workspace toolbar.
2
In the Create New Workspace box, specify the Workspace Name,
Location and Comment. Click OK. By default, a workspace file (with the
extension .mws) is saved in the ImageMaster\Workspaces folder found in
the user’s My Documents directory. If you want to save the file in a
different folder, then browse and open the folder.
3
The Workspace Name appears at the top of the Navigator. The
workspace file is automatically saved.
4.3.2
Opening an existing workspace
To open an existing workspace:
1
Click on the Open icon in the Workspace toolbar.
2
In the Open Workspace box, browse the directory where the workspace
file is located, select its name and click Open.
3
The file is opened in the Workspace window.
You can also go to File > Recent > Workspaces in the menu and choose the
Workspace Name from the list. This is only possible if a workspace file was saved
during the current work session.
If you open a workspace created with an earlier version of ImageMaster, you will
be asked if you want to convert it. If you click Yes, ImageMaster migrates the
workspace so that you can use the new features in ImageMaster 2D Platinum 6.0.
NOTE! Once you open and save a workspace in ImageMaster 2D Platinum
6.0, you are no longer able to work with the file in an older version of
ImageMaster. Be careful not to open version 6.0 workspace files in older
versions, because this can corrupt your workspace file.
4.3.3
Workspace properties
The Workspace Name, Creator, long File Name, Modification Date and Comment
are Workspace Properties. The Comment is helpful because it describes and/or
explains your workspace. It serves as a useful source of information to which a
coworker is referred or as a reminder when reviewing old work. You can add or
modify the Comment (or Workspace Name) while viewing the Workspace
Properties.
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To edit the Workspace Name or Comment:
1
Right click on the Workspace Name at the top of the Navigator.
2
Choose Properties from the contextual menu.
3
In the Edit Workspace Properties box, add or modify the Workspace
Name or Comment. Click OK.
4
The changes to Properties are saved.
4.4
Working with projects
A workspace can contain different projects. A project normally includes all gels,
along with related data, produced and analyzed during the course of a specific
gel study. In principle, a project will include images that are visually comparable
in a straightforward way. Very dissimilar gel images, due to differences in staining
or tissue for example, should better end up in different projects. Images that are
technically related, such as multiplexed or DIGE gel images, are an exception.
Since such images come from the same gel, at least their spot positions are
directly comparable.
4.4.1
Creating a project
To create a project:
1
Right click on the Workspace Name at the top of the Navigator.
2
Choose New Project from the contextual menu.
3
In the Create New Project box, specify the Project Name, Location and
Comment. Click OK. By default, a project file (with the extension .prj) is
saved in the ImageMaster\Workspaces folder found in the user’s My
Documents directory. If you want to save the file in a different folder,
then browse and open the folder.
4
The Project Name and default folders appear in the Navigator. The
project file is automatically saved.
A project is saved whenever you click Save in the Workspace toolbar. You can
remove a project from the current workspace by right clicking on the Project
Name and choosing Delete from the contextual menu. This does not delete the
project file from your hard disk. Therefore the project can be inserted into another
workspace.
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4.4.2
Inserting an existing project
To insert an existing project:
1
Right click on the Workspace Name at the top of the Navigator.
2
Choose Insert Project from the contextual menu.
3
In the Insert Project box, browse the directory where the project file is
located, select its name and click Open. Use the Shift or Ctrl keys to make
multiple selections.
4
The Project Name and the folders, gels, reports and other documents
included in the project appear in the Navigator.
4.4.3
Exporting a project
Exporting a project allows you to save a project together with copies of its gels,
matches, master gels, and other documents (to which new IDs are attributed), so
that you can import it into a new workspace or send it to a colleague. Please note
that the exported gel file structure is based on your project organization. Exported
projects can be included in any workspaces as explained above.
To export a project:
1
Right click on the project to export.
2
Choose Export from the contextual menu.
3
Choose a destination folder for the exported project and click OK.
4.4.4
Project properties
The Project Name, Creator, File Name, Modification Date and Comment are
Project Properties. The Project Comment is helpful because it describes and/or
explains the project. It serves as a useful source of information to which a
coworker is referred or as a reminder when reviewing old work. You can add or
modify the Comment (or Project Name) while viewing the Project Properties.
To edit the Project Name or Comment:
40
1
Right click on the Project Name in the Navigator.
2
Choose Properties from the contextual menu.
3
In the Project Properties box, add or modify the Project Name or
Comment. Click OK.
4
The changes to Properties are saved.
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4.5
Working with gels
The Gels
folder manages your gel images (Figure 4-3). You can easily:
•
Create folders and subfolders in the default structure.
•
Import gel images with .tif, .png, .gel , .img or .scan extensions.
•
Add ImageMaster 2D Platinum / Melanie format gels (version 4 or higher)
with a .mel extension.
•
Import and define DIGE gels.
•
Open images in the Display Zone, in the active worksheet or a new one.
•
See which images have already spots detected on them.
Figure 4-3. The Gels folder.
4.5.1
Creating a subfolder
You can create folders and subfolders in the Gels folder in order to customize the
hierarchical structure and to organize your images.
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DIGE gels must be placed in the designated DIGE Gels folder that is activated in
ImageMaster 2D Platinum DIGE. Each DIGE gel holds one, two or three gel images
and can be processed with the specific DIGE functionalities. The DIGE Gels folder
can not contain subfolders.
To create a subfolder:
1
Right click on the Gels folder or a subfolder.
2
Choose Create Folder from the contextual menu.
3
In the Create Folder box, specify a Name and Comment for the new
folder and click OK.
4
The Folder Name appears in the Gels folder.
Right click on the Gels folder, a subfolder or gel to open a contextual menu from
which you choose an action to be carried out. Detailed descriptions of all the Gels
folder menus are provided in Appendix A.
4.5.2
Importing a new gel
You can Import and automatically convert new gel images with .tif, .png, .gel ,
.img or .scan extensions into ImageMaster format (with the extension .mel). At
this level, you can choose a reduction factor to be applied when importing the
images. Thus, you automatically decrease the image resolution and therefore the
file size of large gel files.
To import a new image:
42
1
Right click on the Gels folder or a subfolder.
2
Choose Import Gels from the contextual menu.
3
In the Import Image box, input the Reduction Factor and File Format
and click OK.
4
In the Import Gels box, 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.
5
In the next Import Gels box, specify the new names and destination
folder for saving the gels in ImageMaster format. You can also add an
extension to all file names, or change an existing one.
6
In the Gel Properties box, specify the Staining (the stain that was applied
to the gel). Click OK.
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4.5.3
The Gel Name appears in the Gels folder or subfolder that was selected
in step 1. The image file is saved with the .mel extension.
Importing a new DIGE gel
DIGE gels must be placed in the designated DIGE Gels folder. This feature is only
available in ImageMaster 2D Platinum DIGE.
To import DIGE gel images:
1
Right click on the DIGE Gels folder.
2
Choose Import DIGE Gel from the contextual menu.
3
In the Import Image box, specify the Reduction Factor and File Format.
Click OK.
4
In the Import Gels box, browse the directory where the image files are
located, select the two or three images belonging to the same DIGE gel,
and click Open. Use the Shift or Ctrl keys to make multiple selections.
5
In the next Import Gels box, specify the new names and destination
folder for saving the gels in ImageMaster format. You can also add an
extension to all file names, or change an existing one.
6
In the Create DIGE box, specify a DIGE Gel Name, the Dye Chemistry
(DIGE Min. or DIGE Sat.) and a Comment. Click OK. By default, the
common part of the image names is suggested as gel name.
7
In the DIGE Gel Properties box, specify the Staining (the dyes that were
applied to each gel). Click OK.
8
In the DIGE Reference box, specify the Standard Gel (typically Cy2 if
using minimal dyes or Cy3 if using saturation dyes). Click OK.
9
The DIGE Gel Name appears in the DIGE Gels folder. The DIGE Gel Image
Names are found below. The gel image files are saved with the .mel
extension.
4.5.4
Adding an existing gel
You can add images that are already in ImageMaster 2D Platinum / Melanie
format (version 4 or higher) to the Gels folder of a project. The Gels folder can only
enclose one copy of each gel file.
To add an existing image:
1
Right click on the Gels folder or a subfolder.
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2
Choose Add Gels from the contextual menu.
3
In the Add Gels box, 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.
4
In the Gel Properties box, verify the Gel Name and Staining (the stain
that was applied to the gel). Click OK.
5
The Gel Name appears in the Gels folder or subfolder.
4.5.5
Adding an existing DIGE gel
You can add DIGE gels that are already in ImageMaster 2D Platinum format to the
DIGE Gels folder of a project. This feature is only available in ImageMaster 2D
Platinum DIGE.
To add an existing DIGE gel:
1
Right click on the DIGE Gels folder.
2
Choose Add DIGE Gel from the contextual menu.
3
In the Add Gels box, browse the directory where the image files are
located, select the 1, 2 or 3 images belonging to the same DIGE gel and
click Open. Use the Shift or Ctrl keys to make multiple selections.
4
In the Create DIGE box, specify the DIGE Gel Name, the Dye Chemistry
(DIGE Min. or DIGE Sat.) and a Comment. Click OK.
5
In the DIGE Gel Properties box, verify the Gel Image Names and Staining
(the dyes that were applied to each gel). Click OK.
6
In the DIGE Reference box, specify the Standard Gel (typically Cy2 if
using minimal dyes or Cy3 if using saturation dyes). Click OK.
7
The DIGE Gel Name appears in the DIGE Gels folder. The DIGE Gel Image
Names are found below.
4.5.6
Moving gels or folders
You can rearrange your gels or subfolders in the Gels folder, to change their
position in the list or move them into another folder. Just drag your gel or folder
to the desired position. It is inserted after the item you drop it on. Whenever there
is a possibility to insert it inside or after a folder, you will be asked for your choice.
4.5.7
Displaying gel images
To view gel images or work on them, they must be opened from the workspace.
You can select and open individual gels, or an entire gel folder. Gels that are in
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subfolders will be opened in separate panes carrying the names of the
subfolders. Each DIGE gel (being the two or three images) is also displayed in a
separate pane.
To open a gel or gel folder:
1
Select one or more gels or folders. Use the Shift or Ctrl keys to make
multiple selections. Right click on one of the gels or folders.
2
Choose Open > In Worksheet from the contextual menu to open the gels
in the active worksheet (or Open > In New Worksheet to open them in a
new worksheet).
3
The selected images are opened in the active worksheet (or a new
worksheet) in the Display Zone.
4.5.8
Spot detection
Once gels are added to the workspace and opened in the Display Zone, you are
ready to detect spots on them. See Chapter 7 to learn more about working with
spots and carrying out spot detection. Note that the gel image icons change
when spots have been detected.
4.5.9
Folder properties
The Name and Comment are properties of subfolders of the Gels folder. The
Comment describes and/or explains the content of the folder. It serves as a useful
source of information to which a coworker is referred or as a reminder when
reviewing old work. You can add or modify the Comment (or Name) while viewing
the Properties of a folder. Note that you cannot modify the name of a subfolder
while its gels are opened.
To edit the Comment or Name of a subfolder:
1
Right click on a subfolder of the Gels folder.
2
Choose Properties from the contextual menu.
3
In the Gel Folder Properties box, add or modify the Name or Comment.
Click OK.
4
The changes to Properties are saved.
4.5.10
Gel properties
The Gel Name and Staining are Properties of any gel image file. You can modify
the Staining while viewing the Gel Properties.
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To edit the Staining:
1
Right click on the image name.
2
Choose Properties from the contextual menu.
3
In the Gel Properties box, modify the Staining. Click OK.
4
The changes to Properties are saved.
The Name, Dye Chemistry and Comment are Properties of DIGE gels. They can
similarly be edited by right-clicking on a DIGE gel (not the individual images) and
choosing Properties from the contextual menu.
4.6
Working with match sets
The MatchSets
easily:
folder manages your match structures (Figure 4-4). You can
•
Create an unlimited number of match sets and submatch sets, enabling you
to match individual gels or gel populations.
•
Define a reference gel or reference match set that serves as the Master for
the matching.
•
Open a match set in a new worksheet, to carry out matching.
•
See which gels or match sets have already been matched.
•
Use your match sets to create classes.
A match set includes gels or populations of gels (in submatch sets) that should be
matched together. This is a major innovation over previous versions of the
software. You are no longer limited to matching gels. You can now match
populations of gels, and what more is, different levels of populations. This is called
population matching.
The concepts of match sets and population matching can best be illustrated with
an example. Imagine a set of samples from bacteria that were cultivated with
either substrate A or with substrate B. In both growing conditions, two treatments
have been 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.
Different possibilities exist to construct your match sets. You could first match the
three gels of each population (Figure 4-5), thus giving the match sets (i.e.
populations) A_T1, A_T2, B_T1, B_T2. You could then match the populations A_T1
and A_T2 to give population A, and do the same to construct population B. Finally,
the match sets A and B can be matched to give match set Bacteria 1.
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Figure 4-4. The MatchSets folder.
At each level, the matches are propagated. This means that once all match sets
have been effectively matched (indicated by a red tick mark), the spots of gel
A_T1_Gel1, for instance, can be directly compared with those of Gel B_T2_Gel3.
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Bacteria 1
A
B
A_T1
A_T2
B_T1
B_T2
A_T1_Gel3
A_T1_Gel2
A_T1_Gel1
A_T2_Gel3
A_T2_Gel2
A_T2_Gel1
B_T1_Gel3
B_T1_Gel2
B_T1_Gel1
B_T2_Gel3
B_T2_Gel2
B_T2_Gel1
Figure 4-5. Example of a hierarchical match set structure. Case 1.
Alternatively you could directly put all gels of population A in a match set, and
create a similar match set for the gels in population B. Populations A and B can
subsequently be matched together (Figure 4-6).
Bacteria 2
A
A_T1_Gel3
A_T1_Gel2
A_T1_Gel1
A_T2_Gel3
A_T2_Gel2
A_T2_Gel1
B
B_T1_Gel3
B_T1_Gel2
B_T1_Gel1
B_T2_Gel3
B_T2_Gel2
B_T2_Gel1
Figure 4-6. Example of a hierarchical match set structure. Case 2.
It is not possible to recommend an ideal matching scheme. Depending on how
you carried out your experiment, what you would like to show in your study, or
how similar your gels are, you may want to opt for a particular match set
structure. You may also desire to carry out several analyses, using different
matching schemes.
You can copy match sets to use them in another configuration. In Figure 4-7, 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). As existing
matches are conserved upon copying match sets, this can save a lot of work.
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Bacteria 3
T1
T2
A_T1
B_T1
A_T2
B_T2
A_T1_Gel3
A_T1_Gel2
A_T1_Gel1
B_T1_Gel3
B_T1_Gel2
B_T1_Gel1
A_T2_Gel3
A_T2_Gel2
A_T2_Gel1
B_T2_Gel3
B_T2_Gel2
B_T2_Gel1
Figure 4-7. Example of a hierarchical match set structure. Case 3.
For each match set a reference gel or reference match set must be defined. This
Reference will be used to create a Master, representing the match set. In Figure
4-5, Figure 4-6 and Figure 4-7, the reference gels or reference match sets have a
red component.
The choice of the reference gel or reference match set is important as it
significantly influences the results of your analysis. This is because spots that are
not in the Reference will also not be present in the Master, unless they are
manually copied there. The rule of thumb is to choose the gel with the most and
the best quality spots as the reference gel. To learn more about the Reference, the
Master, and matching, please refer to Chapter 9.
Please note that the master gel should rather be seen as a spot index. It is not
possible to display quantification values for a master gel.
4.6.1
Creating a match set
There are different ways to create a match set:
To create an empty match set:
1
Right click on the MatchSets folder or a match set.
2
Choose Create MatchSet from the contextual menu.
3
In the Create MatchSet box, specify a Name and Comment. Click OK.
4
The MatchSet Name appears in the MatchSets folder or match set
chosen in step 1.
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To create a match set from a gel folder:
1
In the Gels folder, select one or more folders containing gels with saved
spots. Use the Shift or Ctrl keys to make multiple selections.
2
Left click on one of the folders (a Hand cursor appears). Drag and drop
the folders into the MatchSets folder or into a destination match set. Or
right click on one of the gel folders and choose Copy from the contextual
menu. Right click on the destination match set and choose Paste from
the contextual menu.
3
In the Create MatchSet box, specify a Name and Comment. Click OK.
Repeat for every copied folder.
4
New match sets appear in the MatchSets folder or destination match
set. They contain the gels from the copied gel folders.
Right click on the MatchSets folder, a match set or gel to open a contextual menu
from which you choose an action to be carried out. Detailed descriptions of all the
MatchSets folder menus are provided in Appendix A.
4.6.2
Creating a match set from a DIGE gel
A DIGE gel is an inherent match set. As the different images in a DIGE gel have
identical co-detected spots, ImageMaster automatically creates matches
between the spots in the images. You can easily add a DIGE gel to your MatchSets
folder or insert it into an already existing match set.
To create a match set from a DIGE gel:
50
1
Select one or more DIGE gels (not the individual images) in the DIGE Gels
folder. Use the Shift or Ctrl keys to make multiple selections. Right click
on one of the gels.
2
Choose Create MatchSet from the contextual menu.
3
In the Add DIGE in MatchSet box, select the match set you would like to
add your new match sets to.
4
In the Create MatchSet box, specify a Name and Comment for each
match set to be created. By default, the name of the DIGE gel is used.
Click OK.
5
The new match sets appear in the MatchSets folder and the tick mark in
the gel image icons indicates that matches exist between the images
belonging to a same DIGE gel.
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Alternatively, you can drag and drop, or copy and paste your DIGE gel to the
MatchSets folder to create the corresponding match set.
4.6.3
Adding gels to a match set
There are different ways to add gels to a match set.
To use contextual menus to add gels to a match set:
1
Select one or more images in the Gels folder. Make sure spots were
detected and saved. Use the Shift or Ctrl keys to make multiple
selections. Right click on one of the images.
2
Choose Add in MatchSet from the contextual menu.
3
In the Add Gels in MatchSet box, type in a name if you want to place the
gels in a new match set. Or choose an existing match set from the list.
Click OK.
4
If a new match set is created, specify the reference geI in the following
Add Gels in MatchSet box, and click OK.
5
The gels are added and the reference gel is tagged.
To copy and paste gels to a match set:
1
Select one or more gels in the Gels folder or MatchSets folder. Use the
Shift or Ctrl keys to make multiple selections. Right click on one of the
gels.
2
Choose Copy from the contextual menu.
3
Right click on the destination match set.
4
Choose Paste from the contextual menu.
5
The gels are added.
To drag and drop gels to a match set:
1
Select one or more gels in the Gels folder. Use the Shift or Ctrl keys to
make multiple selections.
2
Left click on the gels (a Hand cursor appears).
3
Drag and drop the gels into the destination match set.
4.6.4
Copying match sets
Match sets can be copied for use in another match set configuration.
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To copy a match set:
1
Select one or more match sets in the MatchSets folder. Use the Shift or
Ctrl keys to make multiple selections.
2
Right click on one of the match sets and choose Copy from the
contextual menu. Right click on the destination match set and choose
Paste from the contextual menu.
3
The copied match sets appear in the designated location, with one or
several characters appended to their names (match set names must be
unique).
4
Right click on a copied match set.
5
Choose Properties from the contextual menu.
6
If necessary, change the match set Name in the MatchSet Properties
box. Click OK. Note that you cannot modify the name of an open match
set.
4.6.5
Setting the match set reference
For each match set, a reference gel or reference match set should be defined. It
is used to create the master image that will represent the match set.
If your match set contains gels, right click on the gel to be used as Reference and
choose Set Gel as Reference from the contextual menu. If your match set contains
submatch sets, right click on the submatch set to be used as Reference and
choose Set MatchSet as Reference from the contextual menu. Icons of reference
gels and reference match sets have a red component.
NOTE! You can still change the Reference as long as the images in your
match set have not been matched. Once they are, the reference image
cannot be changed anymore.
4.6.6
Moving gels or match sets
You can rearrange your gels or match sets in the MatchSets folder, to change
their position in the list or move them into another match set. Just drag your gel
or match set 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 a match set, you
will be asked for your choice.
4.6.7
Displaying match sets
To match gels or match sets, they must be opened in a [MatchSet] worksheet.
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When opening a match set with gels in it, all gels are displayed except for the
Reference. The Master of the match set, recognizable through its red legend, is
shown instead. This is because the master and reference images are the same,
and matches are automatically created between the two. The Reference
therefore no longer needs to be matched. When necessary, the reference gel can
be displayed by choosing Show > Show Reference from the menu.
When opening a match set with submatch sets in it, all master gels of the
submatch sets will be displayed, except for the one that was chosen as a
reference for the opened match set. Instead the master image of the opened
match set is shown.
To open a match set:
1
Select the match set to be opened. Right click on its name.
2
Choose Open from the contextual menu to open the match set.
3
The match set is opened in a new [MatchSet] worksheet in the Display
Zone.
4.6.8
Matching
Once a match set is opened in the Display Zone, you are ready to match the gels
against the Master. See Chapter 9 to learn more about matching. Note that the
gel or match set icons change when matching is completed.
4.6.9
Match set properties
The Name and Comment are properties of match sets. The Comment describes
and/or explains the content of the match set. It serves as a useful source of
information to which a coworker is referred or as a reminder when reviewing old
work. You can add or modify the Comment (or Name) while viewing the Properties
of a match set. Note that you cannot modify the name of an open match set.
To edit the Comment or Name of a match set:
1
Right click on the match set.
2
Choose Properties from the contextual menu.
3
In the MatchSet Properties box, add or modify the Name or Comment.
Click OK.
4
The changes to Properties are saved.
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4.7
Working with classes
The Classes
folder manages your classes (Figure 4-8). You can easily:
•
Create an unlimited number of classes and subclasses.
•
Open classes in a worksheet, to carry out statistical analysis.
Figure 4-8. The Classes folder.
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A class is a set of gels or gel populations that you can compare with other such
entities. In the Classes folder, you state your biological questions. Your goal, by
comparing classes and therefore the gels within those classes, is to find the
protein expression variations between different biological states.
NOTE! To compare gels in a class or within different classes, the gels should
have a common node in the match set that is used to construct the classes.
Without a common node, the gels cannot be matched together and statistical
comparison is impossible. This means that all gels to be compared should
belong to the same higher level match set.
NOTE! The same series of gels may be found in several match sets. Therefore,
several possibilities will exist when adding gels to a class. The decision of
which match set to use can have significant impact on the statistical analysis.
The use of classes can again be illustrated with the example of the bacteria gels
described in the previous section (see Section 4.6). Figure 4-8 illustrates three
possible class setups based on the match set configurations Bacteria 1 (Figure
4-5) and Bacteria 2 (Figure 4-6).
The hierarchical structure of the class Bacteria 1 in Figure 4-8 is based on the
match set Bacteria 1 and uses the same configuration. The class Bacteria 1
contains two sub classes A and B, which in turn have two sub classes each. Using
this configuration you can compare at any moment: class A with class B, A_T1
with A_T2, A_T1 with B_T1 or even A with B_T2.
Still using match set Bacteria 1 as a basis, you can also create a class structure
as in Bacteria 1B (Figure 4-8). This class contains two sub classes T1 and T2 that
each hold the gels corresponding to a specific treatment. It essentially allows you
to compare T1 and T2.
Another option is to use match set Bacteria 2 when defining your classes, but with
the same class configuration as in class Bacteria 1. In this way, you can easily use
the classes A and B, and the subclasses A_T1, A_T2, B_T1 and B_T2 for various
comparisons. However, the results can be more or less different from the first case
discussed above, where the Bacteria 1 match structure serves in the analysis.
NOTE! To see from which match set a gel in a class is derived, you can look at
the MatchSet column in the File Details at the right side of the workspace
window (Figure 4-9). It is also there that you can verify if gels belong to the
same match set (necessary for statistical comparisons).
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Figure 4-9. Selected gels in the Classes folder.
4.7.1
Creating a class
There are different ways to create a class:
To create an empty class:
1
Right click on the Classes folder.
2
Choose Create Class from the contextual menu.
3
In the Create Class box, specify a Name and Comment. Click OK.
4
The Class Name appears in the Classes folder.
To create a class from a match set:
1
56
Select one or more match sets from the MatchSets folder. Use the Shift
or Ctrl keys to make multiple selections.
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2
Left click on one of the match sets (a Hand cursor appears). Drag and
drop the folders into the Classes folder or into a destination class. Or
right click on one of the match sets and choose Copy from the
contextual menu. Right click on the destination class and choose Paste
from the contextual menu.
3
In the Create Class box, specify a Name and Comment. Click OK. Repeat
for every copied match set.
4
New classes appear in the Classes folder or destination class. They
contain the gels from the copied match set.
Right click on a class, subclass or gel to open a contextual menu from which you
choose an action to be carried out. Detailed descriptions of all the Classes folder
menus are provided in Appendix A.
4.7.2
Adding gels to a class
There are different ways to add matched gels to a class:
To use contextual menus to add matched gels to a class:
1
Select one or more gel images in a match set. Use the Shift or Ctrl keys
to make multiple selections. Right click on one of the images.
2
Choose Add in Class from the contextual menu.
3
In the Add Gels in Class box, type in a name if you want to place the gels
in a new class. Or choose an existing class from the list. Click OK.
4
The gels are added to the class.
To copy and paste gels to a class:
1
Select one or more gels in a match set. Use the Shift or Ctrl keys to make
multiple selections. Right click on one of the gels.
2
Choose Copy from the contextual menu.
3
Right click on the destination class.
4
Choose Paste from the contextual menu.
5
The images are added to the class.
To drag and drop gels to a class:
1
Select one or more gels in a match set. Use the Shift or Ctrl keys to make
multiple selections.
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2
Left click on the gels (a Hand cursor appears).
3
Drag and drop the gels into the destination class.
4
The gels are added to the class.
4.7.3
Moving gels or classes
You can rearrange your gels or classes in the Classes folder, to change their
position in the list or move them into another class. Just drag your gel 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 a class, you will be asked
for your choice.
4.7.4
Displaying classes
To carry out statistical analysis on the gels in your classes, they must be opened
in a [Classes] worksheet. You can select several classes from the workspace and
open them simultaneously. Each selected class will be opened in a separate pane
carrying the name of the class. Note that the gels will be opened as part of the
highest level class selected. This means that if you select the classes A (with
subclasses A_T1 and A_T2) and B (with subclasses B_T1 and B_T2) the gels will be
opened in two panes, as part of the classes A and B. If you select the four
subclasses and open them, the gels will be opened in four panes, as part of the
classes A_T1, A_T2, B_T1 and B_T2.
To open a class:
1
Select one or more classes. Use the Shift or Ctrl keys to make multiple
selections. Right click on one of the classes.
2
Choose Open from the contextual menu to open the classes.
3
Each class is opened in a separate pane in the newly created [Classes]
worksheet.
4.7.5
Statistical analysis
Once classes are opened in the Display Zone, you are ready to analyse your gels
and calculate Intra-Class or Inter-Class statistics. See Chapter 10 to learn more
about data analysis.
4.7.6
Class properties
The Name and Comment are properties of classes. The Comment describes and/
or explains the content of the class. It serves as a useful source of information to
which a coworker is referred or as a reminder when reviewing old work. You can
add or modify the Comment (or Name) while viewing the Properties of a class.
Note that you cannot modify the name of an open class.
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To edit the Comment or Name of a class:
1
Right click on the class.
2
Choose Properties from the contextual menu.
3
In the Class Properties box, add or modify the Name or Comment. Click
OK.
4
The changes to Properties are saved.
4.8
Working with reports
The Reports folder manages all tabular report files (with the extension .rpt) that
you produce in ImageMaster. Refer to Chapter 5 to learn more about reporting.
4.8.1
Creating a subfolder
You can create folders and subfolders in the Reports folder in order to better
organize your saved reports.
To create a subfolder:
1
Right click on the Reports folder or a subfolder.
2
Choose Create Folder from the contextual menu.
3
In the Create Folder box, specify a Name and Comment for the new
folder and click OK.
4
The Folder Name appears in the Reports folder.
Right click on the Reports folder, a subfolder or report file to open a contextual
menu from which you choose an action to be carried out. Detailed descriptions
of all the Reports folder menus are provided in Appendix A.
4.8.2
Adding reports
When saving a report, it is automatically inserted into the Reports folder of the
Project it has been created from. However, you can also add report files from disk.
To add reports:
1
Right click on the Reports folder or a subfolder.
2
Choose Add Reports from the contextual menu.
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3
In the Add Report Files box, browse the directory where the files are
located, select the names and click Open. Use the Shift or Ctrl keys to
make multiple selections.
4
The files are added to the Reports folder or subfolder.
4.8.3
Opening reports
You can open a report from the Reports folder by double clicking on it, or by
selecting Open from the contextual menu.
4.8.4
Moving reports or folders
Move folders, subfolders and files within the Reports folder by dragging and
dropping or copying and pasting.
4.9
Working with documents
The Documents folder manages all project-related files other than your gel
images and tabular reports. These can include text files, spreadsheet files,
Microsoft PowerPoint presentations or mass spectrometry data. The Documents
folder can also contain pick lists and graphical files such as histograms, 3-D
views, scatter plots, factor analysis projections, exported gel regions or gel
windows (with .bmp, .tif or .png extensions). When saving a graphical file in
ImageMaster, it is automatically inserted into the Documents folder of the source
Project.
4.9.1
Creating a subfolder
You can create folders and subfolders in the Documents folder in order to better
organize your project related files.
To create a subfolder:
1
Right click on the Documents folder or a subfolder.
2
Choose Create Folder from the contextual menu.
3
In the Create Folder box, specify a Name and Comment for the new
folder and click OK.
4
The Folder Name appears in the Documents folder.
Right click on the Documents folder, a subfolder or file to open a contextual menu
from which you choose an action to be carried out. Detailed descriptions of all the
Documents folder menus are provided in Appendix A.
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4.9.2
Adding files
As mentioned above, any project related file, generated by any software can be
inserted in the Documents folder of the project.
To add files:
1
Right click on the Documents folder or a subfolder.
2
Choose Add Files from the contextual menu.
3
In the Add Document Files box, browse the directory where the files are
located, select the names and click Open. Use the Shift or Ctrl keys to
make multiple selections.
4
The files are added to the Documents folder or subfolder.
4.9.3
Opening documents
You can open a file from the Documents folder by double clicking on it, or by
selecting Open from the contextual menu. The file will be opened in the
associated application.
4.9.4
Moving documents or folders
Move folders, subfolders and files within the Documents folder by dragging and
dropping or copying and pasting.
4.10
Gel and report identifiers
ImageMaster allocates a unique identifier (ID) to each gel and report in order to
assure data consistency, allow reliable identification of gels and reports across a
computer network and enable database integration.
The gel and report IDs in ImageMaster are UUIDs (Universal Unique IDentifiers),
which are 128 bit numbers that are guaranteed to be unique through
combinations of hardware addresses, time stamps and random seeds. These IDs
allow your gels and associated reports to be uniquely recognized. This is because
the gel or report ID always overrides the file name or file location. Thus,
ImageMaster detects, for example, if you delete a gel and replace it with another
one with the same name.
NOTE! This also means that duplicating a gel outside ImageMaster may lead
to confusion, as you will end up with two gels bearing the same ID. For that
reason you should always try to manipulate (delete from disk, save as,
rename) your gels from within ImageMaster.
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Workspace
When you delete, rename or move your gels and associated reports outside
ImageMaster, you will get a message at program startup indicating that the file(s)
can not be found. The corresponding gels and reports will also have a red cross
on them in the Workspace project. To solve this problem, ImageMaster lets you
search for the files, at program startup or with the Search option in the
Workspace contextual menus. You must indicate the folder in which the files are
located. If the software still can not find a file - when it has been overwritten for
instance, the only option is to delete it from the workspace and insert it again.
4.11
Archiving a workspace
It is important to realize that the files included in your workspace can be located
anywhere on your hard disk or network. They are not necessarily all found in the
same directory. Gel and report files are usually shared by different users on one
or several network folders, whereas the workspace files, project files, and
matching data are by default saved in the ImageMaster folder of the user’s My
Documents directory (access to this data should generally be restricted).
In order to make a backup of the entire experiment or to share the files with a
person that cannot access your network, you can save your workspace or just a
single project with all its respective files in a single location.
NOTE! It is not possible to open a workspace/project when you don't have
access to all the necessary folders, and in particular the folder where matches
and other data are stored. Therefore, if a user wants to share his workspace/
project, he has to share his local ImageMaster folder, export his project(s) (see
Section 4.4.3), or provide a backup of his project or workspace (see below).
NOTE! It is good practice to make regular backups of your work using the
functionalities described below, so that you can recover your work at any
time.
4.11.1
Backup / Restore workspace
With the Backup Workspace function, a complete workspace can be archived
including the projects, gels, matches, master gels, reports and other related files.
This backup is written into a single compressed file (with the extension .bkp) that
can be restored when needed. Use the Restore Workspace function to later
retrieve the backup.
To backup a workspace:
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1
Right click on the Workspace Name at the top of the Navigator.
2
Choose Backup from the contextual menu.
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3
In the Backup Workspace box, 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 workspace:
1
Right click on the current Workspace Name at the top of the Navigator.
2
Choose Restore Workspace from the contextual menu.
3
In the Restore Workspace box, browse the directory where the backup
file is located, select its name and click Open.
4
In the Restore Backup box, choose one of the two options for restoring a
workspace. By restoring the files using the original file paths, you return
to your workspace by automatically rewriting the previous file and
folder structure. By restoring the files in a single folder, the workspace is
reproduced in a new directory, for which you have to enter a file name
and location. Click Restore.
5
The workspace is restored.
4.11.2
Backup / Restore project
With the Backup Project function, a single project can be archived including its
gels, matches, master gels, reports and other related files. This backup is written
into a single compressed file (with the extension .bkp) that can be restored when
needed. Use the Restore Project function to later retrieve the backup.
To backup a project:
1
Right click on the Project Name.
2
Choose Backup from the contextual menu.
3
In the Backup Project box, 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
Right click on the current Workspace Name at the top of the Navigator.
2
Choose Restore Project from the contextual menu.
3
In the Restore Project box, browse the directory where the backup file is
located, select its name and click Open.
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Workspace
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4
In the Restore Project box, choose one of the two options for restoring a
project. By restoring the files using the original file paths, you return to
your project by automatically rewriting the previous file and folder
structure. By restoring the files in a single folder, the project is
reproduced in a new directory, for which you have to enter a file name
and location. Click Restore.
5
The project is restored in your workspace.
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Reports
5
Reports
5.1
Introduction
The reports available in ImageMaster not only provide options for displaying
information on selected objects, but also are useful for sifting through data.
Reports can be accessed from the Reports menu, the Analyze menu or sometimes
through displayed reports. Reports are divided into two different menus for the
stated reasons:
•
Reports menu: DISPLAYS only quantitative or qualitative information on
selected objects (gels, spots, labels, annotations, categories and matches).
•
Analyze menu: COMPUTES differences and similarities between gels
(allowing data analysis based on robust statistics, factor analysis, statistical
tests and clustering techniques).
NOTE! Reports are not necessarily in table format. Graphical representations
of data such as histograms, scatter plots and 3D views are regarded as
reports as well.
You can display as many reports simultaneously as you wish. Reports are
Dockable Windows (see Section 3.4.4). Since every report displayed on the screen
is listed in the Window menu, it can easily be located by clicking on its name. In
order to quickly hide all pinned report windows, you can go to Windows >
Minimize All. In order to close all report windows, choose Windows > Close All in
the menu.
5.1.1
Reports menu
From this menu you can display reports by:
3D View
A three-dimensional view of selected gel regions or areas
around selected spots.
Gel
Summarized information about the selected gels, such as
Gel ID, intensity calibration parameters, number of detected
spots, number of annotations, gel resolution and size, etc.
Gel Description
User-defined properties of gels such as sample type, date of
the experiment, operator name, treatment conditions, pH
range, SDS gel percentage, or staining.
Gel Calibration
Summarized information about the selected gels including
file name, ID, file path, calibration coefficients, type of file,
creator and creation date.
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Spot
Specific information about selected spots, such as Spot ID
and coordinates, quantification values, attached labels, etc.
Label
Information on selected labels such as content, category,
Spot ID (if the label is linked to a spot), and annotation
coordinates.
Annotation
Information for selected annotations about chosen
categories and their labels, together with annotation
coordinates and Spot ID (if the annotation is linked to a spot).
Category
Information on all labels belonging to selected categories.
The displayed information is the same as for the report on
labels.
Match
A list of selected matches among a number of selected gels,
in terms of corresponding Spot IDs.
Match Statistics
A list of the matching results (number of matches and
percentage of matches) between the master and the
selected gels.
5.1.2
Analyze menu
From this menu you can display reports and histograms by:
Intra-Class
Information about each selected 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 (such as slope, offset, correlation coefficient and
fitting error) that compare the spot values for two gels.
