PDQuest - Bio-Rad
PDQuest™
User Guide for Version 7.2
Windows and Macintosh
P/N 4000129-16 RevA
PDQuest User Guide
Bio-Rad Technical Service Department
Phone:
800-424-6723
510-741-2612
Fax:
510-741-5802
E-mail:
[email protected]
Notice:
No part of this publication 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 Bio-Rad.
PDQuest and The Discovery Series are trademarks of Bio-Rad Laboratories. All
other trademarks and registered trademarks are of their respective companies.
WASTE text engine © 1993–1997 Marco Piovanelli
Limitations of Liability:
Bio-Rad is not responsible for the misinterpretation of results obtained by following
the instructions in this guide. Whenever possible, you should contact the Technical
Services Department at Bio-Rad to discuss your results. As with all scientific studies,
we recommend that you repeat your experiment at least once before making any
significant conclusions for presentation or publication.
Copyright © 2003 by Bio-Rad Laboratories. All rights reserved.
ii
Table of Contents
1. Introduction ........................................................................ 1-1
1.1. Overview of PDQuest ..................................................................................... 1-1
1.2. Digital Data and Signal Intensity .................................................................... 1-2
1.3. PDQuest Workflow ....................................................................................... 1-4
1.4. Computer Requirements ................................................................................. 1-7
1.5. Installation ...................................................................................................... 1-8
1.6. Software License .......................................................................................... 1-11
1.7. Downloading from the Internet .................................................................... 1-15
1.8. PDQuest Basic .............................................................................................. 1-16
1.9. Contacting Bio-Rad ...................................................................................... 1-16
2. General Operation .............................................................. 2-1
2.1. Graphical Interface ......................................................................................... 2-1
2.2. Keyboard Shortcuts ........................................................................................ 2-5
2.3. File Commands and Functions ....................................................................... 2-7
2.4. Printing and Exporting .................................................................................. 2-20
2.5. Printing ......................................................................................................... 2-21
2.6. Exporting ...................................................................................................... 2-27
2.7. Preferences .................................................................................................... 2-37
2.8. Mouse-assignable Tools ............................................................................... 2-48
3. Viewing and Editing Images ............................................. 3-1
3.1. Windows and Subwindows ............................................................................. 3-1
3.2. Configuring Subwindows ............................................................................... 3-1
3.3. Assigning and Interchanging Images .............................................................. 3-3
3.4. Tiling Windows .............................................................................................. 3-4
3.5. Magnifying Images ......................................................................................... 3-4
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PDQuest User Guide
3.6. Positioning Images .......................................................................................... 3-7
3.7. Hiding Overlays ............................................................................................ 3-10
3.8. Density Tools ................................................................................................ 3-11
3.9. Colors ............................................................................................................ 3-13
3.10. Multi-Channel Viewer ................................................................................ 3-16
3.11. 3D Viewer ................................................................................................... 3-18
3.12. Transform .................................................................................................... 3-21
3.13. Cropping Images ......................................................................................... 3-26
3.14. Flipping and Rotating Images ..................................................................... 3-29
3.15. Filtering Images .......................................................................................... 3-32
3.16. Invert Data ................................................................................................... 3-38
3.17. Text Overlays .............................................................................................. 3-38
4. Detecting and Editing Spots ............................................. 4-1
4.1. Selecting Spot Detection Parameters .............................................................. 4-1
4.2. Automated Detection and Matching ............................................................... 4-9
4.3. Filtered and Gaussian Images ....................................................................... 4-12
4.4. Spot Crosshairs and Ellipses ......................................................................... 4-14
4.5. Adding and Removing Spots ........................................................................ 4-16
4.6. Spot Boundary Tools .................................................................................... 4-19
4.7. Cancelling Spots ........................................................................................... 4-22
4.8. Combining Spots ........................................................................................... 4-23
4.9. Saturated and Faint Spots .............................................................................. 4-24
4.10. Spot Quantity .............................................................................................. 4-24
4.11. Spot Quality ................................................................................................ 4-27
4.12. Spot Parameters ........................................................................................... 4-28
4.13. Finding Spots .............................................................................................. 4-29
5. MatchSets ........................................................................... 5-1
5.1. Creating a MatchSet ........................................................................................ 5-3
5.2. Matching Spots ............................................................................................. 5-12
5.3. Landmarking Spots ....................................................................................... 5-17
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Contents
5.4. Displaying Matches ...................................................................................... 5-20
5.5. Cybergels ...................................................................................................... 5-21
5.6. Adding Spots to the Master .......................................................................... 5-24
5.7. Edit Match Submenu .................................................................................... 5-27
5.8. Matching Summary ...................................................................................... 5-36
6. Analysis Tools .................................................................... 6-1
6.1. Spot Review Tool ........................................................................................... 6-1
6.2. Image Stack Tool ............................................................................................ 6-5
6.3. Scatter Plot Tool ............................................................................................. 6-7
6.4. Replicate Groups ............................................................................................ 6-9
6.5. Group Consensus .......................................................................................... 6-13
6.6. Sample Database ........................................................................................... 6-17
6.7. Normalization ............................................................................................... 6-23
6.8. MrpI Data ...................................................................................................... 6-29
6.9. Standard Spot Numbers ................................................................................ 6-32
6.10. Database Spot Numbers .............................................................................. 6-34
7. Analysis Sets and Annotations ........................................ 7-1
7.1. Analysis Sets ................................................................................................... 7-1
7.2. Annotation Tool ............................................................................................ 7-24
7.3. Browsing Annotations .................................................................................. 7-35
7.4. Creating Annotations from Analysis Sets ..................................................... 7-36
7.5. Printing Annotations ..................................................................................... 7-38
7.6. Transferring Annotations .............................................................................. 7-39
8. Basic Excision Tool ........................................................... 8-1
8.1. Spot Cutter Setup ............................................................................................ 8-2
8.2. Cutting Spots .................................................................................................. 8-9
8.3. Other Spot Cutter Controls ........................................................................... 8-26
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PDQuest User Guide
9. Integrated Excision Tool ................................................... 9-1
9.1. Excision Gel Selection .................................................................................... 9-2
9.2. Integrated Excision Tool ................................................................................. 9-9
10. Mass Spectrometry Analysis .......................................... 10-1
10.1. Setting up a PLGS run ................................................................................ 10-1
10.2. Exporting a MassLynx Worksheet .............................................................. 10-2
10.3. Importing PLGS Results ............................................................................. 10-3
10.4. Mass Spec Score Overlay ........................................................................... 10-5
10.5. Show Identified Spots ................................................................................. 10-6
10.6. Import Mascot Results ................................................................................ 10-7
11. Graphs and Reports ........................................................ 11-1
11.1. Configure Graphs ........................................................................................ 11-1
11.2. Histogram Graphs ....................................................................................... 11-4
11.3. A-B Overlay ................................................................................................ 11-7
11.4. Quantity Table Report ................................................................................. 11-9
11.5. MatchSet Member Report ......................................................................... 11-14
11.6. Scatter Plot Report .................................................................................... 11-16
11.7. Analysis Set Report ................................................................................... 11-17
11.8. Image Report ............................................................................................. 11-19
Appendix A.
Gel Doc EQ ................................................................................ A-1
Appendix B.
ChemiDoc EQ ........................................................................... B-1
Appendix C.
ChemiDoc XRS ......................................................................... C-1
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Contents
Appendix D.
GS-800 Imaging Densitometer ................................................ D-1
Appendix E.
Personal Molecular Imager FX ................................................ E-1
Appendix F.
Molecular Imager FX Family (FX Pro, FX Pro Plus and Molecular
FX) .............................................................................................. F-1
Appendix G.
VersaDoc .................................................................................. G-1
Appendix H.
Calibration and Merging .......................................................... H-1
Appendix I.
Cross-Platform File Exchange ................................................. I-1
Appendix J.
21 CFR Part 11 .......................................................................... J-1
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PDQuest User Guide
viii
Preface
1. About This Document
This user guide is designed to be used as a reference in your everyday use of
PDQuest™. It provides detailed information about the tools and commands of
PDQuest for the Windows and Macintosh platforms. Any platform differences in
procedures and commands are noted in the text.
This guide assumes you have a working knowledge of your computer operating
system and its conventions, including how to use a mouse and standard menus and
commands, and how to open, save, and close files. For help with any of these
techniques, see the documentation that came with your computer.
This guide uses certain text conventions to describe specific commands and
functions..
Example
Indicates
File > Open
Choosing the Open command under the File menu.
Dragging
Positioning the cursor on an object and holding down
the left mouse button while you move the mouse.
CTRL+S
Holding down the Control key while typing the letter s.
Right-click/
Left-click/
Double-click
Clicking the right mouse button/
Clicking the left mouse button/
Clicking the left mouse button twice.
Some of the illustrations of menus and dialog boxes found in this manual are taken
from the Windows version of the software, and some are taken from the Macintosh
version. Both versions of a menu or dialog box will be shown only when there is a
significant difference between the two.
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PDQuest User Guide
2. Overview of Raw 2-D Gel Electrophoresis
Raw 2-D electrophoresis is a method for separating proteins and nucleic acids in a
sample into a two-dimensional pattern of spots in a gel. It combines the techniques of
isoelectric focusing (IEF) with SDS-polyacrylamide gel electrophoresis (SDSPAGE). Since these two separation methods rely on independent properties of
proteins—chemical and physical—this procedure can resolve complex biological
samples with a high degree of specificity and accuracy. The resolved proteins and
polypeptide fractions can be then identified by their molecular weights and charges
(as indicated by their locations in the Raw 2-D gel), as well as by their differential
expression in different samples, proximity to other spots, intensity, etc.
Raw 2-D electrophoresis involves two sequential separations of a sample in
perpendicular directions. The IEF dimension is run first, in tube gels or on
immobilized pH gradient (IPG) strips. After focusing, the tube gel or strip is placed
on top of an SDS-PAGE slab gel and electrophoresed. This technique can resolve
thousands of protein spots in a single sample; these proteins can then be visualized by
metabolic radiolabeling or a variety of staining methods.
Proteomics Applications
Proteomics is the study of protein expression and regulation in cells, tissues, and
entire organisms. Several thousand proteins are expressed at any given moment in an
organism; at the cellular level, dozens of proteins may be expressed and regulated in
fractions of a second.
Two-dimensional gel electrophoresis is a a cornerstone in the study of how proteins
are expressed, regulated, and modified throughout living systems. Developed almost
a quarter of a century ago, Raw 2-D gel technology remains one of the most powerful
techniques for resolving complex mixtures of proteins. The technology has improved
significantly over the past several years, with the advent of IPG strips and simplified
gel running techniques, large-format and cyber gels that allow for greater pH range
and specificity, and new stains and staining techniques. In addition, mass
spectrometry now allows for peptide mass fingerprinting of very small amounts of
protein isolated from gels.
Using a combination of these techniques, pharmaceutical companies can now use
Raw 2-D technology for high-throughput screening of drug compound candidates
using protein targets; research laboratories can study large-scale changes in protein
x
Preface
expression; and companies and institutions can cross-identify and catalog hundreds of
thousands of protein species at the cellular level. This provides an excellent technique
for the study of differential gene expression under various growth conditions. Since
the expression and regulation of individual proteins can be detected,Raw 2-D gels are
an indirect way to monitor gene activity. They allow for the investigation of
quantitative as well as qualitative changes in cellular protein expression.
The environmental conditions of a cell can be changed in order to determine optimal
growth conditions as well as monitor the cell’s response to different stresses.
Environmental conditions that can stress the cell include changes in temperature, pH,
and nutrient availability. Some examples of the chemical stresses that can be placed
on a cell include drug and hormone treatments. Since protein structure and function
are the direct result of gene expression, the loss or change of a protein as detected by
a Raw 2-D gel can be extrapolated back to events occurring at the DNA level.
Many questions encountered when genes are inserted and expressed in bacteria, yeast,
and other cell types can be answered with Raw 2-D analysis: Is the cell making the
protein? Is the cell’s progeny making the protein? Is the protein being made but not
secreted? Have mutations occurred?
Medical Applications
Raw 2-D gels can also have important applications in medical research. For example,
this technique can be used to verify the presence of specific protein markers that are
linked to genetic diseases and disease states. Used in conjunction with other tests,
Raw 2-D gels can be part of medical screening procedures associated with mutations
and teratology linked to genetic damage.
Growth factors are being studied for their role in the regulation of cell growth. Raw 2D gels can be used to evaluate quantitative and qualitative changes in cellular proteins
in response to growth factor stimulation.
Raw 2-D gels allow the visualization of proteins whose expression is altered as the
result of cell transformation, introducing oncogenes into the host genome.
Assessment of phosphorylation, sulfation, or other secondary modifications could
reveal functional protein pathways affected by oncogene expression. This information
could contribute to a better understanding of cell growth and regulation.
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PDQuest User Guide
Sample Experiments
Valuable information can be learned by exposing cells to a set of specific
experimental conditions and subsequently examining their biological response.
A preliminary in vitro experiment that is useful when beginning Raw 2-D gel work is
to radiolabel a cell’s proteins to steady-state with 35S-methionine. Running gels of
these proteins will help to determine the commonly expressed proteins in the cell
under normal conditions.
Subsequently, other amino acids such as 3H-leucine, 3H-proline, 3H-lysine, etc., can
be used to radiolabel the proteins to steady-state. This will label any proteins that do
not contain methionine and were, therefore, not detected in the first experiment, and
will provide preliminary information on the amino acid composition.
Other experiments complementary to Raw 2-D gels include: cell fractionation
procedures, and post-translational modifications (phosphorylation, methylation, etc.).
Data from these experiments can be added to a database, accumulating information
about these proteins.
3. Bio-Rad Listens
The staff at Bio-Rad are receptive to your suggestions. Many of the new features and
enhancements in this version of PDQuest are a direct result of conversations with our
customers. Please let us know what you would like to see in the next version of
PDQuest by faxing, calling, or e-mailing our Technical Services staff. You can also
use Solobug (installed with PDQuest) to make software feature requests.
xii
1. Introduction
1.1
Overview of PDQuest
PDQuest is a software package for imaging, analyzing, and databasing Raw 2-D
electrophoresis gels.
The software runs in a Windows or Macintosh environment and has a graphical
interface with standard pull-down menus, toolbars, and keyboard commands.
PDQuest can acquire images of gels using any of several Bio-Rad imaging systems.
An image of a gel is captured using the controls in the imaging device window and
displayed on your computer screen. The scanned gel can then be cropped, rotated, etc.
using the image editing controls.
Spot Detection, Analysis and Databasing
With Automated Spot Detection and Matching you can select the gels you want to
anlayze, detect spots of interest, create a MatchSet, and match gels all from one
dialog box. The Spot Detection Wizard guides you through the process of identifying
and quantifying the spots in the gel image.
After detection, gels in the same experimental series are placed in a MatchSet for
comparison, statistical analysis, and databasing. Histograms allow you to quickly
compare the quantities of the same spot in all the gels in a MatchSet. Spots can also
be compared qualitatively, organized into user-defined sets for further analysis, and
annotated and databased for easy identification. Spots from different experimental
series can be organized and compared in high-level MatchSets.
PDQuest can be used to simultaneously analyze thousands of spots on hundreds of
gels. Data can be exported to other applications such as spreadsheets for further
analysis.
1-1
PDQuest User Guide
Mass Spec Analysis
PDQuest is part of Bio-Rad’s ProteomeWorks protein analysis package, and controls
Bio-Rad’s ProteomeWorks Spot Cutter. You can cut spots from gels or membranes
using PDQuest, digest them, and perform advanced protein analysis using
Micromass’s mass spectrometry instruments and software. Data from Micromass can
then be imported back into PDQuest to be included in spot annotations.
And More...
Scan files acquired in PDQuest can be analyzed using other Bio-Rad Discovery
Series software applications, such as Quantity One or Diversity Database. Scans can
be converted into TIFF format for easy compatibility with other applications.
1.2
Digital Data and Signal Intensity
The Bio-Rad imaging devices supported by PDQuest are light and/or radiation
detectors that convert signals from biological samples into digital data. PDQuest then
displays the digital data on your computer screen, in the form of gray scale or color
images.
A data object as displayed on the computer is composed of tiny individual screen
pixels. Each pixel has an X and Y coordinate, and a value Z. The X and Y coordinates
are the pixel’s horizontal and vertical positions on the image, and the Z value is the
signal intensity of the pixel.
1-2
Introduction
intensity
Signal intensity of a single pixel
3-D View
2-D View
Fig. 1-1. Representation of the pixels in two digitally imaged spots in a gel.
For a data object to be visible and quantifiable, the intensity of its clustered pixels
must be higher than the intensity of the pixels that make up the background of the
image. The total intensity of a data object is the sum of the intensities of all the pixels
that make up the object. The mean intensity of a data object is the total intensity
divided by the number of pixels in the object.
The units of signal intensity are Optical Density (O.D.) in the case of the GS-700 and
GS-710 densitometers, the Gel Doc EQ and ChemiDoc EQ with a white light source,
or the Fluor-S and Fluor-S MAX MultiImagers with white light illumination. Signal
intensity is expressed in counts when using the Personal FX or FX, or in the case of
the Gel Doc EQ, ChemiDoc EQ, Fluor-S, or Fluor-S MAX when using the UV light
source.
1-3
PDQuest User Guide
1.3
PDQuest Workflow
Acquire the image
Size and orient the image
Spot identification
Spot comparison and
matching
Data analysis
Spot cutting and mass spec
analysis
Publish results
Fig. 1-2. Steps involved in using PDQuest.
Image Acquisition
PDQuest can acquire images of gels using Bio-Rad’s densitometers, storage phosphor
imagers, and camera-based imaging systems.
First you open the acquisition window for your imaging device, and capture the
image using the controls. Gel images are saved on your hard disk, network file server,
or removable storage media. The displayed image in PDQuest is ready for analysis.
Image Sizing and Orientation
In this step, you adjust the size and orientation of the image by using the cropping and
rotating tools on the Image menu.
1-4
Introduction
Spot Identification
Now you are ready to identify the spots in the gel. The Spot Detection Wizard
automates the process of selecting the proper spot detection parameters for your gels.
Using the Wizard, you select the parameters, study the results of spot detection, then
adjust the parameters until you have identified all spots of interest in your gels.
When spots are detected in PDQuest, the original gel image is filtered and smoothed
to clarify the spots, then three-dimensional Gaussian spots are created from the
clarified spots. The end result is three separate images: the original unaltered scan (2D Scan), the filtered and processed scan (Filtered image), and a synthetic image
containing the Gaussian spots (Gaussian image).
2-D Scan
Filtered Image
Gaussian Image
Fig. 1-3. Images created during spot detection.
All spot matching and analysis in PDQuest are performed on Gaussian spots.
What Are Gaussian Spots?
Fuzzy, streaked, or overlapping spots in a 2-D gel can be difficult to accurately
distinguish and quantify. Because the image profile of an ideal spot confirms to a
Gaussian curve, PDQuest uses Gaussian modeling to generate spots that can be
precisely identified and quantitated.
A Gaussian spot is a three-dimensional representation of an original scanned spot.
Gaussian curves are fitted to the scanned spot in the X and Y dimensions, and then
additional modeling is performed to create the final Gaussian spot.
Using Gaussian modeling, you can accurately quantitate overlapping spots, spots in
gel streaks, and multiple spots in dense clusters.
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PDQuest User Guide
Matching and Editing
After you have detected the spots in a gel or set of gels, you are ready to create a
MatchSet. A MatchSet is composed of the Raw 2-D, Filtered and Gaussian images of
the gel(s) in an experiment.
In a MatchSet, the protein spots from the different gels are matched to each other and
are included in a synthetic image called a MatchSet standard. The standard includes
all the information about the spots in the MatchSet.
As you match the spots in a MatchSet, you will correct the results of automatic spot
detection by comparing the Gaussian spots with the original spots in the Filtered
image.
Data Analysis
PDQuest provides a variety of analytical tools to help you determine which spots are
statistically and scientifically meaningful.
You can normalize the spot quantities in different gels for more accurate comparison.
You can define replicate groups of duplicate gels. You can create groups of spots that
are quantitatively, qualitatively, and statistically significant using analysis sets. You
can compare the similarity of gels using scatter plots, and review the quantitation of
individual spots using the Spot Review Tool.
Many of these tools are interactive with the MatchSet, so you can study the actual
spots in the images as you review their quantities and other data.
You can create high-level MatchSets to compare the results of different experiments.
And the powerful annotation tool allows you to annotate your spots, link to Internet
protein databases or other files, and create HTML pages of spot data.
Spot Cutting and Mass Spec Analysis
PDQuest is part of Bio-Rad’s ProteomeWorks protein analysis package, and controls
Bio-Rad’s ProteomeWorks Spot Cutter. You can cut spots from gels or membranes,
digest them, and perform advanced protein analysis using Micromass’s mass
spectrometry instruments and software. Data from Micromass can then be imported
back into PDQuest to be included in spot annotations.
1-6
Introduction
Publish Results
When your analysis is complete, you can print your experimental data or export it to
another system for further analysis.
1.4
Computer Requirements
This software is supported on Windows XP and Windows 2000, or on a Macintosh
PowerPC running MacOS 9.2.2 or MacOS 10.2.6.
The computer memory requirements are mainly determined by the file size of the
images you will scan and analyze. High-resolution image files can be very large. For
this reason, we recommend that you archive images on a network file server or highcapacity removable disk.
PC
The following is the recommended system configuration for installing and running
on a PC:
Operating system:
Windows 2000
Windows XP
Processor:
Pentium ≥ 333 MHz or faster.
RAM:
≥ 256 MB for all Bio-Rad imaging systems.
Hard disk space:
≥ 3 GB
Monitor:
17" monitor, 1024 x 768 resolution (required), True color.
USB port:
Required for the Hardware Security Key (HSK).
Printer:
Optional.
Macintosh
The following is the recommended system configuration for installing and running
on a Macintosh:
1-7
PDQuest User Guide
Operating system:
MacOS 9.2.2, MacOS 10.2.6.
Processor/Model:
PowerPC G3 processor or faster.
RAM:
≥ 256 MB for all Bio-Rad imaging systems.
Hard disk space:
≥ 3 GB
Monitor:
17" monitor, 1024 x 768 resolution (required), Millions of
colors.
USB port:
Required for the Hardware Security Key (HSK).
Printer:
Optional.
Note:
1.5
The default amount of memory assigned to this program on the Macintosh is 128
MB. If the total RAM in your Macintosh is 128 MB or less, you should reduce
the amount of memory assigned to the program to 10 MB less than your total
RAM. With the application icon selected, go to File > Get Info in your Finder to
reduce the memory requirements for the application. See your Macintosh
computer documentation for details.
Installation
Refer to the Installation Guide for detailed information regarding installation of The
Discovery Series.
1.5.a Hardware Security Key (HSK)
The Discovery Series software is password-protected using a Hardware Security Key
(HSK), which is included in your software package. You must attach the Hardware
Security Key to your computer before you can run the software.‘
1-8
Introduction
Fig. 1-4. Hardware Security Key
Before proceeding with installation, plug the HSK into any available USB port on
your computer.
The code for the HSK is EYYCY, which is printed on the key itself. Use this code to
identify the HSK that belongs to The Discovery Series software.
Note:
Initial installation of a network server requires the Hardware Security Key
included in the software package. Installation of an additional Network Client
User to a Network License Server System does not require an HSK. Please refer
to the Network License Installation Guide that ships with Network License.
1.5.b Installing The Discovery Series for Windows
Note:
You must be a member of the Administrators group to install The Discovery
Series software.
Insert The Discovery Series CD-ROM. The installation wizard will start
automatically. (If the CD does not auto-start, click Start in your taskbar then click
Run. In the Open field type d:\setup.exe where d is the letter of your CD-ROM drive.)
Select the software application you want to install. On each panel, click Next when
you are ready to proceed. You must accept the license agreement to continue with
installation.
The installer program guides you through the installation. The installer creates a
default directory under Program Files on your computer called Bio-Rad\The
Discovery Series (to select a different directory, click Browse). The application
program will be placed in the Bin folder inside The Discovery Series folder. An
additional folder for storing sample images is also located in The Discovery Series
folder. User profiles will be created and stored in the Documents and Settings folder
for each user.
1-9
PDQuest User Guide
The installer places a shortcut to the application and user guide on your desktop and
creates a The Discovery Series folder in Programs on your Windows Start menu.
After installation, you must reboot your computer before using an imaging device.
Note:
If you are installing in a Windows 2000 environment, you must start the
application before allowing any other user access to the application.
Uninstalling The Discovery Series from Windows
If you need to uninstall The Discovery Series for any reason, go to Add/Remove
Programs in the Control Panel. Highlight the application you want to remove and
click Remove.
Note:
Uninstalling The Discovery Series software does not remove any system files in
the Documents and Settings folder located in Application Data. If you want to
remove these files as well, you must do so manually.
1.5.c Installing the Discovery Series for the Macintosh
The Mac install process involves installation of the application, dongle drivers, and
Roper support. Insert the Discovery Series CD-ROM into your Macintosh. The TDSMac folder opens on your desktop, displaying the installers for The Discovery Series
applications. Double-click on the installer for your application.
Fig. 1-5. Installation program icon (Macintosh).
Insert the Discovery Series CD-ROM. The TDS-Mac folder opens on your desktop,
displaying the installers for The Discovery Series applications. Double-click the
installer for your application. You must accept the license agreement to proceed with
installation. The ReadMe contains important information about the HSK. Click
Continue.
The installation wizard lists the types of installations in the pull down list. If you want
to install the application and documentation only, select Easy Install from the list.
1-10
Introduction
If you want to install drivers as well, select Custom from the pull down list.
(Remember to also select the application in custom install.)
Note:
If the computer is going to be used to acquire images from Gel Doc EQ,
ChemiDoc EQ, ChemiDoc XRS, or the VersaDoc system, then select the driver
for your particular imaging device now.
Install Location identifies where the application will be installed. If you want to
specify a different location, click the pull-down button.
Click Install to proceed. Once istallation is complete, click Quit. To return to the
installer, click Continue.
Uninstalling The Discovery Series from a Macintosh
Insert The Discovery Series CD-ROM. The TDS-Mac folder opens on your desktop,
displaying the installers for The Discovery Series applications. Double-click the
installer for your application. In the installer screen, select Uninstall from the pull
down list.
1.6
Software License
When the software opens for the first time, you will see a Software License screen
that shows the current status of your software license.
With a new HSK or network license, you receive a 30-day temporary license (“Your
license will expire on _______”). The temporary license is designed to give you time
to purchase the software, if you have not already done so.
1-11
PDQuest User Guide
Fig. 1-6. Temporary license screen.
During the 30-day period, the Software License screen will appear every time you
open the software. To use the software during this period, click on the Run button.
Network license holders can click on the Check License button at any time during the
30-day period to activate their full network license. (If your network license is not
activated when you click on Check License, notify your network administrator.)
HSK users have 30 days to purchase the software and obtain a purchase order number
and software serial number from Bio-Rad. When you have this information, click on
the Check License or Registration Form button in the Software License screen to
register your software.
1-12
Introduction
Fig. 1-7. Software License Registration Form.
Fill out the information in the Software License Registration Form. Be sure to enter
your purchase order number and software serial number under the Purchase
Information tab when registering.
Registering by Internet
If you have Internet access from your computer, click on the Submit via Internet
button to send the Software Registration Form directly to Bio-Rad.
Your information will be submitted, and a temporary password will be generated
automatically and sent back to your computer. Simply continue to run the application
as before.
Bio-Rad will confirm your purchase information and generate a permanent license.
After 2–3 days, click on Check License in the Software License screen again to
update to a permanent password. (The Software License screen will not appear
automatically after the temporary password has been generated; the software will
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PDQuest User Guide
simply open normally. Go to the Help menu and select Register to open the Software
License screen.)
Registering by Fax or E-mail
If you do not have Internet access, click on the Print button in the Software License
Registration Form and fax the form to Bio-Rad at the number listed on the form.
Alternatively, you can enter the contents of the form into an e-mail and send it to BioRad at the address listed in the Registration Form.
Bio-Rad will contact you by fax or e-mail in 2–3 days with a full license.
Entering a Password
If you fax or e-mail your registration information, you will receive a password from
Bio-Rad. You must enter this password manually.
To enter your password, click on Enter Password in the Software License screen. If
you are not currently in the Software License screen, select Register from the Help
menu.
Fig. 1-8. Enter Password screen.
In the Enter Password screen, type in your password in the field.
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Introduction
Once you have typed in the correct password, the OK light next to the password field
will change to green and the Enter button will activate. Click on Enter to run the
program.
1.7
Downloading from the Internet
You can download a trial version of the software from Bio-Rad’s Web site. Go to the
Discovery Series download page at www.bio-rad.com and select from the list of
applications. Follow the instructions to download the installer onto your computer,
then run the installer.
After installation, double-click on the application icon to run the program. The
software will open and the Software License screen will be displayed.
Note:
If you attempt to start the downloaded program and receive an “Unable to obtain
authorization” message, you will need a Hardware Security Key to run the
program. Contact Bio-Rad to obtain a key.
Fig. 1-9. Free Trial screen.
In the Software License screen, click on the Free Trial button. This will open the
Software License Registration Form. Enter the required information (you will not
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PDQuest User Guide
have a purchase order number or software serial number, and can leave these fields
blank) and click on Submit Via Internet.
A free trial password will be automatically downloaded to your computer. This
password will allow you to use the software for 30 days.
If you decide to purchase the software during that period, contact Bio-Rad to receive
a software package and a Hardware Security Key. You can then complete the
registration process as described in the previous sections.
1.8
PDQuest Basic
In order to meet requests for additional software copies at low cost when buying an
imaging system, Bio-Rad has provided a scaled down version of PDQuest called
PDQuest Basic. PDQuest Basic looks and acts exactly like the full version of
PDQuest but with the advanced functions inactive (grayed out).
The Basic version of PDQuest includes the following active functionality: Image
acquisition, Transform, Crop, Flip, Rotate, Text Tool, Basic Excision, Print, and
Export to TIFF and Save. PDQuest Basic also allows you to view previously saved
data created using advanced functionality.
PDQuest Basic does not require a software license. To activate the advanced
commands of PDQuest, contact Bio-Rad for a valid user license.
1.9
Contacting Bio-Rad
Bio-Rad technical service hours are from 8:00 a.m. to 4:00 p.m., Pacific Standard
Time in the U.S.
Phone:
800-424-6723
510-741-2612
Fax:
510-741-5802
E-mail:
[email protected]
For software registration:
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Introduction
Phone:
800-424-6723 (in the U.S.)
+1-510-741-6996 (outside the U.S.)
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2. General Operation
2.1 Graphical Interface
2.1.a Menu Bar
PDQuest has a standard menu bar with pulldown menus that contain all the major
features and functions available in the software.
File Commands Viewing Commands
Toolbars
Fig. 2-1. Main toolbar.
The pulldown menus contain:
•
File—General file commands (Open, Save, New MatchSet, Revert to Saved),
imaging device acquisition windows, and others.
•
Edit—Text overlays, Calibration, Preferences, Mouse commands, and others.
•
View—Toolbars, Zoom Box, View Density, Advanced View, Multichannel View
and others.
•
Image—Image Info, Transform, Advanced Crop, Filter List, and others.
•
Spots—Automated Detection and Matching, Spot Detection Report, Spot
Editing, Crosshairs, Quantity, and Quality and others.
•
Match—Automated and Classic Spot Matching, Edit matching tools, Cybergels
and others.
•
Analysis—Analysis set functions, Annotation tools, MrpI Standards, Replicate
groups functions, and others.
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PDQuest User Guide
•
Identification—Excision (Spot cutting) and Mass Spectrometry analysis tools.
•
Reports—Graphs and Reports. Print and Export.
•
Window—Tile windows, Imitate Zoom, Configure Subwindows and others.
•
Help—Quick Reference Guide, On-line Help, Keyboard Layout, Software
Registration.
Below the menu bar is the main toolbar, containing some of the most commonly used
commands. Next to the main toolbar is a status box which provides information about
cursor selection and toolbar buttons.
2.1.b Main Toolbar
The main toolbar in PDQuest appears below the menu bar. It includes buttons for the
main file commands (Open, Save, Print) and essential viewing tools (Zoom Box,
Grab), and buttons to open the secondary toolbars. See Fig. 2-1., Main toolbar..
Tool Help
Status Boxes
Graph Tools
Fig. 2-2. Tool Help and Status Box
Tool Help
If you hold the cursor over a toolbar icon, the name of the command will pop up
below the icon. This utility is called Tool Help. Tool Help appears on a time delay
basis that can be specified in the Display Preferences dialog box under Edit >
Preferences. You can also specify how long the Tool Help will remain displayed.
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General Operation
2.1.c Status Boxes
There are two status boxes in PDQuest. These appear to the right of the main toolbar.
The first box displays any function that is assigned to the mouse (see section 2.8,
Mouse-assignable Tools). If you select a command such as Zoom Box, the name and
icon of that command will appear in this status box and remain there until another
mouse function is selected or the mouse is deassigned.
The second status box is designed to supplement Tool Help (see above). It provides
additional information about the toolbar buttons. If you hold your cursor over a
button, a short explanation about that command will be displayed in this second status
box.
2.1.d Secondary Toolbars
PDQuest has secondary toolbars that contain icons for groups of associated functions.
You can open these toolbars from the main toolbar or from the View > Toolbars
pulldown menu.
The secondary toolbars can be toggled between vertical, horizontal, and expanded
formats by clicking the resize button on the toolbar itself.
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Horizontal format
Expanded format
Vertical format
Hold cursor over icon
to reveal Tool Help
Click question marks
for on-line help
Click resize button
to toggle format
Fig. 2-3. Secondary toolbar formats and features.
The expanded toolbar format shows the name of each of the commands. Click the
Resize button to access the extended format with direct links to the on-line Help file
for that command.
2.1.e Quick Guide
The PDQuest Quick Guide (accessed from the Help menu) is a tool palette that is
organized to guide you through the major functions of the software. Like the
secondary toolbars, it can be toggled between vertical, horizontal, and expanded
formats by clicking on the resize button.
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General Operation
? opens
on-line Help
Commands are numbered
to indicate sequence for
scanning the gel,
analyzing the image, and
outputting data
Resize button
Fig. 2-4. PDQuest Quick Guide
In the expanded format, the Quick Guide commands are numbered as well as named,
so that the order of operation is clear. Simply follow the steps and the Quick Guide
will lead you through the analysis. Note that many of the buttons open toolbars of
related commands (e.g., Match Tools).
As with the secondary toolbars, you can click the ? next to the name of a function to
display Help.
2.2 Keyboard Shortcuts
Many of the functions may be executed using keyboard commands (e.g., pressing the
F1 key will activate the View Entire Image command). A list of key combinations and
their associated commands will be displayed if you select Keyboard Layout from the
Help menu.
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PDQuest User Guide
Fig. 2-5. Keyboard Layout.
The pulldown menus also list the shortcut keys for the menu commands.
Note:
Mouse-assignable commands behave differently if you assign them using the
keyboard versus selecting them from the menus or toolbars. For example, to use
the Zoom Box command as a keyboard command, position your cursor on the
image where you want to begin to create the magnifying box, then press the F2
key. The command is assigned to your mouse and immediately activated, so that
when you move your cursor over the image, the zoom box is created. When you
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General Operation
click the mouse button once, the defined region is magnified and the tool is
automatically deassigned from your mouse.
2.3 File Commands and Functions
This section describes the basic file commands and functions of PDQuest.
Note:
Print and Export commands are described in Chapter 12.
2.3.a File Types in PDQuest
The types of files supported by PDQuest are:
Raw 2-D scans (.gsc)
Filtered images (.gim)
Gaussian images (.gsp)
Calstrip scans (.csc)
MatchSets (.ms)
Backup scans (.sbk) (Backups cannot be opened in Windows; you must change the
extension to .gsc using Windows Explorer to open the file).
2.3.b MatchSets, Scansets, and Analysis Sets
MatchSets
A MatchSet is PDQuest’S mechanism for comparing and analyzing the spots in an
experiment. Each image in a MatchSet is called a member. A MatchSet contains one
or more separate members in the form of Raw 2-D, Filtered, or Gaussian images.
Using a MatchSet, you can make quantitative and qualitative spot comparisons across
gels, calculate molecular weight/isoelectric point values, annotate spots, group them
into analysis sets, combine the data from multiple gels into replicate groups, and
select spots for excision and mass spec analysis.
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PDQuest User Guide
A MatchSet can consist of one gel or many gels, depending on the type and size of the
experiment. The MatchSet is displayed in a single window, with subwindows for the
images of the member gels, and a subwindow for the MatchSet Master.
Fig. 2-6. Example of a MatchSet.
The commands for creating MatchSets are located under File>New MatchSet, and as
Step 3 of the Automated Detection and Matching dialog.
Scansets
The members of a scanset are three separate files of the same root image. You can
open and close scanset images (i.e., different exposures or versions of the same
image) individually, even though they are loaded into the same window.
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General Operation
Fig. 2-7. Example of a Scanset.
A scanset consists of three separate images of the same gel image. They are
displayed in subwindows as a Raw 2-D Image, as a Filtered image, and as a
Gaussian image All of these are saved as separate images, but they are part of the
same scanset. They have the same root file name, and when you open them they are
loaded into subwindows of the same image window.
To perform an operation on a particular image in a MatchSet or scanset, simply move
your cursor over its subwindow. A green border appears around the selected
subwindow. The border shifts as you move the cursor over different subwindows.
Alternatively, you can select Click to Focus Subwindow under Edit > Preferences >
Display, in which case you must click on a subwindow to select it.
For example, the following image files are all in the same scanset:
Proteins v1 x2.gsc (Raw 2-D scan, exposure 2)
Proteins v1.gim (Filtered image)
Proteins v1.gsp (Gaussian image).
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PDQuest User Guide
Analysis Set
An Analysis set is a set of spots you have chosen to study. Analysis sets allow you to
create groups of spots that are statistically and biologically significant. For more
information see Chapter 7, . Analysis Sets and Annotations.
Fig. 2-8. Analysis Set.
2.3.c Opening Files
The Open command on the File menu or main toolbar opens previously saved images
and MatchSets.
Note:
Due to operating system limitations, it is possible to have image files open in two
or more locations over a network simultaneously. To safeguard your image files
keep them in a protected folder or on your local machine.
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General Operation
Fig. 2-9. Open dialog box.
In the PDQuest Open dialog box:
•
Already open images are marked with an asterisk.
•
File types and icons are listed next to each image name.
•
MatchSets are listed as single files, even though they are really collections of
images.
In the Open dialog box, open an image by double-clicking on its name or selecting the
file name and clicking the Open button. Specify the file type by selecting one from
the Files of Type pulldown menu. Your options include Loadable Images and
MatchSets, Raw Image, Filtered Image or Gaussian Images, MatchSets, or All Files.
To select multiple files to open, use the standard ctrl-click and shift-click key
combinations in Windows, or shift-click the Macintosh. Images in a scanset or a
MatchSet will be loaded into the same window.
Click the Parent Folder button to go up one level in the directory tree. Click the
Desktop icon to open the desktop folder. In the Look In: field, click the default name
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PDQuest User Guide
or down arrow to the right of the field to display a hierarchical tree structure locating
the default drive, folder and file.
Note:
The drive buttons only include mapped drives. If you need to access an
unmapped (network) drive, you can type the drive name directly into the Folder
field using UNC notation (e.g., \\remote server name\shared directory
name\...\file name). Or you can create a shortcut to the drive on your desktop.
To go directly to the folder where your images are stored, click the Sample Images
Folder. To go to the folder containing your MatchSets, click Sample MatchSet Folder.
Specify the locations of these folders in Path Preferences under Edit > Preferences.
An image created in the Windows version of PDQuest can be opened in the
Macintosh version, and visa versa.
PDQuest comes with a selection of sample images. In Windows, these files may be
found in the Discovery Series/Sample Images/2D directory. On the Macintosh, they
are stored in the Sample Images folder in the PDQuest folder. These sample images
are related, and can be used to create a MatchSet.
Opening TIFF Images
The Open command can also be used to import TIFF images from other software
applications.
TIFF images do not contain all of the tagged information that would normally be
included in a Discovery Series image file (e.g., scanner type, date, color, etc.). For this
reason, the Image Info and Print > Image Report commands will not display all such
tagged information from TIFF files.
There are many types of TIFF formats that exist on the market. Not all are supported
by the Discovery Series. There are two broad categories of TIFF files that are
supported:
1.
8-bit Grayscale. Most scanners have an option between line art, full color, and
grayscale formats. Select grayscale for use with the Discovery Series software. In
a grayscale format, each pixel is assigned a value from 0 to 255, with each value
corresponding to a particular shade of gray. Hewlett Packard™, Microtek™, and
Sharp™ each make scanners that produce compatible 8-bit grayscale images.
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General Operation
2.
Note:
16-bit Grayscale. Bio-Rad’s Molecular Imager (storage phosphor) systems use
16-bit pixel values to describe intensity of scale. Molecular Dynamics™ imagers
also use 16-bit pixel values. The Discovery Series understands these formats and
can interpret images from both Bio-Rad and Molecular Dynamics storage
phosphor systems.
PDQuest can import 8- and 16-bit TIFF images from both Macintosh and PC
platforms. PDQuest cannot import compressed images.
TIFF files that are not supported include:
1.
1-bit Line Art. This format is generally used for scanning text for optical
character recognition or line drawings. Each pixel in an image is read as either
black or white. Because the software needs to read continuous gradations to
perform gel analysis, this on-off pixel format is not used.
2.
24-bit Full Color or 256 Indexed Color. These formats are frequently used for
retouching photographs and are currently unsupported in the Discovery Series,
although most scanners that are capable of producing 24-bit and indexed color
images will be able to produce grayscale scans as well.
3.
Compressed Files. The software does not read compressed TIFF images. Since
most programs offer compression as a selectable option, files intended for
compatibility with the Discovery Series should be formatted with the
compression option turned off.
Export Image as TIFF
The Reports>Export to TIFF dialog box contains the settings for exporting a Raw 2-D
scan, Filtered image, or Gaussian image in TIFF format.
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PDQuest User Guide
Fig. 2-10. Export to TIFF dialog.
Note:
See Filtered Images and Gaussian Images for a description of the different image
types.
In the export dialog, first select whether you want to export the raw data image
(Analysis option) or the currently displayed view of the image (Publishing option).
Select the appropriate radio button to publish the export view excluding or including
overlays.
Analysis Export
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General Operation
The Analysis option exports the image data unmodified by any viewing adjustments
you may have made (such as Transform or Zoom). If you select this option, the other
controls in the dialog box will become inactive.
Publishing Export
If you select the Publishing export option, the exported TIFF image will look like the
image as it is currently displayed on the computer screen.
You can specify a resolution for the exported image by selecting 72 dpi (typical
computer screen resolution), 150 dpi or 300 dpi (standard printing resolutions), Same
as scan, or any resolution you specify (up to the resolution of the scan).
If you have log transformed the image, you can specify a Linear transform, or
preserve the Current view.
If your image has a pixel depth of 16 bits, you can compress it to 8 bit data for export
to TIFF by selecting the 8 bit option.
Note:
Note:TIFF images are exported from the Multi-channel Viewer in 24 bit RGB
mode to preserve the colors displayed in the viewer.
Finally, if you are only displaying part of the image due to magnification or
repositioning, you can preserve the Current view or export the Entire image.
Exporting the Image
The size of the pixels in the image and the file size of the image are listed at the
bottom of the dialog. When you are ready to export, click the Export button.
A Save In dialog box will open. The default file name will have a .tif extension, and
the file type will indicate that this is a TIFF image. You can change the file name or
select a different directory to save in.
2.3.d Closing files
File > Close (Alt F4) closes the entire active window, including any subwindows. If
any of the images in the window has changed, you will be prompted to save the
changes before closing.
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PDQuest User Guide
File > Close All closes all open images in all windows. If you have made changes to
any of the images, a message box will open for each unsaved image giving you the
option of saving those changes.
2.3.e Saving Files
File > Save will save a new or old scan, scanset, or MatchSet to your hard disk,
network drive, or other storage media.
Note:
In Windows, new PDQuest images will be given a .gsc extension when they are
first saved.
File > Save As dialog can be used to save scans, scansets, and MatchSets under
different names and different directories. The default name of the file is listed in the
Name field, along with the type of file, for example, MatchSet.
Note that in the case of scansets (see section 2.3.a, File Types in PDQuest), all the
different files in the set must be loaded when you select Save As if you want the root
name to be changed for all of them.
Save All on the main toolbar or File menu saves all images, scansets, and/or
MatchSets that are currently open.
2.3.f
Revert to Saved
File > Revert to Saved will recover the last saved version of the image or MatchSet
you are working on. This is a quick way to undo any alterations you have made since
you last saved it.
Any changes you have made since last saving the file will be lost. (Any open dialog
box will also be closed.) A message box will warn you before completing the
command.
2.3.g Image Info
On the main toolbar, Image> Image Info displays general information about your
image, including the scan date, scanner used, scan area, number of pixels in the
image, data range, and the size of the file. Gaussian image info includes the number
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General Operation
of spots, total data in the spots, and other information. There is also a field where you
can type in a file description or comments.
Fig. 2-11. Image Info box.
The Image Info dialog displays differently, depending on the type of file you are
acquiring information about, for example, Raw 2-D or Gaussian. History lists the
changes made to the image including the date the changes were made. To print the file
information, click Print.
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PDQuest User Guide
2.3.h Reduce File Size
Scan files can be quite large, and computer systems do not have unlimited memory or
storage space. If you are having difficulty loading or storing a particular scan, you
may want to reduce the size of the file by reducing the number of pixels in the image.
(You can also trim unneeded parts of an image to reduce its memory size. See section
3.13, Cropping Images.)
Note:
This command only works on Raw 2-D scans.
This function is comparable to scanning at a lower resolution, in that you are
increasing the size of the pixels in the image, thereby reducing the total number of
pixels and thus memory size.
Note:
Reducing the file size of an image will result in some loss of resolution. In most
cases this will not affect quantitation. In general, as long as the pixel size remains
less than 10 percent of the size of the objects in your image, changing the pixel
size will not affect quantitation.
Select Image > Reduce File Size. The dialog box shows you the size of the pixels in
the image (Pixel Size: X by Y microns), the number of pixels in the image (Pixel
Count: X by Y pixels), and the Memory Size of the image.
As you increase the size of the pixels, the pixel count will decrease, as will the
memory size. You can increase the pixel size in either dimension (see the following
figure for an example). You cannot reduce the pixel size, you can only increase it. You
can reduce the pixel count.
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General Operation
Before:
Pixel size increase confirmation
After:
Pixel Count and Memory Size reduced
Fig. 2-12. Reduce File Size dialog box, before and after pixel size increase.
Note:
Asymmetric pixel reduction (i.e., making pixels smaller in one dimension than
the other) is not recommended for 2-D gels, since you want to resolve spots in
both dimensions.
A pop-up box prompts you to confirm the resolution reduction. If you choose to
reduce in place, the descriptions of objects in the scan will be lost. Only the scan
image is retained. Click Reduce and Copy to make a copy the original file and reduce
the copy’s size, or click Reduce to reduce the file size of the displayed image.
Reducing the file size is an irreversible process. For that reason, we suggest you make
a copy of the image and reduce its file size. That way, if you lose too much resolution,
you can simply delete the copy and try again. Once you are happy with the reduced
image, don’t forget to delete the original. The goal is to save space!
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If you choose to make a copy of the image, you will be asked to enter a name for the
new copy before the operation is performed.
2.3.i
Exiting PDQuest
File > Exit quits the application. You will be prompted to save any unsaved changes
to images or MatchSets.
2.4
Printing and Exporting
The commands for printing images, actual-size images, image reports, and video
printouts are located on the File > Print submenu.
Fig. 2-13. File > Print submenu.
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General Operation
2.5
Printing
•
Page Setup opens a dialog box where you can select the size and source of your
paper, portrait or landscape orientation, and set margins for your report.
•
Print Image (CTRL+P) opens the Print dialog box. You can also open the Print
doalog box by clicing the Print icon in the toolbar.
•
Print Settings opens a dialog box where you can determine header and footer
imformation, whether to print actual size, and border and background
information.
•
Image Report prints the image and information about its scan history, number of
pixels, data range, etc.
•
Video Print prints images and reports to a video printer.
The commands for printing and exporting images, Print Settings, image reports, and
video printouts are located on the File > Print submenu. If you have a MatchSet or
scanset open, you must first select the image you want to print.
Note:
2.5.a
Reports are printed from within the individual report windows.
Printing Images
Print Image prints a copy of the active image window and any image overlays that are
displayed. The Print Image command opens the standard print dialog box for your
operating system. When you are satisfied with all the print parameters, click Print to
send the image to the printer.
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PDQuest User Guide
Fig. 2-14. Print Image dialog box (Windows).
2.5.b
Print Settings
Use the Print Settings command to configure what information to include and how the
image appears each time you print an image. To open Print Settings, click Print
Settings in the File>Print submenu. Any changes made to the Print Settings take
effect immediately. You can also leave the Print Settings dialog box open to quickly
change the settings for images as you print.
Note:
You can have multiple Print Settings dialog boxes open, one for each open
image, with different settings. However, If you use the print command
(file>Print>Print Image or CTRL+P) for an image that does not have the Print
Settings dialog box open, the print format will be based on the last Print Settings
dialog box where changes were made.
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General Operation
Fig. 2-15.
Under the Headers and Footers settings, select what information you want to include
in your prints.
File name - Select File name add the name of the image file to the header of the
print.\
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PDQuest User Guide
Acquisition Info - If Acquisition Info is selected, the date the image was acquired
and the data range of the scan is included in the footer information.
Time printed - To add the date and time the image was printed, select Time printed.
Page number - Select Page number to add the page number to the footer information.
The Pagination settings determine allow you to determine whether to print your
images actual size or fit the images to the page.
Fit Image to Page - This fits the current view of the image to a single page based on
the layout of the image.
Print Actual Size - Select Print actual size to print your images based on the scan size
of the gels.
Note:
If you are using the Gel Doc EQ or ChemiDoc EQ, you must specify the correct
image area size when capturing your images to ensure accurate 1:1 printing. You
can specify the image area size in the acquisition window for the instrument. See
the chapter on each imaging device for more information.
Under Image you can add a border to the image area of the prints as well as a
background. Adding a border and/or background is useful for determining the edges
of faint images.
Click the Page Setup button to open the Page Setup dialog box. Note that, although
you can print directly from the Print Settings dialog box by clicking either Print or
Print Current Page, the print settings affect each image printed using the Print Image
command.
Note:
2.5.c
The Print Settings dialog box attempts to accurately display the image exactly
how it will print. However, in some cases, such as images with large pixel
dimemensions set to Print Actual Size, small differences may be noticed between
the preview window and the actual print.
Image Report
Image Report allows you to print out a single-page report of an image and its
associated information. The format of the report is designed to provide a concise yet
thorough summary of the most relevant features of an image for documentation
purposes.
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General Operation
Fig. 2-16. Master Image Report.
The report includes the following information:
•
The image title.
•
A description of the image if you specified one.
•
The directory location.
•
The filename of the image.
•
The type of imaging device (scanner).
•
Pixel size by X and Y axis as measured in um.
•
The scan (image) area in terms of X and Y axis and the data range as measured in
integers.
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PDQuest User Guide
•
The number of pixels scanned and the memory size as measured in Kilobytes.
•
The Scan history (date and time of the report) and the action(s) performed during
that scan.
•
The imaging area and number of pixels.
•
The intensity range, image color, and memory size.
•
Image background information.
•
Relevant lane and band information.
To print a image report of a particular gel, select Image Report from the File > Print
submenu. This opens the standard print dialog box (see Print Image, above).
Note:
2.5.d
TIFF images do not contain all the tagged information that would normally be
included in an image file (for example, imaging device, scan date, image color,
etc.). For this reason, the Image Report may list this information as Unknown for
imported TIFF files.
Video Printing
The Video Print command allows you to print images and reports on a video printer.
To create a video printout of the active window, select Video Print from the File >
Print submenu.
Note:
Video printing requires installation of the video board and cable that come with
the Gel Doc EQ 1000/2000 and ChemiDoc EQ gel documentation systems. The
video board and cable can also be ordered separately.
Settings for the Mitsubishi P90W/P91W Video Printer
There are three settings for the Mitsubishi P90W/P91W video printer. Set Contrast to
0, Brightness to 0, and Gamma to 5.
The dip switches should stay in the orientation in which they are shipped: Pin 1 is up
(on), and Pins 2–10 are down (off).
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General Operation
2.6
Exporting
You can export gel images to TIFF images, and export MatchSets and Annotations
using the Export submenu in the Reports menu.
Fig. 2-17. Export submenu.
2.6.a
Exporting a TIFF Image
To export a Raw 2-D scan, Filtered image, or Gaussian image as a TIFF image, select
Export to TIFF Image from the File > Export submenu, then click the image in a
MatchSet or scanset.
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PDQuest User Guide
Fig. 2-18. Exporting TIFF Images.
In the export dialog, first select whether you want to export the original image data
(Analysis option) or the currently displayed view of the image (Publishing option).
Note:
Only the Publishing option is available if you are exporting from the MultiChannel Viewer.
Analysis Export Mode
The Analysis option exports the image data unmodified by any viewing adjustments
you may have made (such as Transform or Zoom). If you select this option, the other
controls in the dialog box become inactive.
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General Operation
Publishing Export Mode
If you select the Publishing export option, the exported TIFF image will look like the
image as it is currently displayed on the computer screen. You can choose whether or
not you want to include overlays.
Specify a resolution for the exported image by selecting 72 dpi (typical computer
screen resolution), 150 dpi or 300 dpi (standard printing resolutions), Same as scan,
or any resolution you Specify (up to the resolution of the scan).
If you have log transformed the image, you can specify a Linear transform, or
preserve the Current view.
If your image has a pixel depth of 16 bits, you can compress it to 8 bit data for export
to TIFF by selecting the 8 bit option.
Note:
TIFF images are exported from the Multi-channel Viewer in 24 bit RGB mode to
preserve the colors displayed in the viewer.
Finally, if you are only displaying part of the image due to magnification or
repositioning, you can preserve the Current view or export the Entire image.
Exporting the Image
The size of the pixels in the image and the file size of the image are listed at the
bottom of the dialog. When you are ready to export, click the Export button.
The Export TIFF Image dialog box opens. The default file name will have a .tif
extension, and the file type will indicate that this is a TIFF image. You can change the
file name or select a different directory to save in. Click Save to complete the export.
A confirmation appears in the status box of the top menu bar.
2.6.b Exporting a JPEG Image
The export to JPEG Image command allows you to export a Raw 2-D scan, Filtered
image, Gaussian image, or MatchSet master image as a JPEG image. The Export to
JPEG Image is primarily for exporting your images for use in reports, presentations,
etc. because a JPEG image is considerably smaller in size than a TIFF image. Unlike
TIFF images, JPEG images cannot be opened by TDS applications.
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To export an image as a JPEG, open the File > Export submenu and click Export to
JPEG Image. If you have a scanset or MatchSet open, click in one of the image
subwindows to open the Export to JPEG dialog box. If only a single image is
available in the active window, the Export to JPEG dialog box opens for that image.
Fig. 2-19. Exporting to JPEG
In the Export to JPEG dialog box, under Export mode, use the slider to adjust the
image quality. The higher the quality, the larger the file size.
Click Export to complete the operation. The default file name and location are based
on the original image. You can change the file name or select a different folder to save
in. Click Save to complete the export.
2.6.c
MatchSet Data
Data from a MatchSet can be exported as an ASCII text file for further analysis using
spreadsheet programs. Alternatively, you can export the MatchSet to the Clipboard.
With the MatchSet loaded, select Export MatchSet from the File > Export submenu.
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General Operation
Fig. 2-20. Exporting MatchSet data.
In the dialog, you can export data for each gel in a MatchSet (Spot Data by Gel), or
for each replicate group in the MatchSet (Spot Data by Group).
Under Spot Data by Gel, select the data to export for each spot (position, quantity,
quality, Gaussian size, normalized quantity, and/or peak value).
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The quantities of saturated spots can be estimated, reported as the value -3.0, or
reported in some other way that you specify. If you opt for the latter, click the Other
button and enter the value that should be reported.
If a spot is unmatched in one or more members of a MatchSet, it can be estimated as
the value -1.0 or as a value that you enter in the text box next to the Other button.
For each spot, you can include the SSP number, DSN (if designated), replicate group
ID, and/or MrpI (Peak) value if calculated by selecting the appropriate check boxes.
You can export annotations in all categories or a specified category. If you select
Specified, click the drop-down arrow and select the category from the list.
You can export all analysis sets or a specified analysis set. To specify an analysis set,
click the drop-down arrow and select one from the list.
Under Spots to Export, specify the spots whose data should be exported. You can
choose all the spots in the MatchSet, those spots that are matched in every member of
the MatchSet, or the spots in a particular analysis set. The spot count automatically
adjusts to reflect the total number of spots selected.
Under Output Options, you can include headers for each column in the report. Select
how you want to sort the spot data: by SSP number, DSN, or gel/replicate group.
Select the field separators you want to use to separate columns of data in your
spreadsheets: commas or tabs for Excel.
Your data can be exported as a file or to the clipboard. If you choose the file option
button, clicking Done opens the Export MatchSet Data dialog box, in which you can
enter a name and export location for the file. The file type automatically defaults to an
.xls spreadsheet.
If you choose the clipboard option, clicking Done saves your data to the system
clipboard.
If you have created replicate groups for the MatchSet, you can export the averaged
data for each group. Under Spot Data by Group, select the data to export (Master
position, average quantity, coefficient of variation, standard Gaussian size, average
normalized quantity, normalized coefficient of variation, and/or the counts in the
member gels).
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General Operation
Fig. 2-21. Spot data by group tab.
Basic Export MatchSet
The Basic Export MatchSet dialog allows you to export the individual characteristics
of spots in a MatchSet. This is a more basic version of the standard Export MatchSet
dialog. Data will be exported in ASCII text format.
With the MatchSet loaded, select Basic Export MatchSet from the File > Export
submenu.
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Fig. 2-22. Export MatchSet Data dialog box.
Next to the Data to export options, select the characteristic of the spot that you want
to export.
Specify the spots whose data should be exported. You can choose either all the spots
in the MatchSet, those spots that are completely matched in every member of the
MatchSet or the spots in a particular Analysis Set.
The Spot Count automatically adjusts to reflect the total number of spots selected.
If you are exporting spot quantity data, select either estimated or specified as the way
in which saturated spots and unmatched spots are reported.
The quantitation of saturated spots can be estimated, reported as (-3.0), or reported in
some other way that you specify. If you opt for the latter, enter the value that should
be reported in the Specify field.
If a spot is unmatched in one or more members of a MatchSet, it can be reported as
the minimum spot quantitation value (the minimum quantity needed for a spot to be
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General Operation
detected for the gel). Alternatively, it can be reported as (-1.0) or as a value that you
enter in the text box next to the Specify button.
Next, select the field separators you want to use to separate columns of data in your
spreadsheets: commas or tabs.
Your data can be exported as a file or to the clipboard.
If you choose the File option button, clicking OK opens the Basic Export MatchSet
dialog box, in which you can enter a name and export location for the file.
If you choose the Clipboard option button, clicking OK saves your data to the
clipboard.
2.6.d
Exporting Annotations
You can export MatchSet annotations by category to a text file for inclusion in a
document or spreadsheet.
With the MatchSet open, select Export Annotations from the File > Export submenu.
The box prompts you to select the annotation category or categories to export.
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Fig. 2-23. Export Annotations pop-up box.
Click an individual category name to select it, or shift-click or ctrl-click to select
multiple categories.
When you have made your selection(s), click Export. In the file name dialog, enter a
name for the export file and select the directory to save to.
The data in the text file is tab-delimited. Each row includes the annotation category,
SSP number for a spot in that category, and the associated annotation entry.
Text files can be opened in standard Windows and Macintosh documents and
spreadsheets. If you selected multiple categories to export, the annotations will be
sorted by category.
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General Operation
2.7 Preferences
The Preferences dialog box allows you to customize basic features of your system.
Select Edit > Preferences to open the dialog. Tabs for Misc, Paths, Display, Toolbars,
Application, and Imagers display.
Fig. 2-24. Preferences dialog box.
Click the appropriate tab to access groups of related preferences. After you have
selected your preferences, click OK to implement them. Some of your preferences
will only be implemented by exiting and restarting the program. A message will
notify you if this is necessary.
2.7.a Misc Preferences
Click the Misc tab to access the following preferences.
The Memory Allowance field allows you to specify the amount of virtual memory
allocated for the application at start up. The default value of 512.00 megabytes is
recommended If you receive a warning message when opening the program that the
amount of virtual memory is set too high, you can enter a smaller value in this field.
However, this should be considered a temporary fix.
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If the Enable MassLynx commands checkbox is unchecked, the MassLynx commands
in the Identifications pull down menu are grayed out. If you are using massLynx for
identification purposes, check this box to enable the MassLynx commands.
If the Enable Mascot commands checkbox is unchecked, the Mascot commands in the
Identifications pull down menu are grayed out. If you are using mascot for
identification purposes, check this box to enable the Mascot commands.
The Maximize application window check box determines whether the main window
occupies the entire computer screen when first opened. If this is unchecked, the menu
and status boxes will appear across the top of the screen and any toolbars will appear
“floating” on the screen.
If Enable DOS File Name Parsing is checked, for 8-character filenames ending in two
digits, the final two digits are interpreted as version and exposure numbers. For
example, the file name IMAGE-11.gsc would be parsed as IMAGE ver 1 xpo 1.gsc.
This is designed to enable backwards compatibility for users with DOS image files.
You should only check this box if you are using these image files.
Enable UNIX File Name Parsing is similar to DOS file name parsing. Windows and
Macintosh users are unlikely to run into difficulties with UNIX parsing, therefore this
setting is checked by default.
Multiple Mouse Assignment Mode is designed for backward compatibility with
previous versions of PDQuest. It is not documented in this manual.
2.7.b Path Preferences
Navigate to the Edit > Preferences > Paths tab to set the file paths for various
shortcuts and links throughout PDQuest.
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General Operation
Fig. 2-25. Path preferences Browse button
To set a particular file path, click the Browse button next to the path field and
navigate to the appropriate directory, then highlight the folder where you want to set
the path. Click OK. Click Default to return the path to the default setting.
The Log path Browse button sets the directory where spot detection log files are
saved. Select Spot Detection Report from the Spots menu to access the log files in this
directory.
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Fig. 2-26. Log report from Spots menu.
The Temporary path sets the directory where temporary image files and other files
generated by PDQuest are stored.
If you are using the ProteinLynx Global Server 2.0 for your protein analysis, enter the
IP address of the machine where the ProteinLynx Global Server 2 is located. The port
number should be 2551.If the PLGS 2.0 server is located on the local machine, enter
localhost as the address.
The Mascot Install path sets the directory where your Mascot Install folder is located.
This option is only available if the Enable Mascot commands option is checked in the
Misc tab.
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General Operation
The Mascot Server URL determines where the Mascot server is located. The is
typically an HTTP address. This field is only available if the enable Mascot
commands option is checked in the Misc tab.
2.7.c Display Preferences
Fig. 2-27. Display Preferences
Click the Display tab to access the following preferences.
Zoom % allows you to specify the percentage by which an image moves closer or
farther away when you use the Zoom functions. This percentage is based on the size
of the image.
Pan % determines the percentage by which the image moves side to side or up and
down when you use the arrow keys. This percentage is based on the size of the image.
If you want to apply a uniform background to Gaussian images so they more closely
resemble the original gels, enter a background optical density (O.D.) value in the text
box next to the Gel Spot Bkgd prompt.
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The Default type option lists the three images associated with a MatchSet. Select the
image you wish to have displayed when you create or open a MatchSet.
Note:
If you have Save subwindow configuration selected, this overrides your Default
type selection when you open a MatchSet.