Inter-Class
Central tendency, dispersion and overlapping measures for
classes of gels, computed for all selected matches. Differences
between the spot values in two classes can also be quantified
with Statistical Tests such as the Student t test, Mann-Whitney U
test and Kolmogorov-Smirnov test.
Also look at:
DIGE
Reports and histograms on DIGE gels (display information
such as Volume Ratios and frequency distribution of these
ratios).
Heuristic
Clustering
Classifies sets of gels and highlights significant matches
that may allow identification of different populations and
their characterization by specific spot patterns.
Most of the ImageMaster reports share a certain number of features. One of these
is the standard report toolbar that offers common functionalities such as saving,
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printing, selecting objects or navigating through the gel data. A second feature is
that several reports are editable, more specifically those that display annotation
content and properties. In addition, most of the reports are customizable. This
means that you can suppress or move columns, resize columns or rows, and sort
your columns in descending or ascending order based on their values (as can be
done with spreadsheet software such as Microsoft Excel). Finally, the lines in
tabular reports are numbered, indicating the number of selected objects used to
create the report.
5.2
Report toolbars
5.2.1
Standard tools
Save a report. Enter the File name in the displayed window and
choose the desired format in the Save as type field. Tables can be
saved in the ImageMaster report format (XML file carrying the
extension .rpt), in text format with tabulations separating the
columns (.txt), or as a Microsoft Excel Workbook (.xls). Graphics can
be saved in PNG, TIFF or BMP formats.
Print a report. When you print graphical reports (such as scatter
plots, 3D views, or histograms), the printing window with the normal
printing options is displayed. When you print tabular reports, things
are a bit different. The report table to be printed is first displayed in
your default Internet 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 (find more details about XML and XSL in Chapter 11).
You can subsequently use the print option in your browser to get a
paper printout.
Copy to Clipboard. Export your data directly into another program.
You can, for example, copy tables to spreadsheet software like
Excel, or graphics to programs such as Word or Adobe Photoshop.
To do so, 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 button
in the toolbar or the Copy to Clipboard option in the contextual
menu (available by clicking the right mouse button). Paste directly
into the preferred software.
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5.2.2
Adding comments
Add a Comment to the report. You can add comments to any report
to note relevant information. Since the report comment is available
at any time, it is very convenient to keep information about
selection criteria and other protocols for later use, and in case other
people work with the report. Comments are automatically saved in
the report file (when the latter is saved).
NOTE! Notice that any comment can, but need not, be saved separately
using the Save button in the Comment window. Previously saved comments,
as with any text file, can subsequently be opened and reused by clicking the
Open icon. Comments can also be printed, copied or pasted, as a whole or in
part, using the corresponding icons.
5.2.3
Selecting and navigating
Reports have the property of being navigable, meaning that you may reselect on
the gels one or more items (gels, spots, matches, etc.) that were picked in a report
or histogram. You can also scroll through the items listed in a report,
systematically viewing each of them on the gels by order of appearance.
Double click on a report line or histogram to select and display the corresponding
object on the gel(s). If you want to select several items or go through a list of items
systematically, use the following icons in the report toolbar:
Select on Gels. When clicking on this button, any items
corresponding to the selected report lines or histograms (use Shift
and Ctrl keys for multiple selections) are selected on the open gels.
The open gels are displaced in order to show at least one of the
selected objects
Select Next. You can use this icon to select the following object
listed in the report or histogram. Please note that the open gel(s) are
displaced to show the selected item in the center of the cell in which
the gel is displayed.
Select Previous. You can use this icon to select the preceding object.
in the report or histogram. Again, the open gel(s) are displaced to
show the selected item in the center of the cell in which the gel is
displayed.
An example of how one can navigate through the lines of an Intra-class Report is
shown in Figure 5-1. In order to check the matching results for particular
matches, these matches were selected, and a report on matches was generated.
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(a)
(b)
Figure 5-1. Navigating through the lines of an Intra-class Report. (a) Match 4704 is selected
in the report. When the Select on Gels icon is clicked, the corresponding spots are displayed
on the gels. (b) Select Next displays the following match, with ID 4715, on the gels.
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After selecting the line corresponding to Match 4704, and pressing the Select on
Gels button, all the spots belonging to Match 4704 are selected on their respective
gels. By clicking the Select Next button, the spots belonging to the following
match in the report (Match 4715) are selected on the screen. In this way, one can
go through all the matches in the report. This is just a simple example to illustrate
the use of reports and navigation through data. The possibilities are endless.
The Select on Gels drop down menu gives access to several related options:
•
Select on Gels has the same function as the Select on Gels button.
•
Select on Gels + Reports not only synchronizes the selection from your
report to the gels, but also to any other open reports, including open
histogram windows or 3D views. Imagine, for example, that you have an
open Spot Report and an open Intra-Class Report. If you select a spot in
your Spot Report and then choose the Select on Gels + Reports option,
ImageMaster not only selects and displays the corresponding spot on your
gel, but also selects the corresponding line in the Intra-Class Report. Of
course, this is only true if your spot is part of a match, and if that match was
selected for the creation of the Intra-Class Report.
•
Refine Selection allows you to refine your selection by using an additional
search criterion, applied only to the highlighted items in your table. In an
Intra-Class Report, for example, you could first sort your data based on the
Separability (see Section 5.4.4 for more information on sorting data), select
the 10 matches with highest Separability, and then refine the selection
based on the Ratio. Thus, by keeping matches that have a Ratio higher than
3, one would end up with only 7 selected matches in the example from
Figure 5-2.
•
Select from Gels will select the lines in your report that correspond to the
selected objects on your gels. Please note that ImageMaster scrolls up or
down in the report so that at least one selected line becomes visible.
5.2.4
Displaying related reports
Next to some report specific icons such as those linked with the Histograms,
Measure Histograms or the Factor Analysis functions (which are discussed in the
pertinent sections of this manual), you may find the following button:
In most cases, it allows you to create a Report from Selection, that
is, a new report that only contains the selected lines (or histograms)
from the active report. Thus, you can create a new report, only
containing objects that fit your specific criteria, without first having
to reselect the items on the gel.
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(a)
(b)
Figure 5-2. Refining a selection in your report. (a) The matches in the report were sorted
according to their Separability (in descending order), and the first 10 matches were selected.
This selection was further refined by only keeping the rows for which the Ratio (100%) value
is higher than 3. The result is shown in (b).
However, when you find a drop down menu next to the icon, the ImageMaster tool
tip calls it the Reports icon. In this particular case, the drop down menu gives you
a list with several related reports. These can include:
•
Report from Selection: Generates a new report only containing the selected
lines from the active report (as describe above).
•
Gel Report: Contains summarized information about the gels selected for
the creation of the active report and always includes the master gel, if one
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has been created. It can be used as a legend to the gel index (a,b,c,…) on
histograms. Note that this report is not necessarily the same as the one
obtained by choosing Reports > Gel Report in the menu.
•
Spot Report: Is a modified report on spots that is helpful to compare one
spot against the others in the same match. The usefulness of this report is
described in more detail in the Chapter 10 of this manual.
•
Intra-Class Report: Displays a report on selected matches for a selected
class. It is a kind of reduced report on matches, where only the spot values
of one class are considered.
•
Inter-Class Report: Lists the numerical values corresponding to selected
histograms.
•
Fitting Report / Scatter Report: These reports are only available when
displaying scatter plots and are detailed in Chapter 10 of this manual.
•
DIGE Report: Is a modified report on spots for DIGE gels, which allows to
display Volume Ratios between two selected images of the same DIGE gel,
in addition to the other quantification values for spots.
5.3
Editing report content
Some reports in ImageMaster (on matches, classes, annotations, labels, and
categories) are editable, although to different extents. Within the Label and
Category Reports, you can edit existing annotations on any gel used for the
creation of the report. In the Intra-Class, Inter-Class and Annotation Reports, you
can additionally create new columns corresponding to label categories and add
new labels. However, as the annotations displayed in some of these reports (IntraClass, Inter-Class) are those from the master gel, any modifications are only
applied to the latter. This is not a problem, since you can easily propagate
selected labels to matched spots (see Section 8.6.2). The ability to edit reports
gives you the possibility to easily mark any interesting data from within the report
and to propagate this data to your master gel, resulting in the creation of new
annotations on this master gel.
Exhaustive information about annotations, categories and labels is given in
Chapter 8. However, at this point it is useful to know that you can create different
label categories, which often correspond to columns in your report, and that you
can apply special constraints to the label content in each category. These include
the Data Type (Text, Number or Boolean) and whether each label in the category
should be Unique (as is the case for landmarks) or not.
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The Annotate icon allows you to insert a category as an extra
column by selecting its name from the category list. You can create
a new category by typing its name in the upper field of the
displayed window. In the latter case, you are asked for the category
constraints.
Once this is done, you can directly add new labels to the appropriate cells or edit
existing ones. Just double click in a cell to start typing your label. When finished,
a single click in any cell quits the editing mode. The cell corresponding to the
modified label appears in dark green when the corresponding line is selected or
in gray when it is not selected. You can quickly see that modifications were made
to your report when an asterisk follows the window title.
Subsequently, the Update Gel icon should be used to make the
modifications definitive and to propagate the information to your
gels (which will correspond to the master gel for the Intra-Class and
Inter-Class Reports). Note that the cells of updated labels go back to
their original color. When all labels in your report have been
updated, the asterisk behind the report title also disappears.
NOTE! A word of warning is expressed here. Any changes made to a gel
(addition or modification of labels) by using the Update Gel icon can be
canceled with the undo function. This is not the case for reports. Editing
operations on reports cannot be reversed.
Notice that categories of the type Set, or categories using the Boolean Data Type,
are displayed in the form of check boxes in some reports. Editing these labels
corresponds to checking the box in order to indicate that the item belongs to a Set
(box checked) or not (empty box), or takes the value 0 (empty box) or 1 (box
checked) for a Boolean Data Type.
To check several boxes (i.e. lines) simultaneously:
1
Select the rows you want to change the checked state.
2
Hold down the Shift key while clicking in the box of one of the selected
rows (of course in the relevant column). All selected lines are now in the
same activated state. Click in the check box a second time if the state
should be reversed.
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5.4
Customizing reports
5.4.1
Settings
The presence of the Settings icon indicates that the current report is
completely customizable. This means that you can choose the data
columns to build your personalized report and the order in which
they should be displayed. This is particularly useful when you only
want the essential information to appear for clarity or for printing
purposes.
To customize your report:
1
Click on the Settings icon in the report toolbar.
2
In the displayed window, select the attributes that you would like to add
to the report from the left column (Gels or Master gel list, in the case of
Figure 5-3), and transfer them to the right column (called Visible
attributes list, in the case of Figure 5-3) by pressing the right-pointing
arrow button.
3
To hide columns in the report, select their names in the right column and
transfer the chosen items to the hidden list(s) at the left by pressing the
left-pointing arrow button.
4
Click OK to confirm your choice and view the customized report.
Please note that the order of the items in the list of visible attributes determines
the order of the columns in your report (although the column order can later be
changed, as explained below). Thus, you can manipulate the column order by
adding your attributes in a specific sequence to the list of visible attributes.
Your adapted settings can be saved and used as report templates. For this
purpose, the following options are available from the Settings window:
•
By clicking Save and typing a file name you can save the new report
template.
•
By clicking Load and choosing a file name you can load an existing
template file.
There is no limit to the number of report templates that you can save or use. If you
save a report template in the ImageMaster\Menus folder of the User Documents
directory, it is available at any time from the menu Reports > … Report (the
templates are automatically loaded when ImageMaster starts). If the template is
saved in or loaded from any other folder, it is added to the menu Reports > …
Report > Recent Templates, and is only accessible during the current work session.
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The last report template used is available by choosing Reports > … Report >
Current Template from the menu.
Figure 5-3. Spot Report Settings window. You can add items to, or suppress items from, the
Visible attributes list using the corresponding blue arrow buttons.
5.4.2
Column order
You can reorganize the columns of a table directly in the report window. To do so,
drag the column heading to its new position, that is, to another column to which
it will be inserted to the left.
5.4.3
Column and row size
You can enlarge or reduce the column size. Drag the boundary on the right side
of the column header until the column is the width you want. While dragging, you
will notice that the cursor changes its format. To simultaneously change the
width of all columns, hold the Shift key and then drag the boundary of any
column header. To resize columns so that their whole content is displayed, double
click on their separator. The column to the left of the cursor reverts to its default
size.
You can also alter the row size. In this case, drag the lower border of the row to
reduce or increase its height. To enlarge or reduce all rows at once, hold down the
Shift key and drag the row separator. To resize rows so that their whole content is
displayed, double click on their separator.
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5.4.4
Sorting data
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 data is sorted in descending order and the textual data in
ascending order. When you click once more on the header, the triangle inverts
indicating that the data is sorted 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.
You can sort the items in your table using multiple criteria. Since rows containing
identical values in a sorted column appear together, you can further sort the
items in your table by specifying additional columns for ranking. Note that if you
would like to sort your data by two or more criteria, you should sort by the least
important columns first and finish with your principal criterion. In Figure 5-4 the
principal criterion is the Spot ID of the master gel (Match ID) and the secondary
criterion is the spot Volume.
Figure 5-4. Rows in a Spot Report sorted by two criteria. Spots were first sorted according
to their Volume (in descending order), then according to their Match ID.
5.5
Mouse selection reports
Mouse selection reports (Figure 5-5) are similar to any standard report in terms of
functionality (icons, etc.) The main difference is that they are interactive. The
reports contain only the items you select with the mouse while using the Spot or
Annotation tools. You can select several items at a time, by holding down the Shift
or Ctrl key, but when deselecting them they immediately disappear from the
report.
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Figure 5-5. Mouse selection version of the Intra-Class Histograms. Only the histograms for
currently selected objects (matches 4767 and 4769) are displayed. If the spot from match
4767 is deselected, only the histogram of match 4769 will be shown.
This type of report has the advantage of only displaying information on objects
of interest. Moreover, you do not have to open a new report for each additional
item.
The following report types are available from the Window > Mouse Selection
menu:
•
3D View
•
Spot Report
•
Label Report
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•
Intra-Class Report
•
Intra-Class Histograms
•
Inter-Class Report
•
Inter-Class Histograms
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Gels
6
Gels
6.1
Introduction
This chapter presents ImageMaster operations that relate to gels. You will learn
how to do the following:
•
Change the display of gel images (zooming gels, adjusting the contrast and
looking at intensity profiles or 3D views).
•
Create or verify an intensity calibration.
•
Process your gels (rotating, flipping, scaling, cropping).
•
Save, print and export gel images.
To make any analysis meaningful, it is important to start with good quality image
files. The following paragraphs give some helpful tips on what image format,
resolution and depth should be used to obtain the best results.
6.1.1
Image format
Input formats
Your gels must first be digitized by an imaging device (such as a flatbed document
scanner, camera system, densitometer, phosphor imager or fluorescence
scanner) and then saved in an appropriate file format. The recommended
formats are TIFF (Tag Image File Format), PNG (Portable Network Graphics), GEL
(Molecular Dynamics), IMG (Fuji) and SCAN (Bio-Rad). It is important to remember
that ImageMaster uses the calibration information contained in these image files.
ImageMaster format
After opening your gels for the first time in ImageMaster, they are saved in the
ImageMaster 2D Platinum file format (unless otherwise stated). ImageMaster gel
files use the extension .mel.
6.1.2
Image resolution
The scanning resolution of the 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 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, and this slows down the
gel analysis significantly. A resolution between 150 and 300 dpi is generally
sufficient for gel analysis.
Please note that the 1:1 (or 100%) zoom is recommended when capturing an
image with a scanner. Shrinking the image often leads to a loss of quality or the
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inability to resolve data. Increasing the size of the image is useless because it
does not add any information. Camera-based systems do not capture at a set
scale. When using such systems, you generally end up with different resolutions
in the final images. Although this is not a problem for ImageMaster, working with
identical resolutions certainly facilitates image manipulation (for example,
zooming or alignment).
6.1.3
Image depth
The range of potential gray levels in an image varies according to the image
depth. In the case of an 8-bit image, one pixel has 256 possible gray values (0 to
255). Images scanned with a higher image depth contain more information. A 16bit image (65536 gray levels) will reveal more subtleties. We strongly recommend
an image depth of at least 12 bits for gel analysis.
When carrying out an intensity calibration, the raw pixel values are converted
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. Some
scanning devices produce images that are already calibrated. ImageMaster
takes into account the conversion tables or calibration formulas stored in the files
exported by such devices. If necessary, you can perform a calibration of the
image capture device within ImageMaster, using calibration step wedges or
calibration strips (see Section 6.5).
6.2
Selecting gels and gel regions
Most of ImageMaster's functions can only be performed on selected gels in the
active worksheet. Gels are selected by clicking on their legends, using the Select
menu options or via a report. Gel regions are defined using the Region tool.
Legends
Each image has a legend with its name in the upper left corner. The color of the
legend indicates whether the image is selected (green, or red for a master image)
or not (gray, or pink for a master image). In order to select an image, click on its
legend. Use the Shift or Ctrl keys to make multiple selections.
Select menu
ImageMaster allows you to select gels based on specific criteria. Choose Select >
Gels in the menu and select one of the following options:
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•
All: Selects all gels that are found in the active worksheet. The shortcut for
this option is Ctrl+A.
•
Aligned: Selects only the aligned gels.
•
Inverse selection: Reverses the selection of gels.
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Reports
When you double click on gels, spots, annotations or matches in a report or use
the Select on Gels icon in the report toolbar, the related gels are automatically
selected in the active worksheet. This means that when you double click on a spot
in a Spot Report, the gel containing that spot is selected. When you double click
on a match in an Intra-Class Report, all gels containing that matched spot are
selected.
Region tool
A region is a rectangular area in a gel that is used for different purposes in
ImageMaster. Certain actions can be limited to this region such as the selection
of spots or annotations. You can use a region to preview spot detection
parameters or gray level adjustments. You can also save, print or export a region
as well as display a 3D view of a selected area.
To define a region in a gel:
1
Click on the Region tool in the toolbar.
2
Position the cursor at the top left position of the area you want to select,
hold down the left mouse button, and move the cursor to the bottom
right position. Release the mouse button at the end point.
3
A dashed box outlines the region (Figure 6-1).
Figure 6-1. Region tool. The left gel shows a region during selection (no spots displayed), the
right one after selection.
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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. In order to remove a
region, select the Region tool (if not already done) and double click on the gel.
In order to define the same region on all gels in the active worksheet, hold down
the Shift key while drawing the box on one of the gels. This region is defined in the
other gels based on the corresponding pixels.
6.3
Displaying gels
6.3.1
Moving gels
There are several ways to change positions in gels. You can use the Hand tool,
Show menu options, shortcut keys or scrollbars (see Section 6.3.3). You can also
double click on a gel to move all gels in the active worksheet to the same position.
Hand tool
The Hand tool is the most straightforward means of moving gels.
To move a gel:
1
Click on the Hand tool in the toolbar.
2
Click on the gel and hold down the left mouse button while moving the
cursor. The image changes position.
3
Release the mouse button at the position you want.
In order to move all gels in the active worksheet by the same displacement, hold
down the Shift key while changing the position in one of the gels.
Double click
In order to move all gels in the active worksheet to the same position with the
same magnification, select the Hand tool (if not already done) and double click on
one of the gels. The corresponding position in the different gels is estimated by
interpolating between the surrounding matches, or if such matches do not exist,
between the two nearest common landmarks (i.e. landmarks with the same
name). Finally, when no such landmarks exist, the gels are aligned at the same
location using the X and Y coordinates.
Show menu
ImageMaster allows you to move gels based on specific positions. Choose Show
> Gels > Move in the menu and select one of the following options:
•
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Same location: All gels in the active worksheet are moved to the same
position with the same magnification as the selected gel.
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•
Up: Scroll up in the selected gels
•
Down: Scroll down in the selected gels.
•
Left: Scroll to the left in the selected gels.
•
Right: Scroll to the right in the selected gels.
•
Top Left: Display the top left of the selected gels.
•
Top Right: Display the top right of the selected gels.
•
Bottom Left: Display the bottom left of the selected gels.
•
Bottom Right: Display the bottom right of the selected gels.
6.3.2
Zoom gels
There are several ways to enlarge or reduce the view of gels. You can use the
Magnify tool, Show menu options, Zoom window, Overview option or scrollbars
(see Section 6.3.3).
Magnify tool
To zoom in or out on a gel:
1
Click on the Magnify tool in the toolbar (Figure 6-2).
2
To zoom in, repeatedly click the area of the gel where you want to see
details.
3
To zoom out, right click repeatedly on the gel.
To move all gels in the active worksheet to the same position with the same
magnification, hold down the Shift key while zooming in or out on one of the gels.
Show menu
ImageMaster allows you to zoom in and out on gels based on specific factors.
Choose Show > Gels > Zoom in the menu and select one of the following options:
•
In: Zoom in on the selected gels.
•
Out: Zoom out on the selected gels.
•
1/16: Reduce the original image by a factor of 1/16.
•
1/8 … 16: Use one of the intermediary zoom factors.
•
32: Enlarge the original image by a factor of 32.
•
Fit in Screen: Show the full image in the cell.
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Figure 6-2. Magnify tool. The upper left gel is displayed without zoom factor. The upper right
gel was zoomed 2-fold, whereas the lower gel was zoomed out by a factor of 0.25.
Figure 6-3. Magnifying glass.
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Magnifying glass
To temporarily enlarge an area of a gel:
1
Click on the Magnify tool in the toolbar.
2
Hold down the Ctrl key and click on the region you want to enlarge.
3
The area under the cursor is magnified (Figure 6-3).
Zoom window
The Zoom Window has two functions. First, it can be used to enlarge the area of
the gel under the cursor (Figure 6-4). This is the case when the zoom factor in the
Zoom Window is greater than that of the gel. Second, the Zoom Window can be
used to see the visible area in a larger view of the gel (Figure 6-5). This is the case
when the zoom factor in the Zoom Window is less than that of the gel.
To see the enlargement of the gel area under the cursor:
1
Choose Window > Zoom in the menu.
2
In the Zoom Window, set the zoom factor by clicking the + and - buttons.
The zoom factor must be greater than that of the gel. You can also adapt
the zoom factor of the gel if needed.
3
Position the cursor on the area of the gel where you want to see details.
4
The region under the cursor is magnified in the Zoom Window box.
To see the visible area in a larger view of a gel:
1
Choose Window > Zoom in the menu.
2
In the Zoom Window, set the zoom factor by clicking the + and - buttons.
The zoom factor must be less than that of the gel. You can also adapt the
zoom factor of the gel if needed.
3
Position the cursor on the gel you want to view.
4
The visible area is localized in a green box in the Zoom Window.
The Zoom Window can be resized by dragging a corner or edge. Spots or
annotations are also displayed in the Zoom Window (for zoom factors higher than
or equal to 1).
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Figure 6-4. Zoom Window used to enlarge the region under the cursor. The exact cursor
position is indicated by the green cross.
Figure 6-5. Zoom Window used to localize the visible gel area (represented by a green box)
in a larger view of the gel. The exact cursor position is indicated by a green cross.
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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 6-6). This overview enables you to
easily locate and move to any region you want on your gel.
To display and use the Overview option:
1
Choose View > Overview in the menu. If Overview is checked, then the
option is activated.
2
The overviews appear in the lower right corners of all images in the
Display Zone. The green rectangle in the overview corresponds to the
current view of the gel.
3
Drag the green rectangle to another position to display a new region.
4
To deactivate this option, choose View > Overview. Overview is no longer
checked in the menu. This is the default setting.
Figure 6-6. 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.
6.3.3
Scrollbars
Scrollbars (in blue and grey) are given on the right and bottom edges of each
image. The size of a scroll box indicates the proportional amount of the used area
of the image that is visible in the cell. The position of the scroll box indicates the
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relative location of the visible area within the image. These scrollbars enable you
to:
•
Change the zoom factor of the image. The cursor of the mouse changes to a
double-sided arrow icon when placed over the respective scroll box. Click
the mouse on the edge of either end of the scroll box and change the length
of the box. The image zooms in or out depending upon if you make the scroll
box shorter or longer. The other scroll box scales accordingly. By clicking on
the blue square box at the intersection of the two scrollbars, you can reset
the image to its full image size.
•
Move the visible area of the gel up, down, left or right. Simply place the
cursor of the mouse over the scroll bar, click the mouse and move in the
direction you want.
It is possible to hide the scrollbars by deselecting View > Scrollbars > Show in the
menu.
If you want to view a specific area of your gel image, choose View > Scrollbars >
Adjust. In the Adjust Visible Area window, set the exact horizontal and vertical start
and end coordinates of the area to be displayed. You can do this in terms of
different units: Image coordinate, Percentage and Real value (pI and molecular
weight). Please note that pI_MW annotations have to be defined in order to use
the Real value option.
6.3.4
Transparency
The Transparency mode is used to visualize similarities or differences between the
current gels and the specified reference gel. Two options exist:
•
Image transparency
•
Spots overlapped
Image transparency
With this option (sometimes called dual channel display) turned on, each of the
images is displayed in one of two colors, red and cyan (Figure 6-7). When the pixel
colors of the two superimposed gels are added:
88
•
Overlapping spots appear as shades of gray.
•
Red spots are present only in the current gel.
•
Cyan spots are present only in the specified reference gel.
•
Halos of red or blue around dark spots indicate that the protein is over or
under expressed, respectively, compared to the chosen reference gel.
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This means that the less color you see in the Transparency mode, the more
similar the gels. Of course, this is only true if the gels are correctly aligned (see
Section 6.3.6) and superimposed.
Figure 6-7. Image transparency.
Spots overlapped
With this option, when spots have been detected (see Chapter 7), the visible spots
of the specified reference gel are displayed in blue on the image (Figure 6-8). Thus,
you can easily compare the position and size of the red spots in the current gels,
with the blue spots from the reference gel.
(a)
(b)
Figure 6-8. Spots overlapped with (a) outlined spot shapes and (b) filled spot shapes
(selected spots in the front gel and pair vectors are displayed).
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To activate the Transparency mode in the active worksheet:
1
Choose Show > Gels > Transparency > Show in the menu.
2
In the Transparency Settings window, specify the reference gel, check/
uncheck the desired options and click OK.
3
ImageMaster displays your gels with the chosen options.
NOTE! You can first activate the Transparency mode and then align your gels
with respect to the reference gel. You can even add landmarks in the
Transparency mode. Nevertheless, it is recommended to perform any
operations related to gel alignment (especially the definition of landmarks),
before entering the Transparency mode to avoid a slow down due to the
recalculation of the overlaid images.
You can change the default color to be used for displaying the spots from the
specified reference gel (Overlapped spots). You can do this by going to the Display
tab in Tools > Options.
6.3.5
Grid lines
Display Grid Lines over selected gels to indicate their dimensions in pixels (Image
coordinates). Alternatively, you can show grid lines with pI/MW units, provided
such information 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 against a master gel having pI and MW values. The grid values are then
calculated relative to the master gel.
The software will try to partition the visible area in the Number of subdivisions
entered by the user. The graduations can just be regular subdivisions of the visible
space - in Fixed mode - or a subdivision in terms of real coordinates at whole
multiples - in Adaptive mode. Figure 6-9 shows various grid types, drawn as gray
lines on the gels.
To display grid lines:
90
1
Select the gels.
2
Choose Show > Gels > Grid Lines > Show in the menu.
3
In the Grid Settings box, specify the Type of grid lines to be drawn
(Adaptive or Fixed).
4
Specify the desired grid Units (pI/MW or Image coordinates).
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5
Enter the number of horizontal and vertical subdivisions using the
scrollbars.
6
The grid lines display over your gels (Figure 6-9).
7
To hide the grid lines again, select the gels and choose Show > Gels >
Grid Lines > Hide.
Figure 6-9. Grid lines displayed over gels. The left gel shows grid lines in Image coordinates/
Fixed mode, the middle gel uses Image coordinates/Adaptive mode and the right gel
displays a grid in pI/MW units/Adaptive mode.
NOTE! Displaying grid lines is a helpful way to visualize deformations in
aligned gels (see below for details about aligning images) because the grid
lines are warped in the same way as the image.
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6.3.6
Aligning gels
The Align feature facilitates the visual comparison of images (stacked images in
particular) that demonstrate large variations in protein migration. In fact, gel
alignment essentially warps a gel so that it superimposes better with another
one, thus allowing easy identification of corresponding spots (Figure 6-10). When
aligning gels in a [MatchSet] or [Classes] worksheet, the selected gels are
automatically aligned to the Master (see Chapter 9 to learn more about master
images). When aligning gels in a [Gels] worksheet, a reference gel must be
specified.
Gel alignment is purely a visual tool. It is not used in, and will not improve, the
matching process. Therefore, the only reason to align your gels is to ease their
visual comparison (possibly in combination with the Transparency mode).
To align a selected gel to a master image or specified reference gel, ImageMaster
needs to know which positions in the different gels correspond to each other
(represent the same protein form). This is done by defining landmark annotations
on both the gel to be aligned and the master/reference gel. The alignment
algorithm then deforms the aligned gel to superimpose those annotations that
bear identical labels (in the master/reference gel and the aligned gel). Please see
Chapter 8 to find out how to create such annotations.
NOTE! To carry out an alignment, annotations do not need to be linked with
spots, and therefore spots do not need to be detected. This is what
distinguishes alignment from matching. In matching, the landmark
annotations must be linked to detected spots.
(a)
(b)
Figure 6-10. Gel (a) before and (b) after alignment with the master image. The match vectors
and Landmark annotations are displayed.
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ImageMaster allows you to choose one of the following alignment functions:
•
Global: Authorizes the rotation, translation and scaling of the entire gel
image to find the best possible correspondence between all the landmarks.
•
Exact: Locally warps the image to exactly superimpose all the landmarks.
•
None: Unaligns and restores the images.
Note that the addition of suspect landmarks or a bad distribution of the
landmarks can worsen the alignment results. Therefore, these mistakes should be
kept to a minimum, and the following rules considered when defining landmarks:
•
For alignment using the Global option, landmarks should be well distributed
over the entire gel. This means that they have to cover both the X and Y
directions. On the other hand, when using the Exact option, landmarks
should especially be defined around the distorted regions of the gels.
•
Landmarks should only be defined on clearly corresponding spots.
Moreover, one should differentiate between spots that effectively represent
the same protein form, and spots that could be linked based on biological
arguments (variants of the same protein such as different phosphorylation
states). 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) because in
the latter case the error in the position will be much more substantial.
•
Landmarks should be added gradually, so that you can monitor their
individual influence on the alignment, which is automatically updated after
each landmark addition. Incorrectly placed landmarks may seriously
decrease the alignment quality. If you add all your landmarks at once, you
will not be able to discern which one poses a problem.
•
When a spot is missing on one gel (sometimes happens to border spots), you
should not put a landmark in a hypothetical spot position. Missing
landmarks are not a problem for ImageMaster, as the program just aligns
the landmarks that have the same name and ignores the rest.
To align two or more gels:
1
Bearing in mind the rules enumerated above, define a few annotations
containing labels from the Landmark category on the gels. The master/
reference gel for alignment and other gels should contain the same
landmarks, that is, an identical label inside the same spot.
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2
Select the gels to be aligned (include the reference gel when working in
a [Gels] worksheet).
3
Choose one of the options in the Show > Gels > Align Images menu
(Global, Exact).
4
Specify the reference gel if asked for (only in a [Gels] worksheet).
5
The selected gels are aligned relative to the master/reference gel.
6
If some parts of your gel are still not sufficiently aligned, you can add
extra landmarks to your gels. The alignment is automatically updated.
7
To restore to the original images, choose Show > Gels > Align > None.
8
The original images are restored in place of the aligned ones.
In the event that gels are not aligned but have common landmarks, operations
such as simultaneous gel moving, gel matching, etc. are done based on a simple
interpolation between the two nearest common landmarks (landmarks with the
same name). Otherwise, when the gels are not aligned and have no common
landmarks, the operations are based on the same image location.
6.4
Viewing signal intensity
6.4.1
Loading / unloading gels
The digitized gel is composed of individual pixels, each of which is characterized
by its horizontal and vertical positions (X and Y coordinates) and its signal
intensity (raw pixel value).
The depth of an image (see Section 6.1.3) determines the number of possible pixel
gray values. However, no matter what the original image depth, ImageMaster
remaps an image to 256 gray levels for display. In the normal view of an image,
raw pixel values do not need to be stored in memory. This corresponds to the
default (unloaded) mode in ImageMaster.
For some operations such as spot detection, adjustment of the contrast mapping,
and displaying the Profile, ImageMaster relies on the raw image data. The
program automatically loads the pixel gray values before performing such an
operation. When the operation is done, ImageMaster generally unloads the raw
data in order to free up memory.
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NOTE! The raw image data must be manually loaded in order to display pixel
values in the Cursor Information window. The Loaded mode displays the
original pixel gray values for the image. When your computer has limited
memory, it is best to return to the Unloaded mode as soon as possible.
To load the raw image data:
1
Select the gels.
2
Choose Edit > Gels > Raw Image > Load from the menu.
3
No visible changes occur in the gel, but you may now display the raw
pixel values in the Cursor Information window.
To unload the raw image data:
1
Select the gels. You can also choose Edit > Gels > Raw Image > Select Gels
with Loaded Image in the menu.
2
Choose Edit > Gels > Raw Image > Unload in the menu.
3
The memory used to store image data is liberated and the raw pixel
values are no longer available.
To load the raw image data every time a gel is opened:
1
Choose Tools > Options in the menu.
2
In the Options window, click on the General tab.
3
Check the Keep image in memory box.
4
The raw pixel values for all images are stored in memory by default.
The color depth of your computer screen (go to the Start > Settings > Control Panel
> Display in the Windows menu for more details) affects the memory used by
ImageMaster to display gels. For example, a display using 16 million colors (24
bits) requires three times more memory than one using 256 colors (8 bits).
Therefore, you can also optimize memory usage in ImageMaster by adapting the
color depth of your computer screen.
6.4.2
Cursor information
At any given time, the Cursor Information window can be used to display pixel
values such as the X and Y coordinates or pI and MW estimates. When the original
image data of the gels is loaded, the raw pixel values are shown. Note that if spots
are detected, then the Cursor Information window also displays spot information.
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To display cursor information:
1
Choose Window > Cursor Information in the menu.
2
In the Cursor Information window, click on the Settings icon to specify the
information and display order (see Section 5.4.1 for more details on how
to change, save and load settings). Click OK.
3
Position the cursor over the pixel you want. The Cursor Information
window displays the related information. If the Value field is unloaded,
the raw pixel values are not available. In order to see the raw image
data, choose Edit > Gels > Raw Image > Load in the menu.
6.4.3
Displaying gray levels
A 2-DE gel image is traditionally displayed as a gray level image, where gray
levels represent the signal intensity. Sometimes the displayed gray levels are so
low that small spots are hardly visible. In order to emphasize these very faint
spots, you can adapt the brightness and contrast of the image. You can also
display images using pseudo colors. These types of operations are carried out
with the Adjust Contrast function.
Generally, it is useful to preview the modifications that will be done to your image.
To get such a preview, it is sufficient to select an interesting region in your gel. This
can be done before and during the use of the Adjust Contrast feature. In fact the
Adjust Contrast window can be left open throughout your work session, so that
you can adapt parameters at any time in order to highlight faint spots or to help
in spot editing decisions. This means that you can continue to select gels and
other objects while the Adjust Contrast window is open, and that you can move
the region in your gel at any moment to locally increase the contrast for better
viewing. As you will see below, the Adjust Contrast function may be more
interesting in the preview mode than when its modifications are simply applied to
the gels.
NOTE! Any changes done by the Adjust Contrast function only influence how
the image is displayed on your screen, and do not affect the underlying data,
spot detection and quantitation.
6.4.4
Contrast mapping
As mentioned earlier, modern scanners are usually able to scan 2-DE images with
12 or even 16 bits per pixel, 4096 or 65536 gray levels, respectively. Because
common computer screens are only able to display 256 gray levels, mapping
must be undertaken between the 4096 (or 65536) image gray levels and the 256
screen gray levels. By default, ImageMaster uses a linear mapping function,
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where the lightest point in the image is mapped to 0 (white) and the darkest point
is mapped to 255 (black).
This is illustrated with the gray level histogram in Figure 6-11. The histogram
displays the frequency with which each gray level (from 3524 to 28530) occurs in
the gel image A_T1_Gel1. The low gray levels (at the left of the histogram)
corresponding to the background occur very often, whereas the high gray levels
(at 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.
Figure 6-11. Adjust contrast function.
However, you can choose other mapping functions to accentuate small faint
spots. ImageMaster offers two ways to change the default gray level mapping
and thus improve the visual display of the gels.
You can define the minimum and maximum gray levels (i.e. 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. To accomplish this,
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 displace
the interval by placing your cursor in the middle of the slider, holding down the
left mouse button, and moving it to the left or right.
In Figure 6-12, the histogram shows the gray level distribution of the selected
image. The maximum gray level was set to 9398 by moving the right side of the
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slider to the left. The red transformation function shows that gray level 9398 is
now mapped to the maximum screen gray level (255 or black), and that any
darker pixels will appear as black.