In Gaussian images, specify whether you want to see only single spots or multiple
spots by clicking on the appropriate button under Display combined spots. Note that
valid spots are spots that have not been cancelled. If you elect to display combined
spots, they will appear in the Gaussian image as they do in the Filtered image,
although the quantitation will be combined.
Auto ’Imitate Zoom’ duplicates the zoom and positioning commands made in one
window in all displayed windows. For example, with Auto ’Imitate Zoom’ selected,
Zoom In on one scanset or gel image window and the other open scanset or gel image
windows will also zoom in.
Solid Label Background will automatically display spot labels in a filled-background
box.
Auto ’All Same’ duplicates the zoom and positioning commands made in one
subwindow in all displayed subwindows. For example, with Auto ’All Same’ selected,
Zoom In on one subwindow and the other open subwindows will also zoom in.
By default, a subwindow is selected when you move your cursor over it. If you would
rather click on a subwindow to select it, select Click to Focus Subwindow.
Note:
Click to Focus Subwindow will change the behavior of mouse-assignable tools.
Some commands that were previously mouse-assignable will be performed
immediately on the active subwindow; other commands will require you to click
once in a subwindow to select it, then click or drag again to perform the action.
The Save subwindow configuration saves subwindows in the last order displayed as
well as the last image type displayed, which overrides the Default type selection.
If you are having problems using the 3D Viewer, install the latest drivers for your
graphics card. If, after updating your drivers, you are still having problems, select
Disable hardware acceleration for 3D viewer. This allows PDQuest to emulate a
graphics driver to render a 3D image.
Note:
Disabling hardware acceleration reduces performance for the 3D Viewer.
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General Operation
2.7.d Toolbar Preferences
Fig. 2-28. Toolbar preferences
Click the Toolbar tab to set the behavior and positioning of the secondary toolbars and
Quick Guide.
If the Show Quick Guide check box is selected, the quick guide will open
automatically when you open PDQuest.
If Align Quick Guide with Document is selected, the quick guide will pop up flush
with the edge of your documents. Otherwise, it will appear flush with the edge of the
screen.
The Guides always on top checkbox determines whether the quick guide and toolbars
will always be displayed on top of images, or whether they can be hidden behind
images.
Quick Guide Placement and Toolbar Placement determine which side of the screen
the quick guide and toolbars will first open.
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The Placement Behavior setting determines whether a quick guide or toolbar will
always pop up in the same place and format (Always Auto), or whether it will pop up
in the last location it was moved to and the last format selected (Save Prior).
The Toolbar Orientation option buttons specify whether toolbars will first appear in a
vertical, horizontal, or expanded format when you open PDQuest. In the extended
version, quick access to context sensitive Help displays in the Quick Reference guide.
Tool Help Delay allows you to specify the amount of time (in seconds) the cursor
must remain over a toolbar icon before the Tool Help appears. First-time users may
want to specify a short delay to learn the names of the toolbar functions, while
experienced users can specify a longer delay once they are familiar with the icons.
Persistence determines how long the Tool Help will linger on the screen after you
move the cursor off a toolbar icon.
2.7.e Application Preferences
Fig. 2-29. Application preferences
Click the Application tab to access these preferences.
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General Operation
Enter up to three letters for the MatchSet Name Prefix and a number for the MatchSet
Name Sequence #.
When you create a MatchSet, you can give it a default name. The MatchSet Name
Prefix field determines the prefix of the default name, while the MatchSet Name
Sequence # determines the number added to the prefix to complete the name. The
sequence number increases by 1 each time a new MatchSet is created using the
default name.
Once the prefix and the sequence number have been initially set, you probably will
not need to change them.
If Auto-match after Landmark is selected, PDQuest will auto-match all gels in the
MatchSet each time you place a landmark in all the gels.
Finally, indicate the orientation in which your gels are run by clicking on either the
Acid-Base or Base-Acid button next to the pH Gradient prompt.
2.7.f
Imagers
Checking the name of a scanning device in the Imagers tab allows you to scan using
that instrument.
See the individual appendices on each imaging devices for more details.
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PDQuest User Guide
Fig. 2-30. Imagers.
The Edit > Preferences > Imagers tab contains a list of Bio-Rad imaging devices
supported by the Discovery Series software. These are:
1.
Gel Doc EQ
2.
ChemiDoc EQ
3.
GS-800 Imaging Densitometer
4.
VersaDoc
5.
Personal Molecular Imager FX
6.
Molecular Imager FX
Note:
If you check one or all of these devices they display in the File menu. You need to
restart the program in order for your selections to take effect.
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General Operation
2.7.g Security
Setting the Security preferences for CFR mode requires a valid CFR license. To
modify these settings, you must have administrator privileges. To locate the Security
Preferences, select Preferences from the Edit menu and click the Security tab.
Fig. 2-31. Security Preferences
The following options are available on the Security tab of the Preferences dialog box:
•
Enable 21 CFR Part 11 mode - If this box is unchecked, all other options are
inactive. To activate the other options and CFR mode check this box.
•
Program times out if inactive - When this box is checked, the user must enter
their password to resume using The Discovery Series. The minutes field indicates
the amount of time that must elapse before the application times out.
•
Require login on startup - Check this box to require the user to enter the user's
password when the application opens. This password is the same password as the
one used for logging on to Windows. This is an added security measure to
prevent unauthorized users from opening a TDS application on a machine where
they are not logged in.
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•
Automatically create backup for new images - This option, when selected,
creates a backup file when you save a newly acquired or imported image. Locate
the backup file in the same folder as the original.
See Appendix J, 21 CFR Part 11, for further information.
2.8 Mouse-assignable Tools
Mouse and Mouse Macro functions are designed for backwards compatibility with
previous versions of PDQuest. They are not recommended for new users.
PDQuest has a number of commands that don’t perform actions right away, but
instead assign a function to your mouse (e.g., Zoom Box, Density at Cursor, Add
Spot). Select Mouse from the Edit menu to open the Mouse dialog or Edit > Mouse
macro for a pulldown menu.
Fig. 2-32. Mouse Preferences.
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General Operation
You can choose from up to four groups of mouse functions, and select either two or
three buttons to assign to each group.type the name of the mouse configuration file
into the appropriate field. Lower button options contain. Load, Save, Reset, Delete,
Done.
Note:
Mouse assignable tools selected using the keyboard have slightly different
behavior.
Example of
Mouse-assigned
tool cursor:
Density at
Cursor
Mouse Macro
Pulldown Menu
Fig. 2-33. Mouse Macros and Example of a mouse-assignable tool.
When a mouse-assignable function is selected, the cursor appearance changes. The
name and icon of the function appear in the status box next to the main toolbar (see
section 2.1.c). The status box displays text according to where your mouse was placed
last.
To deassign a function from the mouse, click the toolbar button of the assigned
function or click in the status box displaying the assigned function. You can also
double-click the Hide Overlays button.
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Note:
Some commands in PDQuest become mouse-assignable if you are displaying
multiple subwindows. For example, if you select Spots > Spot Crosshairs with a
single image displayed in a window, crosshairs will simply be displayed.
However, if you select Spots > Spot Crosshairs with multiple subwindows
displayed, the command will be assigned to your mouse and you must click a
subwindow to display the crosshairs in that subwindow.
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3. Viewing and Editing
Images
This chapter describes the tools for displaying images in windows and subwindows,
and magnifying and optimizing images. This chapter also describes the tools for
cropping, flipping, and rotating images and filtering image noise.
These tools may be found on the View, Edit, Image, and Window menus.
3.1
Windows and Subwindows
PDQuest displays the multiple images in a MatchSet or a scanset in subwindows of
the same image window. Subwindows appear as separate, smaller windows within the
main image window. Subwindows are designed to allow you to easily compare and
edit MatchSet and scanset images at the same time.
3.2
Configuring Subwindows
To control how MatchSets and scansets are partitioned into subwindows, click the
Configure Subwindows button on the main toolbar (also Ctrl+F1 and Windows
menu). The Configure Subwindows dialog box will open.
The Loaded Images buttons show the types of files that are open (2D scans, Gaussian
images, MatchSets, etc.) and list how many images are in each type. Click a button to
display only images of that type in the subwindows.
Click All to display all available image types in subwindows.
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Fig. 3-1. Configuring the subwindows of a scanset.
The Quick Config buttons immediately partition the subwindows into the
configuration depicted on the button. (Note that if you have selected a Loaded Images
option, only images of that type will be displayed.)
Otherwise, select a configuration by typing values in the Windows, Rows, and Cols
fields. The Windows field specifies how many subwindows to create. Rows and Cols
specify how to layer the subwindows.
For large MatchSets with many members, you can deselect the Display Images Once
Configured checkbox. Then, when you apply a new configuration, the images will not
be immediately refreshed in the windows. Click in a subwindow to make the image in
that subwindow appear.
Note:
Deselect this checkbox only if you have a slow computer and are displaying
many images.
Click the Apply button to implement your configuration selections.
3-2
Viewing and Editing Images
3.3
Assigning and Interchanging Images
You can use the Assign and Interchange commands to select the image you want to
display in a particular window or subwindow.
Assigning Images
To assign an image to a particular subwindow, select the Assign tool
from the main toolbar or Window menu and click in the subwindow, or
use the Ctrl+A key command in desired subwindow. This will open a
pop-up list of all the images currently loaded in the MatchSet or scanset.
Select an image name from the list; the image will appear in the selected window.
Fig. 3-2. Assign Image lists all the loaded images; select one to display it.
Interchanging Images
Interchange Images switches between the Raw 2-D scan, Filtered image, and
Gaussian image of the same gel within a selected subwindow. With the images
loaded, select the tool from the main toolbar or View menu and click in the
subwindow, or use the F11 key command in the subwindow. The type of image
currently displayed is listed at the bottom of each subwindow.
Interchange All Images (Shift+F11, View menu) switches between the Filtered
images and Gaussian images in all the displayed subwindows of a MatchSet.
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3.4
Tiling Windows
The tile commands under the Window menu are used to arrange image windows on
the screen.
Note:
The tile commands only affect image windows, not subwindows.
Select Tile to resize all open windows so that all the images are maximized. The
windows will be arranged on the screen from left to right and top to bottom.
Tile Vertical will resize all open windows and arrange them in columns across the
screen.
Tile Horizontal will resize all open windows and arrange them in rows across the
screen.
3.5
Magnifying Images
The magnifying tools in PDQuest can be accessed from the main toolbar or View
menu, or by key command.
Note that, by default, if you magnify a region in one image of a MatchSet or scanset,
the same region in all the other subwindows will be magnified. To turn off this
feature, deselect the Auto “All Same” checkbox under Display Preferences (Edit >
Preferences).
Ctrl+F3 will undo your last magnification or other viewing command.
3-4
Viewing and Editing Images
Fig. 3-3. Viewing functions on View menu and main toolbar.
Zoom Box
Use the Zoom Box tool to magnify a specific region of an image, MatchSet, or
scanset.
Select Zoom Box from the main toolbar or View menu, or position your cursor on the
image and press F2. Drag a box on the image, then release the mouse button. The area
you selected will be magnified.
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1. Click Zoom Box button.
2. Drag box on image.
3. Boxed region is magnified to fill window.
Fig. 3-4. Zoom Box tool.
Zoom In/Zoom Out
Zoom In (Alt+F2, View menu and toolbar) will magnify an image, or all the images in
a MatchSet or scanset, by a defined percentage. The percent magnification is
specified under Display Preferences (Edit > Preferences).
Zoom Out (Ctrl+F2, View menu and toolbar) will reduce the magnification by the
same percentage.
View Entire Image
If you have magnified part of an image or moved part of an image out of
view, this command will restore the original, full view. Select View
Entire Image from the main toolbar or View menu and click the image,
or position your cursor on the image and press F1.
3-6
Viewing and Editing Images
Imitate Zoom
Imitate Zoom applies the magnification and positioning of a selected window or
subwindow to all open windows and subwindows. This command only works on
comparable images with similar dimensions.
Note:
Imitate Zoom is different than the Auto “All Same” setting under Display
Preferences, in that is affects all open windows, not just the subwindows in a
single window.
Select a window or subwindow you want to imitate, then select Imitate Zoom from
the Window menu. The other windows will change their display to match that of the
active window
Magnifying Sections of an Image
To magnify one of nine sections of an image or MatchSet/scanset, go the 3x3 Sections
submenu on the View > Advanced View submenu, and select a region (Upper Left,
Middle, Lower Right, etc.) That region will be magnified in the image or MatchSet/
scanset.
Location in Gel
Location in Gel on the View>Advanced View submenu displays a graphical overlay
on a subwindow or all subwindows showing the region that is magnified (green box)
relative to the entire image (blue box). Select the command, then click the image.
3.6
Positioning Images
The image positioning tools in PDQuest can be accessed from the main toolbar or
View menu, or by key command.
Note that, by default, if you reposition one image of a MatchSet or scanset in a
subwindow, all the other subwindows will be repositioned as well. To turn off this
feature, deselect the Auto “All Same” checkbox under Display Preferences (Edit >
Preferences).
Ctrl+F3 will “undo” your last positioning or other viewing command.
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PDQuest User Guide
Grab
Use this tool to drag the image in a window.
Select Grab from the main toolbar or View menu, or position your cursor
on the image and press Ctrl+G. Drag the image, then release the mouse
button. The image will be repositioned.
Swap
The swap command allows you to swap the location of gels in a window. This is
useful for positioning closely related gels next to one another in a window. To use the
swap command, select Grab from the main toolbar or View menu, or position your
cursor on the image and press shift+G. Drag the image to another image, then release
the mouse button. The two images change places.
Arrow Keys
You can shift the displayed region in a subwindow or all subwindows by using the
Arrow keys on your keyboard. With a window or subwindow selected, click an arrow
button. The image will shift in the direction of the arrow click. The amount the image
shifts is determined by the Pan % setting in Display Preferences under Edit >
Preferences.
3-8
Viewing and Editing Images
3.6.a Advanced View
Fig. 3-5. Advanced View submenus.
Centering an Image
You can center an image or images in a MatchSet/scanset quickly and easily using the
At Cursor command (F3 on the keyboard; also on the View > Advanced View
submenu). This is useful if you are comparing the same region of spots on multiple
images and want to center the image windows on the same spot.
Position your cursor on a point on the image, then press F3. The image will shift so
that that point is at the center of the image window.
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Displaying Sections of an Image
To display one of nine sections of an image or MatchSet/scanset, go the 3x3 Sections
submenu on the View > Advanced View submenu, and select a region (Upper Left,
Middle, Lower Right, etc.) That region will be magnified in the image or MatchSet/
scanset.
Pan means to move the camera, (or the focus of your gel), to the right or left, up or
down.
All Windows indicates that the selections you have made in the Advanced View
submenu will be applied to every window in your set.
Location in Gel
Location in Gel on the View>Advanced View submenu displays a graphical overlay
on a subwindow or all subwindows showing the region that is displayed (green box)
relative to the entire image (blue box). Select the command, then click the image.
3.7
Hiding Overlays
To conceal spot markings, graphs, information boxes, etc. in a
subwindow or multiple subwindows, select Hide Overlays from the main
toolbar or View menu and click in the subwindow, or use the F4 key
command in the subwindow.
Note that, by default, hiding the overlays in one image of a MatchSet or scanset will
hide them in all the other subwindows. To turn off this feature, deselect the Auto “All
Same” checkbox under Display Preferences (Edit > Preferences).
Note:
Shift+F4 (Hide All Overlays) will conceal overlays in all subwindows regardless
of whether Auto “All Same” is selected.
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3.8
Density Tools
The tools under the View>View Density submenu and on the Density Tools toolbar
are designed for advanced users when analyzing gels. They provide a quick measure
of the signal intensity of the data in Raw 2-D scans or Filtered images. Note that these
commands will not work on Gaussian images.
Fig. 3-6. Density submenu and toolbar.
Density at Cursor
To display the signal intensity at a point on the image, select Density at
Cursor from the submenu or toolbar and click the image. The pop-up box
will list the intensity in image units (ODs or counts), and will also show the
average intensity for a 3 x 3 image pixel box centered on that point.
Density in Box
To display the total signal intensity inside a specified region on the image,
select Density in Box from the submenu or toolbar and drag a box around
the area you want to measure. The area of the box (in square millimeters),
the total image intensity (volume) inside the box (in image units—ODs or
counts), and the average intensity will be displayed in a pop-up box.
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Plot Density Distribution
Plot Density Distribution displays a histogram of the signal intensity
distribution for the entire image. Select the command from the menu or
toolbar, and click on the image.
Plot Cross-section
To display a cross-sectional intensity trace centered on a point on the
image, select Plot Cross-section from the submenu or toolbar, or use the
Ctrl+T key command, and click or drag on the image. The trace will be
displayed, and will be continuously updated as you move your cursor
over the image. The intensity (in ODs or counts) at the cursor point is listed, as is the
maximum intensity along the lines of the cross-section.
Note:
The sampling width for the density traces is one image pixel.
Click button, then
click or drag on the image.
Fig. 3-7. Plot Cross-section tool.
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Plot Vertical Trace
Plot Vertical Trace plots an intensity trace down a vertical line centered on
the point where you click your mouse. Select the command from the menu
or toolbar, and click or drag on the image.
Note:
3.9
The sampling width for the density traces is one pixel.
Colors
Edit > Colors opens a dialog box in which you can adjust the colors of the image,
windows, buttons, etc.
Fig. 3-8. Colors dialog box.
Selecting a Color Group
Within the Colors dialog box, click the Color Group button to select the colors of the
particular group of objects you want to change (e.g., pop-up boxes, image colors,
etc.).
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Fig. 3-9. List of Color Groups.
Click a color group in the list to select it.
Changing a Color
After you have selected the color group to change, click the specific color button to
change. The Color Edit dialog box will open, allowing you to adjust the red-greenblue (RGB) values of the color you selected.
Fig. 3-10. Color Edit dialog box.
Drag the sliders or enter a value in the fields. The color of the button will change with
your adjustments.
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Saving/Selecting a Defined Set of Colors
After you have changed the colors within color groups, you can save these settings for
future use on other images. The Colormap Name field displays the name of a defined
set of colors and color groups. There are several standard colormaps, or you can
create your own.
To select a predefined colormap, click the Load button. From the list displayed, click
the set of colors you want to apply.
User-defined sets of colors
Fig. 3-11. Selecting a Colormap.
To create your own colormap, adjust the colors within the color groups as described
above and type in a new colormap name. Click OK to apply your changes.
To remove a colormap, click the Delete button. Select the colormap to be deleted
from the displayed list. A pop-up box will ask you to confirm the deletion.
If you want to return to the standard colormap for the selected group, click the Reset
button. All colors will be returned to their default values.
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3.10 Multi-Channel Viewer
You can use the Multi-Channel Viewer to distinguish different types and levels of
fluorescence in a gel that has been imaged at different wavelengths. The MultiChannel Viewer can be used to the merge the information from up to three different
images of the same gel.
Note:
The viewer requires that the images being compared are exactly the size and in
exactly the same position. When capturing the images, you should be careful not
to move the gel between exposures. If your images are not exactly the same size,
you can use the Crop tool to resize them.
With at least one image open, select Multi-Channel Viewer from the View menu. The
topmost open image will be displayed in the viewer window using the Red channel.
Fig. 3-12. Multi-Channel Viewer.
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Note:
The color channel used to display an image in the viewer has no relation to the
filter used when capturing the image. The red, green, and blue channels are
simply designed to distinguish different images.
The image name is displayed in the Red name field at the top of the viewer, and the
Red Channel checkbox will appear selected.
To add another image, make sure the image is open and click the pulldown button
next to the Green or Blue name field. Select the image name from the pulldown list.
Add a third image using the same procedure.
You can reassign the different images to different channels using the pulldown
buttons to the right of the name fields. Select <clear> from the pulldown list to
remove an image from that channel of the viewer.
Viewing Options
To remove a particular color channel from the display, click in the checkbox
associated with that channel to deselect it.
Selecting the Auto-Scale Image When Assigned checkbox will automatically adjust
the brightness and contrast of each loaded image based on the data range in the image.
It invokes the Auto-Scale command from the Transform window when an image is
first opened in the viewer. Note that this setting affects only how the image is
displayed in the viewer, not the actual data.
Note:
If you deselect this checkbox, any images currently displayed will remain autoscaled. click the Transform button in the viewer and click the Reset button in the
Transform window to undo auto-scaling.
Buttons for various viewing tools are included in the Multi-channel viewer.
Commands such as Zoom Box and Grab will change the display of all the images in
the viewer at once.
Note that if you open the Transform window, you can adjust the display of each
channel independently, by selecting the appropriate channel option button in the
Transform window. Similarly, the Plot Cross-Section command will report the
intensity of each channel separately.
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Exporting and Printing
You can export a 24-bit TIFF image of your merged view by
clicking on the Export button. This will open a version of the
Export to TIFF dialog. Note that you cannot export image data
from the Multi-channel Viewer—only the current view of the image (designated as
Publishing Mode in the Export dialog). The colors in the viewer will be preserved in
the exported TIFF image.
You can also print a copy of your merged view to a color or grayscale printer by
clicking on the Print button.
3.11 3D Viewer
The 3D Viewer tool allows you to see a three-dimensional rendering of a portion of
your image. This is important for such instances as determining whether a selected
spot is actually two or more separate spots.
To see a 3D rendering of a portion of your image, select 3D Viewer from the View
menu. Your curser turns into a crosshair. Click and drag your curser over the image
area you would like to view creating a box.
Note:
viewing a large area of your image may reduce performance.
•
To reposition the box, position your cursor at the center of the box. The cursor
appearance will change to a multidirectional arrow symbol. You can then drag
the box to a new position.
•
To resize the box, position your cursor on a box corner. The cursor appearance
will change to a bi-directional arrow. You can then drag that corner in or out,
resizing the box.
•
To redraw the box selection, position your cursor outside the box and click once.
The box disappears, and you can then draw a new box.
To view the selected area, position your cursor inside the box slightly off-center. The
cursor appearance will change to an arrow. Click once to open the 3D viewer.
Note:
If you are having problems using the 3D Viewer, install the latest drivers for your
graphics card. If, after updating your drivers, you are still having problems, go to
the Display tab of the Preferences dialog box and select Disable hardware
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acceleration for 3D viewer. This allows PDQuest to emulate a graphics driver to
render a 3D image. See Section 2.7.c, Display Preferences, for further
information.
Fig. 3-13. 3D Viewer
3.11.a Positioning the Image
Windows
For Windows operating systems, use your mouse to reposition and rotate the image.
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•
Rotate the image - Left click and drag to rotate the image.
•
Reposition the image - Right click and drag to reposition the image.
•
Zoom in/out - To zoom in or out, Click the center mouse button or roll the wheel.
If you do not have a three button mouse or a mouse with a wheel, hold down the
shift key and left click and drag to zoom in or out.
Macintosh
For Macintosh operating systems, use mouse and keyboard combinations to
reposition and rotate the image.
•
Rotate the image - Click and drag to rotate the image.
•
Reposition the image - Ctrl>click and drag to reposition the image.
•
Zoom in/out - Shift>click and drag to zoom in or out.
3.11.b Display Mode
The 3D Viewer window allows you to view the image in three different modes; wire
frame, lighting, and textured.
•
Wire-frame shows the image in a transparent frame view.
•
Lighting shows the image with different areas of light and shadow depending on
the angle of view. Use the slider bar to adjust the intensity of the lighting.
•
Texture gives the image texture.
Use the Scale function to scale the image. This is useful for viewing shallow spots in
the 3D Viewer.
If you lose the image because you moved it too far past the window border, or rotated
it and disoriented the view, click Reset View to return the image to the original view.
Note:
Reset view does not change the scale factor. To reset the scale factor, close the
3D Viewer and click the box again to re-open the 3D Viewer with the original
scale factor.
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3.12 Transform
The Transform controls are used to adjust image brightness and contrast and optimize
the image display. These controls affect the display only, not the underlying data.
Select Transform from the Image menu or main toolbar and click in the subwindow,
or use the Ctrl+B key command in the subwindow.
Fig. 3-14. Transform command.
The preview window in the Transform dialog shows a smaller view of the selected
image. Changes are automatically reflected in the preview window, and are only
applied to the main image(s) when you click OK or (PDQuest only) Apply All.
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Preview Window
Transform Plot
Frequency Distribution histogram
Fig. 3-15. Transform dialog box.
You can use regular viewing tools like Zoom Box and View Entire Image in the
preview window to focus on particular regions of interest. You can also use Grab tool
and arrow keys to move the image within the preview window.
The Frequency Distribution histogram shows the total data range in the image and the
amount of data at each point in the range. In a typical scan, there is a signal spike at
the left (“gray”) end of the histogram due to background noise.
The Transform Plot is a logarithmic representation of how the raw pixel data are
mapped to the pixels of your computer screen.
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Note:
If you are using the Transform window in conjunction with the Multi-channel
Viewer, you can adjust the display of each channel independently by selecting the
appropriate channel option button below the preview window.
3.12.a Transform Controls
Auto-scale
Click the Auto-scale button to optimize the selected image automatically. The lightest
part of the image is set to the minimum intensity (white), and the darkest is set to the
maximum intensity (black). This maximizes the display range for the image. You can
then “fine-tune” the display using the sliders.
Select the Always Auto-scale checkbox to automatically auto-scale every image that
you open. This setting disables the remaining controls in the dialog box; the software
will examine the data range in every image and optimize it accordingly.
Auto All will auto-scale all the images in the selected scanset or MatchSet and close
the Transform dialog.
High/Low Sliders
Use the High and Low sliders to optimize the image manually. Drag the High slider
handle to the left to make weak signals appear darker. Drag the Low slider handle to
the right to reduce image background.
As you drag, examine the Frequency Distribution histogram. Everything to the left of
the low slider will be remapped to minimum intensity, while everything to the right of
the high slider will be remapped to maximum intensity. Position the markers at the
ends of the data range in the histogram, and use the low slider to cut off the “spike” of
background noise.
You can also type specific high and low values in the text boxes next to the sliders.
Clicking anywhere on the slider bars will move the sliders incrementally.
If your image data is in a narrow range (as shown in the histogram), select the Log
High/Low Sliders checkbox. This allows for finer adjustments with the slider handles.
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Range of actual
data in the image
is very limited
Low slider remaps background noise to white
Can better distinguish background
noise from real data
High slider remaps weaker signals to black
Low-High magnifies
area between sliders
Log scaling enables you to
better distinguish peaks
Fig. 3-16. Two views of the Frequency Distribution histogram.
Gamma Slider
Some images may be more effectively visualized if their data are mapped to the
computer screen in a nonlinear fashion. Adjusting the Gamma slider handle expands
or compresses the contrast range at the dark or light end of the range, and this is
reflected in the transform plot and preview window.
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Reset
If at any time you want to return to an unmodified view of the scan data, click Reset.
Applying the Settings
Click OK to apply the selected settings to the image in the preview window. Click
Apply All to apply the settings to all the subwindows in the selected scanset or
MatchSet, or click Auto All to auto-scale all the subwindows. These buttons will
close the Transform window and displayed the transformed image(s).
3.12.b Other Transform Features
Full Scale/Low-High
The Full Scale/Low-High option buttons adjust how the range of image data is
displayed in the frequency distribution histogram and transform plot. They do not
change how the image is displayed in the preview window.
Full Scale adjusts the frequency distribution and transform plot displays so they show
the full intensity range of the image.
Low-High magnifies the range between the low and high sliders. This makes it easier
to view your data if it does not occupy the full intensity range of the image.
Log
The Log checkbox changes the way the data is displayed in the histogram so you can
better discern subtle changes in signal intensity.
Image Max/Min
Image Max and Min display the range of signal intensity in the image.
The image units are determined by the type of scanner used to create the image. In the
case of densitometers, you can select Calibrated Quantity to display your image units
in O.D.s.
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Invert Display
The Invert Display checkbox flips light bands on a dark background to dark bands on
a light background, and visa versa. Once, again, the actual data will not change—only
the image.
Highlight Saturated Pixels
When the Highlight Saturated Pixels checkbox is selected, areas of the image with
saturated signal intensity are highlighted in red. This setting does not apply to
Gaussian images.
3.13 Cropping Images
When matching spots between the gels in a MatchSet, the MatchSet images should be
all the same size and shape. If the original scans of these gels are different sizes, you
will need to crop them before creating the MatchSet.
Note:
You can only crop Raw 2-D scans, not Filtered or Gaussian images.
The crop tools can also be used to reduce the file size of images. (To reduce
the file size of images by lowering the resolution, see Reduce File Size.)
Fig. 3-17. Crop commands.
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Select Crop from the Image menu or toolbar, and drag the cursor on the image,
creating a box. Everything outside the crop box will be deleted.
The size of the crop area (in millimeters and number of pixels) is shown at the bottom
of the crop box, as is the file size of the image inside the crop area.
•
To reposition the crop box, position your cursor at the center of the box. The
cursor appearance will change to a multidirectional arrow symbol. You can then
drag the box to a new position.
•
To resize the box, position your cursor on a box border or corner. The cursor
appearance will change to a bidirectional arrow. You can then drag that border or
corner in or out, resizing the box.
•
To redraw the box, position your cursor outside the box. The cursor appearance
will change back to the Crop tool, and you can draw another box, replacing the
one you just drew.
To complete the crop, position your cursor inside the box slightly off-center. The
cursor appearance will change to a scissors symbol. Click once to perform the crop.
A pop-up box will ask you whether you want to: (1) crop the original image, (2) save
a copy of the area inside the crop box as a separate image, keeping the original image
intact, or (3) cancel out of the cropping operation.
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Fig. 3-18. Crop box and pop-up Crop dialog.
If you select Crop, your cropped image will be displayed in the image window. If you
select the Copy and Crop button, a dialog box will open in which you can enter the
name of the cropped image, its version number, exposure number, and/or file type.
Click OK, and the image window will display both the original image and the
cropped image in subwindows.
3.13.a Advanced Crop Commands
To ensure that your scans are exactly the same size and shape, you can use the tools
on the Image > Advanced Crop submenu to save the crop box from one scan and
apply it to others. These tools also allow you to crop a gel and calstrip out of the same
scan.
To define and save a crop box and apply it to another scan:
1.
Select Define Crop Area from the submenu and drag a crop box on an image.
Position the box as described in Cropping Images.
2.
Select Place Crosshair from the submenu and click a landmark inside the box that
is present in all the gels you want to crop. This could be a spot or some other
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image detail. The crosshair will make it easier to position the box in the other
images so that it encloses the same area.
3.
Select Save Crop Settings from the submenu, enter a name for the current crop
settings in the pop-up box, and click Apply.
4.
Complete the crop action in the current image by positioning your cursor inside
the box slightly off-center and clicking to perform the crop, as described in
Cropping Images.
5.
Open or select the next image you want to crop, select Load Crop Settings from
the submenu, and select the name of the settings you saved. The crop box and
crosshair will appear on the image.
6.
Reposition the crop box so that the crosshair is correctly aligned with the
appropriate image object, then complete the crop as described in step 4 above.
To delete any crop settings you have saved, select Delete Crop Settings from the
submenu and choose the settings to be deleted from the list.
3.14 Flipping and Rotating Images
The matching functions in PDQuest will tolerate minor distortions in the shape and
orientation of images. However, if your images are grossly misaligned, you may have
to flip and/or rotate them. These commands are located on the Image menu and
toolbar.
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Fig. 3-19. Flipping and rotating commands.
Note:
These actions will erase any analysis you have performed on an image. You will
be asked to confirm the flip/rotation before the command is executed.
Flipping
To flip the image right-to-left, select Horizontal Flip from the Image menu or toolbar.
To flip the image top-to-bottom, select Vertical Flip.
90° Rotations
Select Rotate 90 Left, Rotate 90 Right, or Rotate 180 from the Image > Rotate menu
or Image toolbar to perform the specified rotation. You will be asked to confirm your
choice before the command is executed.
Custom Rotation
If you need to rotate your image in increments other than 90°, you can use the Custom
Rotation command.
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Select Custom Rotation from the Image > Rotate menu or Image toolbar. A green
“plus” sign will appear next to your cursor.Click on the image you want to rotate and
a circular overlay with an orange arrow will appear. A small dialog box also will
open, indicating the angle of rotation in degrees and radians.
Fig. 3-20. Custom rotation; the arrow points in the direction of the new top of
the image.
To perform the rotation, position your cursor on the arrowhead and drag. As you drag,
the arrow will rotate and the angle in the dialog box will change. Position the arrow so
that it points in the direction of the new top of the image. You can “fine-tune” your
rotation as much as you like.
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Note:
If you want to center your arrow on a particular point on the image (e.g., to align
along a particular lane), you can use the F3 key command. Position your cursor
on the point on the image you want to center on, and press the F3 key. The image
will move so that the center of the arrow and your cursor point are aligned.
To complete the rotation, click the Rotate button in the small dialog box. A
subwindow will open containing the rotated image. You will then have the option of
renaming your new image and changing the version number.
If you are not satisfied with your rotated image, simply delete it and start over.
Note:
Because an image is composed of square or rectangular pixels, Custom Rotation
has to perform some minor smoothing on the image to turn it at a non-90o angle.
In addition, any analysis performed on the image cannot be rotated and will be
lost.
3.15 Filtering Images
Filtering is a process that removes small noise features on an image while leaving
larger features (like data) relatively unaffected. A wide range of filters are available
for removing different types of noise from images. Depending on the nature of your
data, you will probably need to use only one or two of the available filters. However,
you should experiment with several different filters before selecting the ones that
work best for your images.
The filtering commands are located on the Image menu and toolbar.
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Fig. 3-21. Filtering commands.
Note:
Since filtering is an irreversible process, you will be asked if you want to create a
copy of the original image before you filter. If you are experimenting with
various filters, you should create copies of your image and compare them sideby-side. If you filter the original image and save it, you cannot return to the
original, unfiltered state.
3.15.a Filter Wizard
The Filter Wizard is designed to guide you through the filter selection process. First,
you identify the type of noise in your image. Next, select the size of the filter to use on
that noise. Finally, filter the image.
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To open the Wizard, select Image > Filter Wizard or click the Filter Wizard button on
the Image Tools toolbar.
Fig. 3-22. Filter Wizard dialog box.
The Wizard contains settings for identifying the different types of noise in the image.
It also includes a density distribution histogram of the noise in the image to aid in
filter selection.
Step I: Identify Noise Characteristics
The first step in the Wizard is to identify the type of noise in your image. Examine
both the image and the density distribution histogram, then select one, both, or neither
of the following checkboxes:
•
Salt. This type of noise appears as specks that are lighter than the surrounding
background. The density distribution histogram of this type of noise displays
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noise peaks at the high end of the range (right end of the plot). This type of noise
is common in electronic cameras with malfunctioning pixels. It can also be
caused by dust or lint in the imaging optics or scratches on photographic film.
Salt is a type of outlier noise (see below).
•
Pepper. This type of noise appears as specks that are darker than the surrounding
background. The distribution histogram of this type of noise displays noise peaks
at the low end of the range (left end of the plot). Its causes are similar to those of
salt noise. Pepper is a type of outlier noise (see below).
Next, select one of the following option buttons to describe additional features of
your noise.
•
Gaussian. The distribution histogram of this type of noise has a Gaussian profile,
usually at the bottom of the data range. This type of noise is usually an electronic
artifact created by cameras and sensors, or by a combination of independent
unknown noise sources.
•
Uniform noise. This type of noise appears in the histogram as a uniform layer of
noise across the data range of the image.
•
Outlier noise. This category of noise includes salt and pepper noise (see above).
The distribution histogram of this type of noise displays noise peaks at the high
and low ends of the range.
After you have identified the type of noise, go to Step 2.
Step 2: Select Filter Size
Image noise is filtered by means of a filtering window (or kernel), which is measured
in pixels. This filtering window slides across the image, processing the pixels within
it.
The available filter dimensions range from 3 x 3 pixels to 9 x 9 pixels. To select an
appropriate size, magnify a background region of your image so that you can see the
individual pixels. The filter size you select should be larger than the average noise
feature but smaller than your data features.
Note:
A smaller filter will alter your image less than a larger filter. Large filters can
result in better suppression of noise, but can also blur desirable features in the
image.
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Step 3: Begin Filtering
After you have completed your selections, the filter name and size will be displayed
at the bottom of the Filter Wizard dialog box.
To being filtering, click the OK button. Because filtering is an irreversible process, a
pop-up box will give you the option of filtering the original image, creating a copy of
the image to filter, or cancelling out of this operation.
If you choose to Copy and Filter, you will be asked to enter a new name and/or
version number for the new copy before the operation is performed. Once this
information has been entered, the filtering operation will be performed.
3.15.b Selecting a Filter Directly
If you already know the type and size of filter you want, you can select it directly by
selecting Image > Filters > Filter List. The pulldown menu includes all the filters
available in the Discovery Series.
The types of filters in the Discovery Series are:
•
Weighted Mean. This filter is useful for reducing Gaussian noise; it has little
effect on salt and pepper. It calculates the weighted mean of the pixels within the
filtering window and uses it to replace the value of the pixel being processed.
•
Out of Range Pixel. This filter is useful for suppressing salt and pepper noise; its
effect on Gaussian noise is minimal. This filter calculates the mean of the pixel
values in the filtering window, including the pixel being processed. If the
difference between the mean and the individual pixel value is above a certain
threshold, then the individual value is replaced by the mean.
•
Median. Also useful for suppressing salt and pepper noise, this filter has little
effect on Gaussian noise. It calculates the median value of the pixels within the
filtering window and uses it to replace the value of the pixel being processed. The
median filter produces very little blurring if a small-sized window is selected.
•
Maximum. This filter is useful for eliminating pepper noise in an image (it
worsens the effect of salt noise). It replaces the value of the pixel being processed
with the maximum value of the pixels within the filtering window.
•
Minimum. This filter replaces the value of the pixel being processed with the
minimum pixel value within the filtering window. This filter is useful for
eliminating salt noise in an image (it worsens the effect of pepper).
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•
MidValue. This filter is useful for suppressing uniform noise within an image;
however, it worsens the effect of pepper and salt. This filter replaces the value of
the pixel being processed with the mean of the maximum and minimum pixel
values within the filtering window.
•
Power Mean. This filter is useful for suppressing salt and Gaussian noise within
an image (it worsens the effect of pepper noise). It replaces the value of the pixel
being processed with the power mean of the pixel values within the filtering
window.
•
ContraMean. This filter is useful for suppressing pepper and Gaussian noise
within an image (it worsens the effect of salt). It replaces the value of the pixel
being processed with the contra-harmonic mean of the pixel values within the
filtering window.
•
Adaptive. This filter is useful for suppressing Gaussian noise and salt and/or
pepper within an image. If your image contains a mix of salt and pepper, select
this filter.
To begin filtering, select a filter type from the pull-down list. A pop-up box will ask
you to select a filter size.
Fig. 3-23. Selecting a filter size.
The available filter dimensions range from 3 x 3 pixels to 9 x 9 pixels. (See the
previous section for guidance on selecting a size.)
Because filtering is an irreversible process, a pop-up box will give you the option of
filtering the original image, creating a copy of the image to filter, or cancelling out of
this operation.
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If you choose to copy and filter, you will be asked to enter a new name and/or version
number for the new copy before the operation is performed. Once this information
has been entered, the filtering operation will be performed.
3.16 Invert Data
If your image has light spots on a dark background (i.e., the signal intensity
of the background is greater than the signal intensity of the sample), you
will need to use the Invert Data function before you can analyze the image.
Note:
The Invert checkbox in the Transform dialog box (section 3.12.b, Other
Transform Features) inverts only the appearance of the image.
To invert your image data, select Image > Invert Data. You may need to use the
Transform function to adjust the appearance of your inverted image.
This command is reversible. Select Invert Data again to revert to the original data.
3.17 Text Overlays
If you want to create and display textual notes directly on your image, select Text
Overlay Tools from the View > Toolbars submenu. This will open the Text Overlay
Tools toolbar.
Select tool
Text tool
Line tool
Copy
Paste
Alignment tools
Fig. 3-24. Text Overlay Tools toolbar.
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Viewing and Editing Images
Creating a Text Overlay
To create a text overlay, click the Text Tool, then click on the image at
the spot where you want the text to appear. This opens the Text Overlay
Properties dialog box.
Text color
Background color
Fig. 3-25. Text Overlay Properties dialog box.
To enter your text, simply begin typing in the main field. The buttons in the dialog
allow you to select the properties of your text, including format, alignment, and
justification.
The pull-down boxes (from left to right) allow you to select the font style, font size,
color of the text, and color of the background within the text box.
Once you have typed your text, click OK.
When you exit the Text Overlay Properties dialog by clicking OK, the text you typed
will appear on the image at the spot where you originally clicked.
You can create as many textual overlays as you want.
Editing a Text Overlay
To edit a text overlay, make sure the Text Tool or Select Tool is assigned to your
mouse, then double-click the overlay to open the Properties dialog. The existing text
will be displayed and can be edited.
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Line Tool
You can use the Line Tool on the Text Overlay toolbar to draw a line
between text and an image feature, or between any two points of interest on
your image.
Click the Line Tool button, then drag on your image to create the line. You can create
as many lines as you want.
To resize or adjust a line, make sure the Line Tool or Select Tool is assigned to your
mouse, then position your cursor on one end of the line (marked by a circle) and drag.
To add arrowheads to a line, make sure the Line Tool or Select Tool is assigned to
your mouse, then double-click the middle of the line. A dialog box will pop up with
options to add arrowheads to one or both ends of the line.
Moving and Copying Text Overlays and Lines
You can move, copy, or delete a single overlay/line or a group of overlays/lines within
an image. You can also copy and paste between images.
First, you must select the object(s). Click the Select Tool button on the Text
Overlay toolbar. To select a single overlay or line, click it. To select
multiple objects, either drag a box around them or hold down the SHIFT key
while you click them one at a time. When dragging to select a group of objects, make
sure that you completely surround all the objects to be selected.
Each selected overlay/line will have a green border.
•
To move the selected object(s), position your cursor over the selection and drag.
•
To copy within an image, hold down the CTRL key while dragging the selected
object(s). The copy will be created and dragged to the new position.
•
To delete the selected object(s), press the DELETE key.
•To copy between images, click the Copy to Clipboard button on the
Text Overlay toolbar, then open or select the image you want to copy
to.
• Click the Paste from Clipboard button. The copied object(s) will be
pasted into the new image in the same relative position they were
copied from.
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Viewing and Editing Images
Note:
If you are pasting into an image with a different pixel size (i.e., resolution), you
will receive a message that the placement of the copy may not be exact. Click OK
to complete the paste, then position the pasted objects manually.
Viewing Previously Created Text Overlays/Lines
To display previously created text overlays and/or lines after opening an image, click
the Text Overlay Tools button on the main toolbar.
If you have concealed all your overlays using Hide Overlays, clicking on any of the
buttons on the Text Overlay Tools toolbar will display the hidden text.
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3-42
4. Detecting and Editing
Spots
This chapter describes how to detect and edit spots using PDQuest.
4.1
Selecting Spot Detection Parameters
The Spot Detection Wizard is used to select the parameters for detecting
spots in gel scans.
Because gels vary in stain type, spot intensity, clarity, streaking, etc.,
different parameters are needed to detect spots on different gels. In general, you can
use the same parameters to detect spots on all gels that use the same stain and have
been run and scanned in the same manner.
To select the detection parameters for a particular type of gel, make sure that a
representative gel scan is open and active in PDQuest, then select Spot Detection
Wizard from the Spots menu or quick guide.
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Fig. 4-1. Spot Detection Parameter Wizard.
The Wizard will open, and the name of the scan will be displayed below the preview
window. To select a different image, click the drop down arrow and choose from the
list of open images. Select Show Filtered Image to have the Wizard display the
filtered image after clicking Find Spot Centers in Step 2. The parameter set field
indicates the name of the currently loaded parameter set. If no set is loaded, this field
is blank. Click Load next to the parameter set name to load a different set of
parameters into the Wizard.
You can choose between the Basic and the Advanced tabs. With the Basic tab you can
perform Steps 1 and 2 of the parameter selection process. The Advanced tab offers the
ability to manually adjust the settings in Step 2 and the addition of Optional Controls
to manipulate streaks, backgrounds, smoothing, and speckles.
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Detecting and Editing Spots
Fig. 4-2. Selecting a new image in the Spot Detection Wizard.
The control panel in the Wizard is designed to guide you through the parameter
selection process:
4.1.a Step 1. Give Manual Guidance
In this step, you mark features on the gel scan that will be used to automatically select
the correct spot detection parameters.
1.Click the Faint Spot button, then click on the faintest spot in the scan
that you want to detect. This will set the sensitivity and minimum peak
value parameters.
2.If the smallest spot you want to detect is smaller than the faintest spot,
click the Small Spot button, then click on the smallest spot. This will set
the size scale parameter.
3.Click the Large Spot button, then draw a box around the largest spot
cluster on the image that you want to detect. This sets the radius of the
background subtraction rolling ball and the streak removal rolling disk.
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Fig. 4-3. Image with large, small, and faint spots defined.
4.1.b Step 2. Test Settings
In this step, you first see how many spots are detected based on the settings in Step 1,
then you adjust those settings until all significant spots are identified.
Note:
Before proceeding, if there are streaks in your image, select the Vertical and/or
Horizontal streak removal checkboxes under Optional Controls.
Click the Find Spot Centers button. Noise smoothing, background
subtraction, and (if selected) streak removal will be performed,
and the spot centers will appear marked with crosshairs.
If the Spot Detection Wizard is unable to find parameters, a warning message
displays. This is usually caused by the faint spot being to close to a large spot cluster
or the resolution of your image is too high. If you think the problem may be caused by
the faint spot location, you need to either adjust the sensitivity or select a different
faint or small spot. If the resolution of your image is too high, use the Reduce File
Size command (See Section 2.3.h, Reduce File Size) to reduce the resolution, then
reopen the Spot Detection Wizard and proceed with spot detection.
Review the results of spot detection by studying the crosshairs on the image. The
number of spots detected is displayed in the Spot Count field.
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Detecting and Editing Spots
Fig. 4-4. Spot crosshairs marked in the Spot Detection Wizard.
If all the spots you want to identify have been detected, you can save this parameter
set. If significant spots have been missed, you can change the faint, small, and large
spot selections in Step 1, or change the Sensitivity, Min Peak, and Size Scale directly.
Note:
Adjustments to these settings will automatically redetect spots.
Sensitivity
If significant faint spots in the scan have been missed, you can use the Faint Spot tool
in Step 1 to change your faint spot selection, or you can increase the Sensitivity
setting by typing a number directly in the field (next to the Find Spot Centers button)
or clicking on the adjustment arrows. Higher sensitivity will detect more spots. If
image noise has been detected as spots, decrease the sensitivity or perform additional
speckle removal.
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Min Peak
If increasing the Sensitivity setting or selecting a new Faint Spot still fails to find
some faint spots, you can lower the Min Peak value. This is the minimum peak
intensity that a spot must have to be identified. Again, image noise may be detected as
spots if the Min Peak is too low.
Size Scale
The Size Scale field helps distinguish trends in signal intensity (spots) from random
intensity fluctuations (noise). It uses the size of objects in the image (in pixels) to
determine the nature of those objects. The initial size scale setting is based on the
Small Spot you selected in Step 1.
If a gel image has noticeable noise features, a larger size scale may be preferable. If
smoothing has adequately removed noise, small values are preferred so that small
spots will be detected. You can enter odd values from 3 to 9 in the field or adjust the
value using the arrow buttons.
Gaussian Model During Test
If the Gaussian Model During Test checkbox is selected, two-dimensional Gaussian
fitting will be performed on all spots when you find spot centers. It will take longer to
detect spots; however, spot detection will be more accurate for overlapping spots.
Note:
Fewer spots may be found if Gaussian Model During Test is selected, because
large spots that would be identified as overlapping spot clusters can be correctly
resolved with Gaussian modeling.
4.1.c Optional Controls
Most of the background removal and smoothing settings under Optional Controls are
set automatically based on your selections in Step 1. You should try to Find Spot
Centers first before adjusting these controls.
Note:
If your image has streaks, you should select streak removal under the Streaks tab
when finding the spot centers.
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Detecting and Editing Spots
Streaks
Protein gels frequently display streaking in areas of high spot concentration. Click the
Streaks tab to access the streak removal settings.
To remove vertical and/or horizontal streaks from your image, select the appropriate
checkbox(es). The disk radius size is determined automatically based on the size of
the largest spot you want to detect (Step 1).
The default disk radius size is conservative. If, after you find spot centers, you would
like to remove more streaking from the image, reduce the size of each disk radius by
about half and find spot centers again.
Background
Click the Background tab to access the settings for subtracting background intensity
from the image.
Background subtraction is required for spot detection. Floating Ball is the method
used. The default Radius size is calculated automatically based on the largest spot
cluster you want to detect (selected in Step 1 and displayed in the field at the bottom
of the tab). Although you can change the ball radius, it is highly recommended you
use the default.
Smoothing
Click the Smoothing tab to access settings for performing smoothing.
Image smoothing in your scans is optional but highly recommended. The default state
of the Perform Smoothing checkbox is checked. To deactivate this function, deselect
the checkbox by clicking in it.
The Filter Type is initially set based on the section of the image you boxed in Step 1.
The Kernel Size is set based on the faintest/smallest spot you want to detect.
Note:
You should first attempt to find spot centers using these default settings before
attempting to adjust them.
If you know the type of noise in your images, you can select a different filter type and
kernel size. Section 3.15, Filtering Images, provides additional guidance on filter
selection.
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•
Median. This filter is useful for suppressing salt and pepper noise. It has little
effect on Gaussian noise. This filter produces very little blurring if a small-sized
window is selected.
•
Weighted Mean. This filter is useful for reducing Gaussian noise. It has little
effect on salt and pepper noise.
•
Power Mean. This filter is useful for suppressing salt and Gaussian noise within
an image (it worsens the effect of pepper noise).
•
ContraMean. This filter is useful for suppressing pepper and Gaussian noise
within an image (it worsens the effect of salt).
•
Adaptive. This filter is useful when you have a particularly noisy gel and the
other filters do not perform well.
To adjust the Kernel Size, magnify a background region of your image using the
Zoom Box tool so that you can see the individual pixels. The filter size you select
should be larger than the average noise feature but smaller than the smallest spots.
Speckles
If you received a message upon opening the Spot Detection Wizard indicating the
detection of Ruby Speckles in your image, select the Speckles tab under optional
controls to access the ruby speckle filter settings.
Check the box labeled Apply Sypro Ruby Speckle filter to apply the filter. Use the up
and down arrows to adjust the sensitivity of the filter, or enter a specific sensitivity in
the field. Click the Find Speckles button to locate the speckles in the image.
The speckle count field indicates the number of speckles found in the image based on
the filter sensitivity. To view the speckles in the image, click show Speckles.
4.1.d Targeted Spot Detection
Occasionally the use of the same parameters for all files in a MatchSet produce
inconsistent spot numbers between the gels. To eliminate this problem, select Try to
find the same number of spots on other gels. If checked, then the spot detection
parameters will be varied for each gel so that same number of spots are obtained.
Note that this is before the Gaussian fitting, therefore the actual number of spots may
be slightly different after gaussian modelling is performed.
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Detecting and Editing Spots
4.1.e Saving/Loading Parameters
After you have selected the best detection parameters for the image, save them by
clicking the Save button (next to Parameter Set below the image window). Enter an
appropriate name for the set (e.g., the type of stain used in the gel) in the pop-up box.
Click OK. The parameter set will now be available for processing all similarly
detected gel scans.
To load previously saved parameters, click the Load button and select from the list of
parameter sets. When the parameters are loaded, you can proceed to Find Spot
Centers on the loaded image. (Each parameter set is saved as an “.adp” file in the
Discovery Series <user name>.prm directory. To delete a parameter set, delete its .adp
file.)
4.1.f
Processing Gels
Click the Process All Gels button to close the Spot Detection Wizard and open the
Auto Detection and Matching dialog box.
If you have not saved the current detection parameters, you will be prompted to do so
before you exit the Wizard.
The Quit button will close the Wizard without opening the Auto Detection and
Matching dialog box. If you have not found spot centers, any unsaved parameters will
be lost.
4.2
Automated Detection and Matching
After you have created and saved a set of detection parameters using the Spot
Detection Wizard, you are ready to detect spots on your images. In general, you can
use the same parameters to detect spots on all gels that use the same stain and have
been run and scanned in the same manner.
Click Process All Gels in the Wizard to open the Automated Detection and Matching
dialog box. You can also open the dialog box directly by selecting Automated
Detection and Matching from the Spots menu. All currently loaded gel scans will be
listed in the panel.
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PDQuest User Guide
Fig. 4-5. Automated Detection and Matching
The Automated Detection and Matching tool combines several processing steps into a
single operation.
Step 1 - Select Gels To Analyze
When the Automated Detection and Matching dialog box opens, any open images are
automatically included in the list. Click Add to add unopened images to the list.
The Remove button removes a gel from the list.
The Master button designates the selected gel as the MatchSet master image. If you
do not select a master, PDQuest automatically chooses a master for you based on
which gel has the best average spot quality.
Tip:
To view the average quality for each gel, click the Log file button in the
Matching Summary. See Section 5.8, Matching Summary for further
information.
This gel will be used as the starting point for constructing the eventual Master image.
See Section 5.7, "Adding Spots to the Master" for more details on constructing the
Master image.
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Detecting and Editing Spots
As indicated, you can set the order of the members by dragging them in the list. This
order will be used when displaying the images as well as plotting bar graphs.
Step 2 - Detect Spots on Gels
Step 2 determines the parameters used to automatically process all the images. If you
have processed similar images in the past, select the same parameter set from the
drop-down list. If you cannot use an existing parameter set, click Edit... to launch the
Spot Detection Wizard and create a new parameter set. See Section 4.1, Selecting
Spot Detection Parameters.
Step 3 - Create MatchSet
Enter a name for the MatchSet in the Name field. To assign your MatchSet a default
name, click Auto. The default prefix can be set on the Application tab of the
Preferences dialog box. See Section 2.7, Preferences, for further information.
Enter a description of the MatchSet in the Description field. You can enter further
information about the MatchSet in the Comments field.
The Directory field indicates the folder where the MatchSet will be saved. The
currently listed folder is based on the last time a MatchSet was created using
Automated Detection and Matching. To specify a new location, type in a new path or
click Browse.
Note:
If you want to detect spots without creating a MatchSet, check the "Disable this
step" checkbox at the bottom of Step 3.
Step 4 - Match Gels
This is similar to Step 2 in that you can select existing parameters for matching or you
can create new ones with the Edit... button. However, unlike Spot Detection
parameters, the matching parameters work over a wide range of images and you will
probably obtain satisfactory results with the default parameters. See Section 5.2.a,
Automated Matching, for further information.
Note:
If you want to create a MatchSet without matching spots, check the "Disable this
step" checkbox at the bottom of Step 4.
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PDQuest User Guide
Spot Detection Report
To review the log file created during spot detection, select Spot Detection Report
from the Spots menu. This report documents when spot detection began, which gels
were detected (or failed to detect), which steps were performed, and when spot
detection was completed.
4.3
Filtered and Gaussian Images
When spots are detected in PDQuest, the original gel scan is filtered and smoothed to
clarify the spots, then three-dimensional Gaussian spots are created from the clarified
spots. The end result is three separate images: the original Raw 2-D scan, which
remains unchanged; the Filtered image, which is a copy of the original scan that has
been filtered and processed; and the Gaussian image, which is a synthetic image
containing the Gaussian spots.
Raw 2-D
Scan
Gel image
Gaussian
Image
Fig. 4-6. Images created during spot detection.
All quantitation and other analysis are performed on the Gaussian image. PDQuest
preserves all three images so that at any point you can go back and reexamine the
original scan and the Filtered image to compare the original spots with the synthetic
spots in the Gaussian image.
Note:
Any spots not detected in the Filtered image will not be included in the Gaussian
image. You can add spots to the Gaussian image during spot editing, as described
in following sections.
A Raw 2-D Scan, Filtered image, and Gaussian image of the same gel are part of the
same scanset; they all share the same root file name with different extensions.
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Detecting and Editing Spots
Therefore, performing spot detection on an original Raw 2-D scan named
Proteins.gsc would result in a Filtered image named Proteins.gim and a Gaussian
image named Proteins.gsp
After spot detection, the Raw 2-D scan, Filtered image, and Gaussian image will be
displayed in subwindows of the same window. The file name and type of each image
are displayed at the bottom of each subwindow.
Fig. 4-7. Example of a Scanset.
Note:
Use the Assign tool and Configure Subwindows command on the main toolbar to
arrange images in subwindows. See section 3.1, Windows and Subwindows for
more information about viewing commands.
What Are Gaussian Spots?
Fuzzy, streaked, or overlapping spots in a Raw 2-D gel can be difficult to accurately
quantify. Because the image profile of an ideal spot conforms to a Gaussian curve,
PDQuest uses Gaussian modeling to create “ideal” spots that can be easily identified
and quantitated.
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A Gaussian spot is a precise three-dimensional representation of an original scanned
spot. Gaussian curves are fitted to the scanned spot in the X and Y dimensions, and
then additional modeling is performed to create the final Gaussian spot.
Using Gaussian modeling, you can accurately quantitate overlapping spots, spots in
gel streaks, and multiple spots in clusters.
Note that spots created using the spot boundary tools (section 4.6, Spot Boundary
Tools) are not Gaussian modeled, even though they appear in the Gaussian image.
4.4
Spot Crosshairs and Ellipses
Spot crosshairs mark the centers of spots in the Gaussian image, while spot ellipses
show their shapes. The tools for displaying spot crosshairs and ellipses are located on
the Spots menu.
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Detecting and Editing Spots
Fig. 4-8. Spots menu and Edit Spot toolbar.
Note that, in a scanset, the Gaussian image must be loaded to display crosshairs and
ellipses. With the Gaussian image loaded, you can also display the crosshairs and
ellipses in the Filtered image and Raw 2-D Scan in the same scanset.
Show Crosshairs (F5, Spots menu or main toolbar) is used to display the
crosshairs in a subwindow. Use the tool or the key command in the
subwindow to toggle the display. (In a scanset, the Gaussian image must be
loaded.) To display crosshairs in all subwindows, select Show All Crosshairs
(Shift+F5, Spots menu).
Note:
Spots without crosshairs in the Filtered and Raw 2-D Scan images have not been
detected and are not included in the Gaussian image.
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PDQuest User Guide
Fig. 4-9. Examples of crosshairs and ellipses in a scanset.
Show Ellipses (Alt+F5, Spots menu) shows the shape of each spot generated by
Gaussian fitting. Use the tool or the key command in the subwindow to toggle the
display. Show All Ellipses (Ctrl+F5, Spots menu) shows the spot ellipses in all the
subwindows.
Hide Overlays (F4, main toolbar) will hide the crosshairs and ellipses on the image.
Use the tool or the key command in the subwindow to hide the overlays.
4.5
Adding and Removing Spots
If spot detection has missed some spots or identified too many spots in a cluster, you
can add and remove spots in the Gaussian image.
Note:
You can add and remove spots in the MatchSet and/or the scanset. Note that, after
a MatchSet has been created, subsequent changes in the scanset are not
automatically transferred to the MatchSet, and visa versa.
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Detecting and Editing Spots
Make sure that both the Filtered image and Gaussian image are loaded before adding
and removing spots. In a MatchSet, use Interchange Images (F11, main toolbar) to
toggle between the two images.
The tools for adding and removing spots are located on the Spots menu and Edit Spots
toolbar.
Adding Spots
If a spot in the Filtered image has not been added to the Gaussian image,
select Add Spot from the Spots menu or Edit Spots toolbar, then click the
spot in the Filtered image. The spot will be Gaussian modeled and added to
the Gaussian image.
Note:
It is also possible to add spots on Raw 2-D scans when the Filtered and the
Gaussian images are loaded.
Problem: Spot is undetected in
the filtered image.
Result: Spot is missing in the
Gaussian image.
Solution: Add spot in the
filtered image.
Result: Spot added to Gaussian image.
Fig. 4-10. Adding a spot.
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Note:
Use Plot Cross-section (Ctrl+t, View > View Density submenu) to aid in
accurately positioning new spots.
Note that the spot boundary tools (section 4.6, Spot Boundary Tools) can be used to
add non-Gaussian modeled spots to the Gaussian image.
In a MatchSet, if you add a spot to the Master gel template it is automatically added to
the Master.
Removing Spots
To remove incorrectly positioned or misidentified spots from the Gaussian
image, select Remove Spot from the Spots menu or toolbar, then click
individual spots or drag a box around a group of spots in the Filtered
image. The spot(s) will disappear from the Gaussian image and the spot crosshair(s)
will disappear from the Filtered image.
Note:
It is also possible to add spots on Raw 2-D scans when the Filtered and the
Gaussian images are loaded.
Problem: Too many spot centers
identified in the Filtered image.
Result: Too many spots appear in
Gaussian image.
Solution: Use Remove Spot, then
Add Spot to reposition spot centers.
Result: Correct number of spots in the
Gaussian image.
Fig. 4-11. Removing incorrectly identified spots in a cluster.
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Detecting and Editing Spots
4.6
Spot Boundary Tools
If a spot is saturated, irregularly shaped, or otherwise of poor quality, then Gaussian
modeling will be unable to accurately determine quantity. In these cases, you can
manually define the spot in the Filtered or Raw 2-D image using the spot boundary
tools.
Note:
For most spots, Gaussian modeling results in more accurate quantitation than
defining the spot boundary.
When you define a spot boundary, the region inside the boundary is identified as the
spot and is added to the Gaussian image. Spot quantity is calculated by summing the
intensities of the pixels inside the defined boundary. You can define the boundary of
any spot in a Filtered or Raw 2-D image.
Spots defined using the boundary tools are not Gaussian modeled, even though they
appear in the Gaussian image. However, their quantities can be compared with
regular, Gaussian-modeled spots.
Note:
Creating a spot boundary will overwrite the results of spot detection and
Gaussian modeling. However, regular spot detection will not overwrite any spots
defined using the boundary tools.
You can open the Spot Boundary Tools toolbar from the main menu, or
select commands from the Spots > Spot Boundary Tools submenu.
Fig. 4-12. Spot boundary tools.
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Spot Contour Tool
To use this tool, first magnify the spot you want to quantify in the Filtered image, then
select Spot Contour Tool from the submenu or toolbar.
Click and drag the cursor from the inside of the spot outward to change the shape of
the contour as you move over pixels of different intensity; note that the contour is
created by enclosing all pixels whose intensity is greater than or equal to the intensity
of the pixel at the cursor.
When you release the mouse button, the boundary is created. The contour should
completely surround the spot you want to quantify.
1. Select the contour tool.
2. Drag a contour around
the spot in the Filtered or Raw
2-D image.
3. The contour is created
and the spot is added to the
Gaussian image.
Fig. 4-13. Creating a spot contour.
To edit the boundary, position your cursor on the border with the contour tool. Your
cursor will change to a pencil. Drag across the line; a new yellow line will appear. The
boundary will look blue when not selected, yellow when it its being edited and green
when it is selected.
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Detecting and Editing Spots
Spot Freehand Tool
To use this tool, first magnify the spot you want to quantify (you must be
able to see the individual pixels in the image). Then select the Spot
Freehand Tool from the toolbar or submenu, and click and drag around the
edge of the spot to enclose it in a boundary. When your line crosses itself, a
freehand boundary is created.
If you make a mistake while drawing, simply backtrack with the mouse. The line you
draw should completely surround the spot you want to quantify.
1. Select the freehand tool.
2. Draw the freehand line
around the spot in the
Filtered image.
3. The boundary is created
and the spot is added
to the Gaussian image.
Fig. 4-14. Creating a freehand boundary.
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To edit the boundary, position your cursor on the border and drag across the line with
the freehand tool; a new white line will appear. When you recross the old line, a new
boundary will be created.