Figure 6-12. Remapping of the gray levels. Adjustments are immediately reflected in the
preview region.
Another way to change the default gray level mapping is the use of a non-linear
mapping function. The Bending parameter expands or compresses the contrast
range at the dark or light ends of the range. When the bending parameter is
positive, the image is lighter. The image is darker when the bending parameter is
negative (Figure 6-13).
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(a)
(b)
(c)
Figure 6-13. Region of a gel showing a contrast adjustment. (a) Original image with bending
parameter 0, (b) same area with bending parameter set to -2, (c) same area with bending
parameter set to 2.
To adjust the contrast in an image:
1
Select one or more gels. Draw a region in these gels to get a preview of
the contrast mapping modifications.
2
Choose Show > Gels > Adjust Contrast in the menu.
3
In the Image Display Settings box, select an image from the list. The grey
level histogram of the chosen image is shown and can now be used for
adjusting the contrast.
4
Specify the Unit you want to use (see below) and indicate whether you
want to modify the contrast mapping as a function of the complete
image range or only of the selected region (see below).
5
Change the minimum and maximum gray levels by displacing the slider
borders or by typing valid numbers in the boxes at the lower left and
right corners of the histogram. You can also use the bending scroll bar.
6
These settings are applied to the regions in the selected gels.
7
When you are satisfied with the changes, click OK to apply the settings
to all the selected gels.
All contrast mapping changes are applied to selected gels only and are saved
with the image file. Thus, the next time you open these gels, the modifications are
still active.
In some cases, you may want to save a specific mapping function so that it can
be applied at a later time to other gels with similar gray level properties. To do this,
click on the Save icon in the Image Display Settings window and enter a file name
as well as the desired destination folder. ImageMaster saves the mapping
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function with the extension .glt (Gray Levels Transformation). The mapping
functions stored in the default folder (ImageMaster\Menus folder in the user’s My
Documents directory) are always available from the Open icon in the Image
Display Settings toolbar. It is possible to load the gray mapping function of any
open gel by selecting the gel's name from the Current list in the dropdown menu
attached to the Open icon in the Image Display Settings window.
Unit
Two different units are used for displaying the gray level minimum and maximum:
•
Value: Uses the raw pixel values as displayed in all the reports. When a
calibration is done, these correspond to the calibrated pixel values.
•
Percentage (%): Chooses the scale as a percentage of the total gray level
range in the histogram.
Only in region
By checking the Only in Region box in the Image Display Settings window,
ImageMaster only considers the gray levels that are present in the selected
region.
Only in Region is very useful in combination with the % Unit and the choice of 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 local increase in contrast that is
very useful for viewing very faint spots. This mode can also be combined with the
Gray+Saturation palette in the Colors list.
Pseudo colors
You may want to use a particular color palette instead of the standard gray levels
in your gel. ImageMaster offers color palettes such as Coomassie Blue, Silver
Stain, Hot Iron, Spectrum, etc. A palette that is especially useful is the
Gray+Saturation palette. It corresponds to the standard Gray option, except for
the maximum value that is displayed in red, and the minimum value that appears
in blue. It is helpful when you want to visualize saturated spots (red) or the
background (blue). Of course, you can modify the minimum and maximum gray
levels, as explained above, to decrease the stringency on what is considered as
saturation or background. Finally, you can just inverse the gray levels by
checking the Invert box.
Please note that any color adjustments and the inversion of the gray levels using
the Adjust Contrast function are applied to all selected gels and are not saved
with the gel images.
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To display gels with pseudo colors:
1
Select one or more gels. Draw a region to get a preview of the color
adjustment or inverted gray level modifications.
2
Choose Show > Gels > Adjust Contrast in the menu.
3
In the Image Display Settings box, select one of the color palettes in the
Colors list and/or invert the gray levels by checking the Invert box.
4
The modifications are applied to the defined regions in the selected gels.
5
If you are satisfied, click OK to apply the changes to all gels.
6
To go back to the default gray levels, choose the Gray option from the
Colors list.
6.4.5
Profile
It is sometimes quite difficult to judge whether spots should be split, whether they
are saturated or not, or whether they have some other problems. Sometimes
spots may have a so-called donut structure, with low intensities in the center
compared to the borders. It is very important to identify such problems, as they
will lead to incorrect spot quantitation. However, such errors are difficult to detect
because the human eye is not very sensitive to different shades of gray.
The Profile function can help in such cases. Horizontal and vertical sections on the
gel at the position of the mouse cursor show the intensity variations in the two
directions (Figure 6-14). The Profile visually clarifies the intensity changes in the
gel and can thus assist in making decisions.
Please note that the major advantage of the Profile feature, compared with the
3D View (see below), is that it can be used within the selected image during spot
editing and so on. Thus, you have information at your disposal about the third
dimension (spot intensity) without having to open an additional window, as is the
case with the 3D View option.
ImageMaster relies on the intensity of every pixel in the gel to display the Profile.
For this reason, the raw image data are automatically loaded when using this
utility. If the memory resources on your computer are low, please do not forget to
unload the raw image data when you finish using the Profile function (see Section
6.4.1).
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Figure 6-14. Profile feature. Red curves represent the intensity variations of the gel in the
vertical (left) and horizontal (top) 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.
To view profiles on your gels:
1
Choose Show > Gels > Profile in the menu.
2
Position your mouse cursor over a gel. The horizontal and vertical
profiles at the cursor position are displayed.
3
Once the Profile feature is no longer needed, choose Show > Gels >
Profile from the menu.
6.4.6
3D view
Another way to examine the intensity variations in a gel is by looking at the threedimensional (3D) view of a gel region (Figure 6-15). 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. It can be
rotated in any direction to view the interesting spot(s) from all sides, thus
facilitating spot editing or matching decisions.
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Show
Adjust Contrast
Figure 6-15. 3D View for the active region of gel A_T1_Gel1.
To display the 3D View of a gel region:
1
Select a region in one or more gels. Or select one or more spots.
2
Choose Reports > 3D View in the menu.
3
When both spots are selected and a region is defined, ImageMaster will
ask whether your 3D View should be based on the selected region or
selected spots.
4
ImageMaster displays a 3D View for the region or the area containing
the selected spots.
Figure 6-15 shows the region in gel A_T1_Gel1 and the corresponding 3D View
window. At the top of this window, you see the typical toolbar. You can turn the
gel by clicking on the arrows at the right side of the gel image. By default, the
three axes are displayed: the X axis in brown, the Y axis in blue, and the Z axis in
purple. You can change the point on which the axes are centered by right clicking
on the desired position. This position then becomes the new center of the image.
Note that this is a way to move the view up or down, depending on the position
you click on. The coordinates of this center point are found at the lower right
corner of the image. The angles of rotation in the vertical and horizontal directions
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as well as the zoom factor are displayed at the right side of the 3D View window.
Please note that vertical rotation is limited between 0° and 90° (you cannot look
'under' the gel).
Below the 3D image, the Lighting check box enables you to switch off the light
sources that ensure a proper three-dimensional aspect of the image. The result is
that the gel will appear like a normal gel image when it is looked at with a vertical
angle of 90° (flat position) and that the individual pixels are visible.
NOTE! The pixel gray level is overlaid on the 3D image; the top of the peaks is
therefore always darker. This also means that you can combine the 3D View
with the various color palettes available through the Adjust Contrast feature.
The second box at the bottom of the 3D View window, called Spots Overlay,
should be checked when you want to display the spot positions on the view. Spots
are shown with outlines corresponding to the spot borders or as crosses
indicating the spot centers. To choose one of these options, you should go the
Spot Shape item under the Show drop down menu of the toolbar.
The 3D View module in ImageMaster also enables comparative viewing of
corresponding regions or spots in different gels (Figure 6-16). When using this
feature, the various regions are displayed in a single window using identical
scales and orientations, allowing direct comparison of spot positions and heights.
Figure 6-16. Multiple 3D views for the comparison of corresponding spots or regions in
different gels.
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To display multiple 3D views of several (corresponding) gel regions:
1
Use the Shift key to select the same region in all selected gels, or define
a particular region for each gel. Alternatively, you can select one or more
spots in each gel.
2
Select the gels for which you would like to display and compare the 3D
views.
3
Choose Reports > 3D View from the menu.
4
When spots are selected and a region is defined, ImageMaster asks
whether your 3D View should be based on the selected region or
highlighted spots.
5
ImageMaster displays a single window with side-by-side 3D views for
each active region (or area around selected spots).
Zoom operations and rotations are applied to all gels in a multiple 3D View. Right
clicking on the desired position can change the point on which the axes are
centered. When holding the Shift key at the same time, this change is applied to
all the 3D views in the window.
The 3D View is considered to be a normal report, and, as such, provides most of
the standard icons in its toolbar. More information about the icon functionalities
is found in Chapter 5.
In short, the 3D image can be saved in PNG, TIFF or BMP formats and printed or
copied to the clipboard for insertion into another program. The Select on Gel
Image icon makes it possible to highlight spots on the related gel image, once
they have been selected in your 3D View. To select spots on the 3D View, just left
click on their center or on the tip of their peak when viewed from the side. With
the Select on Gels+Reports option in the Select on Gels drop down menu, the spots
are not only selected in the corresponding gels but also in any open reports.
Finally, the Select from Gels feature does the exact opposite, any spots selected
on the corresponding gel region are highlighted in the 3D View.
The Show icon in the 3D View toolbar groups a series of special features:
•
Show Region on Gel causes the gel to be moved so that the region
corresponding to the 3D View (and more particularly the center of the axes
in the 3D View) becomes visible. This option is useful for finding a specific
point on the gel when no spots were detected.
•
Set Region on Gel not only moves the gel so that the region corresponding
to the 3D View becomes visible, but also reselects the region on the gel. One
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should be careful when using this option because any previously selected
region is lost.
•
Synchronize all 3D Views is used to propagate the orientation (for example,
the Up, Rot and Zoom parameters) of the current view to all other 3D views.
You can perform the same action by holding down the Shift key while
adjusting the center point of the axes in one of the gels (by right clicking).
•
Spot Shape sets the way the spots are displayed.
•
Display axes can remove the axes from the 3D View.
The last icon, Adjust Contrast, enables you to quickly change the gray level
setting of the peaks.
NOTE! An interactive version of 3D View is available from the Window >
Mouse Selection menu. Its functionality is much the same as the standard 3D
View, but it interactively displays the third dimension for the current mouse
selection (region or spots). When you select a new spot on your gel, the 3D
view (as well as that of matched spots in other selected gels) automatically
displays. When you deselect the spot, its 3D view immediately disappears
from the report.
6.5
Calibrating and normalizing gels
ImageMaster offers several possibilities to compensate for image differences
caused by variations in experimental conditions (for example, protein loading or
staining) and scanning properties (such as image depth). This section describes
the available features. The terms normalization and calibration, as used within
ImageMaster, are also clarified. Please note that you need to master the notions
of spots and matches to fully understand the functionalities explained below. You
can refer to the following chapters to learn more about these ImageMaster
objects.
6.5.1
Intensity calibration
Instead of displaying and using the gray levels taken directly from the image
capture device, you can assign real world values, often the optical density (OD), to
the measured pixel values. The advantage of this is that the range of gray values
is the same no matter what the original pixel depth. Some image capture devices
automatically perform this type of calibration. When they do, ImageMaster
generally reads the calibrated intensity values from the saved files. For example,
images acquired with the GE Healthcare ImageScanner using the LabScan 5.0
software, are already calibrated.
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Displaying calibration information
You have different ways of finding out if your images were calibrated and for
viewing the calibration information:
•
Choose Reports > Gel Report to view the calibration function (Calibration)
and calibration units (Unit) for the selected gels. If nothing appears in these
columns, the gels are not calibrated.
•
Choose Reports > Gel Calibration > Report to display a report including the
calibration function, the name of the step tablet used, the name of the
person that did the calibration, and the calibration date.
•
If your gel has been calibrated with LabScan 5.0, you can select the gel and
choose Reports > Gel Calibration > Plot to view the calibration curve. See
below for details about the calibration curve.
Creating a calibration
If you intend to calibrate the image capture device yourself, 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. Normally no calibration needs to be done
when you do reflective scanning. With some equipment, both a transparent and
a reflective calibration strip are provided. When calibrating, be sure to use the
appropriate calibration step tablet.
NOTE! 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
diffused 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.
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To create a calibration once a correct Calibration Tablet File is generated:
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1
Scan the step tablet and import the image file into the ImageMaster
software. If necessary, rotate the image such that the light steps are
displayed at the top.
2
Choose Tools > Calibration Tablet > Create in the menu.
3
The Load Step Tablet Definition box opens. Browse the folder where you
saved the Calibration Tablet File specifically tailored to your step tablet
(see above) and Open the file.
4
A red calibration step overlay appears on the image and the Create
Calibration window is displayed (Figure 6-17).
5
Select the Spot or Annotation tool in the ImageMaster toolbar and adjust
the position of the steps by dragging the overlay while holding the left
mouse button. To help you differentiate the darkest steps, you can
adjust the contrast. Please note that the size of a step on the red overlay
(not the red box, but 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.
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). ImageMaster may
automatically deselect some of the steps (grayed out) because of their
unreliable values, and 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 additionally 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 whether you want to apply this new
calibration. If you answer Yes, ImageMaster applies the calibration to the
image.
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Figure 6-17. 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).
NOTE! When you select a point in the step tablet list (in the Create Calibration
window), the corresponding step becomes automatically highlighted in green
on the step tablet overlay and in the calibration graph. This makes it very easy
to correlate the three sources of information (point in tablet list, point in graph
and step rectangle).
The various icons in the toolbar of the Create Calibration window are used to:
Open another tablet definition.
Save the calibration (with the extension .cal).
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Print the calibration graph.
Copy the calibration graph to the clipboard.
Display related Reports.
Two reports are available from the Reports icon in the Create Calibration window.
These reports can be saved, printed and copied to the clipboard.
•
The Fitting Report displays the coefficients of the regression function.
•
The Calibration Report displays for each step: the step number, the
measured averaged gray level, the calibrated intensity value, and the fitting
error (difference between the curve and the point).
NOTE! Intensity calibration must be performed on a regular basis (once a
month). If you do not calibrate, darker material may not be measured in the
correct proportions to the lighter material.
Controlling a calibration
The Control calibration mode allows you to verify whether you are using a correct
calibration. It requires a different, specially calibrated step tablet (Kodak Step
Tablet no. 3, for instance), which you compare to your previous calibration results.
So 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:
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1
Scan your control step tablet (Kodak Step Tablet no. 3, for instance) and
import the image file into the ImageMaster software. If necessary, rotate
the image such that the light steps are displayed at the top.
2
Choose Tools > Calibration Tablet > Control in the menu.
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.
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 as Windows Notepad so that it contains the
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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.
Applying a calibration
To apply a calibration to newly scanned gel images:
1
Open and select the gels the calibration should be applied to.
2
Choose Edit > Gels > Apply Calibration in the menu. 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 displayed in any reports or in the Cursor
Information window correspond to the calibrated values.
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 ImageMaster.
To remove a calibration from a gel:
1
Select the gels from which you want to remove the calibration.
2
Choose Edit > Gels > Reset Calibration in the menu.
3
You are asked to confirm your choice.
6.5.2
Spot normalization
Spot normalization is a kind of internal calibration that makes the data
independent of experimental variations between gels caused by conditions such
as differences in protein loading or staining. In ImageMaster, this simply implies
the use of the relative Intensity (%Intensity) or relative Volume (%Vol) to quantify
and compare the gel spots. By definition:
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Intensity
- × 100
%Intensity = ------------------------------------n
∑ IntensityS
where IntensityS is the calibrated
intensity of spot S in a gel containing n
spots.
Vol × 100
%Vol = --------------------n
Vol
∑ S
where VolS is the volume of spot S in a
gel containing n spots.
S=1
S=1
These measures take into account variations due to protein loading and staining,
by considering the total intensity or volume over all the spots in the gel. This
means that in a gel 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.
Please note that although the %Vol is a rather efficient measure for evaluating
protein expression differences between gels, the %Intensity is not as relevant to
use.
6.5.3
Intensity normalization using a scatter plot
Intensity normalization may in some cases be beneficial or even indispensable to
obtaining more precise differential expression values. However, if your
experimental procedure is highly reproducible, this image compensation method
is not always needed. Many differential protein expression studies yield good
results without requiring intensity normalization. Improper usage of this tool can
even be detrimental to your analysis. Therefore, clearly understand the
assumptions that are made in the application of this feature before you decide to
use it on your specific set of gels.
The scatter plot in ImageMaster renders information about the relationship
between the spot values from two gels by searching for the linear dependence
between the values from one gel and the values from another gel (see Section
10.2.1). In numerous cases only a relatively low percentage of the spots in the
compared gels are expressed differently. Most of the spot values (especially
Intensity values) evaluated in the two gels should be similar, and therefore the
best-fit line of the scatter plot should be close to identity (Y=X). This assumption
permits your gels to be normalized based on the best-fit line. That is, to correct
the pixel values in one of your gels in such a way that the best-fit line approaches
identity. This normalization therefore minimizes the number of spots that are
expressed differently, and can be used to compensate for sample loading
variations.
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To normalize intensities based on a scatter plot:
1
Select the gels that might need normalization, including the reference
gel to be used for the scatter plots. The gels and the reference gel should
be matched (belong to the same match set).
2
Select all matches.
3
Display the scatter plots by choosing Analyze > Intra-Class > Scatter
Plots. Make sure you choose Intensity as value type. This is important
because normalization does not work with other value types.
4
If the slope in a scatter plot is close to 1, and the offset close to 0, then
normalizing the data is not appropriate. However, if these values are not
close to 1 and 0, then try normalizing your gel using the following
procedure.
5
Select the scatter plots of the gels that need to be normalized.
6
Choose the Fitting Report option from the Reports drop down menu in
the toolbar of the Scatter Plots window, and save this Fitting Report. The
Fitting Report gives the gray slope, gray offset, and correlation values
for the scatter plot best-fit line for each gel as well as the number of
matches that were selected to display the scatter plot. If you only select
some of the lines in the report, you will get the chance to save just those
in the file.
7
Select only the gels to be normalized (make sure the reference gel is
deselected).
8
Import the normalization by choosing File > Import > Normalization from
the menu and selecting the previously saved fitting report. Two different
scenarios can occur. If the fitting data of a single gel was saved in the
report, the normalization will be applied to all or none of the selected
gels, depending on your choice. If the fitting data for two or more gels
was saved, each normalization in the file is applied to the gel with the
corresponding name.
9
Alternatively, you can select the gels one by one. Choose Edit > Gels >
Edit Normalization in the menu, and enter the appropriate Gray Slope
and Gray Offset values (corresponding to those from the scatter plot for
the particular gel).
10 Each selected gel is now normalized. To check this, you can display the
new scatter plots. All slopes should be close to 1 and all offsets close to
0. Normalized gels will have an asterisk after their name in various
reports and displayed spot values correspond to the normalized values.
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11 You can remove the normalization at any time by choosing Edit > Gels >
Reset Normalization in the menu.
Please use caution with respect to this method because if there are many
proteins that are expressed differently, then you can introduce errors and get
identical values for corresponding spots that are in fact widely divergent. This
type of normalization should essentially be restricted to gels that belong to the
same population or that represent the same sample.
For gels with very different protein patterns that present significant disparities in
protein loading and/or staining, you may be able to carry out normalization on
the condition that both gels contain a series of internal standards (proteins for
which the abundance is known to be identical in the two gels). In this case, you
can perform scatter plot normalization as long as only the spots corresponding
to the internal standards were selected when displaying the plot.
6.6
Processing gels
A certain number of functionalities in ImageMaster not only imply the processing
of existing 2-DE images on screen, but also affect the image files on the hard disk.
These options are grouped in the Tools > Gels menu and lead to the creation of
new gels (except for the Delete from Disk and Rename option) that are
automatically opened and displayed in the ImageMaster window. The newly
created gels are also inserted in the Gels folder of the current project in the
Workspace. Some features, such as cropping gels, are not provided in the Tools >
Gels menu because they must be carried out in other ways. Nevertheless, the
processing operations are described below.
6.6.1
Delete from disk
With this function, you not only remove your gels from the screen but also from
your hard disk. The main advantage of deleting gels from within ImageMaster is
that associated matches are suppressed as well.
To delete existing gels:
1
Select the gels to be deleted in a worksheet.
2
Choose Tools > Gels > Delete from Disk in the menu.
3
All selected gels and their associated match files are closed and
removed from your hard disk.
6.6.2
Renaming gels
Since the ImageMaster Workspace checks the consistency between the gel
Name and Gel ID, it is highly recommended to rename your gels (at least those
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that have already been opened and saved with ImageMaster) from within the
program. By doing so, you avoid inconsistencies in the workspace.
To rename your gels:
1
Select the gels to be renamed in a worksheet.
2
Choose Tools > Gels > Rename from the menu.
3
Enter a new gel name for each selected gel.
4
The gels are automatically renamed (gel legend, workspace and file
name).
6.6.3
Rotating gels
When images are scanned in the wrong orientation, they can be rotated to
correct for this. You can typically rotate your gels by 90, 180 and -90 degrees. But
free rotation can also be applied.
To rotate an image:
1
Select the gel to be rotated in a worksheet.
2
Choose Tools > Gels > Rotate from the menu and select the desired
option (90° CW, 180°, 90° CCW, Free).
3
Enter a new file name and destination folder for the rotated gel.
4
If you choose one of the predefined angles, the rotated image is directly
saved to the disk drive and opened in the ImageMaster window.
5
If you select the Free rotation option, the image appears with a grid on
it. 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.
6
Make sure the Region, Spot or Annotation tool is activated. Then click on
any position on the gel 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 6-18). You can also
manually enter a rotation angle in the Rotation Tool dialog box.
7
Press OK to confirm your rotation (spots and annotations are preserved)
or Cancel to return to the initial orientation of the gel.
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Figure 6-18. Rotation Tool. 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.
6.6.4
Flipping gels
Sometimes, images are scanned in the wrong direction, and you may have to flip
them to get their correct mirror image. With ImageMaster, you can flip gels
horizontally or vertically.
To flip your gels:
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1
Select the gels to be flipped (in the same way) in a worksheet.
2
Choose Tools > Gels > Flip from the menu and select the desired option
(Horizontally or Vertically).
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Enter a new file name in the Flipped Gels box. Note that instead of
changing each file name individually, you can add an extension, or
replace the extensions of the original image files. You can also change
the destination folder.
4
The flipped images are saved to the disk drive and opened in the
ImageMaster window.
6.6.5
Scaling gels
ImageMaster lets you create smaller or larger copies of selected gels. This
function is particularly useful for very large images where a reduction in size may
significantly decrease the time and memory required for the analysis.
To reduce or increase the size of your images once they have been
opened:
1
Select the gels to be scaled in a worksheet.
2
Choose Tools > Gels > Scale from the menu.
3
Enter the horizontal and vertical scale factors in the dialog box.
4
Give new file names or modify the file extensions.
5
Click OK. The scaled gels are saved on the hard disk and opened in the
software. All spots and annotations are maintained.
6.6.6
Inverting gray levels
The last option in the Tools > Gels menu lets you change the gray levels of
selected gels by inverting them. 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).
To invert the gray levels of your gels:
1
Select the gels to be inverted in a worksheet.
2
Choose Tools > Gels > Invert Gray Levels from the menu.
3
Enter new file names in the Invert Gels box. Note that instead of
changing each file name individually, you can add an extension, or
replace the extensions of the original image files. You can also change
the destination folder.
4
The inverted images are saved on the hard disk and opened in the
ImageMaster window.
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6.6.7
Cropping gels
With ImageMaster, you can crop your gels. That is, you can create new gels that
only contain the defined regions of selected gels, together with the spots and
annotations that were located in those regions.
To crop gels:
1
Define regions in selected gels using the Region tool. Delimit a particular
area in each gel or use the Shift key to select the same region in all gels.
2
Choose File > Save As from the menu.
3
For each selected gel, enter the destination folder and file name. Click on
Save.
4
ImageMaster asks you if you want to save only the selected area.
Answer Yes.
5
New gels are created on the hard disk and inserted into the current
project. The new gel images contain only the parts of the gels that lie
within the specified regions. Any spots and annotations that were
present in the saved region on the original gels are maintained.
You can also export and import a Selection Box to ensure that the final size of your
cropped gels is identical, even between work sessions. A Selection box is a region
with an anchor attached to it. You position the anchor on an easily recognizable
protein spot, and as the region moves with the anchor, you will always crop a
similar part of the gel.
To export a Selection Box:
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1
With the Region tool, define a region on a gel that you would typically
like to crop.
2
While holding the Alt key, click on a characteristic spot that can easily be
found in all gel images. An anchor appears on the clicked position. Note
that this anchor may be located inside or outside the gel region.
3
If you are not satisfied with the position of the anchor, you can redefine
it by pressing Alt again while clicking on a new location. You can also
remove the entire Selection Box by double clicking in the gel image.
4
Select the gel in which you defined the Selection Box.
5
Choose File > Export > Selection Box to save the Selection Box to a file
with the extension .cpt (Crop Tool).
6
Enter a destination folder and file name. Click on Save.
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To import a Selection Box:
1
Select the gels to be cropped using a Selection Box.
2
Choose File > Import > Selection Box and select the previously saved file.
Click on Open.
3
The Selection Box appears on the selected images.
4
By clicking inside the box, with the Region tool, and dragging it, you can
move the Selection Box to superimpose the anchor on the characteristic
spot.
5
The gels can now be cropped as described above, by using File > Save
As.
6.7
Reporting on gels
6.7.1
Gel report
The Gel Report (Figure 6-19) displays summarized information about the selected
gels, such as image height and width in pixels (Rows and Columns), pixel
dimensions of the scanned image (PixWidth and PixHeight), minimum and
maximum gray levels before (MinGray and MaxGray) and after calibration
(MinValue and MaxValue), as well as the intensity normalization parameters
(Slope and Offset), the calibration function (Calibration) and calibration units (Unit).
It also shows the Gel ID, the full file path of the image, the staining method, the
number of detected or selected spots, the number of defined or selected
annotations, the minimal and maximal values of pI and MW, the match set to
which it belongs, and the class to which the gel was assigned.
To display a Gel Report:
1
Select the gels that should be included in the report.
2
Choose Reports > Gel Report > Current Template in the menu.
3
The Gel Report is displayed.
4
Click on the Settings icon in the report toolbar to customize the report
(see Chapter 5 for more details on report customization).
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Figure 6-19. Gel Report. The gel 94-0002 was normalized as revealed by the asterisk next
to its name, and the values for Gray Slope and Gray Offset.
6.7.2
Gel description report
Besides the above information on selected gels, you can enter experimental data
about your gel images, to be used for later reference by yourself or any
colleagues. This information can include sample type, date of the experiment,
operator name, pH range, SDS gel percentage, or staining. All this data can be
entered in the dedicated Gel Description Report and stored in your gel files (Figure
6-20).
The Gel Description Report is available from the Reports menu. Initially, no
descriptions are defined in the report, and only a column with the gel names is
available.
To define descriptions for your gels:
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1
Select the gels for which you want to add descriptions.
2
Choose Reports > Gel Description Report in the menu.
3
The Gel Description Report is displayed.
4
Click on the Create Labels icon in the Gel Description Report toolbar.
5
In the Gel Descriptions box, click the Add button.
6
In the Add Category box, enter the name of a new description category
and click OK.
7
Set the Category Attribute to Text, Number or Boolean.
8
Continue creating new categories until you are finished.
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9
To suppress an erroneous or unwanted category, select it from the
Categories list and click the Delete button.
10 Click OK when you are finished.
The Gel Description Report now displays a column for each description category.
You can directly add new information in the appropriate cells, or edit existing
data. Just double click in a cell to start typing your information. When finished, a
simple click in any cell ends the editing mode. The modified cells appear in dark
green when the corresponding line is selected, or in gray when not selected
(Figure 6-20). An asterisk in the report's window title reflects the fact that changes
were made to it.
Figure 6-20. Gel Description Report. Gray or dark green cells were modified and need to be
updated on the gel.
Subsequently, the Update Gel icon should be used to make the modifications
definitive and to propagate the information to your gels. Note that the updated
cells return to their original color. If all information is updated, the asterisk next to
the report title also disappears.
Please note that you can keep the list of gel description categories for later use.
To do this, click the Create Labels icon in the Gel Description Report. In the Gel
Descriptions dialog box, the following options are available:
•
Click Save and type a file name to store the current gel description
categories.
•
Click Load and choose a file name to reload an existing category list.
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NOTE! The gel descriptions defined via the Gels Description Report are only
valid for the gels that were selected during the creation of the report. To make
the list of gel description categories a template for a particular workspace,
which should be used for any open gels, you should define the gel description
categories via the Gel Descriptions tab of the Options window (go to Tools >
Options). The procedure is the same as that described above. The only
difference is that the defined gel descriptions are automatically displayed in
any Gels Description Report displayed.
Alternatively, you can define gel descriptions by selecting gels and choosing Edit
> Gels > Add Description from the menu. You will be asked to choose a gel
description category or to type a new one, and to enter the description content.
This option is particularly useful when you want to add identical descriptions to a
series of gels because you can create many descriptions at a time, instead of
having to define each one individually.
Similarly, you can delete all gel descriptions of a certain category for the selected
gels. Select the gels and choose Edit > Gels > Delete Description.
6.7.3
Gel calibration report
If your gels were calibrated, you can find related information in the Gel Calibration
Report (Figure 6-21), which is available from the Reports menu. It displays the gel
name, Gel ID and the full file path of the image, the calibration function, the name
of the step tablet used, the name of the person that did the calibration, and the
calibration date.
Figure 6-21. Gel Calibration Report.
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6.8
Saving, exporting or printing images
6.8.1
Saving gels
In order to be stored in the gel file and thus to be available for future work, any
changes made to a gel have to be saved before exiting the program or closing the
gel. Regularly saving your gels enables you to recover from mistakes (although
the multiple undo function allows you to return to previous analysis states) or to
generate gel files with different names. Modifications are saved in the
ImageMaster 2D Platinum file format, unless otherwise specified by the user.
ImageMaster does not automatically save the changes you make to a gel.
Instead, you can control what data should be saved and when. Nevertheless,
when you close a gel or leave the software, ImageMaster alerts you that
modifications were made and gives you the possibility to save them.
NOTE! Please note that the Save and Save As functionalities in the File menu
apply to gel data, in contrast to the Save feature in the Workspace, which
pertains to the Workspace and Projects, and also saves the software options.
Saving your gels and saving modifications to the workspace (see Chapter 4)
are therefore two distinct operations.
The software also allows you to make regular backups of your work, that is, of the
workspace with all the gel images and related files, and to restore this data when
necessary. Find more details about this in Section 4.11.
Two options are available from the File > Save menu.
•
Worksheet allows you to save all the changes made to the gels opened in
the active worksheet, without giving details or asking for confirmation.
•
Save All allows you to save the changes made to all open gels in all the
worksheets. In this case, confirmation messages give the detail of the
changes made and you have the choice to save them or not.
To save changes made to your gels:
1
Select the gels for which you would like to save changes.
2
Choose File > Save > Worksheet or Save All in the menu.
You may want to save multiple copies of gels or create files of defined regions by
using the Save As command. This is useful when you do not want to overwrite the
previous version of the gel in order to keep track of your analysis, or to return to
a prior stage. Also use this command to crop your gels or to save files in earlier
formats (Melanie II or Melanie 3).
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To save gels with a different name or format:
1
Select the gels to be saved.
2
Choose File > Save As from the menu.
3
For each selected gel, enter the destination folder and file name, and
pick the desired file format from the Save as type list.
To save gel regions:
1
Select regions in the pertinent gels. Define a particular region in each gel
or use the Shift key to select the same region in all open gels.
2
Select the gels for which a cropped region should be saved.
3
Choose File > Save As from the menu.
4
For each selected gel, enter the destination folder and file name, and
pick the desired file format from the Save as type list. Click on Save.
5
ImageMaster asks you if it should save only the selected area. Answer
Yes.
6.8.2
Exporting gels and windows
To files
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). In this case, 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.
To export gels or selected gel regions:
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1
If you want to export gel regions, start by defining them with the Region
tool. Define a particular region in each gel or use the Shift key to select
the same region in all open gels.
2
Select the gels that you would like to export or the gels for which you
would like to export a selected region.
3
Choose File > Export > Image to File from the menu.
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4
For each selected gel, enter the destination folder, file name and file type
in the Export Image As dialog box.
5
If a region was defined, ImageMaster asks you whether you want to
export only the selected area. Answer Yes or No.
You also may want to export a view of the active worksheet.
To export a worksheet:
1
Choose File > Export > Worksheet to File from the menu.
2
Enter the desired folder, file name and file type in the Export Image As
dialog box.
To the clipboard
Alternatively, you can export images to the clipboard for direct pasting into
another software. The procedure is very similar to exporting images to files.
However, you can only export one gel image at a time to the clipboard.
To export a gel or a selected gel region:
1
If you want to export a gel region, first outline it with the Region tool.
2
Select the gel to export.
3
Choose File > Export > Image to Clipboard from the menu.
4
If no region was selected, the entire gel image is sent to the clipboard.
Otherwise, only the region is exported.
5
You can then paste the image into the preferred software.
To export a view of the worksheet to the clipboard:
1
Choose File > Export > Worksheet to Clipboard from the menu.
2
You can then paste the image of the worksheet into the preferred
software.
6.8.3
Printing gels
ImageMaster provides various printing options. Print selected gels or regions of
gels, one image per page, or even the active worksheet. 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.
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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.
To print gels or selected gel regions:
1
To print only regions, start by drawing them with the Region tool. Define
a cropped region in each gel or use the Shift key to select the same
region in all open gels.
2
Select the gels that you would like to print.
3
Choose File > Print > Images from the menu.
4
If a region was selected, ImageMaster asks whether you want to print
only the selected area. Answer Yes or No.
5
The selected gels or gel regions are printed.
To print the active worksheet:
1
Choose File > Print > Worksheet in the menu.
2
The image of the selected worksheet is sent to the printer.
You can change printing parameters such as printer name, paper size, paper
orientation, etc. To do so, choose File > Print > Page Setup from the menu. This
command calls the standard print window where printer-related settings can be
modified.
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7.1
Spots
Introduction
The elementary component of a gel is the Spot, which delineates a relatively tiny
region in the gel where protein is present. This shape is automatically
differentiated by a spot detection algorithm. Each spot in an image is assigned a
unique number when the software identifies it, called the Spot ID. Moreover, a
spot is quantified; its intensity, area and volume are computed.
The ultimate aim of defining spots is to compare protein expression changes
between different gels. Therefore, two major requirements must be met:
•
First, automatic spot detection needs to identify a maximum number of
proteins while minimizing the number of artifacts incorrectly detected as
spots and especially reducing the number of undetected proteins. This
requires finding a good compromise between the sensitivity and the
specificity of the spot detection algorithm. If the automatic spot detection
results are not fully satisfactory, manual spot editing can be attempted (for
non-DIGE gels).
•
A second prerequisite is that the differential expression values are accurate
and reproducible. The goal is not to measure the absolute protein
abundance of each spot in an image. Within a gel, there is no quantitative
relationship between the measurable amounts of protein in different spots.
This is because proteins are colored with dyes that bind differently to
different proteins. What is important, however, is that the relative quantities
of the same protein spots in different gels are correctly evaluated. The spot
detection and quantification methods in ImageMaster were specifically
designed for this purpose. You will learn more on how this is done in the
following pages.
In ImageMaster 2D Platinum 6.0, two different spot detection algorithms are
implemented. The Melanie/ImageMaster algorithm is used for non-DIGE gel
images. DIGE images are co-detected using the algorithm created by the
DeCyder software development team.
7.2
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.
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Through the combination of Spot ID and Gel ID, every spot in any of your
experiments is therefore uniquely identified, thus preventing any confusion. Be
aware that when you edit a spot (making it smaller or bigger) after having
generated a report, the spot values in the existing report are not updated and are
therefore incorrect.
7.3
Detecting spots in non-DIGE gels
The Melanie/ImageMaster spot detection algorithm is optimized to give relevant
biological results with minimum user interaction. Only a few easy to use
parameters need to be defined in order to automatically locate spots in your
image.
NOTE! When you look at a detected spot in the 3D View (see Section 6.4.6),
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. The reason for this
lies in the quantification methodology described in Section 7.3.3.
7.3.1
Procedure
To detect spots efficiently we recommend previewing the spot detection results
on a few small image areas (drawn with the Region tool before or during spot
parameter optimization) so that you can decide whether the parameters should
be adjusted or not. Each change in one of the spot detection parameters is
immediately reflected in the selected region (Figure 7-1) except if you turn the
Auto Preview option off in the Detect Spots window. In this case you need to click
the Preview button to refresh the spot detection preview. The advantage of
manual previewing is that it is quicker to make simultaneous changes to more
than one parameter. Once you find the optimal parameters, you can detect all
spots on the selected images.