Deleting and Moving Spot Boundaries
To remove a spot boundary, you can use the Remove Spot tool, or you can click on
the spot with the Contour, Freehand, or Select tool to select it (the boundary will
change to green) and press the Delete key on your keyboard. The spot will also be
deleted in the Gaussian image.
To reposition a spot boundary, make sure the Contour, Freehand, or Select tool is
active, then position your cursor in the middle of the spot boundary. The cursor will
change to a multidirectional arrow; simply drag the boundary to a new location. The
spot will also appear relocated in the Gaussian image.
Copying Spot Boundaries Between Images
To copy a spot boundary between Filtered images, first select it (the
boundary will change to green), then select Copy to Clipboard from the
submenu or toolbar.
Open or select the image you want to copy to, and select Paste from
Clipboard from the submenu or toolbar. The spot boundary will appear in
the same relative position in the new image. The boundary will also appear
in the corresponding Gaussian image.
Note that the two images should have the same pixel size and approximate
dimensions. You may have to reposition the pasted boundary as described above.
4.7
Cancelling Spots
Cancelling a spot in a MatchSet or scanset preserves its location but does not include
it in matching or any other analysis. This is different than removing a spot from the
Gaussian image entirely.
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Detecting and Editing Spots
Select Cancel from the Spots > More Edit Tools submenu or toolbar and click on the
spot that you want to cancel. It will disappear from the Gaussian image (it will remain
in the Filtered image, but will not be included in any analysis).
To highlight all cancelled spots in the Gaussian image, select Cancelled Spots
(Ctrl+F7) from the Spots > More Edit Tools submenu or Edit Spot toolbar.
To restore a cancelled spot, select Restore from the Spots > More Edit Tools submenu
or toolbar and click on the position of the cancelled spot. The spot will reappear.
To cancel all the spots that have not been matched in a MatchSet, select Cancel
Unmatched Spots from the Match > Edit Matches submenu and click in the MatchSet
gel.
To restore all unmatched cancelled spots in a MatchSet, select Restore Unmatched
Spots from the Match > Edit Matches submenu and click in the MatchSet gel.
4.8
Combining Spots
Combining spots combines the quantitation of a cluster of spots into one spot. This
can be performed on Filtered images in a MatchSet or scanset.
Select Combine from the Spots > More Edit Tools submenu or Edit Spots toolbar, and
then draw a box around the cluster of spots that you want to combine in the Filtered
image. After you draw the box, click on the point in the box where you want the
combined spot to be placed. The cluster will appear as one spot in the Gaussian
image; it will still appear as individual spots in the Filtered image, but the quantitation
will be combined.
To resolve a combined spot into its original cluster, select Un-combine from the
submenu or toolbar and draw a box around the combined spot in the Filtered or
Gaussian image. The spot will be uncombined into its original multiple spots.
Combined Spots (Ctrl+F8, Spots > More Edit Tools submenu, Edit Spots toolbar)
highlights all combined spots in the MatchSet.
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4.9
Saturated and Faint Spots
Saturated Spots
Spot detection may not correctly fit spots in saturated regions of the image. Therefore,
although saturated spots will appear in the Gaussian image, they are not quantified
using Gaussian modeling (their quantity will be reported as zero). You should use the
spot boundary tools (section 4.6, Spot Boundary Tools) to quantify these spots.
To highlight spots that are saturated in the image, use the Saturated Spots command
(Alt+F7, Spots > More Edit Tools submenu, Edit Spots toolbar) in any image
window. The saturated spots will appear highlighted and the number of saturated
spots will be listed at the bottom of the window.
You can also use the Highlight Saturated Pixels in the Transform window to identify
regions of saturation in the image.
Faint Spots
Faint Spots on the Spots > More Edit Tools submenu and Edit Spot toolbar highlights
those spots whose signal falls below a user-defined threshold. Select the command
from the submenu or toolbar, then click in the image. The faint spots will be
highlighted in the image and the number of faint spots will be listed at the bottom of
the image window.
4.10 Spot Quantity
Spot Quantity is the total intensity of a defined spot in a gel image. This corresponds
to the amount of protein in the actual spot in the gel (see section 1.2, Digital Data and
Signal Intensity).
For spots defined using the boundary tools (section 4.6, Spot Boundary Tools), this is
simply the sum of the intensities of the pixels inside the boundary. For Gaussian
spots, it is calculated during spot detection and Gaussian fitting.
The formula for calculating the quantity of Gaussian spots in PDQuest is:
Spot height * π * σx * σy, where:
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Detecting and Editing Spots
Spot height (also known as peak value) is the peak of the Gaussian representation of
the spot. It is measured in ODs or counts/image units2, depending on the type of
imaging device used.
Note:
One image unit (IU) = 100 micrometers or 0.1 millimeter. This scaling factor is
used in to make the numbers in the formula more manageable.
σx is the standard deviation of the Gaussian distribution of the spot in the direction of
the x axis, and σy is the standard deviation in the direction of the y axis. These are
measured in IUs.
Note that this formula results in units of OD * IU2 for images measured in ODs, and
units of counts for images measured in counts.
Note:
This formula results in more accurate quantitation of imaged spots than summing
the intensities of the pixels in the spot. Measuring the area under the Gaussian
model compensates for peak intensity values that approach image saturation, as
well as background intensity that submerges the intensity of a spot at its edges.
To display the quantities of the spots in a single image, use Show Quantity (Alt+F6,
Spots menu) in the image window. To display the quantities of all the spots in all the
subwindows of a selected window, select Show All Quantity from the Spots menu.
Low Quantity Spots
You can define a set of spots that do not meet a threshold quantity level.
From the Spots > Low Quantity Spots submenu, select Define Low Quan. Set, then
click in the MatchSet or scanset Gaussian image. In the pop-up box, enter a threshold
quantity value. All spots below this threshold will be flagged as low quantity.
When you click OK, a pop-up box will list the number of low quantity spots in the
image and their total quantity. The total number of spots in the image and their total
quantity will also be listed.
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PDQuest User Guide
Fig. 4-15. Pop-up box displaying low quantity spot information.
The low quantity spots will appear highlighted in the image.
To re-highlight the low quantity spots in the set, select Spots > Low Quantity Spots >
Mark Low Quan. Spots and click in the image.
To cancel all low quantity spots in the set, select Spots > Low Quantity Spots >
Cancel Low Quan. Spots and click in the MatchSet or scanset Gaussian image.
To restore the cancelled low quantity spots, select Spots > Low Quantity Spots >
Restore Low Quan. Spots and click in the MatchSet or scanset Gaussian image.
Quantity Table
You can display the quantity of a spot in each MatchSet gel (or replicate group) using
the Quantity Table function.
Select Quantity Table from the Spots menu, then click on the spot whose quantities in
different gels you want to compare. Alternatively, position the cursor on the spot and
click Shift+F6. A pop-up box will display the quantities of that spot in table format.
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Detecting and Editing Spots
Fig. 4-16. Quantity table.
If Replicate Group Quantitation is turned on the average quantity of that spot in each
replicate group will be displayed, as will the coefficient of variance (CV) and the
number of gels in the replicate group (Count).
4.11 Spot Quality
Spot quality is a number ranging from 0 to 100 that is calculated based on five
attributes of each spot:
•
Gaussian fit. PDQuest determines how well the spot fits the Gaussian model and
assigns a value based on this fit.
•
X streaking. The software determines how much of the spot is affected by gel
streaking in the x direction.
•
Y streaking. The software determines how much of the spot is affected by gel
streaking in the y direction.
•
Overlap. The software determines the percentage of the spot that overlaps with
another spot or spots.
•
Linear range of scanner. PDQuest determines whether the peak intensity value of
the spot is within the linear range of the scanner.
Each spot attribute is evaluated and weighted to produce a single numerical value for
Spot Quality. If a spot fits the Gaussian model perfectly, has no streaking in the x or y
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PDQuest User Guide
direction, does not overlap with any other spot, and has a peak intensity within the
linear range of the scanner, PDQuest will assign a value of 100 to the spot.
To display the quality of all the spots in a single image, select Show Quality from the
Spots menu and click the image. To display the quality of all the spots in all the
subwindows of a selected window, select Spots > Show All Quality.
Note:
Spots of low quality may be more accurately quantitated using the spot boundary
tools (section 4.6, Spot Boundary Tools). Spots created using the boundary tools
are not assigned a quality value.
Low Quality Spots
You can identify a set of spots that do not meet a threshold quality level. You can then
flag those spots as low quality and/or redefine them using the spot boundary tools
(section 4.6, Spot Boundary Tools).
From the Spots > Low Quality Spots submenu, select Define Low Qual. Set, then
click in the MatchSet or scanset Gaussian image. In the pop-up box, enter a threshold
quality value. All spots below this threshold will be flagged as low quality.
When you click OK, the low quality spots will appear highlighted in the image, and
the number of low quality spots will be displayed at the bottom of the image window.
To re-highlight the low quality spots in the set, select Spots > Low Quality Spots >
Mark Low Qual. Spots and click in the image.
To cancel all low quality spots in the set, select Spots > Low Quality Spots > Cancel
Low Qual. Spots and click in the MatchSet or scanset Gaussian image.
To restore the cancelled low quality spots, select Spots > Low Quality Spots >
Restore Low Qual. Spots and click in the MatchSet or scanset Gaussian image.
4.12 Spot Parameters
The Spot Parameters information box provides a variety of information about an
individual spot, including its quantity, quality, peak value, location in the gel, Mr pI
data (if any), and the sigmas of its Gaussian curve.
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Detecting and Editing Spots
Select Spot Parameters from the Spots menu, then click on a spot in a Gaussian image
to display the data for that spot. Alternatively, position your cursor on the spot and
click F6.
Fig. 4-17. Spot Parameters information box.
4.13 Finding Spots
You can find spots in a MatchSet by SSP number or DSN.
Select Find Spot from the Spots menu (Ctrl+F), click in the MatchSet member or
standard you want to search, and enter the SSP or DSN number in the pop-up box.
Click OK to complete the search. The spot will appear highlighted and numbered on
the image.
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4-30
5. MatchSets
After the protein spots in your gels have been detected, you are ready to compare
spots across gels. To do so, you must create a MatchSet. You can create a MatchSet in
two different ways, with the Match > New MatchSet command, or through the Spot >
Automated Detection and Matching dialog, Step 3. The commands for editing
MatchSets are located in Match > MatchSet Tool and Match >Edit Matches submenu.
A MatchSet is PDQuest’s mechanism for comparing and analyzing the spots in an
experiment. It contains copies of the Gaussian and Filtered images, as well as the
original 2-D scan of the gel(s) in an experiment.
Experiment
Gel 1
Image
Gel 2
Image
MatchSet
Gel 1
Copy
Gel 2
Copy
Master
Gel 3
Image
Gel 3
Copy
Master
Fig. 5-1. How experiments are organized into MatchSets.
A MatchSet can consist of one gel or many gels, depending on the type and size of the
experiment. The MatchSet is displayed in a single window, with subwindows for the
images of the member gels, and a subwindow for the MatchSet Master.
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The Master is a synthetic image that contains the spot data from all the gels in the
MatchSet. All spot comparisons and analysis are coordinated through the Master.
Masters from different MatchSets can be grouped into higher level MatchSets for
further comparison (see section 5.7.c, Higher Level MatchSets).
Using a MatchSet, you can make quantitative and qualitative spot comparisons across
gels, calculate molecular weight/isoelectric point values, annotate spots, group them
into analysis sets, combine the data from multiple gels into replicate groups, and
select spots for excision and mass spec analysis.
You can also use a MatchSet to combine gels with different pH gradients into a larger
cybergel. See 5.5, Cybergels.
Fig. 5-2. Example of a MatchSet. The Master is in the upper left subwindow.
Note:
While MatchSets can contain gels with different quantitation units, quantitative
spot comparisons can only be made between gels that have the same units (i.e.,
spot quantities measured in O.D.s cannot be compared to spot quantities
measured in counts). Qualitative comparisons are still valid.
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Matchsets
5.1 Creating a MatchSet
A MatchSet is created from the Gaussian and Filtered images, as well as the Raw 2-D
scan of the gel(s) in an experiment. There are two ways to create a MatchSet in
PDQuest:
•
Automated Detection and Matching - Automated Detection and Matching
combines several processing steps into a single operation. These steps include
spot detection, creating the MatchSet, and matching spots.
•
New MatchSet command - The new MatchSet command when you are creating a
MatchSet from previously detected images.
These methods for creating a MatchSet are considerably different from each other.
For instance, you cannot use the Automated Detection and Matching tool to create
higher level MatchSets. Instead, you must use the New MatchSet command.
5.1.a Create a MatchSet from Automated Detection and
Matching
The Automated Detection and Matching tool combines several processing steps into a
single operation. Click Automated Detection and Matching from the Spots menu to
open the Automated Detection and Matching dialog box.
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PDQuest User Guide
Fig. 5-3. Automated Detection and Matching
Step 1 - Select Gels To Analyze
When the Automated Detection and Matching dialog box opens, any open images are
automatically included in the list. Click Add to open the Select New Member dialog
box, and navigate to the folder in which your gel images are stored. Use
CTRL+CLICK and SHFT+ CLICK to select multiple images.
The Remove button removes a gel from the list.
The Master button designates the selected gel as the MatchSet master image. If you
do not select a master, PDQuest automatically chooses a master for you based on
which gel has the best average spot quality.
Tip:
To view the average quality for each gel, click the Log file button in the
Matching Summary. See Section 5.8, Matching Summary for further
information.
This gel will be used as the starting point for constructing the eventual Master image.
See Section 5.7, "Adding Spots to the Master" for more details on constructing the
Master image.
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Matchsets
As indicated, you can set the order of the members by dragging them in the list. This
order will be used when displaying the images as well as plotting bar graphs.
Step 2 - Detect Spots on Gels
Step 2 determines the parameters used to automatically process all the images. If you
have processed similar images in the past, select the same parameter set from the
drop-down list. If you cannot use an existing parameter set, click Edit... to launch the
Spot Detection Wizard and create a new parameter set. See Section 4.1, Selecting
Spot Detection Parameters.
Step 3 - Create MatchSet
Enter a name for the MatchSet in the Name field. To assign your MatchSet a default
name, click Auto. The default prefix can be set on the Application tab of the
Preferences dialog box. See Section 2.7, Preferences, for further information.
Enter a description of the MatchSet in the Description field. You can enter further
information about the MatchSet in the Comments field.
The Directory field indicates the folder where the MatchSet will be saved. The
currently listed folder is based on the last time a MatchSet was created using
Automated Detection and Matching. To specify a new location, type in a new path or
click Browse.
Under Member sharing, Copy the originals into this MatchSet’s data folder is selected
by default. This option protects files from being opened or used outside the MatchSet.
This option uses more space and requires you to manage disk space more carefully.
If you choose Do not make copies; Refer to Originals, you will save disk space by not
making copies of the member image files. Note that you can include shared member
files in more than one MatchSet. If you do, edits such as adding or removing spots
will affect all MatchSets that include these files.
Note:
If you want to detect spots without creating a MatchSet, select Disable this step at
the bottom of Step 3.
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PDQuest User Guide
Step 4 - Match Gels
This is similar to Step 2 in that you can select existing parameters for matching or you
can create new ones with the Edit... button. However, unlike Spot Detection
parameters, the matching parameters work over a wide range of images and you will
probably obtain satisfactory results with the default parameters. See Section 5.2.a,
Automated Matching, for further information.
Note:
If you want to create a MatchSet without matching spots, select Disable this step
at the bottom of Step 4.
5.1.b Creating a MatchSet Using New MatchSet
If you have already performed spot detection on a set of images (such as shared
images) and want to use them in a new MatchSet, click New MatchSet from the
Match menu. You must have no images or MatchSets open to open the Create
MatchSet dialog box.
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Matchsets
Fig. 5-4. Create MatchSet dialog box.
At the top of the Create MatchSet dialog, enter a name for the MatchSet in the Name
field. To assign your MatchSet a default name, click the Auto button. The default
prefix can be set on the Application tab of the Preferences dialog box. See Section
2.7, Preferences for further information.
Enter a description of the MatchSet in the Description field. You can enter further
information about the MatchSet in the Comments field.
Next, specify whether you are creating a Level 1 MatchSet or a Higher Level
MatchSet. Level 1 MatchSets are created from the Gaussian images of scanned gels.
Higher Level MatchSets are created from other MatchSets (see section 5.7.c, Higher
Level MatchSets).
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PDQuest User Guide
Click Add to open the Select New Member dialog box, and navigate to the folder in
which your gel images are stored. Use CTRL+CLICK and SHFT+ CLICK to select
multiple images.
Select the images that you want to include in your MatchSet and click Open. The
images will be included in the Members list of the Create New MatchSet dialog.
Raw 2-D and Filtered images are in included in the MatchSet by default. Including
the Filtered images allows you to compare the Gaussian spots with the original spots
while matching, and edit the results of spot detection. (Invariably, during matching
you will notice missed or misdetected spots.)
Under Member sharing, Copy the originals into this MatchSet’s data folder is selected
by default. This option protects files from being opened or used outside the MatchSet.
This option uses more space and requires you to manage disk space more carefully.
If you choose Do not make copies; Refer to Originals, you will save disk space by not
making copies of the member image files. Note that you can include shared member
files in more than one MatchSet. If you do, edits such as adding or removing spots
will affect all MatchSets that include these files.
After you have selected the MatchSet members and options, click Save. You will be
prompted to select the directory where you want to store your MatchSet by the Save
the MatchSet As dialog box. When you have specified the correct file name and
directory, click Save.
As indicated on the dialog box, you can set the order of the members by dragging
them in the list. This order will be used when displaying the images as well as plotting
bar graphs.
5.1.c Selecting the Master
The MatchSet Master is a synthetic image that contains the spot data from all the gels
in the MatchSet. When you select a gel to serve as the Master template, a copy of that
Gaussian image is created and designated the Master. You then match the spots in the
remaining gels with those in the Master, and add the unique spots in those gels to the
Master.
By default PDQuest automatically selects the Master based on which gel has the best
spot quality.
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Matchsets
To view the average quality for each gel, click the Log file button in the
Matching Summary. See Section 5.8, Matching Summary for further
information.
Tip:
To manually select a Master, highlight the gel you want to use and click Master. The
Master icon appears to the left of the Master template gel. The Master template gel
will be used as the starting point for constructing the eventual Master image.
When selecting your Master, choose the gel with the greatest number of spots
and the least streaking.
Tip:
Choosing the gel with the most spots as the template minimizes the number of spots
that you will need to add from the remaining gels.
•
Representative Gel. The MatchSet Master is a representation of all the data in
your MatchSet and is often used in reports. Therefore, you may not want to use
an atypical gel as a template, even if it has the most spots. In this case, we suggest
that you choose the most representative gel in your biological system.
•
Control Gel. Choose a control gel as the MatchSet Master so that it can be
included in every experiment you plan to analyze and used as the Master in every
MatchSet. This makes it easier to compare results between different experiments.
•
Spot Quality. Editing the MatchSet Master and matching other gels to it are
easier when the Master consists of high quality spots.
•
Cybergel. If you are creating a cybergel Master from gels with different pH
gradients, it may be easiest to add spots from each gel in sequence (i.e., use the
gel with the lowest pH as the template). See 5.5, Cybergels.
With your Master created, you are ready to begin matching and editing spots in the
MatchSet (see the following chapter).
5.1.d Displaying MatchSets
A MatchSet is displayed in a single window, with the MatchSet members in different
subwindows. The MatchSet Master initially appears in the upper left subwindow and
has the name Master.
As with scansets, you can configure the subwindows in a MatchSet using the
Configure Subwindows command (section 3.2, Configuring Subwindows), and assign
images to different subwindows using the Assign command (section 3.3, Assigning
and Interchanging Images).
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PDQuest User Guide
The Raw 2-D, Gaussian, and Filtered images (if included) of each member gel are
loaded in the same subwindow. To toggle the images, use Interchange Images (F11)
or Interchange All Images (Shift+F11). The member name and type of image are
listed at the bottom of each subwindow.
Fig. 5-5. Interchange Images
5.1.e Editing MatchSets
The commands for editing a MatchSet are located on the Match > Edit Matches
submenu or the Match > MatchSet tool. The MatchSet Tool dialog is similar to the
Create MatchSet dialog box. (See 5.1, Creating a MatchSet). The exceptions are that
MatchSet Level and Member Sharing are inactive, and you cannot change the
MatchSet name.
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Matchsets
Master icon
Fig. 5-6. MatchSet Tool dialog box
In the MatchSet Tool you can add new images to the MatchSet or remove images
from the MatchSet as well as edit the description and comments. You can also select a
new Master template gel.
Note:
Any annotations, analysis sets, etc. in the MatchSet will be lost if you replace the
Master template gel.
To add a new member to a MatchSet, click the Add button.
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PDQuest User Guide
Removing MatchSet Members
To remove a MatchSet member, select the gel to be removed from the member list
and click the Remove button. Since the Remove command cannot be undone a popup
box asks you to confirm this action.
Note:
Any spots you have added to the MatchSet Master from the removed gel will
remain in the Master.
5.2 Matching Spots
After you have created a MatchSet, you are ready to begin matching spots and adding
unique spots in the gels to the Master.
In a MatchSet, spot data from the member gels are included in the Master via
matching. All the significant spots in your MatchSet should be matched with spots in
the Master.
Note:
If you used Automated Detection and Matching to create your MatchSet,
matching parameters can be set in step 4 of the Automated Detection and
Matching dialog box.
There are two types of matching: Automated Matching and Classic Matching.
5.2.a Automated Matching
PDQuest includes the ability to match spots with no manual assistance. Unlike
previous versions, you do not have to landmark the images before matching.
This facility is the default method used for matching. The previous method is
referred to as "Classic" matching.
Phases of matching
Matching consists of two phases: Primary matching and Extended matching.
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Matchsets
Fig. 5-7. Automated Matching - Primary and Extended options.
Primary Matching
The primary matching phase makes only high-confidence matches. Use the slider to
control the degree of precision. If you specify high precision, a spot must be in
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PDQuest User Guide
exactly the expected position based on its neighbors to be matched. If you lower the
precision, the spot is matched even if it is a short distance away from the ideal
position. Note that lowering the precision increases the number of matched spots but
may match some spots that should not be matched. The more reproducible the gels
are, the higher the precision setting may be set.
Primary matches are shown in green.
Check the Enable overlapped pH-range matching box to create Cybergels. This will
match gels that only partially overlap. Since this can slow down the matching process
considerably, it is not normally set. See Section 5.5, Cybergels, for further
information.
Extended Matching
The extended matching phase makes lower confidence matches. The behavior is
similar to lowering the precision in the primary phase, but the matches are displayed
in a different color. This allows you to keep the precision high in the primary phase
and review only the extended matches.
You have the option of choosing between Classic and Restricted. The Classic method
uses the same algorithm PDQuest has used in previous versions. If you select this
method, the primary phase can be thought of as "auto-landmarking". To manually
adjust the Matching stringency, choose Restricted as your extension method. Then
use the slider just as you would in the Primary phase.
Extended matches are shown in blue.
Getting started
First you must create a MatchSet and designate a Master image. Once this is done,
select Match > Automated Matching.
If you simply want to match all the spots using the default settings, click Match All,
then click Done.
If you want to explore the match setting, select a member from the drop down list.
Then use the sliders to change the settings and click Match. Matches lists the number
of primary matches, while Extensions shows the number of extended matches. Sum
lists the total number of both primary and extended matches.
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Matchsets
Use the default method for matching (extended matching disabled) for high
confidence matches. Classic matching finds to greatest number of matches, but
also increases the likelihood of false matches. With the Restricted method, you
can adjust between the high confidence matches (strict) and the Classic method
(liberal).
Tip:
Use the Configure Sub-windows command in the Window menu to configure the
MatchSet window to display only the master and the member you are matching. Then
Inspect the matches for accuracy and completeness.
Placing a few landmarks in problem areas of your gel(s) may help with Auto
Matching.
Tip:
Once you have obtained the best settings for your images, press Match All to apply
those settings to all members and match all members. You will be prompted for a
name for the settings. When matching subsequent images, you can recall these
settings by name.
5.2.b Classic Matching
By default, Auto Matching occurs immediately after each landmark is placed in all
the MatchSet members. To turn off this feature, clear Auto-match after Landmark/edit
Master in the Applications tab of the Preferences dialog box.
Note:
Most MatchSet gels require at least two landmarks before auto-matching will
occur. The gel that was used as the template for the Master may be auto-matched
after only one landmark is placed.
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PDQuest User Guide
Manual Match
Manual Unmatch
Mark Landmarks
Remove Added Spot
Landmark
Unlandmark
Classic Match All Gels
Show Symbols
Show Offsets
Show Match
Add Spot to Master
Fig. 5-8. Match Menu and toolbar.
Using landmarks, PDQuest automatically matches most of the spots in the MatchSet.
You then review the remaining unmatched spots in the member gels and either
manually match them with existing spots in the Master, cancel them, or add them as
new spots to the Master.
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Matchsets
Each matched spot is marked with a green letter in the Master and the matched gels.
Unmatched spots in the member gels are marked with red circles. Auto-matching
should successfully match most of the spots in the MatchSet.
Fig. 5-9. Matched spots are marked with letters; unmatched spots are circled.
If multiple unmatched spots appear in certain areas of the gels, review these areas
using the Match > Show Offsets (F7) command, and add more landmarks to facilitate
auto-matching.
5.3 Landmarking Spots
Landmarks are required before you can use Classic Matching. They are also used to
correct errors made by Automated Matching. Landmarks are not required for
Automated Matching.
Before you can begin matching spots in the MatchSet, you must landmark key spots
in all the gels. Landmarks are reference spots used to align and position the gels for
matching.
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PDQuest User Guide
When choosing spots to landmark, you should look for spots that are well-resolved
and present in all members of the MatchSet (except when creating a Master out of
gels with different pH gradients; see 5.5, Cybergels). Avoid spots in clusters, in case
the spot detection mechanism has misidentified the peaks in the cluster. Unless you
are creating a Master out of gels with different pH gradients, you should landmark
spots in all corners of the gels, as well as the middle of the gels.
To begin landmarking, first magnify a region of the MatchSet gels and identify a wellresolved spot that appears in all the gels. Study the region under different levels of
magnification to make sure you have identified the same spot in all the gels.
This appears as
one spot in some
gels, two in others.
This spot makes
a better landmark
because it is
well-resolved in
all gels.
Fig. 5-10. Selecting a spot to landmark.
Select Landmark from the Match menu, Match toolbar, or right-click menu, and click
on the spot in the MatchSet Master.
The cursor will jump to the same area in the first member gel. If the landmarking
function can identify the same spot in that member, it will appear marked with a box.
Click it to place the landmark. Landmarked spots are marked with green triangles.
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Matchsets
Fig. 5-11. Landmarking a spot.
If the program is unsure about positioning the landmark, the area in which the spot
was expected will be circled. Locate the appropriate spot and click on it.
As each landmark is placed, the cursor will jump to the next gel. Landmark the spot in
all members of the MatchSet, except when creating a Master out of gels with different
pH gradients; see 5.5, Cybergels.
If you accidentally landmark the wrong spot in a gel, simply go back and click the
correct spot. The landmark will move.
To remove a landmark from all the MatchSet gels, select Unlandmark from the Match
menu or toolbar and click the incorrect landmark in the Master. To remove a landmark
from a single gel, click in that gel.
To highlight all the landmarks in a MatchSet, use the Mark Landmarks command (F8,
Match menu, Match toolbar) in the Master image.
As you add landmarks, study the auto-matching in all regions of the gels. Add more
landmarks to regions where auto-matching has missed spots.
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PDQuest User Guide
The number of landmarks required will vary depending on the number and quality of
the gels in the MatchSet. Gels with well-resolved spots and little distortion will
require fewer landmarks.
5.4 Displaying Matches
Use the following commands to display spot matches on your MatchSet images.
5.4.a Show Symbols
Match > Show Symbols highlights all the different types of matched and
unmatched spots using different labels. Select the tool from the Match
menu or toolbar and click in the Master or member gel subwindow, or use
the F7 key command in the appropriate subwindow.
•
Auto-matched spots are labeled with green letters.
•
Manually matched spots are labeled with purple boxes.
•
Unmatched spots are circled in red. These are also displayed using the Mark
Unmatched Spots command (Shift+F8) on the Match >Edit Matches submenu.
•
Landmarks are labeled with green triangles. These are also displayed using the
Mark Landmarks command (F8) on the Match menu and toolbar.
•
Spots added to the Master are labeled with blue boxes. These are also displayed
using the Mark Added Spots command (Alt+F8) on the Match menu and toolbar.
To highlight individual matched spots, select Show Match from the Match
menu or toolbar and click on a spot in the Master, or use the F9 key command
on the spot. The spot will appear highlighted with a yellow box in all the gels
in which it has been matched. In gels where it is unmatched, a circle will
appear where the spot would be located.
5.4.b Show Vector Offsets
Vector offsets are lines showing the distance and direction between the spot centers in
the member gels and the corresponding spot centers in the Master. They provide a
visual tool for judging gel alignment and distortion and the accuracy of matching.
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Matchsets
Select Show Offsets from the Match menu or toolbar and click in a
subwindow to display the offset lines, or use the Shift+F7 key command in
the subwindow.
Look for the following conditions that indicate possible matching problems:
•
Offset lines that cross each other (this is a key problem).
•
Grossly nonparallel lines in local regions.
•
Lines of unequal length in local regions.
•
Areas of the gel with few offsets; this indicates unmatched regions.
Fig. 5-12. Vector offsets indicate that these two spots have been mismatched.
5.5 Cybergels
PDQuest needs to know the pH gradient of a gel in order to calculate isoelectric point
values. A new feature in this release allows you to combine overlapping areas of gels
and create Cybergels. Cybergels are a combination of gels with different pH gradients
into a larger gel. To create a cybergel select Combine overlapping gels from the
Match menu. Additionally, you can select Match > Automated Matching > Primary
Matching tab. Then check the Enable overlapped pH-range matching box to create a
cybergel.
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PDQuest User Guide
For example, in the illustration below, if the pH gradient set in the lab only goes to 4,
you will only be able to detect proteins that separate at the pH level of 4. Due to the
narrow set of pH ranges (from 3-5, etc.), spots 1, 2, and 3 are the same in gel 1 as in
gel 2. And spots 4, 5, and 6 overlap from gels 2 and 3.
To create a cybergel eliminating overlapping spots, or spots that occur in more than
one gel, you would add spots 1, 2, and 3 from Gel 1 to the Cybergel, and add spots 4,
5, and 6 from Gel 3. The Cybergel is a combination of all the spots in which you are
interested.
Fig. 5-13. Overlapping gels with narrow pH range
To combine gels with narrow-range pHs, select the most acidic gel as your Master
template.
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Matchsets
If you select Match > MatchSet tool you open the Combine Narrow-Range Gels, or
Cybergel dialog.
Fig. 5-14. Combine Narrow-range gels.
Use the Add From Member drop-down list to add successive gels to the Master. Add
additional gels in order of increasing pH. The Master, or Cybergel, becomes a
combination of all gels you added. For more information on selection criteria,
formatting, spot location, and display, 5.6, Adding Spots to the Master
Indicate the orientation in which your gels are run by checking either the Acid-Base
or Base-Acid box under pH gradient in the Edit > Preferences > Application tab.
When you are ready to add spots from the gel, click Proceed. The selected spots will
be added and the gel will be rematched with the Master. If some spots could not be
added because they were too close to existing spots this will be noted in a pop-up box.
You can add these manually.
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PDQuest User Guide
To remove all spots added from the selected gel, click Undo Add. You will be warned
before the action is completed.
Landmarking Gels with Different pH Gradients
If you are combining gels with different pH gradients into the same Master, you will
not be able to landmark the same spot in all the gels. For example, to create a cybergel
from three gels with different pH gradients, you would place landmarks in the
overlapping region of the first two gels, then add the spots from the unique region of
the second gel to the Master. Then you would landmark the overlapping region of the
last two gels.
5.6 Adding Spots to the Master
Recall that all spots initially included in the MatchSet Master come from the template
gel used to create the Master. After you have matched these spots in all the gels, you
are ready to add the unique spots from the other gels to the Master. These may be
spots induced by your experimental protocols, or they may be spots that fall outside
the pH gradient of the current Master.