Please note that you can already view certain properties of spots (for example,
Intensity, Volume or Saliency) in the preview mode while the spots are not yet
detected permanently. Do this by displaying the Cursor Information window (see
Section 7.9) and moving your cursor over a spot or by selecting a spot and
displaying a Spot Report (see Section 7.9). This is particularly useful for entering a
valid Saliency parameter (see Section 7.3.2 ). Obviously, the Detect Spots window
must remain open while doing this.
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Figure 7-1. Adjusting spot detection parameters in real time.
To detect spots automatically:
1
Select the gel images for spot detection.
2
If necessary, choose Show > Spots > Shape > Outlined to see the spot
borders more easily.
3
Click on the Region tool. Draw a rectangle around an area with
representative spots on one or more of the selected gels. Note that you
can still draw, resize or move regions while setting the detection
parameters.
4
Choose Edit > Spots > Detect in the menu.
5
The Detect Spots window appears on the screen and the spots in the
drawn regions of the selected images are detected with the default
parameters. If you do not want the program to recalculate the spots in
the preview regions for each parameter change, turn the Auto Preview
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option off. To manually refresh the preview regions, simply click the
Preview button.
6
Adjust the detection parameters. In particular optimize the Smooth
parameter to detect all real spots and split the overlapping ones.
Subsequently, filter out the noise by changing the Saliency and Min
Area values. See below for more details on spot detection parameters.
7
When you are satisfied with the preview, click OK to detect all spots in
the selected gels using the parameter values you have set. Please note
that you can still change your gel selection at this point.
8
The spot shapes are displayed on the images.
You may want to save the current detection parameters so that they can be
applied to other gels at a later time. To do this, click the Save icon in the toolbar of
the Detect Spots window. Enter a file name and click Save in the Spot Detection
dialog box. ImageMaster saves the detection parameters with the extension
.dpm. By default, they are saved in the ImageMaster\Menus folder in the user’s
My Documents directory. Parameter sets saved in this default folder are always
available from the Open icon in the toolbar of the Detect Spots window.
7.3.2
Spot detection parameters
Spot detection parameters are best adjusted in the following order:
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•
Smooth: First set the Smooth parameter. It fixes the number of times
ImageMaster will smooth the image before detecting spots, using a smoothby-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 (that can be filtered with the Saliency
and Min Area parameters).
•
Saliency: The Saliency 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
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 a Cursor
Information window or Spot Report and look at the saliency value given for
a spot that you would like to suppress. Enter this value in the Saliency field
of the Detect Spots window. The spot detection algorithm then discards all
spots with saliencies smaller than the specified threshold.
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•
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).
7.3.3
Spot quantification
Once spots are detected, ImageMaster automatically computes the amount of
protein present in each spot. Figure 7-2 illustrates the principles of spot
quantification in the Melanie/ImageMaster algorithm. Measuring the protein
quantification values in this way has the advantage of being more robust and
reproducible when calculating protein expression variations (relative
quantification).
•
Intensity: The program 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. It is calculated at an
intermediary height of the spot. More precisely, 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 computed
spot area (expressed in mm2).
•
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 7-2, the measured volume of the spot is hatched. Please
note that the volume values, like the intensities, depend on pixel intensity
calibration (see Section 6.5.1).
ImageMaster additionally calculates the relative intensity (%Intensity) and
relative volume (%Vol) for each spot. These are normalized values that remain
relatively independent of irrelevant variations between images. By definition:
Intensity
- × 100
%Intensity = ------------------------------------n
Intensity
S
∑
where IntensityS is the calibrated
intensity of spot S in a gel containing n
spots.
Vol × 100
%Vol = --------------------n
Vol
∑ S
where VolS is the volume of spot S in a
gel containing n spots.
S=1
S=1
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These measures take into account variations due to protein loading and staining,
by considering the total intensity or 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.
Please note that although the %Vol is a rather efficient measure for evaluating
protein expression differences between gels, the %Intensity is not as relevant to
use.
0.75 * Intensity
Intensity
Intensity
Figure 7-2. 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.
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When detecting spots, ImageMaster also computes the Saliency for each spot
(see Section 7.3.2 ). It is displayed together with the above-mentioned
quantification values in the Cursor Information window or a Spot Report.
7.4
Co-detecting spots in DIGE gels
The co-detection algorithm is designed to simultaneously process 1, 2 or 3
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 comigration benefits
of the CyDye DIGE Fluor dyes. A set of co-run images (2 images in double
detection and 3 images in triple detection) are merged together thereby
incorporating 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.
7.4.1
Procedure
To perform spot detection on DIGE images:
1
Go to the DIGE Gels folder in the Workspace window.
2
Select the DIGE gels to be detected. Right click on one of the gels.
3
Choose Detect from the contextual menu.
4
The images corresponding to the selected gels are loaded and opened
in a new worksheet.
5
In the DIGE Spot Detection window, enter an estimation (see below) of the
Number of Spots present on the images. Click OK.
6
A status window appears showing the progress of the spot detection.
Depending on your computer resources, the spot detection process can
take some minutes.
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7
The spots appear on the gels.
Alternatively, if your DIGE gels are already opened in the Display Zone, then you
can select the gels and go to Edit > Spots > Detect. Enter an estimation for the
Number of Spots to be detected in the DIGE Spots Detection window and click OK.
7.4.2
Spot detection parameter
When detecting DIGE images, you must enter an estimation of the Number of
Spots present on the images. It is recommended that this value be overestimated
to compensate for the detection of non-proteinaceous spots on the image, e.g.
dust particles which can subsequently be excluded from the analysis using spot
filtering (see Section 7.5.2 to learn more about spot filtering with the Refine by
Value feature).
If all the spots have not been 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.
7.4.3
Spot quantification
Numerical data for individual spots are automatically calculated (e.g. volume,
area, intensity, slope and volume ratio) and included in the DIGE Reports (see
Section 7.10.3), Spot Reports (see Section 7.10.1) and Cursor Information window
(see Section 7.9).
•
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 summation of these corrected values.
•
Vol ratio: Volume ratios (volume of first image spot/volume of second image
spot) indicate the change in spot volume between two images.
NOTE! In the DIGE Histogram and DIGE Report, the first and second images
are determined by the order in the workspace or the order in the worksheet.
To obtain the inversed spot ratios, change the order of the gels in the
workspace or the worksheet. In the Spot Report and Cursor Information
window, the first image is the image mentioned in the FileName, the second
image is the DIGE reference image.
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NOTE! When using single detection the volume ratio value is 1.0 for all spots,
since there is no second image.
7.5
Selecting spots
7.5.1
Spot tool
Spots can be selected with the Spot tool as long as they are visible on the image.
Once selected, they are highlighted in green (Figure 7-3), unless the default spot
colors were modified (see Section 7.6.2). If an annotation is attached to a spot, the
annotation is also selected. Similarly, if you select an annotation or label with the
Annotation tool, the linked spot is also selected.
To select a spot, make sure the Spot tool is activated and then click on the spot.
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, position the cursor at the top left position of the
desired region, hold down the mouse button, and then drag the cursor to the
bottom right position. All spots in the designated region will be selected and
highlighted in green.
To deselect all spots, select the images in which you would like to deactivate all
spots, and click in one gel (not on a spot).
7.5.2
Select menu
Additionally, you can select spots by using the items in the Select > Spots menu.
Some of the following are particularly useful for filtering out artifacts (especially
Refine by Value, possibly in combination with Inverse Selection):
•
By ID: Allows the selection of a spot based on its Spot ID. Just enter the
desired spot identifier in the Select Spots by ID dialog box.
•
By Color: When a specific color is assigned to particular spots (using Show >
Spots > Set Color), the spots can be selected by choosing the corresponding
color from the submenu.
•
In Region: This option allows you to select all spots in any regions that were
drawn (with the Region tool) on selected images.
•
From Report File: If any Spot Reports were previously saved, this function
enables the reselection of all spots contained in the specified report file.
•
All: Highlights all spots in the selected images.
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•
Refine by Value: This feature enables the selection of spots based on their
spot value (Vol, Area, Intensity, %Vol or %Intensity), coordinates (X or Y), pI or
MW. Depending on the detection algorithm used, spots can also be selected
based on their Saliency, Volume Ratio or Slope. Make a selection of spots
and then choose Select > Spots > Refine by Value from the menu. Choose the
quantitative value you want to use for your refinement. Indicate the greater
than (>=) and/or lower than (<=) limits of the interval by checking the
appropriate boxes and entering the desired numbers. Spots whose values
are included in the defined interval remain selected.
•
Inverse Selection: If some spots are already selected, this option
deactivates the selected spots and selects all the unselected ones, thus
inverting the selection criteria.
7.5.3
Reports
You can select spots directly from an open Spot Report or DIGE Report. You can
do this by double clicking on a spot in the report, or by selecting one or several
spots and clicking the Select on Gels icon in the report toolbar. In this way, you can
also select spots from an Intra-Class Report, as long as the spots belong to
selected matches, or from reports on Labels, Annotations or Categories if the
spots have linked annotations.
7.6
Displaying spots
7.6.1
Spot shape
Once spots are detected, you can display their shapes on the gels in four different
ways (Figure 7-3), available from the Show > Spots > Shape menu:
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•
Crossed: Draws a cross in the center of gravity of each spot.
•
Outlined: Displays the spot border.
•
Filled: Shows the area of the spot.
•
Outlined/Filled: Shows the spot border for inactive spots (Outlined mode)
and the area of the spot for selected ones (Filled mode).
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(a)
(b)
(c)
(d)
Figure 7-3. Possible spot shapes are (a) Crossed, (b) Outlined, (c) Filled, (d) Outlined/Filled.
Selected spots appear as green crosses, green outlines or green surfaces.
The default spot shapes for newly opened gels can be fixed in the Display tab of
the Options window (accessible by choosing Tools > Options in the menu).
7.6.2
Spot color
By default, unselected spots are displayed in red, selected spots in green and
overlapped spots in blue. You can change these default colors in the Display tab
of the Options window (accessible by choosing Tools > Options in the menu). Click
on the colored box you wish to change and the Color dialog box opens. Choose
the preferred color from the spectrum and click OK.
You can also change the color of a selection of spots as you go.
To change the color of selected spots:
1
Select the spots you want to change the color.
2
Choose Show > Spots > Set Color in the menu and pick one of the three
proposed colors. Please note that the Default option corresponds to the
spot color defined in the ImageMaster Options (accessible by choosing
Tools > Options in the menu) .
3
ImageMaster displays the selected spots with the chosen color.
4
The colors remain displayed this way until you choose Show > Spots >
Set Color > Reset All to revert back to the default spot colors.
7.6.3
Show / hide spots
You may want to hide some spots temporarily, for clarity or illustration purposes,
or perhaps to concentrate only on those spots you consider important. Hiding
spots also corresponds to filtering the spots because you cannot select hidden
spots and they therefore do not appear in generated reports. Hiding spots has the
additional effect of displaying images faster.
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To show or hide particular spots, you can use one of the following options
available in the Show > Spots menu:
•
Show All: Makes all the spots on the selected images visible.
•
Hide All: Hides all the spots on the selected images.
•
In Region: Shows all spots in the region drawn with the Region tool. This
option is handy when all spots were previously hidden.
•
Only Selected: Shows only the selected spots and hides all others. This
feature is helpful when performing a visual inspection of the spot detection
or of the gel analysis results.
•
Hide Selected: Hides the selected spots.
7.6.4
Show / hide spot IDs
You can find out the Spot IDs for specific spots via the Cursor Information window
(see Section 7.9) or a Spot Report (see Section 7.9). However, sometimes you may
want to display the identifiers of selected spots directly on the image.
To show or hide Spot IDs:
7.7
1
Select the spots you want to display the Spot ID for.
2
Choose Show > Spots > Show ID from the menu.
3
The Spot IDs for the selected spots are displayed close to each spot
center.
4
You can hide all Spot IDs again by choosing Show > Spots > Hide All ID.
Adding / modifying spots
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 ImageMaster and to avoid manual editing
as much as possible. At most, spots should be manually separated where
necessary. In spite of these words of warning, you still have the capabilities to
create, modify or delete selected spots in ImageMaster.
NOTE! Spot editing is not allowed on aligned gels (because image warping
may deform spots), and on DIGE gels. For edited spots, the Saliency value
becomes zero.
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7.7.1
Editing spots
In ImageMaster, spot editing is done directly on the image although one needs to
be in the special spot-editing mode.
To modify spots:
1
Select the image you want to work on.
2
Choose Show > Spots > Shape > Outlined to see the spot borders more
easily. It is also recommended to show any hidden spots (Show > Spots
> Show All) in order to prevent the addition of already existing spots (and
thus generating overlapped spots).
3
Choose Edit > Spots > Edit Enabled in the menu (a check mark next to the
option indicates it is activated). The icon of the Spot tool in the main
toolbar also changes to reflect the fact that spot editing is activated
(Figure 7-4).
4
Select the spot you would like to edit with the Spot tool.
5
Carry out the desired spot modifications, as described below.
6
To deactivate this option, choose Edit > Spots > Edit Enabled. Edit Enabled
is no longer checked in the menu.
(a)
(b)
Figure 7-4. Spot tool when (a) spot editing is not activated and (b) spot editing is activated.
NOTE! You are still able to select spots while spot editing is activated.
Nevertheless, we recommend you disable the Edit Enabled option whenever
possible in order to prevent accidental modifications to your spots.
Increase the size of a spot
Outline the area that you would like to add by holding down the left mouse button
while starting at one point on the selected spot and moving to another point
within the spot (Figure 7-5).
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2
1
3
Figure 7-5. Increasing the size of a spot. The green line defines the portion of the spot that
will be added (the numbers indicate the successive positions of the mouse cursor). The plus
sign next to the pen indicates that one is in the 'adding' mode and the pen must start to
draw from a point within the spot border.
Delete part of a spot
In order to erase part of a spot, you should also use the left mouse button, but
now start from outside the initial spot outline and then partially encircle the area
of the spot that you would like to suppress (Figure 7-6). Please note that the
portion of the spot that contains the center of gravity in the initial spot will be
conserved.
1
2
3
Figure 7-6. Deleting part of a spot. The red line outlines the portion of the spot that will be
removed (the numbers indicate the successive positions of the mouse cursor). The minus
sign next to the pen indicates that one is in the 'deleting' mode and the pen must start to
draw from a point outside the spot border.
Split a spot
First, select the spot to split. Only one spot should be selected. Then right click
outside the spot and, while holding down the right mouse button, draw a line
through the spot at the position where it should be split (Figure 7-7).
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2
1
Figure 7-7. Splitting a spot. The grey line shows where the spot will be split (the numbers
indicate the successive positions of the mouse cursor). The cutter indicates that one is in the
‘splitting’ mode.
Merge two spots
Select the first spot. While holding down the Shift key and the left mouse button,
draw the fusion zone starting from within the second spot, going through the first
one, and finishing within the second spot (Figure 7-8).
1
2
3
Figure 7-8. Merging a spot. The green trajectory defines the region where the two spots will
be fused (the numbers indicate the successive positions of the mouse cursor). The plus sign
next to the pen indicates that one is in the 'adding' mode.
Adding spots
When spot editing is enabled, the Spot tool can also be used to add a new spot to
your gel.
To add a new spot to an image:
1
Choose Edit > Spots > Edit Enabled from the menu.
2
Click on the Spot tool.
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3
Double click on the desired location in your image.
4
Use the slider to set the disc radius in the Adding Spot dialog box. The
disc radius, expressed in number of pixels, determines the radius of the
circular spot to be drawn.
5
A new spot (circle) with the specified radius is drawn at the desired
location.
In addition, you can add spots to an image by copying them from other gels,
provided the images were previously matched (see Chapter 9 for more details on
matching). To do this perform the steps described below. Note that ImageMaster
automatically creates matches between the selected spots and the newly
created ones.
To copy spots from one matched image to another:
1
Select spots on a matched image.
2
Select one or more images you want to copy the spots to.
3
Choose Edit > Spots > Copy From Image.
4
The new spots are added to the selected images.
Once new spots are added to an image using either of these methods, you can
make modifications to the newly created spot using the procedures described
above.
Deleting spots
To remove spots from an image, just select the spots you want to delete and
choose Edit > Spots > Delete from the menu.
7.8
MW and pI calibration
If you have a gel with pI/MW standards, ImageMaster can compute
approximated pI and MW values for all the spots/pixels in this image, as well as
any other images matched to it.
The principle is rather simple. You just have to 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 automatically become available from a
Spot Report, DIGE Report or the Cursor Information window (see below). In
addition, pI and MW grid lines can be displayed over the images (see Section
6.3.5).
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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 on the Annotation tool.
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 2 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 the 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 instance, if the pI of a
spot on the extreme right side of your gel is to be determined, the program 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 just 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.
7.9
Cursor information
The Cursor Information window can be used at any time to display information on
pixels (X and Y coordinates, pI and MW estimates or raw pixel values) and spots, if
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spots were detected. Information on spots includes the name of the image on
which the spots were detected, the Spot ID, the coordinates of the center of
gravity (X and Y), and the quantification values (Intensity, Area, Vol, %Intensity
and %Vol). Depending on the spot detection algorithm used, the Saliency, Vol
Ratio and Slope will also be available.
NOTE! In the Cursor Information window, the Vol Ratio given corresponds to:
Volume of the spot under the cursor/Volume of the spot on the DIGE reference
image.
To display cursor information:
1
Choose Window > Cursor Information in the menu.
2
The Cursor Information window (Figure 7-9) appears on the screen.
3
Click on the Settings icon to define the data to be displayed in the
window.
4
In the dialog box, select the attributes to be shown in the Cursor
Information window from the Hidden list (using the Shift or Ctrl keys to
make multiple selections), and transfer them to the Visible list using the
right-pointing arrow button. Similarly, you can remove attributes from
the Cursor Information window by selecting them in the Visible list and
clicking on the left-pointing arrow button. You can also double click on
an attribute to transfer it from one list to the other. Click Apply.
5
The selected attributes are displayed in the Cursor Information window.
6
Position your cursor over the pixel or spot of interest. The Cursor
Information window displays the desired information. If the Value field
shows Unloaded, the raw pixel values are unavailable. In this case,
choose Edit > Gels > Raw Image > Load to load the image data for the
gels.
The order of the items in the Visible attributes list determines the order of the
items in the Cursor Information window. You can therefore adjust the display by
adding your attributes in sequential order to the Visible attributes list.
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Figure 7-9. (a) Cursor Information window and (b) Cursor Information dialog box for
specifying the Settings.
Please note that these settings can be made permanent by going to the Cursor
Information tab in the Options (accessible by choosing Tools > Options in the
menu). Your adapted settings can also be saved and used as templates. For this
purpose, the following options are available from the Cursor Information dialog
box:
•
Save: Allows you to save the new Cursor Information template.
•
Load: Opens an existing template file.
7.10
Reporting on spots
7.10.1
Spot Report
The Spot Report summarizes all relevant information about the selected spots:
their identification number (Spot ID), the name of the image on which they were
detected, the coordinates of their center of gravity (X and Y), the quantification
values (Intensity, Area, Vol, %Intensity and %Vol), pI, MW and all linked labels.
Depending on the spot detection algorithm used, the Saliency, Vol Ratio and
Slope will be available. When working in a [MatchSets] or [Classes] worksheet,
you can also display information on the corresponding spot in the master gel
(Figure 7-10).
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NOTE! When displaying a Spot Report for DIGE images, the Vol Ratio given
corresponds to: Volume of the spot on the image mentioned in the FileName/
Volume of the spot on the DIGE reference image.
The Spot Report can be saved, printed, exported and used to sort through data.
Please see Chapter 5 for more details on these functionalities (available as icons
in a report toolbar) and the possibility of customizing the Spot Report.
Measure Histogram
Figure 7-10. Report on selected spots in selected images.
Measure histogram
By clicking on a numerical column header in the Spot Report and then pressing
the Measure Histogram icon, you can display a histogram showing the
distribution of the values in the selected column (Figure 7-11). The purpose of such
histogram is to graphically summarize the distribution of a univariate data set. It
shows the following:
•
Center (i.e., the location) of the data;
•
Spread (i.e., the scale) of the data;
•
Skewness of the data;
•
Presence of outliers; and
•
Presence of multiple modes in the data.
The Measure icon at the top of the Measure Histogram window allows you to
choose between one of two options:
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•
The Histogram is obtained by splitting the range of the data into equalsized bins (intervals). Then for each bin, the number of points from the data
set that fall into each bin are counted. The horizontal axis represents the
response variable .The frequency (i.e., counts for each bin) is displayed on
the vertical axis.
•
The Cumulative Histogram is a variation of the histogram in which the
vertical axis gives not just the counts for a single bin, but rather gives the
counts for that bin plus all bins for smaller values of the response variable.
Yellow guides move with your cursor and indicate, at the bottom of the window,
the frequency (Y) and the response variable (X) for each bin. The red vertical line
represents the mean value.
Figure 7-11. Measure Histogram.
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7.10.2
DIGE histogram
A DIGE Histogram can be displayed when two co-detected images are selected
(Figure 7-12). It shows data associated with detected spots in the selected
images. Spot data is plotted against log volume ratio on the X-axis, using two Yaxes.
•
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 corresponding drop down menu in the toolbar of the DIGE
Histogram window. A plotted single data point on the histogram represents
an individual protein spot.
Figure 7-12. DIGE Histogram.
The DIGE Histogram is interactive. You can click on the data points representing
spots and select them on the gels and any other open reports. Just select the
spots by clicking on the corresponding points while using the Ctrl or Shift keys for
multiple selections. To select all spots in a region, hold down the mouse button
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and then drag the cursor to define an area. Use the options from the Select on
Gels drop down menu in the toolbar to identify the corresponding spots on the
gels and open reports.
The pull down menu next to the Reports icon in the toolbar can be used to display
information related to the DIGE Histogram:
•
The Gel Report option displays a Gel Report for the two selected gels.
•
The DIGE Report item show the numerical values corresponding to the
spots in the DIGE histogram (see below).
There are two ways of displaying a DIGE Histogram. Depending on which one you
use, all detected spots or only selected spots are included.
To display a DIGE histogram containing all spots:
1
Select two co-detected images in the DIGE Gels folder of the workspace.
Right click on one of the images.
2
Choose Histogram from the contextual menu.
3
A DIGE Histogram containing all spots from the selected gels is
displayed.
To display a DIGE histogram containing selected spots:
1
Select two co-detected images in a [Gels] or [Classes] worksheet.
2
Select spots. Use the Shift or Ctrl keys to make multiple selections.
3
Choose Analyze > DIGE > Histogram in the menu.
4
A DIGE Histogram containing only the selected spots is displayed.
NOTE! In the DIGE Histogram, the Volume Ratio (used to calculate the log
Volume Ratio on the X-axis) corresponds to: Volume of the spot on the first
selected image/Volume of the spot on the second selected image. To obtain
the inversed spot ratios, change the order of the gels in the worksheet.
Measure parameter
• Max Slope: Largest gradient associated with co-detected spots.
•
Area: Number of pixels within the spot boundary.
•
Max Intensity: Largest pixel value associated with co-detected spots.
•
Max Volume: Volume of the largest of the co-detected spots.
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7.10.3
DIGE report
A DIGE report is a special case of a spot report (see Section 7.10.1) enabling you
to display NORMALIZED Vol Ratios (see the note below) for specific combinations
of DIGE images (not only ratios compared to the DIGE reference image). Two
images must be selected to generate a DIGE report. For each Spot ID in the report,
generally two lines will show, one for each gel (Figure 7-13).
NOTE! The reported Vol Ratios in the DIGE report are NORMALIZED, so that the
modal peak of volume ratios is zero (since the majority of proteins are not up
or down regulated). This means that the Vol Ratio is expressed in the range of
1 to 1 000 000 for increases in spot volumes and –1 to – 1 000 000 for
decreases in spot volumes. Values between –1 and 1 are not represented,
hence a two-fold increase and decrease is represented by 2 and –2,
respectively (and not 2 and 0.5 as might have been guessed).
NOTE! In the DIGE Report, the Vol Ratio shown corresponds to: Volume of the
spot on the image mentioned in the FileName/Volume of the spot on the other
selected image.
Measure Histogram
Figure 7-13. DIGE Report
Depending on how you generate the DIGE Report, all the detected spots or only
selected spots appear in the report, as it is the case for DIGE Histograms (see
Section 7.10.2). If you display a DIGE Histogram from the contextual menu in the
workspace, and then select DIGE Report from the Reports icon in the DIGE
Histogram tool bar, all spots will be included. To include only selected spots, select
spots in two gels and choose Analyze > DIGE > Report.
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Annotations
8.1
Introduction
Individual pixels and spots in a gel image can be labeled with annotations. The
annotations are used in functionalities such as alignment, matching, or flagging
spots with their particular characteristics (protein name, database accession
number and so on). Annotations can also be used to mark spots with common
characteristics. Finally, annotations 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 8-1. 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. The label
contents can be any textual data, numerical values or a Boolean value. The user
can also decide that a label within a given category is unique.
As shown in Figure 8-1, each annotation is composed of a basis, which can be a
square or a cross depending on whether the annotation is attached to a spot or
a pixel, a flagpole (by default displayed in cyan) and a flag that consists of a set
of colored labels.
8.2
Predefined label categories
Some predefined label categories are provided with the ImageMaster software.
To help you visually identify the various label categories, they are automatically
created using different background colors, as indicated below, after each
category name:
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•
Ac (pink): This category is provided to hold the protein's accession number
(AC) taken from a user-defined database, and can be the link 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 (see
Section 8.3.1).
•
Landmark (blue): This category is used to mark pixels or spots in the gels as
reference points for the purpose of gel alignment or matching and for the
calculation of corresponding locations between gels. Two annotations are
considered as referring to the same point in different gels when they bear
identical labels (of the same category).
•
pI_MW (green): 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 (probably
because you do not know it).
•
Comment (pale pink): This category is an example of a general category
where users may store their comments. User comments can also be
entered in any other user-defined category (see Section 8.3.1).
•
Set (yellow): This is a generic category used to mark spots having common
characteristics. All labels from a Set category share the same text, or in
other words the same set name. Figure 8-1 exemplifies a set called Pick
containing spots that need to be exported to a spot excision robot. See
Section 8.3.2 for more details.
Please note that the default colors for the different label categories can be
changed. You can do this by going to the Categories tab in the ImageMaster
Options (accessible by choosing Tools > Options in the menu).
8.3
Creating annotations and labels
You can create two types of annotations. The first one, visualized with a cross
basis, is simply connected to a pixel and has the same coordinates as that pixel.
The second type of annotation concerns those that are linked to a spot. These
annotations are marked by a square at their basis, and have the same
coordinates as the spot to which they are linked, that is, the spot's center of
gravity. In addition, the annotations are automatically selected when the linked
spot is selected, and vice versa. (Figure 8-1)
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NOTE! To create a spot-linked annotation, just click somewhere within a spot
during creation (see below). To decrease the chance of missing a spot when
clicking on the gel, you can display spots with the Outlined shape, rather than
the Crossed option.
To create an annotation:
1
Click on the Annotation tool in the ImageMaster toolbar.
2
Double click on the pixel or spot in the gel where the annotation should
be located.
3
In the Create Annotation by Click box, enter the name of a new category
or choose one of the existing categories by clicking on its name.
4
When a new category is created, the Create Category box appears.
Please see Section 8.3.1 to find out more about the parameters
requested.
5
Type the desired label in the next dialog box, and click OK when finished.
6
The annotation is created and its label is displayed on the gel.
Labels can be added to an existing annotation. In fact, one annotation may have
many labels, although 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.
To add a label to an existing annotation:
1
Click on the Annotation tool in the toolbar.
2
Double click at the basis of the annotation to which your label should be
added.
3
In the Create Annotation by Click box, enter the name of a new category
or choose one of the existing categories by clicking on its name.
4
When a new category is created, the Create Category window will
appear. Please see Section 8.3.1 to find out more about the parameters
requested.
5
Type the content of the label in the next dialog box and click OK.
6
The label is added to the existing annotation.
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NOTE! Alternatively, you can add labels by selecting annotations or spots
and choosing Edit > Annotations > Add Label in the menu. This option is
particularly useful when you want to add identical labels to a series of spots
or annotations because you can create many labels at a time, instead of
having to define each one individually.
At any time, ImageMaster allows you to link an annotation to a spot, or do the
opposite. This is helpful because sometimes you may miss a spot to which you
wanted to link a newly created annotation or you may have decided that an
annotation should not be linked to a given spot.
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
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.
8.3.1
Creating label categories
As mentioned above, ImageMaster offers a series of predefined categories.
However, you can also create your own categories, which are always displayed
with gray backgrounds. You can easily do this by adding an annotation using the
procedure described above and typing the name of a new category. You must
enter the constraints for the new category in the Create Category box (Figure 8-2).
Please note that the category constraints can also be modified at a later stage by
going to the Categories tab in the ImageMaster Options (accessible by choosing
Tools > Options in the menu). However, it is better to set the constraints while
creating the category, otherwise you may have to adapt the existing labels to
make them fit the new restraints.
Note that 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.
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Figure 8-2. Create Category dialog box.
Data type
By default, the labels in ImageMaster can contain any character. However, to
ensure coherent annotation data, the label contents can be constrained to only
numerical values or Boolean entities. This is done by fixing the Data Type to one
of the following:
•
Text: Can contain any character.
•
Number: Can only contain numerical values.
•
Boolean: Can only take the values 0 and 1.
Notice that categories using the Boolean Data Type are displayed in the form of
check boxes in most reports (except for the Label and Category Reports). Editing
these labels corresponds to checking the box to indicate whether the item should
take the value 0 (empty check box) or 1 (checked box).
Label categories of the Boolean Data Type are useful to indicate that you checked
your data for specific properties, and that, based on these properties, you decide
to classify your data points into two populations.
Imagine that you found 50 interesting proteins (significant expression variations)
from a preliminary differential analysis. You can add a label of the Boolean type
to each of these 50 protein spots and designate whether yes (value 1) or no (value
0) you want to export them to a spot excision robot. Thus, you can easily select
spots to be picked, but you also have a record of which protein spots were
carefully scrutinized.
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Is unique
When you check the Is Unique box, you indicate to ImageMaster that each label
on a gel within the new category should be unique. That is, no identical labels can
be created. This is the case for the Landmark category, for instance. In order to
prevent confusion as to which spots correspond in different gels, there should
only be one spot marked with a particular label in each of the gels.
When the Is Unique box is checked, ImageMaster will not accept your label when
an identical one already exists. You are asked to enter a new label. This is always
the case for labels of the Landmark category.
External engine
The External Engine field should be used when you want to send HTTP queries to
a CGI script on a server. A CGI (Common Gateway Interface) script is a program or
script file executed on a Web server in response to a user request. It allows, for
example, people from all over the world to query a database that is connected to
the World Wide Web. 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.
Virtually all databases on the Web, and in particular those containing 2-DE and
other protein data (see Chapter 11), use CGI scripts to enable data queries.
ImageMaster takes advantage of this fact by offering 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.
When you subsequently double click on a label of such a category, ImageMaster
opens your default Internet browser and launches an HTTP query that takes the
form of a Web page address.
http://www.expasy.org/cgi-bin/nice2dpage.pl?P02649 The elements are:
•
http://www.expasy.org/ (database HTTP address).
•
cgi-bin/nice2dpage.pl? (database query engine ).
•
P02649 (database accession number).
This example relates to a query of the SWISS-2DPAGE database on the ExPASy
Proteomics Server, using the nice2dpage query engine. As a result, the entry for
Human Apo E, corresponding to the protein with accession number P02649, is
returned in your browser.
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To set the database HTTP address and query engine:
1
In the Create Category box, click on the URL button located next to the
External Engine field.
2
The page http://www.expasy.org/ch2d/2d-access.html, detailing a list
of federated 2-D PAGE databases and servers is displayed. This list
provides the required database query formats (address and query
engine).
3
Copy the desired database address and query engine. The necessary
information generally ends with a question mark (?) or equal sign (=). If
you would like to use the SWISS-2DPAGE, for instance, with the basic
query engine, you should copy the sequence http://www.expasy.org/
cgi-bin/get-ch2d-entry?.
4
Paste the copied information into the External Engine field.
To link to a database that is not federated or that does not contain 2-DE
data:
1
Directly query the database you would like to link your spots to, until you
find a specific protein (or other) entry. For example, you might display
the entry for the protein structure 1BMT in the well-known Protein Data
Bank (PDB).
2
Check in your browser whether you can delete parts of the Web page
address without losing the database entry. In the example of the PDB,
the original address http://www.rcsb.org/pdb/cgi/
explore.cgi?pid=72461033478448&page=0&pdbId=1BMT can be
shortened to http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1BMT.
3
Copy the address of the corresponding Web page in your browser
without including the accession or identification 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 (=). In the
example above, you would copy the string http://www.rcsb.org/pdb/
cgi/explore.cgi?pdbId=.
4
In the Create Category box, paste the copied information into the
External Engine field.
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.
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To set an executable:
1
In the Create Category box, click on the EXE button located next to the
External Engine field.
2
Browse the directory where the executable is located and select the
correct file name (which should carry the extension .exe).
3
After clicking Open, the file, with its complete file path, automatically
inserts into the External Engine field.
Please note that 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 in the active workspace.
8.3.2
Creating sets
The predefined category Set: was created to help you mark spots with common
properties by indicating that they belong to a set. The labels in such a category
do not contain specific information. They only display the name of the set to
which they belong (Figure 8-1).
To create a set:
1
Select one or more spots.
2
Choose Edit > Annotations > Add Label from the menu.
3
In the Add Label box, click on the category called Set: and add a
keyword, which will become the name of the set. For instance, to mark
spots that should be exported to a spot excision robot, the final category
name can be Set:Pick.
4
A label containing the name of the set (for example Pick) is attached to
the selected spots (note that the string Set: is not displayed on the
labels).
Normally, when a new category is created, it is immediately shown on the screen.
Newly created sets are an exception to this rule. A new set is visible or hidden
depending on the current visibility state of the generic category Set:.
8.3.3
Creating specific links
As seen earlier, it is possible to link labels to remote database entries by defining
an external search engine for a particular category (see Section 8.3.1). In addition
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to this functionality, ImageMaster offers additional possibilities to link your gel
objects to Web pages, mass spectrometry data, text files or any other files
located locally or on the Internet. By using specific keywords in the labels of any
category, you can create links to particular objects including Web pages, files and
text.
To create a specific link (for example, to a Web page, file or text), 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 editing mode,
as usual, but the link (Web page, file or text) is automatically opened with the
appropriate software. Alternatively, to open any linked label, choose Show >
Annotations > Linked Data in the menu.
Note that you can define links in any label category but you can only have one
link per label.
Http link:
You can link spots or pixels to specific Web pages. A double click on an http-linked
label will launch your default Internet browser and automatically call the
corresponding Web page.
You can, for instance, create a direct link to the ExPASy Proteomics Server (Figure
8-3). In this case, the label content should contain the string "http:" followed by the
address of the Web page.
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 Database tab. In this case, you only need to give the name and file
extension when creating the link. Alternatively, you can link labels with files
located anywhere on your system. You should then include the complete file path
when referencing the file.
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Figure 8-3. 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).
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Note that you can set a different Annotation folder for each workspace. You can
also 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).
For example, you can link a protein spot to an Excel file containing the amino acid
composition of a protein (Figure 8-3). The label should then consist of the string
"file:" followed by the file name (with its extension).
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 dialog box containing the entire text (Figure 8-3).
Text links are particularly useful to attach the bibliographic references for a spot,
for instance, or any other comment such as the one in Figure 8-3. 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 match sets and classes, folders and projects in the
workspace, as well as to any report that is saved on the hard disk. Additionally,
you can specify gel descriptions (see Section 6.7.2).
8.4
Selecting annotations and labels
8.4.1
Annotation tool
Annotations and labels can be selected with the Annotation tool as long as they
are visible on the gel. The selected labels or annotations are highlighted in green
and displayed in front of the other annotations (Figure 8-4). If the annotation is
attached to a spot, the spot is also selected. Remember that the opposite is also
true; if you select a spot with the Spot tool, the attached annotation is selected as
well.
To select a label, make sure the Annotation tool is activated and then click on the
label. You can select more than one label by using the Shift key.
To select an annotation, activate the Annotation tool and 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.
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(a)
(b)
Figure 8-4. (a) Annotation hidden by some other annotations. (b) Same annotation
displayed in front of all other annotations, after selection.
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 selected region are selected and
highlighted in green, even those that are hidden. To select annotations in more
than one region, hold down the Shift key while selecting additional regions.
To deselect all annotations, select the gels for which you would like to deactivate
all annotations and click on a gel (not on an annotation).
8.4.2
Select menu
In order to select all spots and annotations in the selected gel, choose Select >
Select All in the menu (or Select > Unselect All to deselect all spots and
annotations). You can select specific labels and annotations with the options in
the Select > Annotations menu:
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•
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 Section 8.4.2 for details). For example, to
select all labels containing the string P00, you can either type the
expression .*P00.* and choose the Regular Expression field in the dialog box,
or type P00 and deselect the Regular Expression field.
•
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.
•
By Color: When a specific color was assigned to particular annotations, the
annotations can be selected by choosing the corresponding color from the
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•
In Region: This option allows the selection of all annotations in the active
regions, that is, in all regions that were defined with the Region tool on
selected gels.
•
From Report File: If any report containing information about labels or
annotations (and enclosing the columns X, Y and GelName) was previously
saved, this function enables the reselection of all labels contained in the
selected report file. Reports on Categories, Labels, Annotations and even
Spots can be used.
•
All: This highlights all annotations in the selected gels.
•
Combine: This option can be used to combine the current selection of
annotations with a second criterion based on the Intersection, Union,
Difference or Exclusion of a particular category. Please see Section 8.4.2 for
more information on this feature.
•
Common Labels: This option allows the retrieval of all sets of identical labels
within a gel or among a series of gels. When two or more gels are selected,
this returns the common labels for all the categories chosen in the pop-up
dialog box. When only one gel is selected, this operation corresponds to
selecting packed labels, that is, labels from the specified categories
containing the same content (see Section 8.5.5).
•
Inverse Selection: If some labels are already selected, this option
deactivates the selected labels and selects all previously unselected ones,
thus inverting the selection criteria.
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 8-1.
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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
^
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
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Syntax
Description
Example
$
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 8-1. Regular expressions available to search patterns in labels. Please not that the
term element used in the description indicates a character or a subexpression.
The characters ^.[$()|*+?{\ have a special meaning in certain contexts. You must
exclude them from the expression with a backslash (\) if you want them to be
taken literally. This means that if your labels contain any of these special
characters, you must precede them with a backslash if you want to include them
as normal characters in your search expression. Note that 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 precedence for the regular expressions described above is as shown
in Table 8-2 . 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 order of precedence than alternation.
Escaped characters
\<Special character>
Bracket expression
[]
Grouping
()
Single-character duplication
* + ? {m,n}
Concatenation
Anchoring
^$
Alternation
|
Table 8-2. Order of precedence (from high to low) for regular expressions.
Combining sets of annotations
You can select annotations based on multiple criteria. You can do this by
combining sets of annotations in particular ways. You can select a first set of
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annotations on your gels and then combine this set with a second set according
to one the following schemes (see also Figure 8-5):
•
Intersection: Selects spots that are in the intersection of two criteria.
•
Union: Selects spots that belong to either one criterion or the other.
•
Difference: Selects spots that belong to either of the two criteria, but
excludes those that belong to both of them.
•
Exclusion: Selects spots that are found only in the first criterion and are not
found in the second criterion.
The possibility of combining sets of annotations is particularly useful when you
have different sets defined. Imagine, for example, that you marked interesting
spots found from Factor Analysis with labels of the category Set:Factor Analysis.
Similarly, you marked significant spots emerging from a Student t test with labels
of the category Set:Student t test. You could now select all spots from the first
set by choosing Select > Annotations > By Category from the menu, and picking
Set:Factor Analysis from the Select Labels by Category box. When you
subsequently choose Select > Annotations > Combine > Intersection from the
menu, and pick the category Set:Student t test, only spots that belong to both
sets remain selected.
Set A
Set B
Set A
Intersection
Set A
Set B
Union
Set B
Set A
Difference
Set B
Exclusion
Figure 8-5. Combining sets of spots in ImageMaster.
To combine sets of annotations:
1
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Select annotations according to a first criterion by using the options in
the menu or by picking the desired annotations with the Annotation tool.
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2
Choose Select > Annotations > Combine in the menu, and select one of
the four possible operations (Intersection, Union, Difference or
Exclusion).
3
Choose the label category corresponding to your second criterion in the
dialog box, and click OK.
4
The result of the logical operation is selected according to Figure 8-5.
8.4.3
Reports
You can select labels or annotations directly from a displayed report on
Annotations, Labels, Categories or even Spots (at least for spots that are linked to
annotations). Do this by double clicking on an item in the report or by selecting
one or several items and clicking the Select on Gels icon in the report toolbar.
8.5
Displaying annotations and labels
You can change the way annotations and labels are displayed but these
modifications will not be saved in your ImageMaster file.
8.5.1
Annotation flag position
Sometimes you may want to move an annotation flag because you are preparing
an illustration or just 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 8-6).
(a)
(b)
Figure 8-6. (a) Default and (b) modified annotation flag position.
8.5.2
Annotation flag color
As mentioned inSection 8.2 each predefined label category is automatically
created using a different background color (pink for the Ac category, blue for
landmarks, etc.), to help you visually identify the various label categories. You can
change the default colors by going to the Categories tab in the ImageMaster
Options (accessible by choosing Tools > Options in the menu), selecting an
annotation category, and modifying its Color in the Display properties. The new
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background color is used for all labels of that specific category in the current
workspace.
8.5.3
Annotation flagpole color
Annotation flagpoles can be displayed on the screen using three different colors
other than the default cyan.
To change the color of the annotation flagpoles:
1
Select the annotations for which you would like to change the flagpole
color.
2
Choose Show > Annotations > Set Color in the menu, and select one of
the proposed colors. Please note that the Default option corresponds to
cyan.
3
The flagpoles of the selected annotations are displayed with the chosen
color (after deselecting the spots).
4
The colors remain displayed this way until you choose Show >
Annotations > Set Color > Reset All to revert all selected annotations to
the default cyan, or until you exit ImageMaster.
8.5.4
Show / hide 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 or part of them. ImageMaster not only
gives you the possibility to hide certain label categories; the program also allows
you to hide certain selected annotations (Figure 8-7).
Note that the visibility of an annotation or label plays a role in its selection. As a
rule, annotations and labels can only be selected and manipulated with the
Annotation tool when they are visible on the gels. To select hidden annotations or
labels, the menu options should be used.
To show or hide all annotations and spots in the selected gels, choose Show >
Show All (or Show > Hide All) in the menu. Hide All also hides the match vectors.
To show or hide specific annotations, you can use one of the following options
available in the Show > Annotations menu.
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•
Show All: Makes all the annotations on the selected gels visible.
•
Hide All: Hides all the annotations on the selected gels.
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•
In Region: Shows all annotations in the area selected with the Region tool.
This option is handy when all annotations were previously hidden.
•
For Spots: Shows all the annotations linked to selected spots. This feature is
particularly useful when all annotations were previously hidden.
•
Only Selected: Shows only the selected annotations and hides all the other
ones.
•
Hide Selected: Hides the selected annotations.
(a)
(b)
Figure 8-7. (a) All categories and labels are visible. (b) Only the Ac, pI_MW and Comment
categories are visible, and only for particular spots.
To show or hide specific categories:
1
Select the gels for which you would like to change the category
visibilities.
2
Choose Show > Annotations > Visible Categories from the menu.
3
In the Set Categories Visibility box, the check boxes of the visible
categories are selected; those of hidden categories are empty, whereas
grayed check boxes correspond to categories that take different
visibility states in the various gels (hidden in some gels, shown in others).
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4
To hide a category, make sure its box is unchecked. On the other hand,
select an empty check box to show the corresponding category. You
can click once or twice in a grayed check box to hide or show the
corresponding label category in the selected gels.
When you click on an annotation that has both visible and hidden labels, all of its
labels are displayed on the screen during the time it remains selected (Figure 8-8).
The hidden labels immediately disappear when the annotation is deselected.
(a)
(b)
Figure 8-8. (a) Unselected annotation with hidden labels. (b) When the annotation is
selected the hidden labels become visible.
8.5.5
Pack / unpack categories
By default, each spot is displayed with its own label, even if several labels hold the
same content. However, to declutter the display, you can show a single label for
all identical labels. In ImageMaster, this option is called packing categories (Figure
8-9).
(a)
(b)
Figure 8-9. (a) Unpacked and (b) packed labels for protein P00450 in the Plasma Master Gel
(SWISS-2DPAGE database). The labels belong to the predefined Ac category.
To pack categories:
170
1
Select the gels for which you would like to pack one or more categories.
2
Choose Show > Annotations > Packed Categories in the menu.
3
In the Set Packed Categories box, the check boxes of the packed
categories are selected; those of unpacked categories are empty,
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whereas grayed check boxes correspond to categories that have
different packing states in the various gels.
4
8.6
To pack a category, check its box. Deselect the check box to unpack a
category. Finally, you may need to click once or twice in a grayed check
box to unpack or pack the corresponding category.
Adding / modifying annotations and labels
You can add and modify annotations and labels in different ways using the
Annotation tool, Edit menu options, Reports or File > Import function. The
Annotation tool is helpful if you want to add or modify a single label or just a few
labels. The Edit menu options are more adapted to the creation of a large number
of annotations or labels simultaneously. Reports are useful for editing existing
annotations.
8.6.1
Annotation tool
When double clicking on a label while the Annotation tool is activated, the Edit
Label box is displayed. Change the text in this box to modify your label content.
The Annotation 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.
When you drag an annotation linked to a spot, outside of that spot, ImageMaster
asks if you want to remove the link with the spot. Similarly, when you move an
annotation onto a spot, ImageMaster asks if you want to link it with that spot.
8.6.2
Edit menu
Various options for modifying, copying, pasting, duplicating and deleting labels or
annotations are available from the Edit > Annotations menu. With the exception
of the Modify function, all of these features can be used to edit (add, delete,
modify) several annotations at a time.
Modify
Once an annotation is selected, you can change the content of its labels and add
new categories by choosing the Modify option and entering the desired
modifications in the Edit Label box. Please note that only one annotation can be
selected at a time. When picking a single label, only the content of the selected
label can be modified.
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 simple means of creating a set of
similar annotations in a series of gels, and can be useful for quickly adding
landmarks, for instance. Subsequently, you just need to adjust the annotation
positions.
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To copy annotations from one gel to a set of other gels:
1
Select the annotations you would like to copy to other gels.
2
Choose Edit > Annotations > Copy in the menu. The selected annotations
are copied into memory.
3
Select the gels into which you want to copy the annotations.
4
Choose Edit > Annotations > Paste Annotations in the menu.
5
The copied annotations are pasted to the corresponding locations in the
selected gels. These locations are estimated by interpolation between
the two nearest common landmarks (landmarks with the same name).
When no such landmarks exist, the annotations are placed at the same
image location (X and Y coordinates).
6
Adjust the annotation positions using the Annotation 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.
To copy labels from one gel to a set of other gels:
1
Select the labels you would like to copy.
2
Choose Edit > Annotations > Copy in the menu. The selected labels are
copied into memory.
3
Select the spots or existing annotations into which you want to copy the
labels.
4
Choose Edit > Annotations > Paste Labels in the menu.
5
The copied labels are pasted to the selected spots and annotations.
Copy matched labels
When gels have been matched, labels existing in one gel can be copied to their
corresponding spots in other gels. This is particularly useful when you have
annotated your master gel (for instance, by adding and modifying annotations to
the master gel via an Intra-class Report) and want to propagate the labels to all
matched gels.
To copy matched labels from one gel to a set of other gels:
1
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Select the labels to be copied in the source gel.
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2
Select the source gel and the gels into which you want to copy the
labels.
3
Choose Edit > Annotations > Copy Matched Labels from the menu.
4
The selected labels are copied to the corresponding spots in the other
gels.
Duplicate labels
You can copy selected labels to another category. 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.
To duplicate labels:
1
Select the labels you would like to duplicate (as part of another
category).
2
Choose Edit > Annotations > Duplicate Labels from the menu.
3
In the Duplicate Labels box, pick the recipient category, or enter a new
category name.
4
The selected labels are copied to the chosen category.
5
When a label for the chosen category already exists, ImageMaster asks
whether you want to keep it or replace it with the new one.
Rename category
ImageMaster allows you to rename any category, be it a predefined or userdefined category, or a Set:. In the case of a Set:, the label contents (name of the
Set:) are automatically updated on the gel.
To rename a category:
1
Select the gels for which you would like to rename a category.
2
Choose Edit > Annotations > Rename Category in the menu.
3
In the displayed category list, select the one you would like to rename.
4
Enter the new category name in the next dialog box.
5
ImageMaster immediately applies the modifications to all labels on the
selected gels.
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Deleting annotations and labels
Two possibilities are available from the Edit > Annotations menu for deleting
selected labels or annotations:
•
Delete by Category: With this feature, only the selected labels belonging to
the categories specified from the category list are deleted.
•
Delete: This option deletes all selected labels. Please note that if you select
annotations, all their labels are selected and therefore deleted.
8.6.3
Reports
This section describes the functionalities in ImageMaster that are helpful for
editing annotation and label content in reports that contain information on these
objects (see Section 8.7).
Please see Chapter 5 for details about other standard tools available in the
various report toolbars, including icons to save, print or copy reports or report
lines, to navigate between reports and gels, to refine report selections, and so on.
Creating annotation categories
Annotation Reports, Intra-class and Inter-class Reports contain the Annotate icon
in their toolbar. Clicking on the Annotate icon allows you to insert a category as
an extra column, by selecting its name from the category list (containing all the
categories present in the open gels). You can create a new category by typing its
name in the upper field of the displayed window. In this case, you are asked for
the category constraints.
Adding or modifying labels
Once you created or displayed the desired categories, you can directly add new
labels to the appropriate cells, or edit existing ones. Just double click in a cell to
start typing your label. When finished, a single click in any cell quits the editing
mode. The cell corresponding to the modified label appears in dark green when
the related line is selected, or in gray otherwise. You can quickly see that
modifications were made to your report when an asterisk follows its window title.
Please note that when you want to maintain a current selection while editing
labels, you should hold down the Shift key while double clicking in a cell. You can
release the Shift key as soon as you are in the editing mode.
Notice that categories of the type Set: or categories using the Boolean Data Type
are displayed in the form of check boxes in some reports. Editing their labels
corresponds to checking the box, in order to indicate whether the corresponding
item belongs to a Set: (checked box) or not (empty check box), or takes the value
0 (empty check box) or 1 (checked box) for a Boolean Data Type.
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To check several boxes (rows) simultaneously within a given category
(column):
1
Select the rows for which you would like to change the checked state.
2
Hold down the Shift key while clicking in the check box of one of the
selected rows (obviously in the relevant column). All selected lines are
now in the same checked state. Click in the check box a second time if
the state should be inversed.
Updating gels
Subsequently, the Update Gel icon should be used to make the modifications
definitive and to propagate the information to your gels. Note that the cells of
updated labels return to their original color. When all labels in your report are
updated, the asterisk behind the report title also disappears.
NOTE! You should be aware of the fact that the annotations displayed in the
Intra-class and Inter-class Reports are those from the master gel. This means
that any modifications, including the creation of new columns corresponding
to categories and the addition of new labels, can only be applied to the
master gel. Note that this is not really a problem, since you can easily
propagate selected labels to matched spots (see Section 8.6.2).
8.6.4
Importing labels and annotations
You can import annotations and labels from a file into open gels. You can import
labels from a Label Report, for example, or from any other tab-delimited text file
containing the columns Category, Label, SpotID, X and Y. If the Spot ID is not
known, use -1 in this field and ImageMaster 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 ImageMaster will position the label on the spot with the
corresponding Spot ID.
To import labels into open gels:
1
Select the gels for which you would like to import labels.
2
Choose File > Import > Labels in the menu.
3
Browse the folder containing the desired Label Report (.rpt) or a text file
(.txt) containing the label information. Click OK.
4
ImageMaster asks for confirmation. Answer Yes if you want to proceed.
5
The imported labels are added to the selected gels.
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Similarly, you can import annotations from an Annotation Report or a tabdelimited text file containing the required columns: SpotID, X, Y, and a column for
each category to be imported.
To import annotations into open gels:
1
Select the gels for which you would like to import annotations.
2
Choose File > Import > Annotations in the menu.
3
Browse the folder containing the desired Annotation Report (.rpt) or a
text file (.txt) containing annotation information. Click OK.
4
ImageMaster asks for confirmation. Answer Yes if you want to proceed.
5
The imported annotations are added to the selected gels.
8.7
Annotations and labels in reports
ImageMaster provides various reports including specific information about
annotations, labels, and categories. Intra-Class and Inter-Class Reports can
display the content of annotations and labels attached to spots in the master gel.
As pointed out in Chapter 5, these reports are editable, although to different
extents. In Section 8.6.3 you will find that the editing feature is particularly useful
for adding or modifying annotations and labels.
8.7.1
Label report
The Label Report displays information about selected labels, such as the gel
name, category, label content, Spot ID, and X and Y coordinates (Figure 8-10). The
Label column in this report is editable, thus giving you the possibility to modify the
existing label contents.
Figure 8-10. Report on selected labels in selected gels.
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8.7.2
Annotation report
The Annotation Report displays information on selected annotations in selected
gels. Thus, for each annotation, you will find the gel name, Spot ID, X and Y
coordinates, and the label contents for all chosen categories (Figure 8-11). You
can modify the label contents for each category, or even add new categories.
Figure 8-11. Report on selected annotations in selected gels.
8.7.3
Category report
You can display information about all labels belonging to selected categories and
selected gels. The information displayed in this report (Figure 8-12) is the same as
for a report on labels (Figure 8-10) and the label contents can be edited as well.
Figure 8-12. Report on selected categories in selected gels.
8.7.4
Intra-Class Report
An Intra-Class Report contains information about selected matches and their
comprised spots (Match ID, central tendency, dispersion, spot values, coefficient
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of variation, range ratio, separability, etc.). In addition, they can be used to display
the labels attached to the spots in the master gel. In an Intra-Class Report, you
can add labels or edit existing ones and create new columns corresponding to
categories (Figure 8-13).
Figure 8-13. Report on selected matches in selected gels.
8.7.5
Inter-Class Report
An Inter-Class Report provides statistical values for sets of gels, calculated for all
selected matches, such as central tendency, dispersion and overlapping
measures. In such a report, you can display the labels attached to the spots in the
master gel, add labels or edit existing ones and create new columns
corresponding to categories (Figure 8-14).
Figure 8-14. Report on selected matches in selected classes.
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9.1
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.
To be able to initiate the matching process in ImageMaster 2D Platinum 6.0, gels
must be part of a Match Set. A match set includes gels or populations of gels (in
sub match sets) that should be compared and therefore matched together.
Every match set is represented by a Master. This master image is created by
ImageMaster based on the Reference for the match set, chosen by the user.
Essentially, the Master is a copy of the match set reference and contains the
same spots, which are therefore automatically matched between the two
images. But once other gels in the match set are matched with the Master, it is
possible to copy additional spots from the matched gels to the Master.
As briefly described above, a match set can include gels, but also populations of
gels represented by a sub match set. The principle can be illustrated with Figure
9-1. The gels A1, A2 and A3 are part of Match Set A. This match set is represented
by the master image Master A, based on the selected reference image A1 (in red).
Since Master A is a copy of A1, it contains the same spots. The same is true for
Master B, which represents match set B.
Once all the gels in match set A are properly matched with Master A, and the gels
in match set B are matched with Master B, the match sets A and B can be
compared. To do so, they are included in a higher level match set AB, for which
match set A was chosen as a reference. In practice, comparing match sets A and
B amounts to comparing their master gels, Master A and Master B. The new
representative Master of this match set, Master AB, is an automatically created
copy of the master image from the selected reference match set.
The interesting thing about this procedure is that matches are propagated at
each level. This means that once all match sets (in this case, A, B and AB) are
effectively matched, spots from gel A2, for instance, can be directly compared
with those from gel B3, through the existence of a common node (Master AB) in
the matching tree.
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NOTE! The master images are the nodes for the propagation of matches. If a
protein is not present in the Reference, and so on the Master, no matches will
be formed, even if the protein appears on all other gels (see blue spots in
Figure 9-1). Consequently, the protein will not turn up in the analysis. In the
example of Figure 9-1, choosing one of the other gels (A2 or A3) as the
Reference would have been more appropriate. To solve this problem, spots
from matched gels can be copied to the master gel.
A1
A2
A3
Master A
B1
B2
B3
Master B
A
B
Master AB
Figure 9-1. Population matching and the importance of the master image. Matches are
selected by choosing the corresponding spot, which will appear in green. One match is
selected in match set A. In match set B, all matches are selected. Representative spots in a
population that are not in the reference gel (blue spots) should be manually copied to the
Master.
When several gels are matched to a given Master, this Master provides a unique
numbering scheme for spots across all gels. Each spot in a gel image can in fact
be associated to the corresponding Spot ID in the master gel. The Spot ID in the
master gel is then called the Match ID.
The fact that you are no longer limited to matching gels, but now can match
populations of gels is a major innovation over previous versions of the software.
What is more, you can match different levels of populations. This is called
population matching. The countless possibilities that population matching offers
are further documented in Chapter 4. The current chapter will concentrate on the
matching procedure and the manipulation of matches.
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9.2
Matching
The prerequisites for matching gels or match sets is that spots are detected, the
gels or sub match sets to be matched are added to a match set (see Chapter 4),
and this match set is opened in a worksheet.
NOTE! A DIGE gel is an inherent match set for which the co-run images are
automatically matched (see Chapter 4 to learn how to quickly create match
sets for DIGE gels). To subsequently match different DIGE gels, proceed as
with any other match sets.
NOTE! Instead of the reference image, the Master is displayed in a [MatchSet]
worksheet. As the master and reference images are the same, and matches
are automatically created between the two, the reference image is not
displayed by default. To show the Reference in the match set, choose Show >
Show Reference.
NOTE! The functionalities that are specifically related to editing matches and
reporting on matches are only available when working in a [MatchSet]
worksheet.
9.2.1
Automatic matching
ImageMaster is designed to match gels with minimum user intervention.
Nevertheless, sometimes you may need to help the matching process by
specifying a few starting matches or landmark annotations (see below).
Before adding any landmarks or starting matches, we recommend trying to
match your gels with only a single landmark or possibly none at all. In some
cases, no landmarks will be required. More often, a single landmark is sufficient
for quick and efficient matching. If matching takes a long time and no matches
seem to be found (the counter shown during matching always shows 0 or
increases slowly), you may want to cancel the matching procedure and define a
few landmarks. Subsequently, you can repeat the automatic matching procedure
below.
To match the gels in a match set:
1
Open the match set in a new [MatchSet] worksheet.
2
Select the gels to be matched in the worksheet.
3
Choose Edit > Matches > Match Gels from the menu.
4
If matches already exist, you are asked whether you want to keep them
or not. If you answer Yes, ImageMaster keeps the existing matches and
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just looks for additional ones. If you click No, the matching procedure is
started from scratch (only annotations of the landmark category, if any,
are considered).
5
All selected gels are matched with the master image. Please note that
the matching step might take some time, depending on your computer
capacity.
6
When matching is completed, ImageMaster provides the total number
of matches found.
NOTE! Matches are saved on disk when saving the gel files. However, the
information in the workspace project is necessary to reconstruct the match
sets and retrieve the matches. This is the reason why it is important to
regularly save the workspace (which in turn saves the projects) and why the
workspace is saved by default when closing the software.
9.2.2
Specifying starting matches
When the gels are very distorted or different, automatic matching may fail. In this
case, it can be necessary to define starting matches or landmarks, that is,
corresponding spots that are present in each of the gels to be matched. However,
the number of landmarks or starting matches should be kept to a minimum. Often
a single landmark may suffice to successfully initialize the matching procedure.
NOTE! The wise choice of landmarks or matches is imperative for obtaining
adequate matching results. Landmarks essentially correct global
deformations of gels. Therefore, it is recommended not to put landmarks on
spots in locally distorted regions because this can worsen the matching
results around such regions. To correctly match gels, you should perform a
first matching round using some carefully selected landmarks. If some locally
deformed regions are not properly matched, it is best to delete any wrong
matches that were generated, manually add a few new matches, and then to
carry out a second automatic matching cycle while keeping the existing
matches.
Input matches
When you manually define starting matches (see Section 9.5.1), the matching
algorithm uses these matches as matching seeds. These seeds are not
permanent, however. No difference is made between user-defined matches and
those found by the matching algorithm. If you restart the matching procedure
without taking into account the previously found matches, the manually input
matches are also not considered.
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Landmark annotations
If two or more gels have annotations with identical labels, for example landmark
annotations, you can use the landmarks to initiate the matching process on the
condition that the annotations are linked to spots. The matching algorithm
superimposes those annotations that bear the same labels (in the master gel and
the matched gel) and uses them as matching seeds. Please see Chapter 8 to find
out how to create annotations.
The following rules should be considered when defining landmark annotations:
•
Keep the number of landmarks to a minimum.
•
Only define landmarks on clearly corresponding spots. Protein variants
should definitely not be used as landmarks.
•
Landmarks should be placed on small, sharp spots, rather than on large,
diffuse ones (to reduce the error in the position).
When the annotations are of the landmark category, ImageMaster automatically
takes them into account for the matching procedure. In this case, you just have
to choose Edit > Matches > Match Gels from the menu.
When the annotations belong to any other category, you must first match the
annotations to generate starting matches. Once this is done, you can run the
automatic matching algorithm again by choosing Edit > Matches > Match Gels
from the menu. You must keep the existing pairs when asked for it.
To match annotations:
1
Select the gels for which annotations should be matched.
2
Choose Edit > Matches > Match Annotations from the menu.
3
In the pop-up dialog box, select the desired category to be matched.
4
ImageMaster creates matches between identical labels of the chosen
category.
5
You can now run the automatic matching procedure.
9.3
Selecting matches
9.3.1
Menu options
Selecting matches consists of selecting their corresponding spots. You can select
particular matches by using the items in the Select > Matches menu:
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•
By ID: Allows the selection of matches based on their Match ID.
•
For spots: Selects all spots matched with selected spots in the selected gels.
•
All: Selects all spots matched with spots in the master image.
•
Refine Selection: Refines an initial match selection based on the presence
or absence of matched spots in the selected gels (see below for more
details).
•
Inverse Selection: If some matches are already selected, this option
deselects the selected matches and selects all the unselected ones, thus
inverting the selection criteria.
•
Multiple Matches: Selects only the multiple matches in the selected gels
(see below).
Refining a match selection
ImageMaster allows you to refine an initial match selection based on the
presence or absence of matched spots in the selected gels. By using the sliders in
the Refine Match Selection window (Figure 9-2), you can choose the threshold (X)
for the refinement of the matches corresponding to selected spots:
•
Selected spots is >= X: Keeps matches that have selected spots in at least X
of the chosen gels.
•
Selected spots is < X: Keeps matches that have selected spots in less than X
of the chosen gels.
•
Unselected spots is >= X: Keeps matches that have unselected spots in at
least X of the chosen gels.
•
Unselected spots is < X: Keeps matches that have unselected spots in less
than X of the chosen gels.
Figure 9-2. Refine a match selection based on the number of selected spots in each match.
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For example, suppose you have a set of 6 matched gels, for which you would like
to study the matches that contain at least 3 spots with a relative volume higher
than 0.02. In other words, you would like to analyze spots according to two
criteria. First, the spots should have a relative volume higher than a certain
threshold, in order to select spots that are meaningful in terms of volume. Second,
these spots should be present in at least half of the selected gels. You may select
this special set of spots by performing the following steps:
To filter spots in matched gels:
1
Select your six matched gels. Please note that the master gel will not be
included in the refinement. The reference image must be made visible to
be included in the selection (choose Show > Show Reference).
2
Select all spots in the gels by choosing Select > Spots > All from the
menu.
3
Subsequently, refine the current spot selection based on the relative
spot volume, by choosing Select > Spots > Refine by Value from the
menu, picking the %Vol, and keeping only the spots whose %Vol is >=
0.02.
4
Spots corresponding to this first criterion remain selected.
5
Choose Select > Matches > Refine Selection from the menu, and Keep the
Matches whose Number of Selected Spots is >= 3 (by clicking the first
option and moving the slider to the value 3).
6
The remaining matches meet both specified criteria.
As you may notice, there are endless possibilities for combining selections of
spots and matches. Your own experiments and needs will drive the usage of this
function.
Multiple matches
ImageMaster enables the creation of multiple matches (see Section 9.5 for details
about adding matches). In contrast to a single match, where one spot in a master
gel is matched with exactly one spot in the other gels, a multiple match implies
that a single spot in the master gel can be matched to several spots in another
gel (Figure 9-3). The opposite case (several spots in the Master matched with a
single spot in another gel) is not possible.
The spots from such multiple matches (on a given gel image) are considered as a
single spot in the subsequent data analysis. The calculated quantification values
reflect the size, intensity and abundance of the combined spots.
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Figure 9-3. Multiple matches. One spot in the master gel (spot 3815) is matched with two
spots (spots 3944 and 3951) in another gel. In further data analysis and quantification (e.g.
Vol in the Intra-Class Report), the two spots are considered as being one (the volumes have
been summed).
9.3.2
Reports
You can select matches directly from a displayed Match Report (see Section
9.6.1). You can do this by double clicking on a row in the report, or by selecting
one or more rows and clicking the Select on Gels icon in the report toolbar.
9.4
Displaying matches
A match describes the relationship between individual spots. As mentioned
above, selecting matches corresponds to selecting their constituent spots.
However, this is not always a straightforward way to visualize the correlation
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between spots. ImageMaster therefore provides the following features for easier
visualization of matches.
9.4.1
Show / hide matches
Matches can be displayed as blue vectors that link the locations of the spots in
the displayed gel and the matched spots in the master image (Figure 9-4). These
match vectors are always minimized with respect to the center of the visible gel
area and enable you to evaluate the matching results more easily.
You can change the default color for the match vectors by choosing Tools >
Options from the menu and going to the Display tab.
To display match vectors:
1
Open a [MatchSet] or [Classes] worksheet.
2
Choose Show > Matches > Show Vectors from the menu.
3
Blue vectors are shown between matched spots in the displayed gel and
the master gel.
4
You can hide the match vectors again by choosing Show > Matches >
Hide Vectors.
Figure 9-4. Matched spots in matched gels are linked by blue vectors.
9.4.2
Show / hide match IDs
You can display Match IDs for selected spots on selected gels (Figure 9-5).
Obviously, the gels must be matched to the Master.
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To display Match IDs:
1
Select the spots and gels you would like to display the Match ID for.
2
Choose Show > Matches > Show ID from the menu.
3
To hide the Match IDs again, possibly in specific gels only, select the
desired gels and choose Show > Matches > Hide All ID.
Figure 9-5. Match IDs for selected spots on selected gels.
9.5
Adding / deleting matches
Sometimes it may be necessary to manually add matches. This can be the case
when the matching algorithm needs a few input matches, or when you would like
to add or change matches after the automatic matching procedure.
9.5.1
Adding / deleting matches
To add a match:
188
1
Select a spot in the master image and the corresponding spots in the
other images (you can select several spots in a given image if you want
to create a multiple match). Use the Shift or Ctrl keys to make multiple
selections.
2
Choose Edit > Matches > Add Match from the menu.
3
The new match is created.
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To delete a match:
1
Select the spots for which matches should be deleted.
2
Select the gels from which the corresponding matches should be
deleted. Use the Shift or Ctrl keys to make multiple selections.
3
Choose Edit > Matches > Delete Match from the menu.
4
The matches for the selected spots are deleted.
NOTE! To undo the addition or deletion of matches you should not only select
the gel for which the operation should occur, but also the Master.
9.6
Reports on matches
9.6.1
Match report
ImageMaster can display a Match Report. It shows a list of matched spots in the
selected gels. The first column in such a report displays the Match ID, that is, the
Spot ID in the master image. The following columns display the corresponding
Spot IDs in the matched gels that were selected for the creation of the report
(Figure 9-6).
To display a Match Report:
1
Select the spots that should appear in the Match Report.
2
Select the matched gels you would like to include in the report.
3
Choose Reports > Match Report from the menu.
4
You get a list of spot IDs for all matched spots among the selected gels.
Figure 9-6. Report on selected matches for six matched gels.
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9.6.2
Match statistics report
Another way to evaluate matching results is to generate a Match Statistics Report
(Figure 9-7). This report lists the number of matches (NbMatches) found between
each of the selected gels in the match set and the master image, as well as the
percentage of spots that were matched (PercentMatches), calculated as follows:
2m
PercentMatches = ------------------ng + nm
where m is the total number of matches between the gel and the master image,
ng is the number of spots in the gel and nm is the number of spots in the Master.
Figure 9-7. Report on the match statistics between selected gels and the master image.
To display a Match Statistics Report:
1
Make sure you opened a match set from the workspace.
2
Choose Reports > Match Statistics Report from the menu.
3
A Match Statistics Report is displayed.
9.6.3
Intra-class report
Intra-class Reports contain information about matched spots in a class of gels
(Match ID, central tendency, dispersion, spot values, coefficient of variation, range
ratio, separability, etc.). In Chapter 10 you can find in-depth information about the
Intra-Class Report and its functionalities (Figure 9-8).
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Figure 9-8. Intra-Class Report on matched spots in selected gels.
9.7
Synthetic gels
With the Create Synthetic Gel feature, you can create a composite gel image that
contains only representative spots of a population, or on the other hand shows all
spots present in a series of gels. In the first case, an average gel image is obtained
by averaging the positions, shapes, and optical densities of the matched spots in
a given set of gels. This produces an intersection of all gels, showing only the
spots found in all or at least some of the given images. In the second case, the
unmatched spots are added as well. The synthetic gel can then be considered as
a spot index or a graphical fusion of protein maps.
In reality, you can choose which spots should be included in a synthetic gel. This
is because the synthetic gel will contain all selected spots in the selected gels at
the moment you create the synthetic image. You can therefore filter your spots
carefully before generating the synthetic image.
If spots are matched, only one of the matched spots will be present in the
synthetic gel. Its area corresponds to that of the spot having an area closest to
the average of the matched spots, while the intensity equals the average
intensity of the matched spots. For isolated spots included in the synthetic gel, the
Intensity, Area and Vol values of the original spot are assigned. The %Vol and
%Intensity values are recalculated according to the total Vol and Intensity of the
new synthetic image.
The positions of the spots in the new synthetic gel are based on the positions in
the master image, and therefore the reference image, of the match set. The
resulting synthetic gel normally looks similar to the reference image.
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NOTE! The spots from the reference gel image should be selected as well if
the synthetic gel to be created should take into account the quantification
values of this gel. Therefore, remember to display the reference gel in the
match set by choosing Show > Show Reference, and to select the spots to be
included in the synthetic image. The Master of the match set does not contain
quantification information and will only be used for determining the spot
positions in the new synthetic gel.
9.7.1
Spot filtering
Synthetic gels are created based on selected spots in selected gels. In order to
obtain the cleanest possible synthetic gels, without artifacts or insignificant
spots, you can refine your spot selection using the various filtering options in
ImageMaster. You can perform a type of filtering that consists of selecting, and
possibly eliminating, spots that do not satisfy certain criteria of size, volume, and
so on.
To apply a spot filter:
1
Select all gels to be filtered.
2
Select all spots on the selected gels.
3
Choose Select > Spots > Refine by Value from the menu.
4
You are asked for the value type to be utilized for filtering, and the
thresholds to be employed. Click OK when you finish entering the latter.
5
Only spots meeting the specified conditions are kept.
6
Depending on the applied filter, you can continue to use the current
selection, or on the contrary, delete the selected spots or exclude them
from your analysis (by hiding them, for instance).
A second filter can be applied. You can select matches (that is, the spots in the
matches) that are representative of your population. Spots that are only present
in one of five gels of a match set are not representative of the match set and
should be excluded from the analysis. You can select matches by the number of
spots they contain. For example, select all matches that have at least 4 (or 5)
spots. In other words, you can select spots that are present in at least 4 (or 5) gels.
To apply a match filter:
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1
Make sure the gels have been matched.
2
Select all gels for which a match selection should be refined.
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Select spots or matches (can be the result of a previous spot filter).
4
Choose Select > Matches > Refine Selection from the menu.
5
Enter the threshold for the refinement of matches (see Section 9.3.1). For
instance, you might Keep the Matches whose number of selected
spots is >= 4 (or 5).
6
The remaining matches satisfy the specified criterion. You can obtain all
spots that do not satisfy this criterion by choosing Select > Spots >
Inverse Selection.
NOTE! You should check your results carefully. Detection or matching
problems may cause the exclusion of spots that are actually present in all
gels, or the inclusion of spots that are only present in one or two gels (but
incorrectly matched to other spots). Moreover, when selecting matches, the
program only takes into account the spots that are present in the master gel.
Any spots absent in the Master are not considered, even if they are present in
all other gels.
9.7.2
Creating synthetic gels
Once the spots on your gels have been filtered and only the spots of interest
are selected, you can create a synthetic gel.
NOTE! Synthetic gels can be created from a [MatchSet] or [Classes]
worksheet. When you work in a [Classes] worksheet, the used Master will
correspond to the first common node in the match set structure and the
synthetic gel will automatically be inserted at this level.
To create a synthetic gel:
1
Select the spots and gels to be included in the synthetic gel (see above).
2
Choose Tools > Gels > Create Synthetic Gel.
3
Specify the destination folder and file name in the Create Synthetic Gel
window.
4
The synthetic gel is created (Figure 9-9) and saved, together with the
automatically generated matches (between the synthetic gel and the
master gel).