Auto-Adding Spots
To automatically add the unmatched spots in a MatchSet member to the Master, select
Auto-Add Spots to Master from the Match menu.
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Matchsets
Fig. 5-15. Auto-add Spots to Master dialog.
In the dialog box, select the MatchSet member from the Select Member popup box.
The MatchSet will reconfigure to display the member and Master in adjacent
subwindows, with the unmatched spots selected and their corresponding locations in
the Master highlighted.
Selection Criteria
You can choose from three different options in the Selection Criteria area: Ignore
filter for spots outside current Master, Filter by peak value, and Filter by quality.
Check the Ignore filters for spots outside current master box. if you are adding spots
from a gel with a different PH gradient than the current Master. The Master will be
expanded and all unmatched spots that fall outside the area of the Master will be
added regardless of quantity or quality. See 5.5, Cybergels for more information on
matching gels with different pH gradients.
The second option is Filter by peak value. Use it to add all the unmatched spots or
only those spots that meet a threshold peak value and/or quality value. To set a peak
value threshold, check Filter by peak value and drag the slider to the left, or enter a
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PDQuest User Guide
threshold value directly into the box to the left of the slider. As you adjust the
threshold, spots that no longer meet that threshold will be deselected.
Set the filter range by selecting the Max option button (up to the maximum peak value
in the gel) or the Avg button (up to the average peak value).
The third option in the Auto-Add spot to Master dialog is the Filter By Quality
setting. This setting is similar to Filter by Peak Value, except that it filters by spot
quality.
The number of Spots selected appears below the Selection Criteria. Inside master
shows the number of spots that fall within the area of the current Master image, while
outside master shows the number of spots that fall outside that area.
Check Mark Spots to be added to select the exact position in the master gel you want
to add the spot.
Check Mark add spot location in Master to mark the spot in a member you want to
add to the Master.
Check Auto-configure MatchSet subwindows on launch to automatically configure
the subwindows in the MatchSet to imitate the Master.
You can select the style, color, and symbol for how you want your spots to display by
clicking the Format button.
When you are ready to add spots from the gel, click the Proceed button. The selected
spots will be added and the gel will be rematched with the Master. If some spots could
not be added because they were too close to existing spots you will be prompted to
rematch the gels by a pop-up box. You can add these spots manually.
To remove all spots added from the selected gel, click Undo Add. You will be warned
before the action is completed.
Manually Adding Spots
To manually add spots from a member gel to the Master, first display the unmatched
spots in the gel using Show Symbols (F7) or Mark Unmatched Spots (Shift+F8). Use
the Zoom Box tool to magnify a spot.
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Matchsets
Select Add Spot to Master from the Match menu or toolbar, and click on
the center of the unmatched spot in the member. The cursor will jump to
the same area in the Master (calculated from the surrounding matches).
When you click in the Master, the spot will appear and a match will automatically be
made.
Mark Added Spots
Click Mark Added Spots (ALT+F8) on the menu or toolbar to highlight the
spots you have added to the Master. remove a spot you have added
manually. The cursor displays as a red X which you position over the
unwanted spot.
5.7 Edit Match Submenu
After you have completed adding spots to your master gel the Match > Edit Matches
submenu contains further commands for editing MatchSets. Some of these commands
can also be located in the Match Toolbar (View > Toolbar > Match Tools, or through
the Quick Reference Guide).
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PDQuest User Guide
Fig. 5-16. Edit matches submenu commands.
Manual Matching
After you have landmarked numerous spots and reviewed the results of automatching, you are ready to identify the significant spots that could not be automatched. To highlight these spots in a member gel, use the Mark Unmatched Spots
command (Shift+F8, Match > Edit Matches submenu) in the gel’s subwindow.
Unmatched spots are marked with red circles. Use the Zoom Box tool to study each
unmatched spot in detail in each gel.
If a spot was missed during spot detection, you must add it to the Gaussian image,
then match. If a spot was not included in the Master, you must add it to the Master,
then match. If an unmatched spot is not significant, you can cancel it.
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Matchsets
Otherwise, if auto-matching has missed a spot that should have been matched, you
can landmark it to improve the alignment and modeling of the Master, or you can
manually match it.
Note:
Manual matching will not change the modeling of the Master, and therefore will
not facilitate additional matching.
Select Manual Match from the Match toolbar or Match > Edit Matches
submenu and click on the spot in the Master, then click on the same spot in
all the member images where that spot appears. The spot will be marked
with a purple box to indicate that it has been manually matched.
To unmatch a manual match, select Manual Unmatch from the toolbar or
submenu and click on the manually matched spot in a member image.
Unmatched Spots
Choose Mark Unmatched Spots (Shift+F8) to circle spots you have been unable to
match between gels in red. These are also displayed using the Show Symbols (F7)
command on the Match submenu.
Choose Cancel Unmatched Spots to eliminate unmatched spots from the MatchSet.
To undo this command, select Restore Unmatched Spots.
Use the Zoom Box tool to study each unmatched spot in detail in each gel.
If a spot was missed during detection, you need to add it to the Gaussian image, then
match. If a spot was not included in the Master, you need to add it to the Master. If an
unmatched spot is not significant you can cancel it.
Partial Matches
Partial matches are spots in the MatchSet Master that are not matched in one or more
member images. Review these spots to see if they were undetected, mismatched,
cancelled, or simply not present.
Select Mark Partial Matches from the Match > Edit Matches submenu and click in the
Master. The partial matches will be circled in the Master.
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PDQuest User Guide
Graph Partial Matches displays histograms of each partially matched spot in the
Master. Select the command from the submenu and click in the Master. A missing bar
in the histogram indicates that the spot is unmatched in a MatchSet gel.
Missing bars indicate
that the spots are not
matched in some
members.
Fig. 5-17. Graph Partial Matches; each bar represents the spot’s quantity in a gel.
Note:
Histograms can only be displayed for one region of an image at a time. Use the
Reports > Graph Advance (Alt+.) and Reports >Graph Retreat (Alt+,) commands
to shift between regions. Or you can navigate to the View menu and select the
graph toolbar.
Erratic Spots
Erratic spots are matched spots whose quantities vary significantly between member
gels. To mark these, select Mark Erratic Responses from the Match > Edit Matches
submenu and click in the Master. Enter a fold change factor in the pop-up box. (For
example, a change factor of 3 will highlight spots whose quantity varies by a factor of
3 or more among member images.) When you click Apply the selected spots will
appear circled in the Master.
Graph Erratic Responses displays histogram graphs of each erratic spot in the Master.
Select the command from the submenu, click in the Master, enter a fold change factor
in the pop-up box, and click Apply to display the graphs.
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Matchsets
Histogram bars reveal
a three-fold variation in
spot quantity between
gels.
Fig. 5-18. Graph Erratic Responses; each bar represents the spot’s quantity in a gel.
Note:
Histograms can only be displayed for one region of an image at a time. Use the
Graph Advance (Alt+.) and Graph Retreat (Alt+,) commands to shift between
regions.
5.7.a Colony Matching
If you have a MatchSet containing colony plate images, you can match colonies
quickly and easily using the Colony (two-point) Match command on the Match > Edit
Matches submenu.
Unlike the regular matching function, you only need to define two landmarks on your
member images to use this command.
Note:
Also unlike the regular matching function, this command automatically corrects
for any rotational errors in the imaged plates (common in colony plates). You do
not need to perform additional image rotation.
After you have created a MatchSet and defined two landmarks in the member images
(see above), select Colony (two-point) Match from the submenu and click on a
member image to automatically match the colonies. The other matching commands
also work in colony plates.
Removing Matches
To remove all automatic matches from a member image, select Remove Auto
Matches from the Match > Edit Matches submenu and click on the member image.
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PDQuest User Guide
Manual matches and landmarks will not be removed. Because this command is
irreversible, you will be prompted to complete the operation.
To remove all matches (automatic and manual), as well as landmarks, from a member
image, select Remove All Matches from the Match > Edit Matches submenu and
click on the member image. Because this command is irreversible, you will be
prompted to complete the operation.
5.7.b Redetecting Members
The Redetect Members command allows you to redetect member gels of a MatchSet.
Redetecting members is useful if you find that, after you ran Automated Detection
and Matching, either too many or not enough spots were detected in one or more of
the gels. In either case this usually can be traced to incorrect spot detection
parameters.
Note:
Redetect Members is useful for large numbers of spots. If the number of missed
or erroneously detected spots is small, use the Edit Spots tools instead. (See
Section 4.5, Adding and Removing Spots, for further information.)
To redetect members of a MatchSet, select Redetect Members from the Spots menu,
click Redetect in the Matching Summary, or right click on an image and select
Redetect Members from the context menu. A dialog box opens listing all the member
gels of the currently active MatchSet. Select the image, or to select multiple images,
use SHIFT-CLICK or CTRL-CLICK.
The image used as the master is identified by the Master icon. If your MatchSet
contains analysis sets or annotations, redetecting the master member will delete all
annotations and analysis sets. After you click Go, a warning dialog box displays
requiring confirmation of deletion of annotations followed by a warning dialog box
requiring confirmation of deletion of analysis sets. If you want to keep your analysis
sets and annotation, click Cancel.
Note:
Although you can redetect the master template gel, you cannot select a different
gel as the master template using Redetect Members. To choose a different master,
see Section 5.1.e, Editing MatchSets.
Once you have chosen which gels to redetect, click Go, and the Spot Detection
Wizard opens. Make any necessary changes to your parameters, then click Process all
gels. The progress of spot detection is displayed on the screen. Once the gels have
been redetected, PDQuest automatically proceeds with matching.
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Matchsets
5.7.c Higher Level MatchSets
PDQuest allows you to create MatchSets from MatchSets, so you can build a database
by comparing and summarizing the results of a series of experiments.
Higher level MatchSets are MatchSets created from the Masters of lower level
MatchSets. A lower level MatchSet might be a MatchSet created from your original
gels (a level 1 MatchSet), or it could be another higher level MatchSet. Many
experimental protocols can be designed to take advantage of higher level matching.
For example, one experiment may find all the proteins that change during cell growth
in culture, while another may identify nuclear proteins by cell fractionation. These
two experiments can be analyzed individually in level 1 MatchSets, each with their
own Masters. These two Masters could then be combined into a higher level
MatchSet with its own Master. Data from the two experiments can now be compared,
and the higher level MatchSet might shed light on growth-related proteins found in
the nucleus.
Higher level MatchSets are actively linked to the MatchSets used to create them. If
you edit spots in the member images of a level 1 MatchSet, those changes will take
immediate effect in the higher level MatchSet.
For example, when working with a higher level MatchSet, you may notice that a spot
is present in one member and not the other. You could open the level 1 MatchSet with
the missing spot and see if that spot in present in any of the gels. If it is, you could add
it to the Master of the level 1 MatchSet and it would appear in the corresponding
member in the higher level MatchSet, ready to be matched.
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PDQuest User Guide
The masters of the two
level 1 MatchSets become the members of
the higher level matchsets.
Fig. 5-19. Example of a higher level MatchSet.
Creating a Higher level MatchSet
Use the Create Matchset dialog, Match > New MatchSet to create Higher level
MatchSets the same way you created Level 1 MatchSets. Under MatchSet level select
the radio button for Higher level MatchSets. Add entire MatchSets as members,
instead of individual gels. Higher level sets are similar to Level 1 MatchSets (see
section 5.1, Creating a MatchSet), except:
1.
You select the Higher Level option button instead of Level 1.
2.
You select MatchSets as members instead of images.
Note that you cannot include Filtered images in a higher level MatchSet, because
higher level MatchSets are made up of Masters, which do not have Filtered images.
Because higher level MatchSets are linked to the lower Level MatchSets they are
created from, they should not be moved from the file directory in which they are
created. If you want to create a higher level MatchSet to be shared among a group of
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Matchsets
users, you should move the lower-level MatchSets to a shared file server and create it
there.
Note:
If a higher level MatchSet and/or its constituent MatchSets have been moved
from their original location, you can edit the higher level MatchSet file to make it
openable. First open the MatchSet folder using Windows Explorer or Macintosh
Finder, then open the descript file (a text file) inside the MatchSet. In the
descript file, the path links to the lower-level MatchSets are shown. You can
change these paths to the new location to make the higher level MatchSet
openable.
Fig. 5-20. Creating a higher level MatchSet.
You cannot create a Higher Level MatchSet from the Automated Detection and
Matching dialog, nor from the Edit MatchSet dialog.
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PDQuest User Guide
Click Add to open the Select Member dialog where you can highlight the MatchSets
you want to include. Click open and they will display in the Members list. Select one
Matchset Master gel to use as the MatchSet Master.
Note:
The MatchSets you select must be writable (i.e., not locked or read-only). See
your Macintosh or Windows documentation for instructions on making files
writable.
When you have made your selections, click the Save button.
The higher level MatchSet is displayed in a single window with subwindows, just like
a level 1 MatchSet.
You can add members to, remove members from, and reorder members in higher level
MatchSets just like level 1 MatchSets. These commands are described in 5.1,
Creating a MatchSet.
Landmark and match spots in a higher level MatchSet just as you do in a level 1
MatchSet. See 5.3, Landmarking Spots and 5.2, Matching Spots.
You will be prompted to select a template to use as the Master for the Higher level
MatchSet. This is similar to selecting the Master template in a level 1 MatchSet
Note that when you select the template for the Master, you will be prompted to copy
the Standard Spot Numbers from that template into the higher level Master. Click Yes
to copy the numbers, or No to assign new numbers. You can also transfer numbers
from the MatchSet member of your choice later.
5.8 Matching Summary
The Matching Summary dialog box contains information regarding the gels
associated with the current MatchSet. Open the Matching Summary dialog by
selecting Matching Summary from the Match menu.
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Matchsets
Fig. 5-21. Matching Summary dialog
Member information lists each gel in the MatchSet and information regarding each
gel including total number of spots in the gel and the number of gels matched to the
master. The Match rate 1 column lists the percentage of matched spots relative to the
total number of spots on the gel. The Match rate 2 column lists the percentage of
matched spots on the gel relative to the total number of spots on the master.
To change the reference gel for the correlation coefficient, double click the gel name
in the list.
To view the log file for a gel, click the log name in the gel table.
MatchSet Information
MatchSet information contains information regarding the total number of spots
matched to all gels and the overall mean coefficient of variation of the replicate
groups.
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PDQuest User Guide
Replicate Group and Class Information
The Replicate group and class information lists the replicate groups and classes with
the number of members in each group or class, the number of spots matched to all
members and the mean coefficient of variation.
Other Features
From the Matching Summary dialog, you can:
Redetect - Opens the Redetect Images dialog box, where you can select images that
you want to redetect. (See Section 5.7.b, Redetecting Members, for further
information.)
Normalize - This opens the Normalization dialog box allowing you to normalize the
data in the Matchset. (See Section 6.7, Normalization, for further information.)
Rematch - This opens the Matching dialog allowing you to rematch the gels in the
MatchSet. (See Section 5.2.a, Automated Matching, for further information.)
View the Scatter plot - Opens the Scatter plot viewer. (See Section 6.3, Scatter Plot
Tool, for further information.)
Report - Opens the Matching Summary Report viewer. The Matching Summary
Report displays the Matching Summary table in a spreadsheet format. From the
viewer you can either print or export the table to be used in reports or presentations.
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6. Analysis Tools
The tools on the Analyze menu are designed to help you identify and compare spots
of interest in a MatchSet. Analysis sets and annotations are covered in the following
chapter.
Fig. 6-1. Analyze tool and submenus.
6.1
Spot Review Tool
The Spot Review Tool displays histograms of all the spots in a MatchSet. The bars in
each histogram represent the spot’s quantity in each member of the MatchSet. If
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PDQuest User Guide
Replicate Group quantitation is selected, the bars represent the spot’s quantity in each
group. See11.2, Histogram Graphs for a more complete description of histograms.
This tool is interactive, so you can use the graphs to identify spots in the MatchSet for
matching and editing or create an analysis set out of selected spots.
With a MatchSet open, select Spot Review Tool from the Analyze menu or View >
Toolbars > Graph Tools toolbar. The review tool will open and the MatchSet will be
reconfigured alongside it. This automatic window configuration can be set under the
report options (see below).
Fig. 6-2. Spot Review Tool.
6-2
Analysis Tools
At the top of the review tool window, select All Spots to display histograms of all the
spots in the MatchSet or Analysis Set to display histograms of only the spots in a
specified set. If you select the analysis set option, a pop-up list of available analysis
sets will be displayed. Select the set you want to display from the list.
You can reorder the graphs by clicking the Sort By buttons. You can sort by Standard
Spots SSP number (default) or Database Spot (DSN) (if defined). If you select Trend,
the graphs are arranged based on a linear regression model of spot presence and
quantity in each member. If you select Quantity, the graphs are arranged starting with
spots of greatest quantity to spots of least quantity.
To scroll through the pages of histograms, click First, Prev, Next, or Last with the red
arrows above the graphs.
If you click on a histogram of a spot, that spot will be magnified and highlighted in all
the MatchSet gels. You can then detect and match the spot using the other tools in
PDQuest.
If you click on a histogram, the histogram itself will be highlighted by a blue
background, indicating that it is selected. You can select more than one histogram at a
time. To deselect a histogram, click on it again.
Note:
To highlight and magnify spots in the MatchSet without selecting the histograms,
shift-click each histogram.
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PDQuest User Guide
Fig. 6-3. Reviewing spots using the Review Tool.
You can create an arbitrary analysis set (section7.1, Analysis Sets) out of spots whose
histograms are selected. First, select the spots by clicking on the histograms. If you
make a mistake, click a selected graph to deselect it or click the Clear Selections
button to deselect all the graphs.
After selecting all the graphs of interest, click the Create Analysis Set button and
enter a name for the set in the pop-up box. Click Create to create the set. You can then
select the set in the Spot Review tool as described above. To remove the set of graphs
you selected for an analysis set click Clear Selections.
Click the Options button in the Spot Review tool to change the display options of the
report. When you click a spot graph, that spot will be centered in the subwindow
(Center images on selected spot), highlighted (Highlight spot on images), and
magnified (Auto-zoom images) by default. Also by default, the review tool window
and MatchSet window will be displayed side-by-side when you open the tool
(Configure windows on launch). Deselect these options to turn off one or all of these
functions.
To close the Spot Review tool window, click Done.
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Analysis Tools
6.2
Image Stack Tool
The Image Stack Tool allows you to scroll through the images in a scanset or
MatchSet layered on top of one another. Using this tool, you can easily compare spots
that appear, disappear, or change size in gels run under different experimental
conditions. You can flag these spots and save the flagset using the new Save Flag Spot
Set command.
Note:
Your images should be close to the same size with spots in the same relative
positions to use this tool.
With all your MatchSet or scanset images open, select Image Stack Tool from the
Analyze menu to open the stack tool.
Fig. 6-4. Image Stack Tool.
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PDQuest User Guide
In the stack tool window, all available gels are listed in the Select gels to display field.
To select an image to display, click on a gel name. The name is highlighted with an
arrow and the image appears in the window.
Click on another gel name to display that image.
Using the Playback controls you can step forward or backward through the images in
the stack tool. First, select some or all of the gel names using standard SFT+click or
CNTRL+click key commands. When multiple names are selected, the back and
forward Step arrow buttons become active. Click the arrow buttons to scroll through
the list of selected gels. Your current gel selection is highlighted with an arrow in the
gel display window.
Alternatively, click the Auto checkbox next to the arrow buttons to begin
automatically scrolling through the list. You can adjust the auto-scroll speed using the
Slow-Fast slider. To stop auto-scrolling, deselect the checkbox.
You can rearrange the order of the images with the image order arrows.
Buttons for various viewing tools are included in the stack tool. These commands will
change the display of all the images in the stacker at once (e.g., zooming in on one
image will magnify the same relative area in all the images).
With the image flagging tools, it is possible to compare spots across gels and flag
spots of interest manually. There are two new commands: Save flagged spot set and
Clear spot flags. Place your cursor on the spot you want to flag and use CNTRL+click
to flag the spot. The cursor becomes an orange circle that shows which spot will be
flagged (marked for stacking). Use Clear spot flags to delete any spots you added. Or
use SFT+ CNTRL+click to unflag spots.
If the spot is flagged with a red cross it needs to be added to the Master gel. If the spot
is flagged with a green cross that means the spot has been matched to the Master gel.
If images flicker back and forth between the gel members in two windows the
flickering cursor becomes a green or red X. The Save flag spot set button prompts you
to add flagged spots to the Master. If you click Yes, a pop-up box prompts you to
name and describe the flagged spot set. The flags will then stay in the MatchSet when
you close the Image Stack tool.
To display the spot crosshairs on the images in a MatchSet check the Show Crosshairs
box. Flags are treated the same as crosshairs. If you restart the program (reopen the
MatchSet) the flags are still there.
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Analysis Tools
Note:
6.3
Spot flags only affect the images you select for display from the list of members.
Scatter Plot Tool
The Scatter Plot Tool shows the relatedness of two gels, replicate groups, or classes in
a MatchSet. It can also be used to print scatter plots for all the gels, replicate groups,
and classes in a MatchSet, or a selected analysis set in a MatchSet.
With a MatchSet open, select Scatter Plot from the Analyze menu. When the scatter
plot window opens, the first two gels in the MatchSet are selected by default. Choose
whether you want to view gels, replicate groups, or classes. Next, select different gels,
replicate groups, or classes to compare from the pull-down list buttons next to the
X-Axis and the Y-Axis fields.
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PDQuest User Guide
Fig. 6-5. Scatter Plot dialog.
The quantity of each spot in the first item (X-axis) is plotted on a log scale against its
quantity in the second item (Y-axis). If the spot’s quantity is the same in both, its point
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Analysis Tools
will fall on the black center line in the graph (slope = 1.00). If the quantities are not
the same, the point will fall above or below the center line.
The regression line generated from the plot is shown in green. This is displayed when
the Regression Line checkbox is selected.
If you select the 2-fold, 3-fold, or 4-fold buttons, parallel red and blue lines will
appear on the graph. All spots between these lines have quantities in the two items
that fall within the selected fold-factor range.
The correlation coefficient for the regression line is displayed on the graph. A
coefficient of 1.00 indicates that the two items are perfectly similar, while a low
coefficient value (e.g., 0.40) indicates that the two are not very similar. The number of
spots that appear in both is also listed on the graph.
If you click a point in the graph, that spot will be highlighted and magnified in the
actual MatchSet.
To print the currently displayed scatter plot, click the Print button in the window.
To print scatter plots comparing every gel in the MatchSet with every other gel, click
the Print All button. This opens the Scatter Plots Report dialog. Enter a title,
researcher, and institution and click Done. This opens the report viewer.
The report viewer allows you the option of printing or reformatting the report. Click
reformat report button to return to the Scatter Plots Report dialog.
6.4
Replicate Groups
Since most experiments are carried out in duplicate, data analysis can be performed
on each gel individually or on sets of duplicate gels grouped together. The Replicate
Groups function allows you to group sets of duplicate gels and determine the average
quantities of their protein spots.
Note:
All duplicate gels must be in the same MatchSet before you create a replicate
group.
To make a replicate group, make sure the MatchSet with the duplicates is open and
active, then go to the Analyze menu, open the Replicate Groups submenu, and select
Create Group.
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Fig. 6-6. Replicate Groups submenu.
In the dialog box, enter a name for the replicate group, then click each duplicate gel in
the list to be included in the group. The gel names will appear highlighted. Click Go
to create the group.
Fig. 6-7. Create Replicate Group dialog box.
In the upper left status bar in PDQuest, you will see the total number of gels in this
group. Repeat this procedure to designate other groups in the MatchSet.
To delete a replicate group, select Delete Group from the Analyze > Replicate Groups
submenu, then select the group to delete from the list.
To edit a replicate group, select Edit Group from the submenu. In the dialog, add or
remove gels from the list.
To Display Replicate groups you need to specify which groups you want to display
from the pop-up box.
To rename a replicate group, select Rename Group from the submenu, select the
group to rename in the dialog, then enter a new name for the group in the pop-up box.
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Quantitating a Replicate Group
After you have created a replicate group, you can calculate the average spot quantities
in the members of the group by selecting Spots > Show Quantity (F6). Place your
cursor in each image for which you want to see spot quantity. Quantity will not
display for the Master image.
Spot quantity displays as blue numbers with a text box stating whether the quantity is
normalized.
Fig. 6-8. Spots > Show Quantity
Replicate group quantitation can also be displayed using the Quantity Table function
(Shift+F6, Spots menu).
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Fig. 6-9. Quantitation table displaying replicate group quantitation.
Click the spot whose quantitation you want to see. If you select the All Replicate
Group as your graph configuration (located in the Report menu), the average value of
that spot’s quantitation will be displayed.
Fig. 6-10. Select graph configuration.
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6.5
Group Consensus
The Group Consensus tool displays a list of all replicate groups associated with the
current MatchSet. The purpose of the Group Consensus tool is to use spot matching
information across all of the gels within the replicate group to find possible spot
detection errors. Ideally spots in a replicate group would match across all gels in the
group. As this is not always the case, the Group Consensus tool allows you to easily
edit spots in the replicate group to achieve consensus across all gels.
When the Group Consensus Tool window opens, it will be configured alongside the
MatchSet window. The MatchSet window displays the images belonging to the
selected replicate group.
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Fig. 6-11. Replicate Group Consensus
At the top of the Group Consensus tool is the Consensus Summary listing all replicate
groups in the MatchSet. The columns represent the number of spots found on one gel,
two gels, three gels, and so on depending on the number of gels associated with the
replicate group. The number in the 1 Gel column represents the number of spots
found on only one gel; the number in the 2 Gels column represents the number of
spots found on two gels, etc. The column representing the greatest number of gels
lists the number of spots found on all gels. This is the column that represents total
consensus. Ideally, only this column should have a non-zero value. The Ambiguous
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column lists the number of spots that appear on one gel and can be matched to more
than one spot on one or more of the other gels.
Fig. 6-12. Consensus Summary Table
Use the Cancel, Review, and Accept buttons to organize the different Consensus
Summary cells according to your review criteria. Marking a cell Cancel will color
code it red and remove it from automatic inclusion in the list of spots to be reviewed
with the Individual Spot Tools later. Similarly, marking a cell Accept will color code
it green and remove it from automatic inclusion in the review list. Note that any cell,
even those marked Cancel or Accept, can be manually selected for review later
Click Update Consensus to update the Consensus Summary table with any manual
changes made in the Review tool. Rematch will rematch all the spots in the replicate
group.
Note:
If you rematch the gels in a replicate group, you will lose all prior matching.
The Global actions and Review tool sections of the Group Review affect the cell you
have selected in the Consensus Summary table (highlighted in blue). The global
Actions affects all the spots in the selected cell, while the Individual Review tool
allows you to review each spot individually and add or remove the spot (represented
by a blue triangle in the gel images).
6.5.a Review Individual Spots
The Review Individual Spots section of the Group Consensus allows you to
individually review each spot in the group. The active group field shows the spot
number of the highlighted cell in the Consensus Summary table. Use the arrows to
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cycle through the spots in the active group. The Review Tool displays a quantity
graph of the selected spot. The bars in the graph represent the spot’s quantity in each
member of the group where it appears.
To select a different group to review, select a different cell in the Consensus Summary
table.
To select a spot in a gel, click the button labeled Select Spot in Gel then click on a
spot in one of the gel images. The spot is automatically selected and the group to
which it belongs is highlighted in the Consensus Summary table.
To see spot number in the gel, check the box labeled, “Display SSPs”. When this box
is checked, the spot number appears below the marker in the gel.
6.5.b Achieving Consensus
The following actions are used to make changes to the replicate group to achieve
consensus across all members in a group.
Automatic
Auto Add – Allows you to automatically add the selected spot to all the gels.
Clicking Auto Add will add the spot marker to the gels where it does not appear. A
red circle indicates where the spot marker is added.
Auto Remove – removes the selected spot from the gels.
Auto Cancel – Cancels the spot in the group. This does not remove the spot from the
list, but it does remove it from your analysis.
Manual
The Spot edit actions allow you to manually add or remove, cancel or restore,
combine or un-combine spots on the images in the replicate group. Click update
under the Consensus Summary table for the changes to take effect.
When you have finished making your changes to the consensus groups, click Update
under the Consensus Summary table.
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6.5.c Global Actions
Global actions are much the same as the adding and removing of individual spots but
are performed on all the spots in the cell.
Auto Add All - allows you to automatically add all the spots of the selected cell to all
the gels. For instance, a replicate group of three gels may contain 15 spots located in
two of the gels. Clicking Auto Add All will add the spots to the gels where they do
not appear.
Auto Cancel All – Cancels all the spots in the cell. This does not remove the spot
from the list, but it does remove it from your analysis.
Auto Remove All – removes the selected spots from the gels.
Since the global actions affects multiple spots on multiple gels, PDQuest warns you
before proceeding with the action.
Performing a global action causes the consensus summary table to automatically
update. There is no need to manually update the Consensus Summary table.
6.6
Sample Database
The Sample database of PDQuest allows you to track gel - sample associations. With
the sample database you can attach attributes to samples. This is important for
tracking two different gels of the same sample type. For instance, your MatchSet may
contain a series of E. coli samples but with different treatments such as temperature.
6.6.a Associating Samples to Gels
To associate samples to gels, select Analyze>Describe Samples>Enter Sample
Attributes.
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Fig. 6-13. Enter Sample Attributes dialog box
Adding and Editing Samples
There are various ways to add samples to gels. To quickly add sample names to all
your gels at once, click Auto-generate. A sample name is then associated with each
gel in your MatchSet based on the gel name. Otherwise you can click in a cell and
type a new name for the sample. Another way to add a sample is to click Add located
under Sample. This opens the Create New Sample dialog box where you can enter
more in depth information about the sample.
Fig. 6-14. Adding a Sample
If the sample name exists in your database, click the drop-down arrow and select the
name from the list. If not, type in a new name. In this dialog box you can also enter a
description and the name of the individual from which the sample came. The date the
sample was created is automatically entered, but this can be changed.
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If you want to edit an existing sample, click the sample name in the list and click Edit
under Sample. This opens the Edit Sample Dialog box.
Fig. 6-15. Editing a sample
Adding and Editing Attributes
Once you have created gel - sample associations, you can add attributes to the
samples. To add a new attribute, click Add under Attribute. This opens the Enter
Attribute dialog box.
Fig. 6-16. Entering an Attribute
Enter a name and description for the attribute. If an attribute exists in your database,
click the drop-down arrow and select it from the list.
Once the attribute has been added to the table, you can add a value by clicking the cell
and entering a value for each sample. You can also click Select Value and select a
value from the list. Attribute values are known as classes. (See Section 6.6.b, Classes,
for further information.)
To edit an attribute, click Edit under Attribute.
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6.6.b Classes
Attributes are variables that cannot be measured, but must be expressed in a
qualitative manner. A class is a group of gels with the same attribute value. An
example of this might be a MatchSet of six gels. Some of these gels may be treated or
untreated. By attaching these values to your sample in the sample database, you can
refer to these sample as a group of treated or untreated samples. You can also create
analysis sets based on the attribute values. (See Section 7.1, “Analysis Sets” for
information on creating analysis sets.)
Fig. 6-17. Sample Report showing attributes and values
All of the samples in the above table have the attributes treated, temp, and color. The
samples with the attribute value yes in treated are considered members of a class. The
same goes for the samples with the attribute Temp and the same value. A gel can
belong to as many classes as it has attributes.
Once you have associated samples to gels and added attributes to them, you can use
the classes when creating analysis sets. See Section 7.1, “Analysis Sets” for
information on creating analysis sets.
Importing a Sample Database
If you are working with MatchSets that have similar gel types, you can import a
sample database from one MatchSet into the other or by importing a text file of
samples. If you choose to copy a database from another MatchSet, any samples
already in the current MatchSet’s sample database will be overwritten. If you import
from a text file, the samples are added to your database.
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To import a sample database from a text file, click import then click Import. This
opens the Select a File dialog. Locate the file you want to import and click Open. The
samples, and any attributes attached to the samples, are added to the sample list.