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Figure 9-9. A synthetic gel (Synthetic gel AT1 AT2) and its six contributing gels. The classes
A_T1 and A_T2 were originally opened in a worksheet to create the synthetic gel. Therefore,
Master_A was used for the matching (first common node in the match set structure, see File
Details) and the synthetic gel was automatically inserted at this level (in match set A).
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10 Data analysis
10.1
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 set), placed in classes,
and opened in a [Classes] worksheet. Please refer to Chapter 4 to learn more
about the workspace and the definition of classes.
Data analysis can be carried out at two different levels. Protein expression
changes within a class of gels are studied with the intra-class statistics tools.
These tools include:
•
Scatter plots, to analyze gel similarities or experimental variations.
•
Descriptive statistics of central tendency and dispersion, which can be
calculated and displayed in Intra-Class Reports and Histograms.
•
Factor analysis, enabling the identification of similar gels, and of spots that
are characteristic for a particular population of gels.
•
Heuristic clustering, an artificial intelligence technique to automatically
classify sets of gels and highlight significant protein spots.
ImageMaster can also help find significant protein expression between classes of
gels. For this purpose, two complementary statistical methods for inter-class
analysis are available:
•
Overlapping measures, which summarize each class of gels as an interval
and measure the overlap between these intervals. The overlapping values
can be displayed in Inter-Class Histograms and Reports.
•
Statistical tests, which assess the difference between populations and give
an estimate for the significance of the difference (that is, how high is the
probability that this difference is only due to chance).
NOTE! The statistical tools in ImageMaster include all the options that the
expert user might expect. Nevertheless, those who are less familiar with the
statistical methods can simply use the default settings. Whenever possible,
this manual indicates how the statistical data can be used and what
precautions should be taken into account. However, keep in mind that the
results are generally only indicative, and should always be checked carefully.
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NOTE! For all data analysis functionalities described in this chapter, you must
choose the spot quantification value on which the calculations should be
based. You can choose between Intensity, Area, Volume, %Intensity, and
%Volume. In addition, the Saliency is available for gels detected with the
ImageMaster algorithm, and the Volume Ratio and Slope for spots detected
with the DeCyder co-detection algorithm. For conventional 2-DE gels, the
%Volume or Volume are generally indicated. For DIGE gels, the Volume Ratio
should be selected in order to use the Internal Standard.
10.2
Intra-class statistics
10.2.1
Scatter plots
In order to analyze gel similarities or experimental variations such as disparities
in stain intensity or sample loading, one can produce Scatter Plots for matched
spots (Figure 10-1). Scatter plots give an idea of the relationship between the spot
values (for example Intensity, Vol and %Vol) from two gels by searching for the
linear dependence between the spot values of one gel (variable X) and the
corresponding values in a reference gel (variable Y).
Select on Gels
Reports
Scatter Plot
Studied spot value type
Regression line
Regression line equation
Correlation coefficient
Number of matches
Figure 10-1. Scatter graphs for matched spots.
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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 extent,
by the values of the other gel) while 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:
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
about 20% higher in the reference gel.
1.0 × x + 0.2
and Corr = 0.95
indicates that almost all spot values are
shifted by +0.2 with respect to the
reference gel.
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 check for possible reasons to
explain why your values are systematically biased. Stain intensity variations,
differences in protein loading or image acquisition problems are examples of
typical causes.
To display scatter plots:
1
Make sure your gels are opened in a [Classes] worksheet.
2
Select a reference gel and the gels for which a scatter plot should be
displayed.
3
Select matched spots to be included in the scatter plots. Generally, all
matched spots are selected.
4
Choose Analyze > Intra-Class > Scatter Plots.
5
Select one of the spot value types (Intensity, Vol, Area, %Intensity, %Vol,
Vol Ratio) to be plotted.
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NOTE! To show the correlation between the spot quantities in the gels, the
Intensity, Vol or %Vol values are most indicated, both for conventional 2-DE
gels and DIGE gels.
The Scatter Plots window shows a scatter plot for each selected gel versus the
specified reference gel, together with the best fit line, correlation coefficient and
the number of matches displayed. If only one gel is selected (other than the
reference gel), a single scatter plot is drawn. This scatter plot is completely
resizable when the Scatter Plots window is in floating mode. You can drag the
corners or borders of the window to make it smaller or larger. Selection of two or
more gels other than the reference gel displays two (or more) scatter plots in one
window. In this case, all scatter plots will have a fixed size and use the same scales
on the X and Y axes.
The scatter plots are interactive. You can click on the points representing the
matched spots and select them on the gels and other open reports. Just select
matched spots by clicking on the corresponding data points while using the Ctrl
or Shift keys for multiple selections. To select all data points in a region, hold down
the mouse button and then drag the cursor to define an area. Use the options
under the Select on Gels icon to identify the corresponding spots on the gels and
open reports. You can also display a new scatter plot showing only the selected
data points by clicking on the Scatter Plot icon.
As usual, you can save and print the graphics using the corresponding icons in
the Scatter Plots window toolbar or copy plots to the clipboard for use in other
applications. Information related to the scatter plots can be found in the Reports
menu icon in the toolbar (Figure 10-1):
•
The Gel Report gives information on the gels used in the scatter plots.
•
The Fitting Report displays the slope and offset values of the regression
line, the correlation coefficient and the number of data points (matched
spots) for each plot.
•
The Scatter Report displays the corresponding spot values in the gels and
the error in relation to the regression line for each pair of matched spots.
This error can be used, for example, to verify abnormalities between
matched spots.
10.2.2
Descriptive statistics
Statistical analyses of the spot values within a class of gels allow you to analyze
variations in protein expression among gels. ImageMaster provides the most
commonly used descriptive statistics of central tendency and dispersion, which
can be calculated and displayed in the Intra-Class Reports and Histograms.
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These descriptive statistics are in fact numbers that summarize the spot values
from a match. The central tendency allows you to localize the central value
representing the data, whereas the dispersion indicates how closely the data
points fall around the center. Based on these measures you can isolate matches
composed of spots whose quantification values are unusual. The detected
variations can be due to an inadequate detection or matching operation, or can
result from protein expression changes among gels. Therefore, these statistics
are not only useful for analyzing extracted data, but also for verifying process.
The descriptive statistics can be chosen with the following interface (Figure 10-2):
Figure 10-2. Statistics common to the Intra-Class Report, Intra-Class Histograms, InterClass Report and Inter-Class Histograms.
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 (or midrange) values are very sensitive to extreme values (outliers) and can
be seriously contaminated by a single observation. On the other hand, the
median is highly resistant to outliers. A compromise between mean (or midrange)
and median is given by the trimmed mean (or 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.
All of the above central tendencies can be obtained through the selections and
the percentage slider available in the dialog box. The slider allows you to remove
outliers. A 100% value means that all the spot values available in a match will be
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. Therefore:
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•
by just selecting Mean, the arithmetic mean is calculated, that is, the sum of
all the sample values divided by the sample size.
•
by just selecting Midrange, the midpoint of the sample value is calculated,
that is, the middle location between the two sample extremes.
•
to obtain a Trimmed Mean (or Midrange), you should select Mean (or
Midrange) and discard the desired percentage of outliers with the
percentage slider.
•
to obtain the Median, you should select Mean and discard 50% of the values
at each of the extremities, that is, select 0% in the percentage slider.
•
by selecting Reference..., the value from the spot in a specified reference gel
is taken as the central tendency.
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 many others.
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.
•
by selecting Mean Squared Deviation (M.S.D.), the square root of the
average squared difference of each sample value to the center location is
calculated.
•
by selecting Mean Absolute Deviation (M.A.D.), 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 since the
differences are not squared.
•
by selecting Half-range Size, the difference between the largest and the
smallest values divided by two is calculated.
NOTE! Notice that absent spots, represented by zero values, are also
considered in the calculation of statistics (for both central tendencies and
dispersion).
Examples
• The Mean 100% and the Mean Squared Deviation 100% are the most
commonly used statistics (Figure 10-3, a). Notice that the standard deviation
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is the Mean Squared Deviation multiplied by N, where N is the sample size.
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 10-3, b).
•
The Midrange 100% and Half-range 100% define an interval that includes
all sample values (Figure 10-3, c).
•
The Midrange 50% and Half-range 50% are known as order statistics and
interquartile ranges (Figure 10-4).
(a)
(b)
(c)
Figure 10-3. 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 10-4. Histograms showing the effect of suppressing outliers. Midrange and halfrange values are given for (a) 100%, (b) 80%, (c) 50% and (d) 33%.
10.2.3
Intra-Class Histograms
Intra-Class Histograms and Reports provide valuable tools for looking at unusual
matches within a class. Analyzing protein expression changes, checking spot
detection or verifying matching operations are just a few of the numerous
potential usages. One can also compare spots visually or even navigate through
matches. At this point, it should be noted that Intra-Class Histograms and Reports
are dual elements; they have a reciprocal relationship. They are not only
complementary tools to interpret the information, but are also designed to
effortlessly switch between them. Starting from an Intra-Class Report you can
easily display the corresponding histograms and vice versa. Histograms are a
more visual way to look at matched spots.
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To display Intra-Class Histograms:
1
Select the matches to be studied (on the gels or on an Intra-Class
Report).
2
Select the gels to be included in the histograms. The gels can belong to
different classes, but the class information will be ignored for intra-class
statistics.
3
Choose Analyze > Intra-Class > Histograms.
4
In the pop-up list, select the value type (Intensity, Area, Vol, … ) to be
displayed.
5
Choose the desired statistics (central tendency and dispersion values,
see Section 10.2.2) in the subsequent dialog box.
Figure 10-5 shows histograms for some selected matches. The sliders at the
bottom and the right side of the window allow you to see other matches. If many
matches are selected, you may have to use the little page slider at the bottom
right corner to see the matches displayed on additional pages.
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 delimit
the range defined by [Central value - Dispersion, Central value + Dispersion].
The Match ID displayed at the bottom of each histogram allows you to keep track
of the matches you are analyzing.
The histograms can of course be selected as with any other ImageMaster object
and saved, printed or exported to other software (by using the corresponding
icons). To select a histogram, simply click on it. To select many histograms, use
the Shift or Ctrl keys.
After having selected the corresponding histograms, you can also re-select
matches on the gels by using the Select on Gels icon. Navigating through the
matches is accomplished using the Select Next and Select Previous buttons. Other
selection possibilities (for example, Select on Gels + Reports, Select from Gels) are
associated with the Select on Gels icon. Please see Chapter 5 for their detailed
description.
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Sort by similarity Displayed value
Range extremes
Central tendency
Gel index
Match ID
Page slider
Figure 10-5. Intra-Class Histograms.
Additional information on the gels and matches is available from the Reports drop
down menu in the toolbar (Figure 10-5):
•
The Gel Report is a legend for the gel index on the histograms.
•
The Intra-Class Report item displays the numerical values corresponding to
selected histograms.
•
The Report from Selection function creates a new histogram window that
only contains the histograms that are selected in the active window.
NOTE! You can use the Intra-Class Report to change the order of your
histograms (sort your data in the report and then re-display the
corresponding histograms) or to refine your histogram selection (see Section
10.2.4).
In addition to the functions explained above, ImageMaster provides the following
options in the histograms window to enhance the visual comparison of protein
expression changes within matched gels.
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Normalization
By default, the histograms display the raw spot values. Nevertheless, to simplify
comparisons across matches, it can be convenient to normalize the spot values
relative to their intra-class statistics. The following normalizations can be chosen
from the Displayed Value list (Figure 10-5):
Value
Raw spot value.
Relative
Spot value – Central tendency
This normalization places the central tendency values to 0, and
if the Adaptive Gradations option (see below) 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 below), 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.
Normalized
Spot value – Central tendency-----------------------------------------------------------------------------Dispersion
This normalization is a compromise between the two
normalizations described above and is sensitive to all values.
Adapt gradations
To highlight spot variations inside a match, the histogram gradations are adjusted
according to the spot values in each match. However, in order to display an
identical gradation in all histograms, you can deselect the Adaptive Gradations
check box at the bottom of the Histograms window (Figure 10-6).
(a)
(b)
Figure 10-6. Histograms on matches with (a) adaptive gradations set individually for each
histogram and (b) set according to the minimum and maximum values in all histograms.
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Sort values
Additionally, to highlight and classify spot variations, you can display the spot
values sorted in ascending order by clicking in the Sorted Values check box at the
bottom of the Histograms window (Figure 10-7).
(a)
(b)
Figure 10-7. Histograms on matches with (a) unsorted values and (b) with values sorted in
ascending order.
Sort by similarity
ImageMaster provides an easy way to look for similar histograms in the IntraClass Histograms window. Select one histogram and click on the Sort by Similarity
icon in the toolbar (Figure 10-5). A new histogram window is displayed, in which
the selected histogram is depicted in the first position, and the other histograms
follow according to a similarity criterion. This similarity criterion is based on the
sum of the squared spot value differences for each gel G. The general formula is:
N
2
Similarity = ∑ ( v i – v j )
G=1
Where vi is the spot value for match i, vj is the spot value for match j, and N is the
number of gels displayed in the histogram. The result generally is that histograms
with similar shapes appear together (Figure 10-8). It must be noted that, when
using raw spot values, very abundant spots predominantly influence the
similarity criterion. To prevent this, it is better to use the Sort by Similarity option
in combination with normalized spot values (see above).
Figure 10-8. Histograms displayed according to their shape similarity.
10.2.4
Intra-Class Report
Similar to the Intra-Class Histograms, the Intra-Class Report describes numerical
data from matches (i.e. their matched spots), as well as their corresponding
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statistical values. In addition to the central tendencies and dispersion quantities
described earlier (Section 10.2.2), other measures for the analysis of matched
spots within a class of gels are available in the Intra-Class Report (Figure 10-9):
Figure 10-9. Additional values that can be added to the Intra-Class Report.
•
The Coefficient of Variation (Coef. Variation) 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 is the maximum value divided by the smallest value in the
sample specified by the percentage slider (used to suppress outliers from
the calculated statistics, see Section 10.2.2).
•
The Separability 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.
To display an Intra-Class Report:
1
Select a given number of matches (possibly all) to be studied.
2
Select the gels to be included in the histograms. The gels can belong to
different classes, but the class information will be ignored for intra-class
statistics.
3
Choose Analyze > Intra-Class > Report.
4
In the pop-up list, select the value type (Intensity, Area, Vol, … ) to be
displayed.
5
Choose the desired statistics (central tendency and dispersion values,
see Section 10.2.2) and the additional values to be calculated in the
subsequent dialog box.
Figure 10-10 shows a typical Intra-Class Report. The sliders at the bottom and
right side of the window allow you to see the rest of the table. The Match ID is
followed by the desired statistical values, the coefficient of variation (optional), the
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range ratio (optional), the separability (optional), and finally the spot values from
each gel. These spot values can be normalized as described for Section 10.2.3, in
order to simplify the comparison between matches. To do this, just select the
desired type of normalization (Value, Relative, Ratio or Normalized) in the
Displayed value list (Figure 10-10).
A number of standard functionalities are available from the Intra-Class Report
toolbar (see Chapter 5 for details). Selected lines (use Shift or Ctrl keys for multiple
selections) can be saved, printed or exported to other software. You can view your
data on gels or related reports using the selection and navigation buttons. The
Intra-Class Report supports the display and editing of annotation content, and
therefore offers the Annotate and Update Gel functions. These functions enable
you to add new categories and to propagate label modifications to your master
gels. Finally, you can customize the content of the Intra-Class Report by using the
Settings option.
Factor Analysis
Histograms
Reports
Update Gel
Settings
Annotate
Displayed value
Figure 10-10. Intra-Class Report.
By clicking on the Histograms icon in the toolbar (Figure 10-10), you can display
the histograms of matches that were selected in the report. Please note that the
order of the histograms in the resulting Intra-Class Histograms window will be the
same as that of the selected matches in the Intra-Class Report. You can take
advantage of this characteristic to organize your histograms in a certain way.
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The Reports icon in the toolbar (Figure 10-10) allows you to display reports related
to the matched spots:
•
The Gel Report item is a reduced version of the original report on gels that
can be used, for example, as a legend for the histograms.
•
The Spot Report is a modified report on spots that is useful to compare one
spot against the others in the same match (see the possible applications in
the paragraph below).
•
The Report from Selection function creates a new report that only contains
the matches that are selected in the active report.
Spot report
In this report, the measures available for quantifying the spot values relative to
their match are the same as above (Value, Relative, Ratio or Normalized). You can
use these values to select spots that are outliers, or on the contrary, to select
spots that are representative of a match:
Representative spots
• In order to select the most representative spots of a match, one could, for
example, pick out the spots whose values are close to the mean (with less
than 20% difference). To do this, choose the Mean 100% as central
tendency, and then select all spots that have a Ratio (Displayed value list)
between 0.8 and 1.2.
•
Another possibility is to choose spots with a Median (Statistics) Ratio
(Displayed value list) of 1, which will select the spots that are in the middle of
the match.
Outliers
• Outliers can be selected by choosing values that are outside the range given
by the mean ± two times the standard deviation. In fact, with a normal
distribution, at most 5% of the values should be outside this interval. In
practice, you can find such outliers by using the Mean (100%) and Mean
Squared Deviation (100%), and then choosing all spots with a Normalized
value (Displayed value list) higher than 2.
208
•
When you calculate the median, and choose Ratio values (Displayed value
list) larger than 2, you select spots that are at least 2 times higher or lower
than half of the spots from the match.
•
The Midrange (100%) and Half-range (100%) statistics, combined with a
Normalized value (Displayed value list) of 1, allow you to select spots that
are the highest or the lowest in their match.
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The Midrange (50%) and Half-range (50%), combined with a Normalized
value (Displayed value list) superior to 2, will select spots that are at least 2
times higher or lower than 75% of the spots from the match.
NOTE! In order to restrict the number of selected matches, you can remove
the matches that contain too many outliers. You can also use this
functionality to search for unusual gels. If a gel contains more outliers than
the others, it may be atypical or abnormal. You should determine the reasons
for this abnormality before continuing the analysis.
10.2.5
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. In particular, it can be hard to
assess whether different sample populations exist and to characterize their
different protein expression profiles. Factor analysis helps here, by finding a way
to condense 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, please see
Appendix C for references. In the following paragraphs, you learn how to perform
a factor analysis and how to interpret the results. Your attention is also directed
to some critical points of this analysis.
To carry out a factor analysis:
1
Click the Factor Analysis icon in the Intra-Class Report toolbar (Figure
10-10).
2
If any matches are selected, the program asks whether you want to use
only the selected rows or not. Please see further on in this section to
judge which of the options is most applicable.
3
The program displays a Factor Analysis Report with the lines
corresponding to the axes that can be drawn in the Factor Projection
Plot. Select 2 axes to be displayed in the plot (the first two ones,
generally).
4
Click the Plot Projection icon in the Factor Analysis Report toolbar.
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5
A dialog box lets you choose the number of matches to be displayed.
Please note that the selected number does not influence the factor
analysis results. It just improves the clarity of the plot by reducing the
number of visible matches.
6
The Factor Projection Plot is displayed. It is completely resizable when in
floating mode. You can drag the corners or borders of the window to
make it smaller or larger.
Interpreting a factor analysis
The following explanations about how to interpret a factor analysis are illustrated
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, matched and added
to a class. The class was opened in a [Classes] worksheet, all the gels and
matches were selected, and an Intra-Class Report was displayed.
The Factor Analysis Report (Figure 10-11) was obtained by clicking on the Factor
Analysis icon in the Intra-Class Report toolbar. It summarizes the variance
accounted for by successive axes (or factors), expressed as a percent of the total
variance. Thus, factor 1 accounts for 83.7% of the variance, factor 2 for 7.5%, and
so on. The report also lists the coordinates for each gel projected on these axes.
Figure 10-11. Factor Analysis Report.
The number of factors equals the number of gels being analyzed. Factor analysis
cannot be performed with less than two gels and so at least two factors are
always calculated. Of course, the more gels you use, the more reliable the results
are likely to be, and the more factors will be calculated. Since the first factors are
generally far more important for characterizing gels and matches than any
subsequent ones, the factors are ranked in order of importance.
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Figure 10-12. Factor Projection Plot. Note that the options in the lower left corner allow you
to display only matches or only gels. The blue curve represents a part of the correlation
circle; its form is linked to the scale of the axes. Points corresponding to matches can be
selected on the plot (in green) and highlighted on the gels or any open reports.
In the current context of gel image analysis, it has been noted that the first two
axes are usually the most significant ones for finding gels and matches that
behave similarly. Figure 10-12 shows the Factor Projection Plot obtained when
the first two axes in Figure 10-11 were selected. This plot displays the projection
of each match (red or green cross) and each gel (blue vector) on the two factorial
axes. In the present example, only the 20 most significant matches are displayed
on the projection plot. 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, i.e. protein spots, are not significant in classifying the gels. The further
away a spot is from the origin, the more important it is likely to be in terms of
characterizing the gels.
To find a possible meaning for a given factor, one should first identify the matches
that largely contribute to this factor. The Factor Projection Report (Figure 10-13, a),
available through the Reports item in the Factor Projection Plot, is used for this
purpose. When sorting the matches in this report according to their contribution
to the first axis, one discovers that matches 4825, 4715, 4704, 4898 and 4784
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appear at the top of the table. Interestingly, these matches are those with the
highest relative volumes, as found from the Intra-Class Report that is accessible
in the Factor Projection Report toolbar. In fact, the first axis is generally correlated
with protein abundance.
(a)
(b)
Figure 10-13. Factor Projection Report with (a) matches ranked based on their contribution
to the first axis and (b) ranked based on their contribution to the second axis.
Please note that the Factor Projection Report also contains a Quality measure for
each match. This number gives you an appreciation of whether a match is well
represented on the factorial subspace. It tells you how close the distance of the
projection is to reality. Indeed, matches with very similar behavior (similar
expression profiles across gels) will be close in space. However, when projected
onto a two-dimensional subspace, matches that are actually far apart may
appear together. It is therefore important to look at the Quality measures to judge
whether matches are effectively close. If the values are high for both matches, the
chance is great that they are indeed nearby and have a similar behavior. If one of
the matches has a low value, any interpretation becomes tentative.
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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 10-12, for instance, one can observe
that the matches 4844, 4903, 4691, 4767 and 4839 are close to the gels belonging
to T1. The histograms in Figure 10-14 show that these matches have high spot
values for the T1 gels and low values for the T2 gels. Match 4704, on the other
hand, is 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 populations. 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.
The Factor Projection Plot is interactive. You can click on the points representing
the matches and select them on the gels and other open reports. Just select
matches by clicking on the corresponding points while using the Ctrl or Shift keys
for multiple selections. To select all matches in a region, hold down the mouse
button and then drag the cursor to define an area. Use the options in the Select
on Gels drop down menu to identify the corresponding spots on the gels and open
reports. You can also display a new factor projection plot showing only the
selected matches by clicking on the Plot Projection icon.
You can save and print the graphics using the corresponding icons in the Factor
Projection Plot window toolbar or copy the plot to the clipboard for use in other
applications. Information related to the Factor Projection Plot can be found in the
Reports drop down menu in the toolbar:
•
The Factor Analysis Report summarizes the variance accounted for by the
successive axes, expressed as a percent of the total variance. The report
also lists the coordinates for each gel projected on these axes (Figure 10-11).
•
The Projection Report displays the contribution of each match to the two
axes displayed in the corresponding Factor Projection Plot, as well as a
Quality measure that gives you an appreciation of whether the projection of
the match is well represented on the factorial subspace (Figure 10-13).
•
The Intra-Class Report for the selected matches, shows the individual
quantification values for each spot in the match and allows to display
related Intra-Class Histograms (Section 10.2.3).
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Comments on factor analysis
Factor analysis is used to examine the protein expression pattern within each
match. The quality of the factor analysis 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 the Select > Matches > Refine Selection function from the
menu, as described in Section 9.3.1). 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 classes among the gels and allows
identification of matches (i.e. matched spots) that are characteristic of these
classes. However, one should be very critical when analyzing factor analysis
plots, as 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.
Figure 10-14. Intra-Class Histograms ranked based on their contribution to the second axis.
Matches 4844, 4903, 4691, 4767 and 4839 are characteristic for population T1, match 4704
is characteristic for population T2. The gel legends are given in the corresponding Gel
Report.
10.2.6
Heuristic clustering
ImageMaster proposes a powerful analysis method to automatically create
classes of gels and highlight significant protein spots. Heuristic clustering, a
machine learning algorithm based on artificial intelligence, is used to describe the
characteristic spots of 2-D gels while using heuristics to speed up the search. The
algorithm provides the possibility to blindly classify similar gels into two or more
classes and to determine the characteristic spots (i.e. matches) of each class.
These characteristic spots often correspond to differently expressed proteins.
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The number of classes that are formed is dependent on the user input.
ImageMaster first asks for the minimum and maximum number of classes, for
example 2 and 4, respectively. It then splits the gels in 2, 3 and 4 classes,
compares the resulting classifications and, according to a similarity function,
shows the best one.
The classification step can also be repeated for each of the classes in order to find
subclasses. In this way, a descending hierarchical classification tree is produced.
The depth of classification, or the number of sublevels, is established by the
Classification Level parameter that must be defined by the user.
The number of characteristic spots is determined by the Sensitivity parameter.
This defines the gap between two classes (the difference between the highest
spot value in one class and the lowest value in a second class, Figure 10-15). The
smaller this parameter, the smaller the accepted difference between the class
intervals in a match. Additionally, the number of spots considered in the
classification is higher.
Sensitivity
Interval Class 1
Match
min
max
Interval Class 2
min
max
Figure 10-15. Sensitivity parameter.
To search for classes with the heuristic clustering algorithm:
1
Select the matches (possibly all) to be considered in the classification.
2
Select the gels to be classified. The gels can belong to different classes,
but the class information will be ignored for the heuristic clustering.
3
Choose Analyze > Heuristic Clustering > Do Clustering.
4
In the pop-up list, select the value type (Intensity, Area, Vol, … ) to be
considered.
5
In the subsequent dialog box, define the Classification Level, the
Minimum and Maximum number of classes, and the Sensitivity
parameter.
Depending on your computer capacity, this could take time, especially when the
maximum number of classes is high, and many matches and gels were selected.
When the heuristic clustering process is finished, ImageMaster automatically
creates default classes in the workspace and opens them in a new [Classes]
worksheet. The matches that were found to be characteristic for one of the
classes are shown in an Inter-Class Histograms window. You can learn more
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about Inter-Class Histograms in Section 10.3.3. They display for each class
formed, the central tendency (blue horizontal line) and the dispersion interval (red
vertical line). To find out which gels compose each class (designated with a letter),
you can select the Gel Report item from the Reports icon.
NOTE! The matches that stay selected on the gels after the heuristic
clustering analysis are those that were considered significant in the analysis
(and are therefore present in the Inter-Class Histograms).
Plot
The resulting classification can be visualized in a dendrogram, which represents
the classes and their gels in a descending classification tree (Figure 10-16). To plot
such a dendrogram, choose Analyze > Heuristic Clustering > Plot.
By using the icons from the toolbar in the Heuristic Clustering Plot window, the
dendrogram image can be saved (in .png, .bmp or .tif format), printed, or copied
to the clipboard for use in other applications.
10.3
Inter-class statistics
10.3.1
Specifying classes
To identify protein expression variations between populations of gels, you need
to specify what gels belong to which population by creating classes. There are
several possibilities for doing this. The first one requires that you already know the
populations in your set of gels. This is the case, for example, when you are
comparing gels from healthy tissue extracts with those from disease-associated
samples. You can then define the classes yourself. Classes are created in the
Workspace (see Chapter 4).
When you have no preliminary knowledge of the populations in the set of gels,
you can implement a heuristic clustering analysis (see Section 10.2.6) and
possibly draw conclusions from a factor analysis study (see Section 10.2.5). When
doing heuristic clustering, classes are automatically defined, and you can
analyze them with the tools described below.
10.3.2
Overlapping measures
As for the intra-class analysis, the spot values of each protein class can be
summarized by two statistical descriptors, which are the central tendency and
the dispersion (see Section 10.2.2). In addition to these descriptors, ImageMaster
computes overlapping measures between the class intervals, which are defined
by the ranges [Central value - Dispersion, Central value + Dispersion]. These
ranges quantify the overlap between the spot values from two classes, and thus
gauge how different the protein expression changes between the classes really
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are. These measures of overlap take into account both the difference between the
central tendency in each population and the dispersion.
Figure 10-16. Dendrogram (with two classification levels) representing the classes found
and their corresponding gels. In the example, gels were run on samples from bacteria
grown on two different substrates (A and B), and for which two different treatments (T1 and
T2) were tested. The substrate populations were found by the clustering algorithm. But the
different treatments were not correctly recognized through heuristic clustering (each time
a gel from T1 has been clustered with the gels from T2). This could be due to the way the
match sets were defined though.
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In the Inter-Class Histograms and Reports, the displayed statistical descriptor or
overlapping measure needs to be selected from the Displayed value list in the
toolbar:
•
Center: Raw central tendency of the current class.
•
Dispersion: Raw dispersion value of the current class.
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 (for the calculation of the difference or ratio between central
values, for example) set the dispersion percentage slider to 0%.
218
•
Gap: Maximum difference between the range of the current class and the
range of one of the other classes (in the example of Figure 10-17, c-b in the
case of Class A). Negative values indicate overlapping intervals whereas
positive values are non-overlapping class ranges.
•
Ratio: Maximum ratio between the lower limit of one of the other classes
and the upper limit of the current class (in the example of Figure 10-17, 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 (in the example of
Figure 10-17, (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 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.
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0
2
6
8
1.5
Class A a
b
c
2
Class B
Gap:
Ratio:
Normalized:
0
OVERLAPPING CLASSES
4
10
2
d
Class A
Class B
2.0 [c-b]
1.4 [c/b]
1.5 [(c-a)/(b-a)]
2.0
1.4
2.0
4
6
8
0.75
Class A a
b
c
Class B
Gap:
Ratio:
Normalized:
d
0.5
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 10-17. 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.
Please note that the above mentioned formulas only apply to Class A for these
particular examples. Their presence in this manual is only to illustrate the
principles of the overlapping measures used in ImageMaster. Many different
cases (and therefore formulas) exist.
Observe that in the Inter-Class Histograms and Reports the number 1000000
characterizes the cases where the protein is completely absent from a class (in
this case ImageMaster cannot compute ranges). A value of 0 for the Ratio or
Normalized measures indicates that the particular class is entirely covered by
another one.
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NOTE! For non-expert users, it is recommended to compare only two classes
at a time in the Inter-Class Report (see Section 10.3.4). This is because only in
this case one gets the direct relationships (difference, ratio or percentage
overlap) between the two classes. As indicated in their definition, the Gap,
Ratio and Normalized values always calculate the MAXIMUM difference, ratio
or percentage, respectively, with respect to ANY of the other classes. This
means that when the Ratio values for three classes (e.g. A, B, C) are compared,
for instance, the software calculates the ratio of A with respect to B and of A
with respect 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 (i.e. protein
marker) that distinguishes the current class from any of the other classes.
Once such protein markers are found, the complementary tools in the
software, such as the Inter-Class Histograms (see Section 10.3.3), can be used
to study the match in more detail.
10.3.3
Inter-Class Histograms
As with intra-class statistics, you can visually investigate the statistical and
overlapping descriptors of classes by displaying Inter-Class Histograms.
To display Inter-Class Histograms:
1
Select the matches to be studied (on the gels or on a report).
2
Select the gels to be included in the analysis.
3
Choose Analyze > Inter-Class > Histograms.
4
In the pop-up list, select the value type (Intensity, Area, Vol, … ) to be
displayed.
5
Choose the desired statistics (central tendency and dispersion values,
see Section 10.2.2) in the subsequent dialog box.
By default, the displayed histograms (Figure 10-18) show the raw central
tendency (blue horizontal line) and the dispersion interval (red vertical line) for
each class. Visualizing the various overlapping measures is possible by changing
the Displayed value in the list at the top of the Inter-Class Histograms window.
The Inter-Class Histograms can be selected as with any other ImageMaster
object and saved, printed or exported to other software using the icons in the
toolbar (Figure 10-18). You can also re-select the corresponding matches on the
gels by using the Select on Gels drop down menu, and systematically navigate
through matches with the Select Next and Select Previous icons. Finally, you can
display more complete Inter-Class + Intra-Class Histograms (see below) for
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selected histograms. These mixed histograms display the individual spot values
in addition to the statistical descriptors for each class.
Inter-Class+Intra-Class Histograms
Reports
Displayed value
Dispersion interval
Central tendency
Class index
Match ID
Figure 10-18. Inter-Class Histograms
The Reports drop down menu in the toolbar (Figure 10-18) allows you to display
information related to the Inter-Class Histograms:
•
The Gel Report item is a reduced version of the original report on gels to be
used, for example, as a legend to the classes.
•
The Inter-Class Report (see Section 10.3.4) lists the numerical values
corresponding to the selected histograms.
•
The Intra-Class Report displays a report on selected matches for a selected
class. It is a reduced Intra-Class Report, where only the spot values of one
class are considered. The information displayed is that which is usually
found in an Intra-Class Report (see Section 10.2.4).
•
The Report from Selection function creates a new report that only contains
the lines that are selected in the active report.
NOTE! Analogous to the Intra-Class Histograms and Reports, the Inter-Class
Histograms and Reports are dual elements; they have a reciprocal
relationship. You can therefore use the Inter-Class Report to change the order
of your histograms (sort your data in the report and then re-display the
corresponding histograms) or to refine your histogram selection.
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Adapt gradations
By default, the histogram gradations are adjusted according to the spot values in
each match. However, in order to display an identical gradation in all histograms,
you can deselect the Adaptive Gradations check box at the bottom of the
histograms window (Figure 10-19).
(a)
(b)
Figure 10-19. Inter-Class Histograms with (a) adaptive gradations set individually for each
histogram and (b) set according to the minimum and maximum values in all histograms.
Sort values
To simplify the visual search for non-overlapping intervals, you may want to
classify the displayed class values in ascending order, by clicking in the Sorted
values check box at the bottom of the histograms window (Figure 10-20).
(a)
(b)
Figure 10-20. Inter-Class Histograms with (a) unsorted values and (b) values sorted in
ascending order.
Inter-Class + Intra-Class Histograms
ImageMaster can draw more detailed Inter-Class Histograms for selected
matches. These histograms not only display the class intervals but also show the
individual spot values in each class. They can therefore be considered as a kind
of mixed representation with properties from both the Intra-Class and Inter-Class
Histograms.
To display Inter-Class + Intra-Class Histograms:
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1
Select the matches to be studied (on the gels or on a report).
2
Select the gels to be included in the analysis.
3
Choose Analyze > Inter-Class > Histograms.
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In the pop-up list, select the value type (Intensity, Area, Vol, … ) to be
displayed.
5
Choose the desired statistics (central tendency and dispersion values,
see Section 10.2.2) in the subsequent dialog box.
6
In the toolbar of the displayed Inter-Class Histograms window, click on
the Inter-Class+Intra-Class Histograms icon.
10
Reports
Dispersion interval
Central tendency
Spot values
Class separation
Figure 10-21. Inter-Class + Intra-Class Histograms.
As shown in Figure 10-21, the Inter-Class + Intra-Class Histograms display all the
individual spot values in each match, separated for each class by vertical gray
lines. The classes are characterized by their central tendency (blue horizontal line)
and dispersion interval (bounded by the outer red lines).
The information, icons and functions enclosed in these specific histograms are
similar to those described in the Intra-Class Histograms (Section 10.2.3) and InterClass Histograms (Section 10.3.3). The only point to be emphasized here is that
selecting the Sorted Values check box at the bottom of the Inter-Class + IntraClass Histograms window not only implies the sorting of the classes according to
their central value but also the sorting of the spot values inside each class (Figure
10-22).
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(a)
(b)
Figure 10-22. Inter-Class + Intra-Class Histograms with (a) unsorted values and (b) values
sorted in ascending order. The classes are sorted according to their central value, and the
spot values within each class are also sorted.
10.3.4
Inter-Class Report
ImageMaster allows you to generate specific reports in which you can
numerically display one of the statistical descriptors or overlapping measures for
each match and class, as well as the maximum value among all the classes.
To produce an Inter-Class Report:
1
Select the matches to be studied (on the gels or on a report).
2
Select the gels to be included in the analysis.
3
Choose Analyze > Inter-Class > Report.
4
In the pop-up list, select the value type (Intensity, Area, Vol, … ) to be
displayed.
5
Choose the desired statistics (central tendency and dispersion values,
see Section 10.2.2) in the subsequent dialog box.
The Inter-Class Report (Figure 10-23) displays the descriptor selected from the
Displayed value list for each class and gives the maximum value among all the
classes (Max Column). It is thus possible to differentiate one class from the others
according to a match.
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Histograms
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Displayed value
Figure 10-23. Inter-Class Report.
As usual, the icons in the Inter-Class Report toolbar allow you to save, print or
export your report, and navigate through the data on the gels. The Settings icon
is used to customize your table display (for example, show the annotations in the
master gel for all matches listed in your table). You can also create additional label
categories in the master gel and update any modifications made to labels by
using the Annotate and Update Gel buttons. The items in the Histograms icon are
used to produce an Inter-Class Histogram or an Inter-Class + Intra-Class
Histogram. Finally, you can generate related reports with the Reports icon:
•
The Gel Report item can be used as a legend for the classes and their gels.
•
The Intra-Class Report displays an Intra-Class Report (see Section 10.2.4)
on the selected matches for a selected class.
•
The Report from Selection function creates a new report that only contains
the lines that are selected in the active report.
10.3.5
Statistical tests
ImageMaster provides three statistical tests: two-sample t test, 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 whether the data
were sampled from populations with different means or whether the populations
have the same mean with the observed difference being a coincidence of random
sampling.
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In fact, there is no way to definitely conclude which of the two possibilities is true.
All you can do is 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! ImageMaster provides qualitative indications about different protein
expressions. In order to state the results as probabilities, the data points
(protein values in the case of ImageMaster) must respect the restrictive
assumptions made by the various statistical tests (see below). If an
assumption is not met even approximately, the significance levels and the
power of the test are invalidated. Matters are made even worse when several
assumptions are violated. Since this is often the case in studies of protein
expression, ImageMaster only displays the numerical values for each test
(and not the probabilities) and lets the user search in the appropriate tables
for the corresponding significance levels. If the appropriate test was chosen
and the assumptions were met, the probabilities found can be used and the
results accepted. If this is not the case, the numerical values given by
ImageMaster still allow you to classify the differences between the sample
means according to their relative significance.
Two-sample t test
The two-sample t test is generally used to determine whether the mean (or
median) of a variable differs between two populations. It is based on the following
assumptions:
•
The data are continuous (not discrete).
•
The data follow the normal (Gaussian bell-shaped) probability distribution.
•
The variances of the two populations are similar.
•
The two samples are independent. There is no relationship between the
individuals in one sample as compared to the other (as there is in a paired t
test).
The t test statistic is based on the difference between the mean values ( X ) of the
two classes, normalized by the standard deviation (s). For this test, the number of
degrees of freedom equals the total sample size (n1 + n2) minus 2. The t ratio is
calculated as follows:
( X1 – X2 ) n1 + n2 – 2
t = -------------------------------------------------------------2
2
1
1
⎛ ----⎞
+ ----- ( n 1 s 1 + n 2 s 2 )
⎝n
n ⎠
1
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If the t ratio is larger than a certain threshold, chosen according to a confidence
level and the sample size, you can conclude that the difference between the two
populations is statistically significant.
NOTE! Only the t ratio is given, and not the probability p of not being wrong
when concluding that the two populations have significantly different means.
This is for reasons of consistency and to make the user more aware of the
implications of his or her analysis. In numerous cases, the restrictive
assumptions are not met and the statistics will not be valid. Nevertheless, this
in no way restricts the utility of the t test results, notably for classifying the
spot differences by their potential significance. You should always check your
hypotheses by visual inspection of the spots since results may be due to
artifacts in the spot detection and/or matching steps.
If the data for the samples to be analyzed come from populations whose
distribution violate the assumption of normality, then nonparametric tests like the
Mann-Whitney or Kolmogorov-Smirnov tests can provide a better analysis.
Mann-Whitney or Wilcoxon test
As indicated above, the Mann-Whitney U test or rank sum test is the
nonparametric substitute for the two-sample t test when the assumption of
normality is not valid. It is equivalent to the Wilcoxon rank sum test. Once again,
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, ImageMaster 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 MannWhitney test statistic, U. The formula for UX is as follows (The formula for UY is
obtained by replacing X by Y):
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nX ( nX + 1 )
U X = W X – -------------------------2
In fact, 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 10-24, 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 (Figure 10-24).
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 10-24. Two examples to illustrate the Mann-Whitney and Kolmogorov-Smirnov
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.
In fact, ImageMaster displays the smaller of the two calculated U values in the
Statistical Tests report. The lower this 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.
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Caution should be used when analyzing the results of a Mann-Whitney test. First,
the assumptions are often violated. This is the case, for example, when spots are
completely absent in one of the classes (in that case you have repeated values of
0). Moreover, if you have small samples, the Mann-Whitney test is meaningless. In
fact, 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.
Kolmogorov-Smirnov test
The Kolmogorov-Smirnov test tries to determine if two data sets differ
significantly. In other words, it is used to test whether or not two samples may
reasonably be assumed to come from the same distribution. It has the advantage
of not making an 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). Note however, that this generally comes at a price. 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 Kolmogorov-Smirnov 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. In the second example from Figure 10-24, 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 10-25):
It is clear that no data lie strictly below 0.034, 17% = 0.17 = 1/6 of the data is
strictly smaller than 0.045, 33% = 0.33 = 2/6 of the data is strictly smaller than
0.056, 50% = 0.50 = 3/6 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 10-25). The Kolmogorov-Smirnov test statistic
D is then defined as the maximum distance between the empirical distribution
functions (EDF) of the two samples. In the example cited here, D is 0.63 (0.83-0.20).
If D is greater than a particular decision limit (critical value found in a KolmogorovSmirnov table), there is a statistically significant difference between the two
samples. However, the test provides no insight as to what causes the difference.
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Fraction of data points to the left of
Spot value z
Empirical Distribution Plot
1.00
0.80
0.60
D
0.40
Class X
0.20
Class Y
0.00
0.02
0.04
0.06
0.08
0.1
0.12
0.14
Spot value z
Figure 10-25. The empirical distribution plot for the spot values of match 613 (Figure 10-24),
in Classes X and Y. The Kolmogorov-Smirnov statistic D corresponds to the maximum
distance between the two empirical distribution functions.
Statistical tests report
The Statistical Tests report (Figure 10-26) contains, for each selected match, the
desired statistical values, which quantify the differences between the means of
two classes. These values should be considered as qualitative indications of the
variations in protein expression between two populations, and only rarely (in ideal
cases) should be used to calculate probabilities and draw quantitative
conclusions. In addition, one should always check the results by visual inspection
of the spots, since the conclusions may be erroneous due to inaccuracies in the
detection or matching steps.
To obtain a report of statistical test results:
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1
Select the matches to be studied (on the gels or on a report).
2
Select the gels to be included in the analysis.
3
Choose Analyze > Inter-Class > Statistical Tests.
4
In the pop-up list, select the value type (Intensity, Area, Vol, … ) to be
displayed.
5
Choose one or more of the statistical tests for which you would like to
see the results displayed.
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Histograms
Figure 10-26. Statistical tests report.
The icons in the Statistical Tests report toolbar allow you to save, print or export
your report, and navigate through the data on the gels. The Histograms icon is
used to produce Inter-Class Histograms. Related reports can be selected from the
Reports drop down menu:
•
The Gel Report item can be used as a legend for the classes and their gels.
•
The Report from Selection function creates a new report that only contains
the lines that are selected in the active report.
NOTE! In order to focus your analysis on the most significant spot differences
between classes, you can sort the values in the report by clicking on the
column headers. This way, you can re-select the match that meet your own
criteria of statistical value.
NOTE! The given statistical values are useless when the samples (classes) do
not consist of more than two gels. In any event, your objective should always
be to work with the largest possible sample sizes.
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11.1
Converting into ImageMaster 2D Platinum format
11.1.1
From earlier versions of ImageMaster and Melanie
ImageMaster recognizes gel files that were saved with ImageMaster 2D Platinum
(4.9 or 5) and Melanie (2, 3, 4 or 5). If you add files from these versions in the
Workspace Gels folder, you will recover spots and annotations, but no match
data. However, you can convert entire analysis sets (such as a single folder
containing all matched gels) into the current ImageMaster file format. This way
all match information is recovered without having to redo the gel matching.
To convert ImageMaster 2D Platinum or Melanie data:
1
Choose File > Import > ImageMaster 2D Platinum or Melanie Data in the
menu.
2
In the Batch Files Conversion box, indicate the Source Folder containing
your gels and associated match files in one of the old format, by
browsing to the desired directory.
3
Provide a Destination Folder name where your analysis set should be
saved in ImageMaster 2D Platinum format. You can manually add the
name of a new subfolder, if needed.
4
Click OK.
ImageMaster then creates new gels and match files in ImageMaster 2D Platinum
6.0 format and saves them to the given destination folder. These files are now
ready to be added to the Workspace in ImageMaster.
11.1.2
From ImageMaster 2D Elite
Any 2D experiments from ImageMaster 2D Elite (or Phoretix 2D Elite) can continue
to be analyzed by the new ImageMaster 2D Platinum project.
It is important to note that you can convert and use spot shapes and
quantification values, matching information, 1D and 2D calibration results,
annotations, spot filters, and much more from the Elite versions. However, you are
not allowed to edit imported spots or compare quantification values from
imported gels with new data obtained from the ImageMaster 2D Platinum
software. This is because spot detection and especially spot quantification are
done very differently in the two software versions, thus rendering any
comparisons inappropriate. You can still compare your old gels with a new set of
gels analyzed by the latest software, although you must redetect the spots and
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redo the matching. All other information such as annotations and 1D or 2D
calibrations are preserved.
To convert ImageMaster 2D Elite experiments:
1
In ImageMaster 2D Elite, make sure you have synchronized the spot
numbers. This is necessary to correctly export the match information.
2
Export the experiment in XML format (by choosing File > Import/Export
XML > Save Experiment from the ImageMaster 2D Elite menu). Save the
XML file in the same folder as your experiment.
3
Start ImageMaster 2D Platinum 6.0 and choose File > Import >
ImageMaster 2D Elite Experiment in the menu.
4
Browse the folder containing your Elite experiment, and select the saved
XML file.
5
You are asked to enter the Source Folder containing the experiment gels
(indicated by default if you saved your XML file in the experiment folder)
as well as the Destination Folder for saving the new project in
ImageMaster 2D Platinum format. Note that you can create a new
subfolder by adding the name manually.
6
Click OK to confirm your folder settings.
7
The import operation takes a few seconds.
ImageMaster then creates new gels and project files in ImageMaster 2D Platinum
6.0 format and saves them to the given destination folder. These files are now
ready to be added to the Workspace in ImageMaster.
Elite experiments
All gels in the original experiment are converted into the ImageMaster 2D
Platinum format and inserted into a new workspace and project bearing the
same name as the experiment (see Chapter 4). The generated project can contain
several match sets, depending on whether or not the initial experiment contained
average gels. A separate match set is created for each Average Gel.
User Defined Fields in an experiment are converted to properties of a project and
stored in the project file. However, they are currently not supported by the
software. The Comments field in the Experiment Header Information is converted
to a Comment for the project.
Gel images
The gel images (originally in .tiff, .gel or .img format) are treated as 16-bit TIFF files
and inserted in the newly created project folder. In the case of an .img file, an
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accompanying .inf file should be present in the experiment folder. When the .inf
file is missing, a warning message is displayed and import is aborted. In that case,
try creating a valid .inf file (resolution and gel size information must be known)
and do the import of the experiment again.
If the images were initially calibrated, the calibration is also applied to the
converted gel images. Similarly, if the Invert Intensities option in the Elite
Experiment was turned on, the TIFF image is inverted upon conversion of the gel.
If a master gel was defined in the original experiment, this status is also
maintained in the new master file (tagged in red).
The Name, Title, Comments and User Defined Fields in the Gel Header box of the
Elite Experiment are transferred to Gel Properties in the ImageMaster 2D Platinum
files.
Average gels
In the ImageMaster 2D Platinum project, an average gel from the Elite Experiment
becomes the master of a match set that contains the gels that were composing it.
Spots
Converted spots have spot shapes and quantification values extracted from the
experiment file. As spot areas and the associated volumes in ImageMaster 2D
Elite are calculated in terms of pixels (and not mm2), the Compute area in mm2
box in the Quantification tab of the ImageMaster Options (accessible by choosing
Tools > Options in the menu) is not checked to reflect this fact. Please note that it
is prohibited to edit spots from imported files. If you need to compare imported
gels with new ones, you must do an automatic spot detection in ImageMaster 2D
Platinum.
Comments on spots (previously visible in the Edit Spot Fields box) are converted
to spot annotations of the Comment category. Any User Defined Fields for spots
(entered through the Edit Spot Fields box or Measurements window) are
converted to annotations of a category with the name of the original User
Defined Field. Note that User Fields of type Document are converted to
annotations that contain a file link (the label content is file:file path).
Filtered spots (not displayed in the Elite version) are imported into ImageMaster
2D Platinum, but are flagged with annotations of type Set:Filtered_Spot. You can
easily select these spots (Select > Annotations > By Category) and decide if you
want to keep, delete or hide them. Similarly, all spots that were selected for
picking are flagged with annotations of type Set:Picked_Spot.
Matches
If gels were matched in the original experiment, the spot matches are converted
into ImageMaster 2D Platinum matches.
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1D and 2D calibrations
1D calibration steps are converted to annotations of the pI_MW category. For pI
calibrations, the labels contain the pI value, followed by a space and the number
-1 (indicating that no MW information was available for this annotation). For MW
calibrations, the labels contain the number -1 (indicating that no pI data was
available), followed by a space and the MW expressed in Daltons (Da). Using this
data, ImageMaster 2D Platinum automatically displays computed pI and MW
values for all the spots in the gel (and any matched gels).
2D calibration information, available from the Protein List in ImageMaster 2D Elite,
is also converted to annotations of the pI_MW category when proteins are
assigned to spots. In this case, the labels contain the pI value, followed by a space
and the MW expressed in Da.
Protein lists
Information from the Protein Lists in ImageMaster 2D Elite is converted to spot
annotations. The pI and MW values are transferred to annotations of the pI_MW
category (see above). Accession numbers appear under the Ac category.
Annotations
Annotations from ImageMaster 2D Elite are converted to annotations (of
category Annotation). Please note that annotation formats such as font, style,
size, color, etc. are lost.
11.1.3
From Twain compatible scanners
ImageMaster can also acquire images directly from TWAIN compatible scanners.
You must indicate the scanning source (all TWAIN compatible scanners attached
to your PC are automatically recognized by ImageMaster) and then launch the
scan. 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 can be added to the
Workspace.
To scan one or more gel images using a TWAIN compatible scanner:
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1
Choose File > Import > Twain > Select Source in the menu.
2
Select the appropriate image capture device from the given list. This
only needs to be done once (unless you want to change to a new image
capture device).
3
Choose File > Import > Twain > Acquire in the menu.
4
The scanner software automatically starts.
5
Set the suitable parameters for the scan.
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Launch the scan.
11.2
Exporting and importing ImageMaster 2D Platinum
data
11.2.1
XML format
To make the vast amounts of data that can be obtained with ImageMaster
available for processing by other applications or to import information coming
from external sources, ImageMaster uses the common XML format. XML is also
exploited for saving reports.
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 Internet Explorer (version 5 or
higher), Netscape (version 6 or higher) and Mozilla (version 1.4 or higher). 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 a history or script are printed from ImageMaster. The software 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. Notice that XSL stylesheets
can also be used to convert an XML file into another XML file.
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NOTE! Because the XSL stylesheets are specific to the browser you use, you
will find that different versions (both for printing reports and scripts) 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, script or
history. Look at which template works with your browser, and delete the other
one. In this way, the software automatically opens the remaining file and does
not ask you to make a choice.
11.2.2
ImageMaster reports
The default mode for saving reports in ImageMaster is in XML format. Reports
saved in this format carry the extension .rpt and are restored to their original
tabular form, on the condition that they were inserted into a Workspace. When
reopened in ImageMaster, these reports can be manipulated, sorted, customized
and edited as with any newly displayed report.
Outside the ImageMaster program, the content of ImageMaster reports can be
viewed with a browser. However, only a color-coded raw view of the root and child
elements is available (generally not very useful). The main interest in XML format,
besides being used directly by the ImageMaster software, is that external
applications can easily extract necessary data. Moreover, the files can be
converted to other user-defined formats.
Please note that the classical reports in ImageMaster contain particular data
types, and to export the comprehensive information of an analysis set, the
creation of several reports would often be necessary. This is why ImageMaster
offers an additional feature for exporting gel data.
11.2.3
Export gel data
ImageMaster allows the export of all gel-related data, except for the gel image
itself, into a single XML file. This XML file can include all available information on a
set of gels, together with spots (shape, quantification, aligned coordinates),
annotations, and match information (Figure 11-1).
To export gel data to an XML file:
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1
Select all objects (gels, spots, annotations) that should be exported to an
XML file. No preliminary selection is necessary if all gels in the active
worksheet should be exported, potentially with all their spot, annotation
and match data.
2
Choose File > Export > Gel Data to XML in the menu.
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3
Select the data to be exported from the Export Gel Data to XML File
dialog. See below for more details about the items that can be exported.
4
If objects are selected, ImageMaster asks whether you want to save
only the selected objects or not. If you answer Yes, just the selected
objects are exported. No means that you want to export all information
on all open gels.
5
Enter a name and destination folder for the file to be generated.
6
ImageMaster creates a file on the hard disk that can be read by other
applications. You can also view the contents of the file with your
browser.
11
Figure 11-1. Export Gel Data to XML File dialog box.
The following items can be exported to the XML file:
Gels: Minimal exported items for gels are gel IDs, gel names, and summarized
information about gels as present in the Gel Report. This includes image height
and width in pixels, pixel dimensions of the scanned image, minimum and
maximum gray levels (raw and calibrated), intensity calibration parameters,
number of spots, number of annotations, gel class and match set assignments. In
addition, the following data can be added to the exported file:
•
Descriptions: User-defined gel descriptions, as entered in the Gel
Description Report.
•
Pixel Annotations: Exports all annotations placed on individual gel pixels.
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Spots: Minimal exported properties for spots are X and Y coordinates on the gel,
and Spot ID. In addition, the following data can be added to the exported file:
•
Quantification: All quantification values as appearing in the Spots Report.
•
Annotations: Annotations linked to spots.
•
pI - MW: Calculated pI and MW values, if available.
•
X-Y align: Aligned spot coordinates, if the gels were aligned.
•
Shape: Spot shape descriptions using directional chain codes.
Matches: Exports all match information between all or selected spots in the
exported files.
11.2.4
Import gel data
Similar to the export function, gel data can be imported from an XML file
previously generated by ImageMaster or produced by another application (the
file should contain the appropriate tags, recognized by ImageMaster):
To import gel data:
1
Select a single gel you want to input data from an XML file.
2
Choose File > Import > Gel Data from XML from the menu.
3
Browse the appropriate folder and pick the desired file name.
4
If data from several gels is present in the file, ImageMaster asks you to
specify the gel from which data should be imported.
5
Select the data to be imported from the Import Gel Data from XML File
dialog box. See below for more details about the items that can be
imported. Some options may be inactivated if the necessary information
is not present in the opened file.
6
ImageMaster imports the desired information.
The following items can be imported from an XML file (Figure 11-2):
Gels: You can import the following data from existing gels:
240
•
Calibration: Intensity calibration parameters (Slope and Offset).
•
Descriptions: User-defined gel descriptions, entered in the Gel Description
Report.
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Spots: Imports spots. Note that spot shape descriptions using directional chain
codes need to be present in the file.
Annotations: Imports both pixel and spot annotations.
NOTE! When several types of objects are imported at the same time, all
import operations should take place correctly. If this is not the case (because
of ambiguous or erroneous file formatting), none of the objects are imported.
Figure 11-2. Import Gel Data from XML File dialog box.
11.3
Exporting to spot excision robots
Please note that in addition to exporting spots to an excision robot, it may be
useful to annotate the exported spots. This allows you to easily select them later
on, for adding experimental data (such as mass spectrometry information).
11.3.1
Bruker Proteineer SP spot picker
ImageMaster can export spot coordinates directly to the Bruker Proteineer SP
spot picker. For more details about this functionality, please see the
documentation provided by Bruker Daltonics.
To export a spot coordinate file to the Bruker Proteineer SP:
1
Select all spots to be cut with the spot excision robot.
2
Choose File > Export > Spots to Picker > Bruker Proteineer SP in the menu.
11.3.2
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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analyzed gel image and the coordinates of the actual gel located on the spot
picker (Figure 11-3).
Once the gel has been digitized and analyzed with ImageMaster 2D Platinum, 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 of 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. If a reference marker is well
detected during the automatic spot detection process (nice round spot perfectly
centered on the marker), you can just add an annotation on the marker spot. The
basis of such an annotation is displayed as a small square, and its coordinates
correspond to the center of the spot. If a reference marker is not well detected
(irregular shape or consisting of several spots), it is better to delete the existing
spot(s) and create an annotation on the pixel that is in the center of the marker.
This kind of pixel annotation has a crossed basis and its coordinates correspond
to the pixel it is attached to. Note that the two 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:
242
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
from the menu.
4
Click on the Annotation tool in the main toolbar.
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.
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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
11-3).
12 Choose File > Export > Spots to Picker > GE Healthcare Ettan.
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).
14 The Ettan Spot Picker can now read the exported files.
Figure 11-3. 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).
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11.3.3
Genetix GelPix spot picker
To export a spot coordinate file to the GelPix spot picker:
1
Select all spots to be cut with the spot excision robot.
2
Choose File > Export > Spots to Picker > Genetix GelPix in the menu.
3
Enter a file name and destination folder.
4
The spot picker subsequently reads the saved file.
11.3.4
Genomic Solutions ProPic spot picker
The ProPic spot picker produces a TIFF file of the entire gel holder area with a
resolution of 1035 x 1317 pixels, each pixel representing approximately 330 x 330
microns of the image area. This ProPic image can be analyzed with ImageMaster,
and spot coordinates in the image can be exported back to the ProPic spot picker
in a predetermined file format. The ProPic software subsequently translates the
image X, Y coordinates into ProPic robot X, Y coordinates by using a robot map.
Since the robot mapping process assumes that each X, Y coordinate in the image
always corresponds to the same position on the robot bed, the ProPic image must
remain in its original form. It can never be resized, cropped or rotated.
The ProPic image is a picture of the current state of the gel from which the spots
are to be picked. It is not necessarily the image used to determine the spots to
pick. One can select spots from any analytical image (of the same gel) obtained
from a different resolution system and analyzed with ImageMaster. However, the
analytical image needs to be aligned to the ProPic image so that ImageMaster
can export the aligned X and Y coordinates of the selected spots. Please keep in
mind that proper alignment is crucial at this stage, especially for images of "old"
gels that underwent significant shape change since they were originally imaged
and analyzed, and for gel images that were acquired on a high-resolution system.
To export a spot coordinate file to the ProPic spot picker:
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1
Analyze your analytical gel image with ImageMaster and select the
spots you want to export to the spot picker. Annotate these spots (with
a category of type Set:) so that you can easily re-select them at a later
stage.
2
Place your gel on the ProPic robot bed, produce the ProPic image and
open it in ImageMaster. Use the gel image as is, meaning do not resize,
crop, flip or rotate the image.
3
Align your analytical gel image to the ProPic image, using a sufficient
number of landmarks (see Section 6.3.6 for details on how to align gel
images).
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Select all spots to be cut with the spot excision robot from the analytical
image.
5
Make sure only the analytical gel image is selected.
6
Choose File > Export > Spots to Picker > Genomic Solutions ProPic.
7
Enter a file name and destination folder. The file automatically is given a
.tds extension (TwoDSpotlist).
8
ImageMaster exports the aligned X and Y coordinates for each selected
spot. That is, the coordinate system of the ProPic image will be used.
9
The spot picker subsequently reads the saved file.
11.4
11
Connecting to external protein databases
A key advantage of the ImageMaster program is its ability to link spots on gel
images to protein data in 2-DE or other databases. 2-DE 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. ImageMaster can work in conjunction with several specialized 2DE databases, but is particularly compatible with data from the SWISS-2DPAGE
database that is accessible over the Internet. If your computer has access to the
World Wide Web, 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 SWISS-2DPAGE databases, please
contact GeneBio (www.genebio.com).
11.4.1
Setting the database
A detailed description of how to query a remote database through the Internet is
given in Chapter 8. In short, these queries use the label (holding a protein
accession number taken from a 2-DE database) of a particular category (linked to
a specific database) to interrogate the database. The HTTP query must be
composed of:
•
The database HTTP address (for example, http://www.expasy.org/).
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•
The database query engine (for example, cgi-bin/nice2dpage.pl?).
•
The database accession number (for example P02649).
The database HTTP address and query engine are entered as constraints to the
annotation category. Go to the Categories tab in the ImageMaster Options
(accessible by choosing Tools > Options in the menu), and type them in as one
string in the External Engine field (for example, http://www.expasy.org/cgi-bin/
nice2dpage.pl?). A list of federated 2-D PAGE databases, with the required
database query formats, can be found at http://www.expasy.org/ch2d/2daccess.html.
11.4.2
Querying the database
The database accession number (for example P02649) is entered as a label of the
particular category linked to a spot. It identifies the protein corresponding to that
spot.
When you subsequently double click on the label while the Annotation tool is
selected, ImageMaster opens your default Internet browser and launches a
query that consists of the concatenation of the HTTP address, query engine and
accession number (for example, http://www.expasy.org/cgi-bin/
nice2dpage.pl?P02649). As a result, the entry for the protein with the given
accession number opens in your browser (Figure 11-4). In the case of the above
example, this would be the entry for Human Apo E (Gels).
11.4.3
SWISS-2DPAGE master gels
SWISS-2DPAGE master gels, or reference maps, contain annotations for all
proteins corresponding to entries in the SWISS-2DPAGE 2-DE database. Each
annotation contains the accession number (in the Ac category) of the database
entry, as well as the entry's short name (in the ProteinName category). For
example, the Human Plasma protein map possesses the Ac label P02768 and the
ProteinName label ALBU_HUMAN, for each spot that corresponds to albumin.
In most 2-DE databases the accession number is the unique entry identifier, while
the protein name can vary. It is therefore strongly recommended to use
accession numbers for your queries. Nevertheless, ImageMaster provides a way
to automatically update the protein names in your gels, if you use the SWISS2DPAGE accession numbers.
To update ImageMaster labels with database protein names:
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1
Select a gel containing labels with valid SWISS-2DPAGE accession
numbers.
2
Select all labels you want to update.
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Choose Edit > Annotations > Load Protein Name from SWISS-2DPAGE.
4
For each selected label, ImageMaster then requests its short name from
the SWISS-2DPAGE database (using the Ac as identifier) and then
updates the ProteinName label accordingly.
11
Figure 11-4. The SWISS-2DPAGE entry for human Apo E. Entries from this database contain
full protein names, bibliographic references, annotations (such as protein function,
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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12 Safety, control and automation
12.1
Introduction
Discover how ImageMaster lets you monitor the operations carried out on your
images and automate various analysis steps in this chapter. Although the three
options associated with these features - namely Undo/Redo, History and Script
- may seem quite independent, they in fact share a number of common interface
elements and properties that are described below.
12.1.1
Action descriptors
Each operation carried out in ImageMaster is described by a generic name called
Action Descriptor. The Action Descriptor is used in Undo/Redo, History and Script
to identify a specific operation so that you can act on it (for example, undo the
operation or copy it from a history to a new script). Nearly all operations executed
are displayed in the Action Descriptor list, which you can find in the Undo/Redo,
History and Script windows. However, there are some exceptions to this
depending upon the option used. These exclusions are specified in the ensuing
sections.
12.1.2
Displayed actions
The number and type of operations included in the Undo/Redo lists and History
can become very large and diverse, thus making it difficult to find the actions of
interest (when deleting or copying to a new script, for instance). In order to reduce
the number of displayed actions, four major types of operations were defined:
•
Edition: Covers the functionalities available under the Edit menu, this kind of
operation renders permanent modifications to the image data.
•
Selection: Comprises the options from the Select menu. These allow the
selection of specific objects or the refinement of an existing selection. Once
selected, you can perform further actions on the chosen objects.
•
Display: This type of operation is typically found in the View, Analyze, Report
and Window menus. It generally displays new windows (for example
reports), modifies the Display Zone (layout or stack settings) or manages the
visual relationships between images (for example alignment).
•
Show: The features available from the Show menu belong to this type of
operation. They act as a kind of visual filter or can be considered as nonpermanent modifications to the gels because nothing is saved with the
image file.
All ImageMaster functions are classified into one of these types so that three
different filter criteria can be applied to the items in the Action Descriptor list.
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These filter criteria and the type of actions they include are listed in the table
below:
Edition
Selection
Display
Show
All
X
X
X
X
Without Display
X
X
Only Edit
X
Each of these filters has a specific context:
•
All is interesting if you want to list all types of operations carried out during
your work session.
•
Without Display is particularly useful when generating scripts.
Modifications to the display (move image, etc.) are not important and often
cannot be scripted.
•
Only Edit is used when you want to track permanent changes that have
been made to the images and image data.
An additional filter can be combined with any of the above three filters. When the
Only Gels Action box is checked, only actions that operate on selected images
are displayed. Thus, you can exclude operations that act at the level of the
program window (for example, layout settings) or that result in the generation of
reports. By default, this box is not checked and therefore all operations (in the
filter types above) are listed.
12.1.3
History and script navigators
The Action Descriptor lists in the History and Script windows have the form of a
navigator, in which some Action Descriptors are preceded by a + or - node allowing
the item to be expanded or collapsed, and an icon indicates whether it can be
scripted or not:
The corresponding action does not require any user-dependent
input (such as the selection of a region) and can therefore be
scripted.
Actions identified by this icon cannot be scripted because they
require user intervention. Even if selected in the History navigator,
they are not copied to any newly-generated script.
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Once an action is expanded, you see one or both of the following icons:
Identifies a Parameter. This represents a major property of the
action. For instance, the Select Rows from Report action is
characterized by a Window parameter (describing the title and
type of the report), the Sort parameter (indicating the column in
which the report data was sorted), and the Selection parameter
(containing information about which items in the report were
selected).
Identifies an editable Value that characterizes an action or an
action parameter.
12.1.4
Gel selection
The listed actions only include the operations relevant to the gel images selected
when first opening the History window or activating the Undo or Redo functions.
So even if the gel selection changes and the History is refreshed, the Action
Descriptors only apply to the originally selected gels. Therefore, if you want a
history for one or more images that were not selected during the creation of the
current history, you must select the image(s) in question and open a new History
window. Similarly, if you want to undo a general action (for example, spot
detection) on specific images, you should first select the desired gels and then
choose the Undo function from the menu.
12.2
Undo / redo
ImageMaster allows you to cancel any unwanted modifications. You can return
to an earlier state of your analysis by using the Undo option (Figure 12-1). You no
longer lose whole parts of your analysis simply because you made an error and
could only recover a previously saved state. Moreover, ImageMaster not only
offers a multiple Undo function, it also enables you to restrict the canceled
actions to specific images. Please keep in mind that actions can only be undone
for images that were selected when the operation was originally performed, even
if the gel selection subsequently changed.
Each Action Descriptor in the Undo/Redo list is preceded by the time at which the
action was carried out. This information helps you to identify the particular
sequence of actions that you would like to cancel.
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Figure 12-1. Undo window.
To undo the last action performed:
1
Select the images for which you would like to cancel the most recent
operation.
2
Choose Edit > Undo from the menu.
3
The last action is selected by default.
4
Click OK.
To undo several successive actions:
1
Select the images for which you would like to cancel a certain number
of operations.
2
Choose Edit > Undo from the menu.
3
Select a prior action to be undone.
4
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 obviously reapply the actions.
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To redo specific actions:
1
Select the images for which you would like to redo a certain number of
canceled operations.
2
Choose Edit > Redo from the menu.
3
Select the action to be redone.
4
Click OK. The selected action and any preceding actions are redone.
All operations performed can be reversed with the exception of the following nonexhaustive list:
•
Modifications to the Workspace cannot be canceled.
•
It is not possible to undo operations such as opening, saving and printing
files.
•
Changes that only apply to all or none of the gels cannot be canceled (for
example, displaying the profile or changing the color palette).
•
Direct actions on reports (such as the creation, edition or closure) cannot be
undone. However, when selections or modifications in a report are
propagated to the corresponding images, they are included in the undo list
and can therefore be canceled.
12.3
History
Please keep in mind that actions are only displayed for images that were selected
when the History window was first opened, even if the gel selection subsequently
changed and the History was refreshed.
To open a History window:
1
Select the images for which you would like to display a history.
2
Choose Edit > History > Show from the menu.
3
You can place a marker in the History by choosing Edit > History > Insert
Marker from the menu, or clear the list of actions by choosing Edit >
History > Clear.
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Figure 12-2. History window.
The History window contains a toolbar and a list of actions (Figure 12-2). The
functionality of each of the icons is as follows:
Save the History in an XML type file with extension .hst.
Print the History. You can choose to print only selected actions.
Note that before printing the History first displays in your default
Internet browser. The XSL stylesheet located in the Template\Script
folder of the ImageMaster installation directory is used to transform
the XML formatted History into an attractive document (find more
details about XML and XSL in Chapter 11). You can then use the print
option in your browser to get a paper copy.
Copy the selected actions for subsequent pasting into an open
script.
Refresh the History. All actions are collapsed and the list scrolls
down until only the most recent operations are visible.
Display the Gels Report. Note that the report only includes the
images selected when the History window was first opened. This
tool is useful for checking which images are included in the current
History.
Copy the selected actions to a New Script.
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Similar to the Undo/Redo options, there are some actions that are not included in
the History. The following is a non-exhaustive list of exceptions:
•
Modifications applied to the Workspace are not logged.
•
Operations that require file manipulations such as opening, saving and
printing files are currently not recorded with the History function.
•
Direct actions on reports are not included in the History navigator, except
when selections or modifications in the report are propagated to the
corresponding images.
12.4
Script
The Script function automates parts of the analysis process. ImageMaster
routinely carries out a sequence of instructions when you run a script. You are
empowered to encode desired actions simply by cutting and pasting from the
History to the Script windows, without ever needing any programming
knowledge (Figure 12-3). Several features in ImageMaster make it possible to
modify a script to best suit your immediate requirements.
Figure 12-3. Script window.
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To create a script:
1
Carry out the desired sequence of operations on a selected set of
images.
2
Choose Edit > History > Show to display the History window.
3
Copy the required actions to a new script by selecting the actions and
clicking the New Script icon in the History window toolbar.
4
A Script window appears on your screen.
The Script window contains a toolbar and a list of actions. The functionality of
each of the icons is as follows:
Open an existing script. Browse the directory where the script file
(.srt) is located and select the file name. The drop down menu gives
you access to a list of scripts opened during the current work
session and scripts that were previously saved in the default
ImageMaster\Scripts folder in the user’s My Documents directory.
Save the script. You are given the choice of saving only the selected
actions. Scripts saved in the default ImageMaster\Scripts folder in
the user’s My Documents directory can be accessed directly using
the Open icon.
Print the script. Choose to print only the selected actions. Note that
before printing the script is first displayed in your default Internet
browser. The XSL stylesheet located in the Template\Script folder of
the ImageMaster installation directory is used to transform the XML
formatted script into an attractive document (find more details
about XML and XSL in Chapter 11). You can then use the print option
in your browser to get a paper copy.
Cut the selected actions from your script.
Copy the selected actions for subsequent pasting in a script.
Paste the previously copied actions (from any History or Script
window) below the currently selected action.
Run the script. Be sure to select all the images that should be run
with the script.
The originally encoded script sometimes needs to be modified because you
forgot to do an operation, would like to suppress an action, or want to adapt some
parameters to a specific set of gels. The following tools and possibilities are
available for modifying your script:
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•
Move the actions in a script by dragging and dropping them into another
position. They are placed below the point of your cursor.
•
Copy actions from any history or script using the Copy tool and paste them
into your script using the Paste tool. Pasted actions are inserted at the end
of your script or below the currently selected action, but can be moved
simply by dragging and dropping them into another position.
•
Suppress one or several actions in your script by selecting the actions and
clicking the Cut icon.
•
Modify the values that characterize the actions or action parameters. To do
so, right click on the Value icon and choose Modify from the contextual
menu. Enter a new value.
•
Oblige ImageMaster to ask for user input while the script is running. It is
practical, for example, if you want to postpone the choice of statistics until
the moment you actually run the script. To do this, right click on the Action
Descriptor and choose Set User Action from the contextual menu. The script
will stop at the designated point and display a standard dialog box relating
to the operation, thus enabling you to adjust your input. Once all the user
definitions are made, click the OK button and the script will continue to run.
12
The Tools menu in the ImageMaster program menu bar offers additional
functionality for creating or handling scripts.
To create a script:
1
Choose Tools > Script > New from the menu.
2
Code your new script by copying specific actions from a history or other
script using the tools described above.
3
Save your new script using the corresponding icon in the Script window
toolbar.
To run an existing script:
1
Choose Tools > Script > Run from the menu.
2
Browse the directory where the script file is located and select its name.
3
ImageMaster may ask you for approval to run the script (see below).
Answer Yes.
4
The script runs immediately, stopping at each operation that was set as
a user input action or when an illogical or impossible operation is
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encountered (for example, Select All Matches when images were not
matched).