The configuration of the file you import is important. Sample names and attributes
(including values) must be separated by a comma or tab. The text file should resemble
the following example:
sample1,temp=30 deg,dilution=2-fold,color=red
sample2,temp=37 deg,dilution=none,color=blue
sample3,temp=42 deg,dilution=2-fold,color=neg
sample4,temp=45 deg,dilution=none,color=no color
The sample name must come first followed by the attributes. In the example given,
the attributes are temp, dilution, and color. Each attribute is then given a specific
value for each sample. For example, Sample1 contains the attributes temp, dilution,
and color with the values for each being 30 deg, 2-fold, and red.
To copy the sample database of another MatchSet into the current MatchSet, click
Import, and then click Copy. A warning dialog box requires you confirm your request
to overwrite the current database. Click OK. This opens the Select Files to Import
dialog box. Locate the MatchSet containing the database you wish to import and click
Open. If the MatchSet sample database you are importing contains sample pairs, these
will also be imported. (See Section 6.6.c, Sample Pairs, for further information.)
Viewing Reports
There are two types of reports you can view in the Sample Database. Click View
Report to view a report of the samples associated with the gels in the current
MatchSet. The report shows the sample names and the attributes values of each
sample. Click View Classes to view the Classes Report, which lists the attribute
values and the samples that contain them in each column.
6.6.c Sample Pairs
Sample Pairs are used for statistical analysis of sample proteins. To create sample
pairs, select Analyze>Describe Samples>Pair Samples. This opens the Pair Samples
Dialog.
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Fig. 6-18. Creating Sample Pairs
To create a sample pair, highlight a sample inn the left column and a sample in the
right and then click Pair Selections. The Right sample turns red to indicate it is paired
with the selected sample in the left column. To un-pair a selection, highlight the
sample pair you wish to un-pair and click un-pair selection. If the sample you select in
the left column is already paired to a sample, pairing it with another sample will
automatically un-pair the selected sample with original sample.
You can also import sample pairs by clicking Import. This opens the Select file to
Import dialog. Locate the file you wish to import and click Open. The format of your
file must be:
sample1,sample2
sample3,sample4
Each row in the text file represents a pair separated by a comma.
Upon importing the sample pairs, PDQuest attempts to locate samples in your list
which match the pair in the file. If a match for a pair cannot be found it is simply not
imported. Only those pairs containing samples in your database are imported.
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Note:
Importing sample pairs will overwrite any pair selections you have already
created containing samples that match imported pairs.
To view a report of your sample pairs, click View Report. This opens the report
viewer listing each sample and its pair. Samples not paired are not listed. From the
viewer you can either print or export the table to be used in reports or presentations.
6.7
Normalization
When comparing gels in a MatchSet, there is often some variation in spot size and
intensity between gels that is not due to differential protein expression. This variation
can be caused by a number of factors, including:
•
Inconsistent cell numbers (cell densities) in the sample prep,
•
Inconsistent sample prep efficiency due to variations in reagents, protocols, etc.,
•
Handling errors resulting in some sample loss during prep,
•
Pipetting errors during sample prep,
•
Sample loss during gel loading,
•
Inconsistent staining times between gels,
•
Inconsistent labeling efficiency with radioisotopes,
•
Inconsistent excitation/emission efficiencies between different fluorescent tags,
•
Inconsistent detection energy sources between gels during image acquisition, and
•
Inconsistent exposure times between gels during image acquisition.
To accurately compare spot quantities between gels, you must compensate for these
nonexpression-related variations in spot intensity. This is called normalization.
With a MatchSet open, select Normalize from the Analyze menu.
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Methods
Fig. 6-19. Normalize dialog box for member images.
In the dialog box, check the Enable Normalization box to activate normalization for
the MatchSet.
Normalization Formula
Normalization in PDQuest is performed for each gel in a MatchSet using the
following formula:
Normalized spot quantity = Raw spot quantity * Scaling factor * Pipetting error
compensation factor (if any) / Normalization factor
Raw spot quantity is the unnormalized quantity (intensity) of each spot,
Scaling factor is a factor you select to give a meaningful value (see below), and
Normalization factor is calculated for each gel based on the normalization method
you select (see below).
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Pipetting error compensation is only available if you have calibrated the gels using
Calstrips and entered the number of counts loaded in the gel into the Gel Record. See
Appendix J.
After you have selected the settings described below, click Done.
6.7.a Scaling Factor
The scaling factor is a constant by which the normalized quantity is multiplied to give
a more meaningful value. A common scaling factor is 106, giving units of parts per
million (PPM).
Under Scaling, specify the scaling factor that you would like to use by clicking on
PPM (x 1000000), Percent (x 100), or User Specified. If you select User Specified,
enter your own scaling factor and units in the appropriate fields.
6.7.b Normalization Methods
The normalization method you select will depend in part on how much you know
about your gels and how they were prepared. In the Normalization dialog box, select
from the options for your Basis or denominator. For example, if you can readily
identify housekeeping proteins on your gels, the Total quantity in analysis set method
is preferable. If you know that each gel was loaded with a different amount of sample,
you can select Specified Value and enter the known variation factor.
Or, check the Total quantity in analysis set option and select your set of housekeeping
proteins from the pulldown list.
Total Quantity in Analysis Set
This normalization method involves identifying a group of protein spots that are
present in all MatchSet members and are conserved (i.e., their quantities should not
vary between samples). These are proteins that you know are expressed in a very
consistent manner at all times within a cell, with little or no regulation. They are
commonly referred to as housekeeping proteins.
Step 1:
Create an arbitrary analysis set containing several of these housekeeping proteins
from different regions of the MatchSet Master. To create an arbitrary analysis set,
select Analysis Set Manager... from the Analyze menu.
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Step 2:
Click Create then select Arbitrary form the Select Set type dialog. This opens the
Arbitrary Analysis Set template.
Fig. 6-20. Create an Analysis Set.
Step 3:
To add spots to this set, click Add spots to set and click an appropriate (i.e.,
housekeeping protein) spot. The cursor displays as a green cross. The spot will be
highlighted, indicating that it has been added to the set. If you want to remove a
spot from the analysis set, click Remove spots from set and click a highlighted
spot. Click Save. The new analysis set is added to the list.
See section 7.1 for more information on creating analysis sets.
The total quantity of the valid spots in this analysis set will be displayed for each
MatchSet gel in the table at the bottom of the dialog box.
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The raw quantity of each spot in a gel will be divided by the total quantity of the spots
in the analysis set in that gel, according to the normalization formula.
Note:
The advantage of selecting multiple housekeeping proteins is that occasional
minor variations in the size and/or intensity of a particular spot will be averaged
out when the group of spots is considered is a whole.
Total Quantity in Valid Spots
In this normalization method, the raw quantity of each spot in a member gel is divided
by the total quantity of all the spots in that gel that have been included in the Master
(see the normalization formula).
Select the Total Quantity in Valid Spots option button in the Normalization dialog to
activate this method. This normalization method assumes that few protein spots
change within the experiment, and that the changes average out across the whole gel.
It can be useful if very little information is known about the possible source of sample
variation.
Total Density in Gel Image
In this method, the raw quantity of each spot in a member gel is divided by the total
intensity value of all the pixels in the image (see the normalization formula).
Select the Total Density in Gel Image option button to activate this normalization
method. This model assumes that the total density of an image (i.e., background
density plus spot density) will be relatively consistent from gel to gel. It can be useful
if very little information is known about the possible source of sample variation.
Specified Value
In this method, the raw quantity of each spot in a member gel is divided by a userdefined normalization factor for that gel.
Select the Specified Value option and enter a normalization value for each gel in the
table at the bottom of the dialog box. This value will be used as the denominator in
the normalization formula.
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This model is useful if you want to correct for known variations in samples across
gels (e.g., the amount of cells that went into an experiment, the amount of extract
loaded, the length of time the images were detected, etc.).
Counts Loaded from Gel Record
In this method, the raw quantity of each spot in a member gel is divided by the total
counts loaded into that gel, as specified in the Gel Record when you performed
calibration.
Note:
This method is only available if you have performed calibration on your
MatchSet gels. (See Appendix J.)
The counts loaded in each gel are listed in the table at the bottom of the Normalization
dialog; this value will be used as the denominator in the normalization formula. Select
the Counts Loaded from Gel Record option button to activate this normalization
method.
This method assumes that few protein spots change within the experiment, and that
the changes average out across the whole gel. This model can be useful if you want to
correct for known variations in the amount of sample loaded across gels, as measured
prior to loading with a scintillation counter.
If you select this method, you can compensate for pipetting errors in all MatchSet
members by selecting the Pipetting Error Compensation checkbox.
Pipetting Error Compensation
When this checkbox is selected, the software will attempt to correct for minor
systematic errors inherent in scintillation counting, pipetting, and exposing films. The
normalization process is based on the assumption that these minor errors affect all
proteins equally. It also assumes that few protein spots change within the experiment
or that when changes do occur, they do not alter the random distribution from one
sample to the next.
When you select this checkbox, a pop-up box will display the calculated
compensation value for each MatchSet member. These values will also appear in the
in the table at the bottom of the Normalize dialog.
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6.8
MrpI Data
In a MatchSet, you can enter the molecular weight and isoelectric point values for
your known protein spots. With this data, PDQuest can calculate MrpI values for all
the spots in the MatchSet.
The functions needed to enter and display MrpI data are found in the Analyze > MrpI
Standards submenu and the MrpI Tools toolbar.
Fig. 6-21. MrpI commands.
Note:
PDQuest needs to know the pH gradient of the gel in order to calculate isoelectric
point values. To enter the correct orientation, go to Edit > Preferences >
Applications tab and click either Acid-Base or Base-Acid next to the pH Gradient
prompt.
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Entering MrpI Values for Known Spots
To determine MrpI values for all the spots in the MatchSet, you first need to enter the
values for a few known spots.
Select Enter MrpI Data from the Analysis > MrpI Standards submenu or toolbar, then
click on a spot in the MatchSet Master whose molecular weight and/or isoelectric
point values are known. A dialog box prompts you to enter the values for that spot.
Fig. 6-22. Pop-up box in which MrpI data are entered.
Type the molecular weight first, followed by a space, followed by the isoelectric
point. Click OK when you are done.
If you make a mistake entering the data, you can select Enter MrpI Data again and
click the spot. The values you entered will appear in the dialog box and you can make
the necessary corrections.
If you want to remove the MrpI data for a spot, select Remove MrpI Data and click
the spot.
Displaying MrpI Data
To see the proteins for which you have entered MrpI data, you can use either the Mark
MrpI Spots or Display MrpI Data option. The first option highlights the spots for
which you entered data. The second option displays MrpI values in red next to the
spots.
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Analysis Tools
For a graphical display of the overall trend of MrpI values across the entire gel, first
make sure the Master subwindow is active, then select Plot MrpI Grid from the
Analyze > MrpI Standards submenu or toolbar.
Fig. 6-23. MrpI grid with values in the Spot Parameters dialog.
A grid of MrpI axes will appear over the Master. You can use the grid to verify that
the values that you entered make sense; you can also use it to estimate the MrpI
values of proteins of interest.
To see the exact MrpI values of a spot, select Spot Parameters from the Spots menu or
toolbar and click the spot, or use the F6 key command on the spot. The MrpI values
are displayed in the pop-up box.
Note:
If the isoelectric point value of the spot falls outside the highest and lowest
known values, the pI value will not be calculated. Empirical results have shown
that extrapolated pI values are unreliable, due to gel distortions and other factors.
Transferring MrpI Data in a Higher Level MatchSet
Transfer MrpIs allows you to apply the molecular weight and isoelectric point values
from one higher level MatchSet member to the other members. This makes it easier to
correlate data from the member MatchSets. (See section 5.7.c, Higher Level
MatchSets for more information on higher level MatchSets.)
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With your higher level MatchSet open, select Transfer MrpIs from the Analyze >
MrpI Standards submenu. In the first pop-up box, select the MatchSet member you
want to copy from; in the second box, select the member you want to copy to. You
will be asked to confirm the transfer.
Repeat the procedure to apply the values to all the higher level MatchSet members.
6.9
Standard Spot Numbers
Standard spot (SSP) numbers are unique numbers assigned to each spot in the
MatchSet Master. In general, spots in the lower left region of the image have low SSP
numbers, while spots in the upper right region have high SSP numbers.
Note:
SSP numbers are increasingly important when you begin comparing spots
between different MatchSets using higher level MatchSets.
SSP numbers are automatically assigned when you create the MatchSet Master. To
create SSP numbers, PDQuest first divides the Master into rows and columns, placed
so that there are approximately the same number of spots in each section and that no
section has over 100 spots. The first two digits of a spot’s SSP number correspond to
its section’s X and Y coordinates. Spots within a section are numbered sequentially.
Displaying SSP Numbers
The spot number tools are located on the Analyze > Spot Numbers submenu.
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Fig. 6-24. Spot number submenu.
With the MatchSet open and active, select Plot SSP Grid (CNTRL +F9) from the
submenu to display the SSP grid on the Master. Numbered vertical and horizontal
grid lines will appear on the Master.
The grid is helpful for locating spots by SSP number. However, it is an approximation
and should only be used as a guide. If enough spots are added to a single grid block to
use up the 99 available numbers, numbers will be assigned from other blocks.
Display SSPs displays SSP numbers next to every spot in the active window. You
may need to zoom in on the image to be able to read them.
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Display All SSPs displays SSP numbers next to every spot in every open window.
Transferring SSP Numbers in a higher level MatchSet
Transfer SSPs is used to copy the SSP numbers from one higher level MatchSet
member to the others. This makes it easier to correlate data from the member
MatchSets.
With the higher level MatchSet open, select Transfer SSPs from the submenu. In the
first pop-up box, select the MatchSet member you want to copy from; in the second
box, select the member you want to copy to. You will be asked to confirm the transfer.
When the transfer is complete, the number of SSP numbers transferred will be listed
in an alert box. Repeat the procedure to apply the numbers to all the members in the
MatchSet.
6.10 Database Spot Numbers
Database spot numbers (DSNs) are an alternative to the SSP numbers. The DSN
system provides a more flexible way to number protein spots in a Master in that it
indicates the general location of each spot. A coordinate grid can be established for
each axis, based on molecular weight and isoelectric point. The grid coordinates are
identified with letters (A–R, excluding I and O because they look like numbers). The
letters are intended to represent specific MrpI ranges so that the numbering between
different gels can be comparable. These ranges are as follows:
Mr values:
Mr values (cont):
pI values:
0-10=A
11-14=B
15-19=C
20-25=D
26-34=E
35-46=F
47-63=G
64-86=H
87-117=J
118-158=K
159-215=L
216-293=M
294-398=N
399-541=P
542-736=Q
737-1000=R
0-2=A
2-3=B
7.0-7.5=J
7.5-8.0=K
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3-4=C
4-5=D
5.0-5.5=E
5.5-6.0=F
6.0-6.5=G
6.5-7.0=H
8.0-8.5=L
8.5-9.0=M
9-10=N
10-11=P
11-12=Q
12-14=R
DSNs have the format ABnn. “A” is the molecular weight grid letter, “B” is the
isoelectric point grid letter, and “nn” is the number of the spot within that section of
the grid.
Assigning DSNs
After you have entered MrpI values for known spots, you can create a DSN grid using
this data by selecting Assign DSN Grid from the Analyze > Spot Numbers submenu.
This automatically assigns a DSN number to each spot in the Master.
Plot DSN Grid
Plot DSN Grid Counts
Display DSNs
Fig. 6-25. DSN grid, counts, and numbers.
To display the grid (this does not occur automatically when the grid is created), click
in the Master and select Plot DSN Grid. A grid of vertical and horizontal axes will
appear, with the letter assignments of each axis displayed in the window margins.
To see the spot assignments, select Display DSNs. To read the numbers, you will
probably need to zoom in on a smaller region of image.
Transfer DSNs is used to copy the DSN numbers from one higher level MatchSet to
another See section 5.7.c, Higher Level MatchSets.
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Transferring DSNs in a Higher level MatchSet
Transfer DSNs is used to copy the DSNs from one higher level MatchSet member to
the others. This makes it easier to correlate data from the member MatchSets.
With the higher level MatchSet open, select Transfer DSNs from the submenu. In the
first pop-up box, select the MatchSet member you want to copy from; in the second
box, select the member you want to copy to. You will be asked to confirm the transfer.
When the transfer is complete, the number of DSNs transferred will be listed in an
alert box. Repeat the procedure to apply the numbers to all the members.
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7. Analysis Sets and
Annotations
Analysis sets and annotations are tools to help you organize and analyze your spot
data.
Analysis sets allow you to create groups of spots that are statistically and biologically
significant. You can also create sets of spots to cut using Bio-Rad’s ProteomeWorks
Spot Cutter.
Annotations allow you to add notes, comments, and other pertinent information to
spots. Using annotations, you can create links to protein databases or files, import
mass spec data, and store spot information in HTML format for easy portability and
uploading to Web sites.
7.1 Analysis Sets
The analysis set tools allow you to create and study sets of proteins that are
statistically and scientifically significant. You also use analysis sets to identify spots
to cut using Bio-Rad's ProteomeWorks Spot Cutter.
There are six different kinds of analysis sets:
Qualitative Analysis Sets:
Are composed of spots that are present in one gel but
not in another (i.e., spots that have been turned “on” or
“off”).
Quantitative Analysis Sets: Are composed of spots whose quantitation has increased
or decreased “x” fold (where x is a user-determined
value) or whose quantitation has changed above or
within the fold change factor that you specify.
Statistical Analysis Sets:
Are composed of spots that are significant based on the
statistical test that you select.
Arbitrary Analysis Sets:
Are composed of spots that you manually select.
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Boolean Analysis Sets:
Are created by comparing two or more other analysis
sets. For example, set C could be made up of those spots
that are present in both set A and set B.
Matching Analysis Sets:
Are composed of spots which are either unique to one
member, or are present in all members.
Analysis Sets in Higher Level MatchSets
When you create a higher level MatchSet in PDQuest, analysis sets created in the
members are automatically transferred as part of the higher level MatchSet. In
addition, you can create analysis sets in the higher level MatchSet. There are three
types of analysis sets you can create in a higher level MatchSet: Arbitrary Analysis
Sets, Boolean Analysis Sets, and Matching Analysis Sets:
Note:
Transferred analysis sets cannot be edited.
7.1.a Analysis Set Manager
The Analysis Set Manager is a convenient tool, which allows you to create and apply
multiple analysis sets. With the Analysis Set Manager you can create and apply one or
more analysis sets at a time, edit one analysis set at a time, quickly switch analysis
sets on and off, and delete an analysis set. The Analysis Set Manager also allows you
to determine when to apply the analysis sets, which gels to mark, and what overlays to
include in your analysis.
To open the Analysis Set Manager, select Analyze>Analysis Set Manager....
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Fig. 7-1. Analysis Set Manager
The top portion of the analysis set lists the analysis sets associated with the current
MatchSet.
Mark - A check in this box indicates the analysis set is currently applied to the
MatchSet. To hide an analysis set, clear the box. Click the column header to organize
the list according to marked sets.
Sym - indicates the spot symbol for the analysis set on the gels you specify. Click the
symbol to open the Spot Symbol Format dialog. From this dialog you can choose
style, color, and symbol.
Analysis Set Name - Indicates the name of the analysis set. Click the column header
to organize the list according to the analysis set name. An up arrow in the column
header indicates ascending order, while a down arrow indicates descending order.
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Type - Indicates the type of the analysis set. Click the column header to organize the
list according to the analysis set type. An up arrow in the column header indicates
ascending order, while a down arrow indicates descending order.
Count - Indicates the number of spots that match the analysis set criteria. Click the
column header to organize the list according to the Count. An up arrow in the column
header indicates ascending order, while a down arrow indicates descending order.
Edit - Click the button under the Edit heading to either edit the analysis set or delete it
from the list.
Analysis sets are automatically evaluated when you open the MatchSet, when you
open the Analysis Set Manager, and if you make any edits to an analysis set. If you
have On opening MatchSet selected as your Auto-evaluate option, click Evaluate
after any spot editing.
The Mark spots on options allow you to determine on which gels to display the
analysis sets. The Master gel option displays the analysis sets on the master only. The
Operand gels option displays the analysis sets on the master image and the gels being
compared in the analysis sets. All gels displays the analysis sets on all the gels.
In the Analysis Set Manager you can determine what to display in the MatchSet
subwindows. Select Window labels to display the analysis set names in the
subwindows where they are displayed. Select SSP numbers to display the spot
numbers included in the analysis sets in the MatchSet images. Click Format to
determine how to display the spot numbers. Graphs displays in the master image the
graphs of those spots included in the currently active analysis sets.
Tip:
It is best to view the graphs when the MatchSet windows displays only the
master image. See Section 3.2, Configuring Subwindows for further information.
To print a report of all your analysis sets, click Print. This opens the Analysis Set
Report dialog box. See Section 11.7, Analysis Set Report for further information.
Click Done to close the Analysis Set Manager.
7.1.b Creating an Analysis Set
To create an analysis set, click Create in the Analysis Set Manager. This opens a
dialog for you to select the type of set you wish to create.
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Fig. 7-2. Select the Analysis Set type you wish to create.
In the Select Set Type dialog, select the type you want to create by clicking the name.
This opens the Analysis Set Template for that analysis set type. As you apply criteria
for an analysis set, the results appear in the gel or gels you specify in the Analysis Set
Template. This is helpful in that you can analyze data directly from the template
without saving the analysis set to the Analysis Set Manager.
See the following sections for information pertaining to each type of analysis set.
Editing and Deleting Analysis Sets
If you want to edit an analysis set that already exists, click on the button below the
Edit heading in the Analysis Set Manager and select Edit from the list. This opens
the Analysis Set template dialog with the current settings for the selected analysis set.
Make any necessary changes, including changing the name if you choose. Click Save
when you have finished making changes to the analysis set.
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To delete a saved set, click on the button below the Edit heading in the Analysis Set
Manager and select Delete from the list. This permanently removes the analysis set
from the list.
7.1.c Qualitative Analysis Sets
Qualitative analysis sets contain spots that were not detected in one gel but were
detected in the other. This test would detect a protein that was expressed under
experimental conditions but was not expressed in the control sample, or visa versa.
Note:
We strongly recommend that all the spots in such an analysis set be doublechecked for accuracy. It is possible that a protein might actually be present in
both gels but was missed during matching.
To create a Qualitative Analysis Set, select Qualitative from the Select Set Type
dialog.
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Fig. 7-3. Qualitative Analysis Set dialog.
Enter a name, or accept the default name, then enter a description of the analysis set.
Note that while the name of the set is required, the description is not.
Note:
If you have Normalization enabled, normalization parameters will be used for
spot quantities. See Section 6.7, Normalization, for further information.
Qualitative Analysis Set Options
The following options help refine the analysis set:
Estimate missing spots:
If this box is left unchecked, then the spot must be
detected in 100% of the gels in the group for it to be
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included in the analysis. If the box is checked, the
spot must be detected in >50% of the gels in the group
to be included. This is important in qualitative
analysis because the analysis is based on whether
spots appear in one group or class and do not appear
in another.
Estimate saturated spots:
Note:
This option estimates the total peak value for a
saturated spot.
These options are only available for groups and classes.
Choose whether to compare gels, replicate gels, or classes (See Section 6.6, Sample
Database, for information on classes). Select the items you want to compare. If you
click A the pop-up box displays a list available for analysis. Select what you want to
compare from the list and their names display in the A or B field.
Now select whether you want to highlight spots that were on in A/off in B (On in A)
or on in B/off in A (On in B).
Finally, you need to tell PDQuest what “turned on” or “turned off” means. How many
times greater than the minimum detectable spot should the quantitation be for a spot
to be included in this analysis set? Should it be ten-fold greater than the minimum? If
so, enter 10.00 next to the Fold over minimum sensitivity prompt.
Click on the Save button at the bottom of the dialog box to add the analysis set to the
ASM.
Be sure to enter a name for the set. If you forget, a pop-up box will remind you to do
so.
As with all other types of analysis sets, when the Analysis Set Template is open, you
can view the results in the MatchSet immediately. To the right of the dialog, you can
specify the spot marker, on which gel(s) to show the spots, and what information to
show on the gel(s).
When you open the Analysis set Template, whatever analysis sets you have selected
in the ASM become inactive. To view these sets in the Analysis set Template, check
the box labeled, “Display Selected sets.”
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7.1.d Quantitative Analysis Sets
Quantitative analysis sets allow you to perform fold change analyses. To create a
Quantitative Analysis Set, select Quantitative from the Select Set Type dialog.
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Fig. 7-4. Quantitation Analysis Set dialog
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Enter a name, or accept the default name, then enter a description of the analysis set.
Note that while the name of the set is required, the description is not.
Note:
If you have Normalization enabled, normalization parameters will be used for
spot quantities. See Section 6.7, Normalization, for further information.
Quantitative Analysis Set Options
The following options help refine the analysis set:
Estimate missing spots:
. Otherwise the spot will be reported as missing or
excluded from calculations, as appropriate. This
option is only available on replicate gels and classes.
Estimate saturated spots:
If a spot is saturated and therefore cannot be
accurately quantified, an estimate of its value is made
by fitting a gaussian to the sides of the spot and
extrapolating the peak value on that basis. The
volume is calculated based on the extrapolated
gaussian.
Include qualitative changes:
This option includes allows the inclusion of spots
“turned on” in one gel, but “turned off” in the other
based on the minimum fold over background value
you enter.
Note:
Estimate missing spots and estimate saturated spots are only available for groups
and classes.
Choose what you wish to compare (gels, replicate gels, or classes, See Section 6.6,
Sample Database, for information on classes) and enter your selections for
comparison (e.g., control gels versus experimental gels).
After you have chosen what to compare, you need to decide what kind of quantitative
changes you want to examine. You can select Above upper limit, Below lower limit,
Between limits, or Outside limits.
Depending on your method choice, the graph to the right changes to reflect the
method. The green portions of the bars correspond to the spots that will be included in
your analysis set.
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To view the scatter plot, click the Scatterplot button. Selecting your limits with the
scatterplot is similar to the histogram in that you use the slider bars. The difference
here is you are given a view of which spots will be included or excluded from the
analysis set.
Do you want to look for a protein whose quantitation went up under the experimental
conditions as compared to the control? If so, click the Above Upper limit button.
A represents all matched spots in sample A and B represents the corresponding
matched spots in sample B. Comparisons are made using spot quantity only.
Positional comparisons, etc., are not possible.
The comparison is performed as follows: A threshold value is established by
multiplying and/or dividing A by the factor you specify. B is then compared to the
threshold.
For example:
•
If you ask to create a set by clicking on the Above upper limit button and enter a
factor of 2.0, the set will include spots whose quantity in B is at least twice that of
the corresponding spot in A.
•
If you click on the Above Upper Limit button and enter a factor of 0.5, the set
will include spots whose quantity in B is greater than half the quantity of the
corresponding spot in A.
•
Clicking on the Below Lower Limit button and entering a factor of 0.5 (-2.0 fold)
will find spots whose quantity in B is less than half the quantity of the
corresponding spot in A.
•
Creating a set by clicking on Below lower limit and entering a factor of 0.33 (-3.0
fold) finds all the spots whose quantity in B is less than one-third the quantity of
the same spot in A.
•
If you are interested in finding the spots whose quantitation has significantly
changed either way (either increased or decreased), select the Outside limits
option. The proteins found in this test have increased in quantitation above the
upper limit or decreased below the lower limit. If you enter an upper limit of 2.0
and a lower limit of 0.33 you will find spots whose quantity in B is more than
twice the quantity in A or less than one-third the quantity in A.
•
To find spots whose quantitation in B has not changed significantly from A, click
on the Between limits button. Enter factors to define the upper and the lower
limits. If, for example, you enter an upper bound factor of 1.10 (+1.1 fold) and a
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lower bound factor of 0.90 (-1.1 fold), the analysis set will include spots whose
quantity in B is between 10% up or 10% down relative to the quantity of those
spots in A.
Click on the Save button at the bottom of the dialog box to add the analysis set to the
ASM.
Be sure to enter a name for the set. If you forget, a pop-up box will remind you to do
so.
As with all other types of analysis sets, when the Analysis Set Template is open, you
can view the results in the MatchSet immediately. To the right of the dialog, you can
specify the spot marker, on which gel(s) to show the spots, and what information to
show on the gel(s).
When you open the Analysis set Template, whatever analysis sets you have selected
in the ASM become inactive. To view these sets in the Analysis set Template, check
the box labeled, “Display Selected sets.”
7.1.e Statistical Analysis Sets
Statistical analysis sets are composed of spots from either replicate groups or classes
(See Section 6.6, Sample Database, for information on classes) found to be significant
according to the statistical test that you specify.
To create a statistical analysis set, select Statistic from the Select Set Type dialog.
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Fig. 7-5. Statistical Analysis Set
Statistical analysis sets only work with replicate groups or classes because the tests
require multiple gels.
Enter a name for the analysis set, or accept the default name, then enter a description
of the analysis set. Note that while the name of the set is required, the description is
not.
Note:
If you have Normalization enabled, normalization parameters will be used for
spot quantities. See Section 6.7, Normalization, for further information.
Select whether you want to compare replicate gels or classes, then choose which
members to compare by clicking on the drop down buttons for A and B.
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Indicate the statistical test you want to perform by clicking the button next to the
name of the test.
Refer to the following table to determine the number of gels per group for you to run
a specific test.
Table 1: Statistic Analysis Types
Minimum
# of
sample
pairs
Maximum
# of
samples
per group
or class
Test
Replicate
Groups
Classes
Minimum
# of gels
per group
Student T
Yes
Yes
2
NA
N/A
MannWhitney
Yes
Yes
4
NA
20
Wilcoxon
No
Yes
6
6
25 (class
only)
Partial
Least
Squares
Yes
Yes
2
NA
NA
Student’s T-test
Student’s T-test is possibly the most popular of all statistical tests. The test compares
two mean values to determine whether the two groups from which the averages were
calculated could stem from a common population. The following is computed from
the observations of the two groups: the two sample sizes, the respective mean values,
and the respective standard deviations. Use the radio buttons under Significance to
determine the confidence level. Click Table to view a table listing degrees of freedom
and critical values.
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Mann-Whitney Signed Rank Test
The Mann-Whitney Signed Rank test uses non-parametric statistics which converts
all values to ranks and operates on the ranks. This type of test does not make
assumptions about the underlying statistical distribution as opposed to the Student’s
T-test which does make certain statistical assumptions about the data. Use the radio
buttons under Significance to determine the confidence level. Click Table to view a
table listing degrees of freedom and critical values.
Wilcoxon Paired-Sample Test
The Wilcoxon Paired-Sample Test is used to test the null hypothesis that the
population median of the paired differences of the two samples is zero. The
Wilcoxon paired-sample test is a nonparametric test that can be used if you don't want
to assume anything about the underlying statistical distribution. Because the
observations are paired, the Wilcoxon paired-sample test is more powerful than the
Mann-Whitney test.
The Wilcoxon test is only available if you are comparing classes. You must have at
least six samples per class. Follow these steps to create sample pairs in order to use
Wilcoxon test:
Step 1:
Create samples in sample database (See Section 6.6, Sample Database).
Step 2:
Create an attribute for these samples. Enter values for this attribute. Each
attribute-value pair defines a class. For example, an attribute could be
"treatment" with values of "treated" or "untreated". This defines two classes
"treatment=treated" and "treatment=untreated". See Section 6.6, Sample
Database, for further information on attributes.
Step 3:
For the Wilcoxon test, the two classes should have same number of samples and
there should be at least 6 samples in each class.
Step 4:
Pair the samples in the two classes. (See Section 6.6.c, Sample Pairs)
Step 5:
Once you have created your sample pairs, go to the Analysis Set Manager. Click
create and select Statistic from the Select Set Type dialog.
Step 6:
Choose to compare Classes and select the select the classes by clicking on the
drop down buttons for A and B.
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When you have finished pairing your samples, return to the Statistical Analysis Set
Template and choose the Wilcoxon Paired Sample Test. Use the radio buttons under
Significance to determine the confidence level. Click Table to view a table listing
degrees of freedom and critical values.