To require confirmation before running a script:
1
Go to Tools > Script > With Confirmation in the menu. If With Confirmation
is checked, then the option is activated. This is the default setting.
2
To deactivate this option, choose Tools > Script > With Confirmation. With
Confirmation is no longer checked in the menu.
To run a recently used script :
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3
Go to Tools > Script > Recent in the menu.
4
Choose one of the scripts from the list (only possible if scripts were used
or created during the current work session).
5
The script runs immediately (ImageMaster may ask for confirmation).
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Appendix
A.1
Contextual menus
Contextual menus are available throughout the Workspace and Display Zone. In
the Workspace, right click on a file or folder to open a contextual menu from
which you choose an action to be carried out. In the Display Zone, access
contextual menus by right clicking worksheet or pane banners, gel legends or
inside a gel image. These contextual menu options are often duplicates of main
menu options but are an alternative and quicker way to perform the action.
A.2
Workspace contextual menus
A.2.1
Workspace name
•
New Project: Creates a project in the workspace.
•
Insert Project: Adds an existing project to the workspace.
•
Backup: Archives the workspace file (with the extension .bkp).
•
Restore Workspace: Returns an archived workspace to its previous state.
•
Restore Project: Returns an archived project to its previous state.
•
Properties: Displays the workspace properties: Workspace Name, Creator,
File Name, Modification Date and Comment.
A.2.2
Project name
•
Export: Exports a project with copies of its gels, matches and documents.
•
Backup: Archives the project file (with the extension .bkp).
•
Delete: Removes the project from the active workspace.
•
Properties: Displays the project properties: Project Name, Creator, File
Name, Modification Date and Comment.
A.2.3
Gels folder
•
Add Gels: Adds gels that are already in ImageMaster format (with the
extension .mel).
•
Import Gels: Adds new gels that are not already in ImageMaster format.
•
Create Folder: Creates a subfolder into the Gels folder.
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Paste: Pastes previously cut or copied items into the Gels folder.
Folder
• Open > In Worksheet: Opens all gels contained in the folder in the active
[Gels] worksheet in the Display Zone.
•
Open > In New Worksheet: Opens all gels contained in the folder in a new
[Gels] worksheet in the Display Zone.
•
Add Gels: Adds gels that are already in ImageMaster format (with the
extension .mel).
•
Import Gels: adds new gels that are not already in ImageMaster format.
•
Create Folder: Creates a subfolder into the selected folder.
•
Delete: Removes the selected folder.
•
Cut: Cuts the selected folder in order to paste it.
•
Paste: Pastes previously cut or copied items into the selected folder.
•
Properties: Displays the folder properties: Name and Comment.
Gel
• Open > In Worksheet: Opens the selected gels in the active [Gels]
worksheet in the Display Zone. You can also double click on a file to open it
in the active [Gels] worksheet.
260
•
Open > In New Worksheet: Opens the selected gels in a new [Gels]
worksheet in the Display Zone.
•
Add in Matchset: Adds the selected gels in an existing match set or in a new
one. If you would like to create a new match set, enter a new name.
Otherwise, you can choose from the other match sets available in the
proposed list.
•
Delete: Removes the selected gels from the folder. Please note that it does
not remove the file from your hard disk. The image is only deleted from the
active project.
•
Search: Searches for the gel on the hard disk (in the indicated directory)
when a gel with the appropriate ID is not found at the usual location. This is
the case when a red mark covers the gel icon, generally indicating that you
moved the original gel file or replaced it with another one.
•
Cut: Cuts the selected gels in order to paste them.
•
Properties: Displays the gel properties: Gel Name and Staining.
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A.2.4
DIGE Gels folder
•
Add DIGE Gel: Adds DIGE images that are already in ImageMaster format
(with the extension .mel) to define a DIGE gel.
•
Import DIGE Gel: Adds DIGE images that are not already in ImageMaster
format to define a DIGE gel.
•
Paste: Pastes previously cut or copied items into the DIGE Gels folder.
DIGE gel
• Open > In Worksheet: Opens the selected DIGE gels in the active [Gels]
worksheet in the Display Zone. You can also double click on a file to open it
in the active worksheet.
•
Open > In New Worksheet: Opens the selected gels in a new worksheet in
the Display Zone.
•
Detect: Performs spot detection on the selected DIGE gels. You are asked to
estimate the Number of Spots to be found in the gels.
•
Create MatchSet: Creates a new match set that will include the selected
DIGE gels as sub match sets. The name of the DIGE gel is input as the default
Name.
•
Delete: Removes the selected gels from the folder. Please note that it does
not remove the file from your hard disk. The DIGE gel is only deleted from
the active project.
•
Copy: Copies the selected gels into memory. You can then paste them into
other folders of your workspace.
•
Properties: Displays the DIGE properties: Name, Dye Chemistry and
Comment.
DIGE gel image
• Open > In Worksheet: Opens the selected DIGE images in the active [Gels]
worksheet in the Display Zone. You can also double click on a file to open it
in the active [Gels] worksheet.
•
Open > In New Worksheet: Opens the selected DIGE images in a new [Gels]
worksheet in the Display Zone.
•
Histogram: Shows data associated with detected spots in two selected
images from the same DIGE gel. Spot data (intensity, area, volume or slope)
is plotted against log volume ratio. The blue curve represents the frequency
distribution of the log volume ratios.
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•
Add in Matchset: Adds the selected DIGE images in an existing match set or
in a new one. If you would like to create a new match set, enter a new
name. Otherwise, you can choose from the other match sets available in the
proposed list.
•
Search: Searches for the gel on the hard disk (in the indicated directory)
when a gel with the appropriate ID is not found at the usual location. This is
the case when a red mark covers the gel image icon, generally indicating
that you moved the original image file or replaced it with another one.
•
Copy: Copies the selected images into memory.
•
Properties: Displays the DIGE gel properties: Gel Name and Staining.
A.2.5
MatchSets folder
•
Create MatchSet: Creates a new match set into the MatchSets folder.
•
Paste: Pastes previously cut or copied items into the MatchSets folder.
MatchSet
• Open: Opens the selected match set in a new [MatchSet] worksheet.
•
Create MatchSet: Creates a sub match set within the selected match set.
•
Delete: Removes the selected match set from the folder.
•
Copy: Copies the selected match set into memory.
•
Cut: Cuts the selected match set in order to paste it.
•
Paste: Pastes previously cut or copied items into the selected match set.
•
Properties: Displays the match set properties: MatchSet Name and
Comment.
SubMatchSet
• Open: Opens the selected match set in a new [MatchSet] worksheet.
262
•
Set MatchSet as Reference: Defines the selected match set as the reference
to be used when matching two or more match sets. The match set icon is
tagged red.
•
Create MatchSet: This option is available only if the sub match set is empty,
It creates a sub sub match set within the selected sub match set.
•
Delete: Removes the selected match set from the folder.
•
Copy: Copies the selected match set into memory.
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•
Cut: Cuts the selected match set in order to paste it.
•
Paste: Pastes previously cut or copied items into the selected match set.
•
Properties: Displays the match set properties: MatchSet Name and
Comment.
Gel
• Open: Opens the selected gels with the corresponding master in a new
[MatchSet] worksheet.
•
Set Gel as Reference: Defines the selected gel as the reference to be used
when matching two or more gels. The gel icon is tagged red. A master gel is
automatically created by duplicating the reference gel when opening the
match set in a worksheet.
•
Delete: Applies to non-DIGE images only. Removes the selected gels from
the match set. Please note that it does not remove the file from your hard
disk. The image is only deleted from the active project.
•
Add in Classes: Adds the selected gels in a class. If you would like to add the
gels to a new class, then enter a new name. Otherwise, you can choose
from the other classes available in the proposed list.
•
Copy: Copies the selected gels into memory.
•
Properties: Displays the gel properties: Gel Name and Staining.
A.2.6
Classes folder
•
Create Class: Creates a new class in the Classes folder.
•
Paste: Pastes previously cut or copied items into the Classes folder.
Class
• Open: Opens the selected class in a new [Classes] worksheet.
•
Create Class: Creates a subclass within the selected class.
•
Delete: Removes the selected classes from the Classes folder.
•
Cut: Cuts the selected classes in order to paste them.
•
Paste: Pastes previously cut or copied items into the selected class.
•
Properties: Displays the class properties: Name and Comment.
Gel
• Open: Opens the selected gels in a new [Classes] worksheet.
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•
Delete: Removes the selected gels from the class. Please note that it does
not remove the file from your hard disk. The gel is only deleted from the
class in the active project.
•
Copy: Copies the selected gels into memory.
•
Cut: Cuts the selected gels in order to paste them.
•
Properties: Displays the gel properties: Gel Name and Staining.
A.2.7
Reports folder
•
Open: Opens all the reports contained in the Reports folder in dockable
windows.
•
Add Reports: Inserts one or more reports from the hard disk into the Reports
folder.
•
Create Folder: Creates a subfolder within the Reports folder.
•
Paste: Pastes previously cut or copied items into the Reports folder.
Folder
• Open: Opens all the reports in dockable windows.
•
Add Reports: Inserts one or more reports from the hard disk into the
selected folder
•
Create Folder: Creates a subfolder within the selected folder.
•
Delete: Removes the selected folder.
•
Copy: Copies the selected folders into memory.
•
Cut: Cuts the selected folders in order to paste them.
•
Paste: Pastes the previously cut or copied folders or reports in the selected
folder.
•
Properties: View or edit the Name or Comment for the folder.
Report
• Open: Opens the selected reports in dockable windows. You can also double
click on a report file to open it.
•
264
Delete: Removes the selected reports from the folder. Please note that it
does not remove the file from your hard disk. The report is only deleted from
the active project.
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•
Search: Searches for the report on the hard disk (in the indicated directory)
when a report with the appropriate ID is not found at the usual location. This
is the case when a red mark covers the report icon, generally indicating that
you moved the original report file or replaced it with another one.
•
Copy: Copies the selected reports into memory.
•
Paste: Pastes the previously cut or copied items below the selected report.
•
Properties: Displays the report properties: Path, Type, Message, and
Comment. The Type item tells you whether your report is a Spot Report, Gel
Report, Match Report, etc. The Message gives information on the spot value
or statistics that were used to generate the report, or other data depending
on the report type. The Comment field displays the report comment that
was defined by the user.
A.2.8
Documents folder
•
Open: Opens all the files, contained in the Documents folder, with the
appropriate software, depending on the extensions. As in Windows Explorer,
files with unrecognizable extensions are not opened.
•
Add Files: Inserts one or more files from the hard disk within the Documents
folder.
•
Create Folder: Creates a subfolder within the Documents folder.
•
Paste: Pastes the previously cut or copied items in the Documents folder.
Folder
• Open: Opens all the files with the appropriate software, depending on the
extensions. As in Windows Explorer, files with unrecognizable extensions are
not opened.
•
Add Files: Inserts one or more files from the hard disk within the selected
folder.
•
Create Folder: Creates a subfolder within the selected folder.
•
Delete: Removes the selected folders (with their files).
•
Copy: Copies the selected folders into memory.
•
Cut: Cuts the selected folders in order to paste them.
•
Paste: Pastes the previously cut or copied items in the selected folder.
•
Properties: Displays the folder properties: Name and Comment.
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File
• Open: Opens the selected files with the appropriate software, depending on
the extensions. You can also double click on a single file to open it.
•
Delete: Removes the selected files from the folder. Please note that it does
not remove the file from your hard disk. The file is only deleted from the
active project.
•
Copy: Copies the selected files into memory.
•
Paste: Pastes the previously cut or copied items below the selected file.
•
Properties: Displays the file path.
A.3
Display zone contextual menus
A.3.1
Worksheet, pane or legend
•
Close Images: Closes one or more selected gel images. This operation can
also be carried out by selecting the applicable images and going to File >
Close Images or press Ctrl+W.
•
Close All: Closes all worksheets available in the Display Zone. This operation
can also be carried out by going to File > Close All.
•
Save Worksheet: Saves all the changes made to the gel images displayed in
the active worksheet.
•
Pane: Show Vectors, Hide Vectors, Stacked pane, Tiled pane.
•
Select: All, Aligned, Inverse Selection.
A.3.2
Image
Gels
• File: Close Images, Close All, Save Worksheet.
•
Select: All, Aligned, Inverse Selection.
•
Report: Generates a Gel Report.
Spots
• Edit: Detect, Edit Enabled, Copy from Image, Delete.
•
266
Show: Shape, Show All, Hide All, In Region, Only Selected, Hide Selected,
Show ID, Hide All ID, Set Color.
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•
Select: By ID, By Color, In Region, From Report File, All, Refine by Value,
Inverse Selection.
•
Report: Generates a Spot Report.
Annotations
• Edit: Add Label, Modify, Copy, Paste Annotations, Paste Labels, Duplicate
Labels, Delete, Delete by Category, Copy Matched Labels, Link with Spot,
Unlink from Spot.
•
Show: Show All, Hide All, In Region, For Spots, Only Selected, Hide Selected,
Visible Categories, Packed Categories, Linked Data, Set Color.
•
Select: By Content, By Category, Combine, By Color, In Region, From Report
File, Common Labels, All, Inverse Selection.
•
Report: Label Report, Annotation Report, Category Report.
Matches
• Edit: Add Match, Delete Match.
•
Show: Show ID, Hide All ID, Show Vectors, Hide Vectors.
•
Select: By ID, For Spots, All, Refine Selection, Inverse Selection, Multiple
Matches.
•
Analyze: Intra-Class Report, Intra-Class Histograms.
•
Match Report: Generates a Match Report.
•
Zoom: Zoom in or out on the selected images or regions. You can also
choose a factor to reduce or enlarge the selected images or regions by.
•
Show All: Makes all spots and annotations visible in the selected gel images.
•
Hide All: Hides all spots and annotations in the selected gel images. It also
hides all match vectors in the selected gel images.
•
Select All: Activates all spots and annotations in the selected gel images.
•
Unselect All: Deactivates all spots and annotations in the selected gel
images.
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Appendix
B
Appendix
B.1
Shortcut keys
Several menu options can be activated by keyboard shortcuts. These are
indicated at the right-hand side of the corresponding menu option. Please note
the logic behind the key combinations:
Ctrl is used for handling gels.
Shift is used for handling spots.
Alt is used for handling annotations.
Ctrl + Shift is used for handling 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
Shift+Z
Edit > Redo
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B.2
270
Gel shortcuts
Shortcut
Menu Command
Ctrl+A
Select > Gels > All
Ctrl+F
View > Switch
Ctrl+I
Show > Gels > Grid Lines > Show
Ctrl+J
Show > Gels > Grid Lines > Hide
Ctrl+N
Select > Gels > Inverse Selection
Ctrl+S
File > Save > Worksheet
Ctrl+W
File > Close Images
Ctrl+<Up>
Show > Gels > Move > Up
Ctrl+<Down>
Show > Gels > Move > Down
Ctrl+<Left>
Show > Gels > Move > Left
Ctrl+<Right>
Show > Gels > Move > Right
Ctrl+1
Tools > Toolbar > Hand
Ctrl+2
Tools > Toolbar > Magnify
Ctrl+3
Tools > Toolbar > Region
Ctrl+4
Tools > Toolbar > Spot
Ctrl+5
Tools > Toolbar > Annotation
<Page Up>
View > Pane Layout > Next
<Page Down>
View > Pane Layout > Previous
Shift+<Up>
Show > Gels > Zoom > In
Shift+<Down>
Show > Gels > Zoom > Out
F1
Show > Show Reference/Master
F2
Show > Show All
F3
Show > Hide All
F4
Select > Select All
F5
Select > Unselect All
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B.3
Spot shortcuts
Shortcut
Menu Command
Shift+A
Select > Spots > All
Shift+B
Select > Spots > In Region
Shift+E
Edit > Spots > Edit Enabled
Shift+H
Show > Spots > Hide All
Shift+I
Show > Spots > Hide Selected
Shift+J
Show > Spots > Show ID
Shift+K
Show > Spots > Hide All ID
Shift+N
Select > Spots > Inverse Selection
Shift+R
Show > Spots > In Region
Shift+T
Show > Spots > Only Selected
Shift+X
Edit > Spots > Delete
Shift+Y
Show > Spots > Show All
Shift+1
Show > Spots > Shape > Crossed
Shift+2
Show > Spots > Shape > Outlined
Shift+3
Show > Spots > Shape > Filled
Shift+4
Show > Spots > Shape > Outlined/Filled
Shift+5
Show > Spots > Set Color > Purple
Shift+6
Show > Spots > Set Color > Orange
Shift+7
Show > Spots > Set Color > Brown
Shift+8
Show > Spots > Set Color > Default
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B.4
272
Annotation shortcuts
Shortcut
Menu Command
Alt+A
Select > Annotations > All
Alt+B
Select > Annotations > In Region
Alt+C
Edit > Annotations > Copy
Alt+D
Edit > Annotations > Delete by Category
Alt+E
Edit > Annotations > Modify
Alt+F
Edit > Annotations > Add Label
Alt+H
Show > Annotations > Hide All
Alt+I
Show > Annotations > Hide Selected
Alt+J
Show > Annotations > Visible Categories
Alt+K
Show > Annotations > Packed Categories
Alt+L
Edit > Annotations > Link with Spot
Alt+N
Select > Annotations > Inverse Selection
Alt+O
Show > Annotations > For Spots
Alt+P
Edit > Annotations > Paste Annotations
Alt+R
Show > Annotations > In Region
Alt+T
Show > Annotations > Only Selected
Alt+U
Edit > Annotations > Unlink from Spot
Alt+V
Edit > Annotations > Paste Labels
Alt+X
Edit > Annotations > Delete
Alt+Y
Show > Annotations > Show All
Alt+4
Show > Annotations > Set Color > Purple
Alt+5
Show > Annotations > Set Color > Orange
Alt+6
Show > Annotations > Set Color > Brown
Alt+7
Show > Annotations > Set Color > Default
Alt+8
Select > Annotations > By Color > Purple
Alt+9
Select > Annotations > By Color > Orange
Alt+0
Select > Annotations > By Color > Brown
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B.5
Match shortcuts
Shortcut
Menu Command
Ctrl+Shift+A
Select > Matches > All
Ctrl+Shift+G
Edit > Matches > Add Match
Ctrl+Shift+H
Show > Matches > Hide Vectors
Ctrl+Shift+J
Show > Matches > Show ID
Ctrl+Shift+K
Show > Matches > Hide All ID
Ctrl+Shift+M
Select > Matches > For Spots
Ctrl+Shift+N
Select > Matches > Inverse Selection
Ctrl+Shift+U
Edit > Matches > Delete Match
Ctrl+Shift+Y
Show > Matches > Show Vectors
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Appendix
C
C.1
Appendix
Software references
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.
ExPASy Molecular Biology Server (2003) The Melanie 2-DE analysis software.
[Online] http://www.expasy.org/melanie.
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.
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Appendix
C.2
Statistical methods
Armitage P and Berry G (1987) Statistical methods and medical research. Oxford,
London: Blackwell Scientific Publications.
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.
C.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.
ExPASy Molecular Biology Server (2003) [Online] http://www.expasy.org.
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: 1082-1093.
276
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Appendix
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.
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 twodimensional differential gel electrophoresis proteomics technology. Proteomics 1:
377–396.
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Index
IX
Index
Numerics
2-DE gel electrophoresis ..................................................................................................................................... 7
3D view
display axes ..............................................................................................................................................106
synchronize all ........................................................................................................................................106
A
Ac category ..........................................................................................................................................................152
accession number .............................................................................................................................................152
action descriptors .............................................................................................................................................249
activate ImageMaster ........................................................................................................................................18
adapt gradations
inter-class histograms .........................................................................................................................222
intra-class histograms .........................................................................................................................204
adjust contrast .......................................................................................................................................................96
analyze menu ............................................................................................................................................. 23, 65
annotation ...............................................................................................................................................................12
add ................................................................................................................................................................171
add labels ..................................................................................................................................................153
combine sets ...............................................................................................................................163, 165
copy ..............................................................................................................................................................171
create ...........................................................................................................................................................152
create label categories ........................................................................................................................154
delete ...........................................................................................................................................................174
display .........................................................................................................................................................167
flag position ..............................................................................................................................................167
flagpole color ...........................................................................................................................................168
hide ...............................................................................................................................................................168
import ..........................................................................................................................................................175
link with spot ............................................................................................................................................154
modify .........................................................................................................................................................171
pack categories .........................................................................................................................163, 170
report ..............................................................................................................................................176, 177
select ............................................................................................................................................................161
select by category .................................................................................................................................162
select by content ....................................................................................................................................162
show .............................................................................................................................................................168
tool .........................................................................................................................................24, 161, 171
auto hide ...................................................................................................................................................................29
automatic spot detection ..............................................................................................................................128
B
backup workspace ................................................................................................................................... 62, 63
batch files conversion .....................................................................................................................................233
Bruker Proteineer SP spot picker ................................................................................................................241
C
calculated
MW ................................................................................................................................................................143
pI ....................................................................................................................................................................143
calibration .............................................................................................................................................................106
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IX
Index
control ......................................................................................................................................................... 110
export graph ............................................................................................................................................ 110
open ............................................................................................................................................................. 109
print graph ................................................................................................................................................ 110
report ........................................................................................................................................................... 110
save .............................................................................................................................................................. 109
strip ............................................................................................................................................................... 106
category ................................................................................................................................................................ 151
create .......................................................................................................................................................... 174
create sets ................................................................................................................................................. 158
data type ................................................................................................................................................... 155
external engine ....................................................................................................................................... 156
is unique ..................................................................................................................................................... 156
rename ....................................................................................................................................................... 173
report .............................................................................................................................................. 176, 177
central tendency ............................................................................................................................................... 199
class ................................................................................................................................................................. 12, 54
add gels in workspace ............................................................................................................................57
create in workspace ................................................................................................................................56
heuristic clustering ................................................................................................................................ 215
open ................................................................................................................................................................58
properties .....................................................................................................................................................59
comment category .......................................................................................................................................... 152
contextual menus ................................................................................................................................................28
gel image ................................................................................................................................................... 266
legend .......................................................................................................................................................... 266
pane banner ............................................................................................................................................. 266
worksheet banner ................................................................................................................................. 266
contrast mapping ................................................................................................................................................96
control
calibration ................................................................................................................................................. 110
cursor information ..................................................................................................................................95, 143
D
data analysis ....................................................................................................................................................... 195
differential gel electrophoresis ........................................................................................................................ 8
DIGE .............................................................................................................................................................................. 8
add gels to workspace ...........................................................................................................................44
histogram .................................................................................................................................................. 148
import gels in workspace ......................................................................................................................43
internal standard ......................................................................................................................................10
report ........................................................................................................................................................... 150
dispersion .............................................................................................................................................................. 200
display zone ............................................................................................................................................................24
displayed actions .............................................................................................................................................. 249
dockable ...................................................................................................................................................................28
windows ........................................................................................................................................................28
documents ..............................................................................................................................................................60
add files to workspace ...........................................................................................................................61
create folder in workspace ...................................................................................................................60
open ................................................................................................................................................................61
E
edit
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IX
menu ...............................................................................................................................................................23
edit spots ...............................................................................................................................................................139
eLicensing ................................................................................................................................................................18
exit ImageMaster ..................................................................................................................................................21
export ......................................................................................................................................................................237
gel data to XML .......................................................................................................................................238
F
factor analysis ....................................................................................................................................................209
interpret ......................................................................................................................................................210
report ...........................................................................................................................................................209
file
link .................................................................................................................................................................159
menu ...............................................................................................................................................................23
fitting report ............................................................................................................................................110, 196
floating ......................................................................................................................................................................29
floating license .......................................................................................................................................................18
folder
properties ......................................................................................................................................................45
further reading ....................................................................................................................................................276
G
GE Healthcare Ettan spot picker ................................................................................................................241
gel ................................................................................................................................................................................11
add to workspace .....................................................................................................................................43
align .................................................................................................................................................................92
anatomy ........................................................................................................................................................11
calibrate ......................................................................................................................................................106
calibration report ...................................................................................................................................122
create folder in workspace ...................................................................................................................42
crop ...............................................................................................................................................................118
delete ...........................................................................................................................................................114
description report ..................................................................................................................................120
erase ............................................................................................................................................................114
export ...........................................................................................................................................................124
gray levels ....................................................................................................................................................96
grid lines ........................................................................................................................................................90
IDs .....................................................................................................................................................................61
import in workspace ................................................................................................................................42
invert gray levels ....................................................................................................................................117
load image data ........................................................................................................................................94
move ...............................................................................................................................................................82
open ................................................................................................................................................................45
print ..............................................................................................................................................................125
properties ......................................................................................................................................................46
rename ........................................................................................................................................................114
rotate ...........................................................................................................................................................115
synthetic .....................................................................................................................................................191
unload image data ...................................................................................................................................94
zoom ...............................................................................................................................................................83
gels folder .................................................................................................................................................................41
Genetix GelPix spot picker .............................................................................................................................244
Genomic Solutions ProPic spot picker .....................................................................................................244
graphical user interface ....................................................................................................................................22
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IX
Index
grid lines ...................................................................................................................................................................90
H
hand tool ....................................................................................................................................................... 24, 82
hardware requirements ....................................................................................................................................17
help
menu ...............................................................................................................................................................23
online ................................................................................................................................................................ 2
user manual .................................................................................................................................................. 2
heuristic clustering ........................................................................................................................................... 214
histograms
inter-class .................................................................................................................................................. 220
inter-class and intra-class ................................................................................................................. 222
intra-class .................................................................................................................................................. 201
history ..................................................................................................................................................................... 253
copy to new script ................................................................................................................................. 254
navigator .................................................................................................................................................... 250
print .............................................................................................................................................................. 254
refresh ......................................................................................................................................................... 254
save .............................................................................................................................................................. 254
http link .................................................................................................................................................................. 159
I
ImageMaster
2D Elite ........................................................................................................................................................ 233
2D Platinum 5.0 ....................................................................................................................................... 233
DIGE .................................................................................................................................................................15
modules .........................................................................................................................................................15
window ..........................................................................................................................................................22
images
close ................................................................................................................................................................27
depth ...............................................................................................................................................................80
format .............................................................................................................................................................79
resolution ......................................................................................................................................................79
select ...............................................................................................................................................................27
stack ................................................................................................................................................................27
import ..................................................................................................................................................................... 237
gel data to XML ....................................................................................................................................... 240
installation ...............................................................................................................................................................17
intensity normalization ................................................................................................................................... 112
inter-class
histograms ................................................................................................................................................ 220
overlapping measures ......................................................................................................................... 216
report .............................................................................................................................................. 178, 224
specify ......................................................................................................................................................... 216
statistics ..................................................................................................................................................... 216
intra-class
histograms ................................................................................................................................................ 201
normalize ................................................................................................................................................... 204
report .............................................................................................................................................. 190, 205
isoelectric point calibration .......................................................................................................................... 142
K
keyboard shortcuts .............................................................................................................................................23
282
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IX
L
label ............................................................................................................................................................................12
add ................................................................................................................................................................171
copy ..............................................................................................................................................................172
delete ...........................................................................................................................................................174
display .........................................................................................................................................................167
duplicate .....................................................................................................................................................173
import ..........................................................................................................................................................175
modify .........................................................................................................................................................171
report ...........................................................................................................................................................176
select ............................................................................................................................................................161
landmark
annotations ...............................................................................................................................................183
category .....................................................................................................................................................152
legend ........................................................................................................................................................................27
link objects to external data .........................................................................................................................158
M
magnify tool ................................................................................................................................................. 24, 83
master image ......................................................................................................................................................179
match ............................................................................................................................................................12, 179
add ................................................................................................................................................................188
delete ...........................................................................................................................................................188
display .........................................................................................................................................................186
hide ...............................................................................................................................................................187
ID ....................................................................................................................................................................187
refine selection ........................................................................................................................................184
report ...........................................................................................................................................................189
select ............................................................................................................................................................183
sets ...................................................................................................................................................................13
show .............................................................................................................................................................187
show ID .......................................................................................................................................................187
statistics report .......................................................................................................................................190
vectors .........................................................................................................................................................187
matching ...............................................................................................................................................................179
annotations ...............................................................................................................................................183
automatic ..................................................................................................................................................181
landmarks ..................................................................................................................................................183
start ..............................................................................................................................................................182
matchsets ...................................................................................................................................................46, 179
add gels in workspace ............................................................................................................................51
copy and paste ..........................................................................................................................................51
create in workspace ................................................................................................................................49
master image ...........................................................................................................................................179
open ................................................................................................................................................................52
properties ......................................................................................................................................................53
reference image ...........................................................................................................................52, 179
measure histogram ..........................................................................................................................................146
Melanie ...................................................................................................................................................................233
menu bar ..................................................................................................................................................................23
min area .................................................................................................................................................................131
molecular weight calibration .......................................................................................................................142
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IX
Index
N
next .............................................................................................................................................................................27
node-locked license ............................................................................................................................................18
normalization ............................................................................................................................ 106, 111, 204
O
outliers .................................................................................................................................................................... 208
P
panes .........................................................................................................................................................................26
close ................................................................................................................................................................26
select ...............................................................................................................................................................26
stack ................................................................................................................................................................26
pI_MW category ................................................................................................................................... 143, 152
pinned ........................................................................................................................................................................28
population matching ....................................................................................................................................... 179
predefined label categories .......................................................................................................................... 151
previous ....................................................................................................................................................................27
profile ...................................................................................................................................................................... 101
projects .....................................................................................................................................................................39
add ...................................................................................................................................................................40
create .............................................................................................................................................................39
insert ...............................................................................................................................................................40
open ................................................................................................................................................................40
properties .....................................................................................................................................................40
view .................................................................................................................................................................35
protein databases ............................................................................................................................................. 245
query ............................................................................................................................................... 156, 246
pseudo colors ...................................................................................................................................................... 100
Q
query databases ............................................................................................................................................... 156
R
redo .......................................................................................................................................................................... 251
reference
maps ............................................................................................................................................................ 246
region tool ..................................................................................................................................................... 24, 81
regions .......................................................................................................................................................................11
regular expressions .......................................................................................................................................... 163
report .........................................................................................................................................................................59
3D view ..........................................................................................................................................................65
add to workspace .....................................................................................................................................59
create folder in workspace ...................................................................................................................59
export .......................................................................................................................................................... 238
gel .....................................................................................................................................................................65
gel calibration .............................................................................................................................................65
gel description ............................................................................................................................................65
IDs ....................................................................................................................................................................61
inter-class .................................................................................................................................................. 224
open ................................................................................................................................................................60
statistical tests ........................................................................................................................................ 230
toolbar ............................................................................................................................................................67
reports menu ............................................................................................................................................... 23, 65
284
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representative spots ........................................................................................................................................208
restore workspace ...............................................................................................................................................63
S
saliency ..................................................................................................................................................................130
scatter
plot ...................................................................................................................................................112, 196
report ...........................................................................................................................................................196
script ........................................................................................................................................................................255
create ...........................................................................................................................................................256
navigator ....................................................................................................................................................250
open .............................................................................................................................................................256
print ..............................................................................................................................................................256
run .................................................................................................................................................................256
save ..............................................................................................................................................................256
select
menu ...............................................................................................................................................................23
next ..................................................................................................................................................................68
on gels ............................................................................................................................................................68
previous .........................................................................................................................................................68
selection box
export ...........................................................................................................................................................118
import ..........................................................................................................................................................119
set category .........................................................................................................................................................152
setup wizard ...........................................................................................................................................................17
shortcuts ................................................................................................................................................................269
annotations ...............................................................................................................................................272
gels ................................................................................................................................................................270
matches ......................................................................................................................................................273
spots .............................................................................................................................................................271
show menu ..............................................................................................................................................................23
smooth ...................................................................................................................................................................130
software
features ............................................................................................................................................................ 1
references ..................................................................................................................................................275
requirements ...............................................................................................................................................17
sort
by similarity ..............................................................................................................................................205
values in inter-class histograms .....................................................................................................222
values in intra-class histograms .....................................................................................................205
spot .............................................................................................................................................................................12
add ................................................................................................................................................................141
automatic detection .............................................................................................................................129
borders ........................................................................................................................................................128
color ..............................................................................................................................................................137
delete ...........................................................................................................................................................142
delete part .................................................................................................................................................140
detection ....................................................................................................................................................127
detection parameters ..........................................................................................................................130
display .........................................................................................................................................................136
edit ................................................................................................................................................................138
export ...........................................................................................................................................................241
filter ...............................................................................................................................................................136
hide ...............................................................................................................................................................137
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IX
Index
ID ....................................................................................................................................................... 127, 138
increase size ............................................................................................................................................. 139
merge .......................................................................................................................................................... 141
pickers ......................................................................................................................................................... 241
preview of detection ............................................................................................................................. 128
report .............................................................................................................................................. 145, 208
save detection parameters ............................................................................................................... 130
select ............................................................................................................................................................ 135
shape ........................................................................................................................................................... 136
show ............................................................................................................................................................. 137
split ............................................................................................................................................................... 140
tool ......................................................................................................................................................24, 135
spot filtering ......................................................................................................................................................... 192
start ImageMaster ...............................................................................................................................................21
statistical tests .................................................................................................................................................... 225
Kolmogorov .............................................................................................................................................. 229
Mann-Whitney ........................................................................................................................................ 227
report ........................................................................................................................................................... 230
Smirnov ....................................................................................................................................................... 229
two-sample t test ................................................................................................................................... 226
Wilcoxon .................................................................................................................................................... 227
statistics
descriptive ................................................................................................................................................. 198
inter-class .................................................................................................................................................. 216
intra-class .................................................................................................................................................. 196
methodology ............................................................................................................................................ 276
status bar .................................................................................................................................................................28
step tablet ............................................................................................................................................................. 106
control ......................................................................................................................................................... 110
SWISS-2DPAGE gels ......................................................................................................................................... 246
synthetic gels ...................................................................................................................................................... 191
create .......................................................................................................................................................... 193
system requirements .........................................................................................................................................17
T
tabbed groups .......................................................................................................................................................30
text link ................................................................................................................................................................... 161
three-dimensional view .................................................................................................................................. 102
toolbar .......................................................................................................................................................................24
tools menu ...............................................................................................................................................................23
Twain compatible scanners ......................................................................................................................... 236
U
undo ......................................................................................................................................................................... 251
unique identifiers ..................................................................................................................................................61
un-pinned .................................................................................................................................................................29
UUIDs .........................................................................................................................................................................61
V
view menu ...............................................................................................................................................................23
W
window menu ........................................................................................................................................................23
workflow ........................................................................................................................................................ 13, 33
286
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worksheets ..............................................................................................................................................................25
close ................................................................................................................................................................26
rename ...........................................................................................................................................................25
select ...............................................................................................................................................................25
workspace
archive ............................................................................................................................................................62
backup ................................................................................................................................................ 62, 63
classes ............................................................................................................................................................54
create ..............................................................................................................................................................36
documents ...................................................................................................................................................60
gel icons .........................................................................................................................................................34
gels folder .....................................................................................................................................................41
hierarchy .......................................................................................................................................................37
matchsets .....................................................................................................................................................46
navigator .......................................................................................................................................................35
open ................................................................................................................................................................36
properties ......................................................................................................................................................39
reports ............................................................................................................................................................59
restore ............................................................................................................................................................63
save .................................................................................................................................................................36
structure ........................................................................................................................................................37
toolbar ............................................................................................................................................................35
window ...........................................................................................................................................................35
X
XML format ...........................................................................................................................................................237
XSL stylesheet .....................................................................................................................................................237
Z
zoom window .........................................................................................................................................................85
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ImageMaster 2D Platinum User Manual 11-0034-38 Edition AA
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 this User Manual, as well as on the
ImageMaster 2D Platinum software, belong to the Swiss Institute of
Bioinformatics. No part of this User Manual may be reproduced or transmitted
in any form or by any means, electronic or mechanical, including photocopy,
recording or any information storage or retrieval system, without permission in
writing from the Swiss Institute of Bioinformatics.
© 1992-2005 Swiss Institute of Bioinformatics - All rights reserved.
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ImageMaster 2D Platinum 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
copyright, and all commercial users of these databases are required to
purchase a database license from Geneva Bioinformatics (GeneBio) SA. Please
contact GeneBio at [email protected] for more information.
Geneva Bioinformatics (GeneBio) SA
Avenue de Champel 25, CH-1206 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 (http://www.apache.org).
© 1999-2003 The Apache Software Foundation - All rights reserved.
www.amershambiosciences.com
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Cy, CyDye, Ettan, Typhoon, DeCyder, ImageMaster, LabScan and
ImageScanner are trademarks of GE Healthcare Ltd. GE tagline and GE
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SYPRO is a trademark of Molecular Probes Inc.
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All goods and services are sold subject to the terms and conditions of sale of
the company within GE Healthcare which supplies them. GE Healthcare
reserves the right, subject to any regulatory and contractual approval, if
required, to make changes in specifications and features shown herein, or
discontinue the product described at any time without notice or obligation.
Contact your local GE Healthcare representative for the most current
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imagination at work
User Manual 11-0034-38 AA
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