Partial Least Squares Test
The Partial Least Squares (PLS) test is similar to the Student’s T-test with the
exception that the PLS is a multivariate statistical discriminant analysis technique.
The Partial Least Squares test looks at all spots together whereas the other tests
included in the Statistical analysis set are univariate, which look at spots individually.
In addition to being multivariate as opposed to univariate, the Partial Least Squares
test includes other options for refining your test.
•
Prefilter noisy data - Select this option to pre-filter out some spots which seem to
be noise and are not expected to be significant in discriminating the two classes.
•
Iteratively remove insignificant spots - This will execute an iterative algorithm to
remove insignificant spots and with each iteration a new PLS model is built,
which typically converges after a few iterations (10 max).
•
Compute confidence values - This allows you to use statistical confidence values
(90%, 95%, etc.) rather than thresholds to determine significant spots. To
manually determine the threshold, clear this option. Then use the slider under
Significance to determine the threshold. The graph represents the spots in
increasing order of significance from left to right.
•
Scale spots to ignore size difference - This will standardize (normalize) spot
values for each spot by taking mean and standard deviation of the values across
all gels for a spot, and then subtracting the mean and dividing by the standard
deviation.
Class Separability depicts the likelihood that the classes being analyzed are
significantly different from each other on a scale of zero to one hundred.
When you are finished, click Save, and the new analysis set is added to the Analysis
Set Manager with significant protein spots highlighted in the MatchSet.
As with all other types of analysis sets, when the Analysis Set Template is open, you
can view the results in the MatchSet immediately. To the right of the dialog box, you
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can specify the spot marker, on which gel(s) to show the spots, and what information
to show on the gel(s).
When you open the Analysis set Template, whatever analysis sets you have selected
in the Analysis Set Manager become inactive. To view these sets in the Analysis set
Template, check the box labeled, “Display Selected sets.”
7.1.f
Arbitrary Analysis Sets
Arbitrary analysis sets are composed of any group of spots that you choose. To create
an arbitrary analysis set, select Arbitrary from the Select set type dialog.
Fig. 7-6. Arbitrary Analysis set
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No Spots
The No spots option creates an empty analysis set to which you can add any spots you
choose. To manually add spots to the analysis set, Click the Add spots to set button.
Click on a spot to add it to the set. The spot will be highlighted, indicating that it has
been added to the analysis set. To remove a spot, click the button labeled Remove
spots from set, then click a spot in the image to remove the spot.
All Spots in Member
The All spots in member option will create an analysis set containing all the spots in a
selected member that are matched to the Master. Click the drop down button to the
right of the member field to display a list of the members from which to choose. The
member you chose displays in the Member field. The analysis set will include all the
spots in the member you selected that are matched to the Master.
Saturated Spots
Saturated spots will create an analysis set of all spots that are saturated in any
member.
Importing Spots from External Analysis
PDQuest allows you import a set of spots from another MatchSet or a spot list
exported from some other analysis application.
Standard Spot (SSP) numbers are unique numbers that are automatically assigned to
each spot in the MatchSet standard when you create the standard. Selecting Import
list of SSPs from text file option opens the Select file to import dialog. Navigate to the
directory where the Standard Spot Numbers text file is located.
As with all other types of analysis sets, when the Analysis Set Template is open, you
can view the results in the MatchSet immediately. To the right of the dialog, you can
specify the spot marker, on which gel(s) to show the spots, and what information to
show on the gel(s).
When you open the Analysis set Template, whatever analysis sets you have selected
in the ASM become inactive. To view these sets in the Analysis set Template, check
the box labeled, “Display Selected sets.”
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7.1.g Boolean Analysis Sets
Boolean analysis sets are formed by combining two or more previously defined
analysis sets using Boolean operators. You can create Boolean sets that include:
1.
The intersection of two or more sets of spots (proteins present in all sets).
2.
The union of two or more analysis sets (all spots in all sets).
3.
Spots that are unique to set A plus spots that are unique to set B.
4.
Spots that are found only in set A.
5.
Spots that are found only in set B.
Note:
Boolean analysis of more than two analysis sets is not available for options 3, 4,
and 5.
Set A
Set B
Boolean
AND
Set A
Set B
Boolean
OR
Fig. 7-7. Venn diagrams depicting the AND and OR boolean operations.
To create a Boolean set, select Boolean from the Select Set Type dialog.
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Analysis Sets and Annotations
Fig. 7-8. Boolean Analysis Set
Enter the name, or accept the default name, then enter a description of the analysis
set. Note that while the name of the set is required, the description is not.
In the Compare section, add at least two analysis sets to use in your analysis. If you
choose more than two sets, then you will only be able to choose between spots present
in all sets or spots unique to all sets. All other choices will be inactive.
Select the Boolean operator in the Method section.
When you click Save, the new analysis set appears in the ASM and the spots that
meet the criteria that you specified will be highlighted.
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As with all other types of analysis sets, when the Analysis Set Template is open, you
can view the results in the MatchSet immediately. To the right of the dialog, you can
specify the spot marker, on which gel(s) to show the spots, and what information to
show on the gel(s).
When you open the Analysis set Template, whatever analysis sets you have selected
in the ASM become inactive. To view these sets in the Analysis set Template, check
the box labeled, “Display Selected sets.”
7.1.h Matching Analysis Sets
Matching Analysis Sets are composed of spots either unique to one member, or are
present in all members. To create a matching analysis set, select matching from the
Select Set Type dialog.
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Analysis Sets and Annotations
Fig. 7-9. Matching Analysis Set
There are two types of Matching analysis sets. The first option, Spots matched to
every member, finds spots that exist in all members of the MatchSet. For obvious
reasons, this option does not allow you to select a member. If you select Spots unique
to member, the Member field becomes available. Select the member from which you
wish to identify spots. This option will create an analysis set of all spots that are not
matched to any other member in the MatchSet.
Click Save. The new analysis set appears in the ASM and the spots that meet the
criteria that you specified will be highlighted.
As with all other types of analysis sets, when the Analysis Set Template is open, you
can view the results in the MatchSet immediately. To the right of the dialog, you can
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specify the spot marker, on which gel(s) to show the spots, and what information to
show on the gel(s).
When you open the Analysis set Template, whatever analysis sets you have selected
in the ASM become inactive. To view these sets in the Analysis set Template, check
the box labeled, “Display Selected sets.”
7.2 Annotation Tool
You can add notes, comments, and other pertinent information to each protein spot in
a MatchSet using annotations. Annotations can be used to describe spots, group them
into categories, and reference additional information in other files or Internet
databases. You can import MassLynx mass spec data into annotations. Spot
annotation data is automatically stored in HTML format for easy portability and
uploading to Web sites.
7.2.a Opening the Annotation Tool
Spots are annotated using the Annotation Tool. The tool can be opened by selecting a
spot or spots in the MatchSet, or you can open the tool with no spots selected.
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Analysis Sets and Annotations
Fig. 7-10. Annotation Tool.
To open the tool with spot(s) selected, select Annotation Select Tool from the
Analysis menu, and click on a spot in the MatchSet Master or drag a box around a
cluster of spots. You can also right-click on a spot in the Master and select Annotation
Tool from the dropdown menu. The Annotation Tool will open with the SSP
number(s) of the selected spot(s) highlighted.
Alternatively, select Annotation Tool from the Analysis menu or toolbar. The tool will
open with no spots selected.
Note:
If you are annotating a MatchSet for the first time, you will be prompted to add a
list of standard categories when you first open the tool.
Using the tool, you select a category for each annotation, select the spot(s) that you
want to annotate in that category, and then type the annotation entry. Note that the
workflow is flexible; e.g., you can select the spot(s) first, then select a category.
However, each annotation entry must be placed in a category and have at least one
associated spot.
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7.2.b Annotation Categories
Annotations are organized in categories. Some examples of annotation categories are
protein name, amino acid composition, binding properties, translational regulation,
etc.
You first select the category, then enter the annotation. A single spot may be
annotated in multiple categories, depending on the amount and type of information
you know about it.
Most categories contain simple text annotations. Specialized categories can be used to
link spots to Internet protein databases or open files in other applications.
PDQuest includes a number of standard categories. To add these to the Select
Category list, click the Std button below the list. The standard categories will appear
in the list.
Creating and Editing Categories
To create a new category, click the Add button below the Select Category list.
Fig. 7-11. Adding an URL category.
In the dialog box, enter a Name for the category and any Description you want to
include. If this category will contain textual annotations, the Text option button
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should be selected. You can also create specialized categories using the URL arg and
Filename buttons (category types are described below).
When you click Done, the category is created and will appear selected in the Select
Category list.
To edit an existing category, select the category in the list and click the Edit button.
You can then change the category type and description. Note that the annotation entry
may be specific to a particular category type; changing the type may render the entry
meaningless.
To delete a category, select it, then click Delete. You will be warned before the
category and all its entries are deleted.
URL Categories
This type of category provides a link to an Internet protein database or other Web site;
then the annotation entry references the specific file or Web page for a particular
protein. You can create different URL categories to link to different databases,
including Swissprot, Unigene, and GeneCards.
To create a URL category, open the Add Category dialog box, enter a name for the
category, then select the URL arg option button and type a name for the database or
Web page in the Link Name field. This is the name that will appear in the Browse
Type the entire link address in the Link URL field including http://, any arguments,
with a %s where the individual spot annotations will go. For example, Swissprot is
http://www.expasy.ch/cgi-bin/sprot-search-de?%s.
When you are finished creating the category, click Done. When you create an
annotation entry for a spot in this category, it should be the exact format necessary to
complete the URL link to the page in the database for the specific protein information
you are referencing.
To open a URL link, right-click on the spot and select Browse Annotations. The
HTML page for the spot will open, and the URL category name will be displayed as a
link. Click on this link to access the protein information in the database or Web site.
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File Name Categories
This type of category provides a link between spots and files on the user’s computer,
network, or removable disk drive. For example, you can open a Protein Probe file
linked to a spot from within PDQuest.
To create a file name category, open the Add Category dialog box, enter a name for
the category (e.g., Protein Probe), then select the Filename option button. In the File
Extension field, type the extension of the file type you want to link (e.g., Protein
Probe files have the extension "prp"). Use the Browse button next to the Files in
Folder field to select the directory where the files in this category located.
Fig. 7-12. Editing a text category.
When you are finished, click Done. In the annotation entry for a spot in this category,
enter the exact file name you want to associate with the spot. This file must be located
in the directory you specified in the Add Category dialog.
To open the file linked to a spot, right-click on the spot in the MatchSet and select
Find File For. In the pop-up box, select the appropriate category name. This will
launch the application associated with that file and open the file.
You can associate different files with the same spot, but they must be linked using
different file name categories.
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Analysis Sets and Annotations
Protein Name Category
You can create a special category for identifying spots by their protein names instead
of by SSP numbers in the Annotation Tool.
Open the Add Category dialog box and type "protname" in the Name field. Click
Done to create the category. In the annotation entry for a spot in this category, type the
name of the protein for that spot.
When you select the Sort: Name option under the Select Spot(s) list in the Annotation
Tool, the spots will be listed alphabetically by their protein names.
7.2.c Selecting a Category
After you have created a category, you can select it. Click on the category name in the
Select Category list. Click the Show Description checkbox to display the description
of each category in the list.
All the spots that have been annotated in a selected category will be
marked in the Select Spot(s) list.
7.2.d Selecting Spot(s)
In the Select Spot(s) list, click on a spot SSP number to select it, or SHFT-CLK or
CTRL-CLK -click to select multiple spots that will have the same annotation entry.
Alternatively, click the Select Spots in Image button and click on a spot in the
MatchSet Master to select it. To select multiple spots with the Select Spots tool, ctrlclick or drag on the spots in the Master.
Selected spots are highlighted in the list. You can sort the spot list by SSP number or
Name. Names of spots are specified using a special category called protname, which
you either create or add as a standard category. You type the protein name of a spot as
an annotation entry in this category, and you can sort by it using the Name sort option.
Auto-select Same Entry will automatically select spots in the list that have the same
entry in a specified category. With the checkbox and category selected, click on a spot
in the list or the MatchSet; the other spots with the same entry will be selected too.
All categories containing annotations for the selected spot will be marked in the
Select Category list. If multiple spots are selected, no categories will be marked.
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7.2.e Entering an Annotation
After you have selected an annotation category and a spot or multiple spots, type the
annotation in the Annotation Entry field.
If you are entering an annotation in a URL category, it should be the exact format
necessary to complete the URL link to the page in the database for the specific protein
information you are referencing.
If you are entering an annotation in a file name category, the entry should be the exact
file name you want to link to the spot.
If you are entering an annotation in the protname category, the entry should be the
protein name you want to associate with the spot.
To apply an annotation entry, click the Apply Entry button. The entry will be
automatically applied if you select another spot or category to annotate.
To delete an annotation entry, select the entry text and press the Backspace or Delete
key. Then click Apply Entry or select another spot to apply the deletion.
7.2.f
Importing Annotations
The Import Annotations tool allows you to import annotations from a text file. This is
useful for adding a large number of the same annotations to multiple MatchSets. The
import annotations tool is fairly flexible with the exception that the first column must
contain the SSP numbers.
To import annotations, click Analyze>More Annot Tools>Import Annotations... This
opens the Import Annotations dialog.
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Analysis Sets and Annotations
Fig. 7-13. Import Annotations Tool
Select a file to import by clicking the folder icon to the right of the field. The next two
fields are informational only. If you have the First line is Header checked, then the
Header row appears in the Header line field. The first data line shows your first line of
data (2nd line of text in your file if you have first line is header selected). If your text
file is tab delimited (columns separated by tabs) the tabs appear as boxes between the
data.
Confirm Overwrites - If any annotations will change when you click import, you
will get a warning message asking you to confirm the change.
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Fig. 7-14. Confirm Overwrite Dialog
If you do not want to overwrite what is there already, click no and the import will skip
that annotation and continue. If you click yes, any data will be overwritten.
If the Confirm overwrites box is unchecked, changes are made to existing information
without a warning.
Column delimiter - This option determines how columns are separated in your text
file. Currently your only options are comma and tab. Your text file must use one of
these two delimiters in order to be imported.
Relate Column to Category - This field shows a list of columns in your text file.
Whatever is in the first line of your file appears in parentheses next to the column
number. To relate a column to a category, highlight the column in the Relate column
to category field and click the down arrow to select a category. To add a new category,
click the Add button. (See spot Annotation Category Editor for more information.)
Any column that does not have a category relationship will not be imported. If the
Show description box is checked, the category description appears beside the
category name. Clear this box if you do not need to see the category description.
Save Settings - The save settings button allows you to save the import settings of the
Import annotations dialog. The save settings option saves First line header and
Confirm overwrite selections, the delimiter type, and the column to category
relationship for each column. Saving the import annotation settings is useful tool
especially if you plan on adding many annotations from different files. The Save
Settings option saves your settings in a simple text file.
Load Settings - To load a saved settings file, click the Load Settings button and
locate the settings file you wish to load. Click OK.
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Analysis Sets and Annotations
Click Import when you are ready to import your annotations. A progress box appears
indicating the number of additions and changes that have been made.
Fig. 7-15. Changes made indicator
Click OK.
7.2.g Reviewing Annotations in the MatchSet
The Image Options in the Annotation Tool allow you to highlighted selected spots in
the MatchSet Master. You can also display actual annotations on the Master image.
Note:
When reviewing annotations in the MatchSet, it is useful to shrink the annotation
tool window. Click the arrow button in the lower left-hand corner of the
Annotation Tool to collapse the tool; only the reviewing functions will be
displayed.
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Click here to expand tool.
Fig. 7-16. Annotation Tool in reviewing mode.
Highlight Target Spot will highlight the spot(s) that are selected in the Annotation
Tool. The spots will be marked with
Mark All Annotated Spots will mark all annotated spots in the MatchSet. Click the
Format button to select the style and color of the markings.
Mark Spots in Category will mark the spots that are annotated in the selected
category. Click the Format button to select the style and color of the markings.
Show Spot Annotations will display all the annotations in the selected category next
to the annotated spots in the MatchSet. Click the Format button to select the style and
size of the annotations.
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Analysis Sets and Annotations
7.3 Browsing Annotations
You can click on an annotated spot in PDQuest and open an HTML page displaying
all the annotation information for the spot. You must have a Web browser installed on
your computer to use this command.
Right-click on an annotated spot in the MatchSet Master and select Browse
Annotations, or select the command from the Analysis > More Annot Tools
submenu or Analysis toolbar and click on a spot in the Master. The default
browser will open and display a page containing all the annotation information for the
spot, listed by category. The MrpI (if calculated) and protein name (if entered) of the
spot are listed at the top of the page.
Fig. 7-17. Example of the annotation browser.
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If you have created an annotation link for the spot in a URL category (see section
7.2.b, Annotation Categories), that link will appear in the page under the Link
column. Click on this link to open the database or Web site specified by the category
and link to the protein information.
Use the Print command in the browser window to print the annotation information.
The displayed page is a temporary file that can be saved by using the browser’s Save
As command. You can then include saved pages on your Web site or e-mail them to
colleagues.
7.4 Creating Annotations from Analysis Sets
Analysis sets are used to group spots in a MatchSet that are statistically and
scientifically meaningful. After you have created an analysis set, you can create an
annotation describing the spots in the set.
Note:
You can create annotations for individual spots or other groups of spots using the
Annotation Tool.
With the MatchSet open, select Create Annotation from Set from the Analyze > More
Annot Tools submenu. The dialog box will open.
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Analysis Sets and Annotations
Fig. 7-18. Create Annotation from Analysis Set dialog box.
In Step 1, select the analysis set from the pulldown menu. The number of spots in the
set will be displayed in the Spot Count field.
In Step 2, select an existing annotation category from the pulldown list, or create a
new one by clicking on the Create Category button. See Annotation Categories for
more information on creating categories. The initial spot count in the category will be
displayed in the Spot Count field.
In Step 3, enter the annotation for the spots in the set. Note that this annotation will be
applied to all the spots in the analysis set.
If you are creating an annotation in a specialized category (i.e., a URL or file name
category), make sure the entry is in the appropriate format.
When you have entered the annotation, click Add. An alert box will notify you how
many spots were annotated in the category. If you are editing a category entry or want
to add the entry to spots that were added to the set, click Update. If any spots were
added to or removed from the analysis set, clicking replace will remove the entry
from the original spots and create new entries for the new list of spots in the set.
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7.5 Printing Annotations
The functions for printing spot annotations are located on the Print Annotations
submenu of the Analysis > More Annot Tools submenu. You can also print
annotations from the Browse Annotations page.
Fig. 7-19. Print Annotations submenu.
Using the commands in this submenu, you can print annotations for individual spots,
the protein spots in an analysis set, or all the spots in the MatchSet Master. You can
also print annotations for particular categories or all categories.
Printing Annotations for Individual Spots
To print the annotations for a particular spot, select One Spot, Specify Categ’s or One
Spot, All Categ’s, then click on the spot in the Master. Note that Browse Annotations
will also print out all the annotations for a particular spot.
If you select One Spot, Specify Categ’s, a pop-up list will prompt you to select
annotation categories to print. Once you have made your selections, click Done.
Printing Annotations for Analysis Sets
To print annotations for spots in a specific analysis set, select Analysis Set, Specify
Categ’s or Analysis Set, All Categ’s from the Print Annotations submenu. A list of
analysis sets will be displayed. Click on the name of the set whose annotations you
want to print.
If you selected Analysis Set, Specify Categ’s, a pop-up list of categories will appear.
Make your selection(s), then click Done.
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Analysis Sets and Annotations
Printing Annotations for All Spots
All Spots, Specify Categ’s prints selected annotation categories for all the annotated
spots.
All Spots, All Categ’s prints the annotations in all available categories for all
annotated spots.
7.6 Transferring Annotations
If a member of a higher level MatchSet includes annotations, you can transfer all or
some of them to another higher level MatchSet member or to the higher level
MatchSet Master.
Note:
Only annotations for spots that are matched in the higher level MatchSet will be
transferred. If no spots are matched between the members, only categories will be
transferred.
With the higher level MatchSet open, select Transfer Annotations from the Analysis >
More Annot Tools submenu.
In the dialog box, select the member MatchSet that you want to transfer from using
the From MatchSet pulldown list.
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Fig. 7-20. Transferring annotations.
Select the member MatchSet that you want to transfer to using the To MatchSet
pulldown list. Note that when you select the second MatchSet, the total spots in each
MatchSet will be listed, as well as the number of spots that have been matched
between the MatchSets (in the higher level MatchSet).
Next, select whether you want to transfer annotations in All Categories or just one
category. If you select Specified Category, select a category from the pulldown list.
If the two MatchSets share categories, selecting Replace Categories will replace each
duplicate category and all its entries in the "to" MatchSet. Replace Entries will
replace only the entries for shared spots in the "to" MatchSet. If neither checkbox is
selected, the transfer will only add new entries; shared entries and categories will not
be replaced.
Selecting Confirm Before Replacing will prompt you before each category/entry is
replaced.
Click Done to complete the transfer. An alert box will list how many categories and
entries were transferred. You can review the transferred annotations using the
Annotation Tool.
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8.
Basic Excision Tool
The Basic Excision Tool is used to control Bio-Rad’s ProteomeWorks™ spot cutter
and ProteomeWorks™ Plus spot cutter system for basic spot cutting operations. If
you are cutting spots from MatchSets for mass spec analysis, use the Integrated
Excision Tool.
Before using this tool, make sure the spot cutter is connected with the computer, is
switched on and has warmed up, and make sure the camera is connected and has been
turned on. Alternately, if you are not connected to a spot cutter you will be prompted
to use a simulated cutter/camera. Answering Yes to the prompt also opens the Basic
Excision dialog.
Note:
See the spot cutter hardware manual for instructions on installing the platform,
camera, and associated hardware and software.
Select Basic Excision Tool from the Identification menu. The software will connect
with the spot cutter platform and camera, and the Basic Excision Tool dialog box will
open.
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PDQuest User Guide
Fig. 8-1. Basic Excision Tool dialog.
The Basic Excision Tool includes a window for viewing the spot cutter camera image
and a control panel for running the spot cutter. The Setup buttons are in the lower left
hand corner of the dialog.
8.1
Spot Cutter Setup
Before you can use the spot cutter for the first time, you must focus the camera and
adjust the cutting tip height. You must also adjust the spot cutter settings and calibrate
the instrument.
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Basic Excision Tool
Calibrate cutter
spot cutter setting
Move head to lower left
Focus cutter camera
Set cutting tip height
Move head to upper right
Fig. 8-2. spot cutter setup
8.1.a
Note:
Focus Cutter Camera
If you are using a lens filter, make sure that it is installed before focusing. If you
add or remove the lens filter after focusing, you will need to refocus.
The Focus Cutter Camera dialog box controls the camera exposure setting. Focusing
and aligning the camera are described in the ProteomeWorks spot cutter hardware
manual.
Click the Focus Cutter Camera button to open the dialog. The platform light
on the spot cutter will turn on and the camera will focus on a small region at
the center of the gel or membrane and begin taking continuous exposures.
The exposures will appear in the Focus window at the rate of about 2 per second.
At the top of the dialog, click Auto-Expose to reset the exposure time of the camera.
The Time field shows the time of each exposure in milliseconds. The Ceiling field
shows the highest pixel value in the focused region. The Levels field shows the levels
of gray scale in the image. All of these settings are automatically determined by the
software, based on the position and aperture of the camera lens.
Manually adjust the camera lens and position as described in the hardware manual to
optimize the image. When you are satisfied with the image and the levels of gray
scale, click Stop to close the Focus dialog.
8.1.b
Set Cutting Tip Height
Click the Set Cutting Tip Height button to adjust the distance the cutting tip
drops down to cut a spot. The adjustment dialog box will open, and the
cutting tip will move to the center of the gel.
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Click on Lower Tip to drop the cutting tip to its lowest position. In this position,
manually adjust the cutting tip height as described in the hardware manual until it
presses lightly against the surface of the cutting mat.
Raise Tip raises the cutting tip. Test Cut performs a test cut in the center of the gel.
Click Done to accept the settings and return the cutting tip to home position.
The cutting tip height only needs to be set once, unless the spot cutter gets out of
alignment.
8.1.c
Spot Cutter Settings
Click the spot cutter Settings button to open the settings dialog box for the
cutter.
Fig. 8-3. spot cutter settings.
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Basic Excision Tool
Cutter Model
Select the spot cutter model you will be accessing for your cut run. This setting most
likely will not change unless you have regular access to both types of spot cutters.
There are slight differences between the two types. For instance, because the
ProteomeWorks Plus spot cutter has a larger platform and cutting tip range, it is not
necessary to crop the left margin. The ProteomeWorks Plus spot cutter also allows
you to cut backed gels. See “Step 3. Set Cutter Options” on page 8-17.
Microtiter Plate
Specify the microtiter plate size you are using by clicking on 96 wells or 384 wells.
Specify the order in which you want to the load the wells on the microplate by
selecting A1, A2, A3, … or A1, B1, C1, … This function is useful for matching a
partial plate to the load order of a digest robot.
Well A1 position
The fields and buttons in this section are used to calibrate the position of the
microplate for proper loading of samples. The number fields contain the X and Y
coordinates (in millimeters) of well A1 on a plate in the spot cutter plate holder. Once
you have set the position of this well, the cutter robot will automatically calculate the
positions of the remaining wells.
To set the well A1 position, position your cursor in the number in the X or Y
coordinate field, click to place the cursor in the number field, and press Enter on your
keyboard. The tip will move to that position without attempting to lower into the well.
If the tip is not centered over the well A1 on the microplate, you will need to adjust
the numbers in each field.
You can either type a new value directly in each field, or (for small adjustments) use
the arrow buttons to adjust the tip by fractions of a millimeter. The cutting tip will
move after you press Enter or when you click the arrow buttons.
Note that the arrow buttons are oriented as if you were facing the front of the spot
cutter; also, if you are facing the cutter, higher numbers in the first (X) field will move
the cutting tip to the right, while higher numbers in the second (Y) field will move the
cutting tip toward the back of the instrument.
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When you are satisfied with the position of the cutting tip, click Test to lower the tip
into the well. The tip should be centered inside the well.
Click Park to return the tip to its home position.
Wash Station 1 position
Calibrate the position of Wash Station 1 on the cutting platform by setting the cursor
in one of the Wash station fields and pressing enter. Then follow the steps described
above for Well A1. Once you have set the position of station 1, the cutter robot will
automatically calculate the positions of the remaining wash stations.
Miscellaneous Settings
Specify the diameter of the cutting tip by selecting 1.0 mm or 1.5 mm. Note that the
cut request circles that you make on the image reflect the approximate size of the
cutting tip, and will change based on the tip size selection.
If you are using the ProteomeWorks spot cutter, you will need to crop the left margin
of your image. Crop Left Margin repositions the image in the Excision Tool window
to the left. Enter a value (in screen pixels) in the field to reposition the image. Note
that you must click Done to close the settings dialog and then acquire a new image for
this change to take effect.
Note that cropping the left margin does not change the raw image, only the portion of
the displayed image. It is important to crop so that you can see the right edge of the
gel holding area of the platform in order to maximize the area available for cutting.
Serial Port
In the dialog box, select the communications port to the spot cutter by clicking on
COM 1 or COM 2.
8.1.d
Spot Cutter Calibration
Before you can begin excising spots, you must calibrate the spot cutter. This
procedure requires a coomassie R-250 stained PVDF membrane.
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Basic Excision Tool
Note:
Before calibration, you should focus the cutter camera and align the wash station
position. You should make sure the spot cutter camera image is properly centered
using the Crop Left Margin setting.
Note:
If you are using the lens filter for UV light, make sure that it is installed BEFORE
calibration. If you add or remove the lens filter after calibration, you must
recalibrate.
Click the Calibrate Cutter button. A series of dialog boxes will guide you
through the calibration procedure. The first dialog box will prompt you to
specify whether you have installed the lens filter or not. To use in visible
mode only, calibrate with the lens filter not installed. The lens filter must be
INSTALLED if you are using the fluorescent spot cutter in fluorescent and visible
modes. The lens filter is not required if you are using the spot cutter only in visible
light mode.
Step 1:
The second dialog box prompt for flat field calibration requires that you ensure
the stage is completely empty and dry. This includes the cutting mat, which must
be removed. Press Ready to proceed. If you have previously performed this step
you can skip to Step 2, although this is not recommended.
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PDQuest User Guide
Fig. 8-4. spot cutter calibration step 2.
Step 2:
The third dialog requires the calibration template grid pattern supplied with the
PDQuest system. Place the grid on top of the cutting mat, which should have
been previously installed on the cutter. Make sure the grid is flat and aligned with
the stage edges. Click Ready to proceed. You may also skip this step, return to the
previous dialog, cancel the action, or ask for help.
Step 3:
The fourth dialog prompts you to place the coomassie stained PVDF membrane
on the mat. Click Yes or No.
At any point in the procedure you can move back to repeat a step.
Note:
Calibration creates several parameter and image files in the Install.prm directory
in the Discovery Series directory on your hard drive. These files should not be
moved, deleted or altered, or calibration will be affected.
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Basic Excision Tool
When calibration is complete, you can proceed with normal use of the spot cutter.
Once the cutter is calibrated, you do not need to repeat the procedure during normal
day-to-day use. You must recalibrate if:
•
The instrument is moved.
•
You install or remove the lens filter after calibration.
8.2
Cutting Spots
After you have focused the camera and calibrated the spot cutter, you are ready to
begin cutting spots. The Basic Excision tool control panel is designed to guide you
through the procedure.
1.
Acquire Image. In this step, you capture an image of your gel or membrane using
the spot cutter camera.
2.
Specify Cuts. In this step, you identify the spots in the image that you want to cut.
3.
Select the Cut Run Options. These options can be adjusted for each cut run.
4.
Set Wash Options. Select the wash stations to use after each cut by clicking on
one or more checkboxes.
8.2.a
Step 1. Acquiring the Image
Before acquiring an image, select the light option appropriate for your gel/membrane/
stain type:
•
Light on (gels).
•
Light off (PVDF membranes). Ambient light will produce a better image for
PVDF membranes.
•
UV on (fluorescent gels). UV light is only available for the Fluorescent spot
cutters.
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PDQuest User Guide
Fig. 8-5. Light settings.
If UV light is selected, specify an exposure time in seconds in the field. Also, make
sure that the UV lamp switch on the back of the cutter box is on.
Note:
For Sypro Ruby stains, an exposure time of 20-30 seconds is recommended.
When you have selected a light option, click Acquire Image.
Note:
For UV exposures, there will be a 15-second delay while the lamps heat up
before the exposure begins.
The camera will take a single image of the gel or membrane on the platform, then a
background image to adjust for image background. When the exposure is complete,
the image will appear in the window.
To adjust the brightness and contrast of the image, open the Transform
window by clicking on the button to the right of the image window.
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Basic Excision Tool
Transform window
Coomas color selected
Main window in Basic excision
tool is transformed after spots
have been selected for cutting
Fig. 8-6. Transform window with Coomas color selected.
The colored box displayed on the image shows the boundary of the cutting area. The
cutting head cannot cut spots outside this area. To select another spot to cut, position
your cursor in the center of the colored box and drag it over to the new spot.
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Spots selected outside the cutting area cannot be cut. Either remove the cut, or locate
it inside the cutting area.
8.2.b
Step 2. Specifying Cuts
In this step, you click on spots in the image to identify which spots to cut.
Use the Zoom Box tool on the right of the Basic Excision Tool to magnify each spot.
When clicking on spots in the image, select the Snap to Peak checkbox to
automatically center the cut circle on the spot peak (calculated from the spot intensity
in the image).
To make a single cut in a spot, make sure the Multiple Cuts/Spot checkbox
is unchecked, then click the Add Cut button and click on the spot in the
image that you want to cut.
Fig. 8-7. Adding cuts.
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Basic Excision Tool
If this is the first cut added, the Enter Plate Information dialog box will open first.
when all the pertinent information is added, the cut will be added to the image.
Fig. 8-8. Enter Plate Information dialog box.
Enter a name for the microplate. If you have a barcode scanner attached, and there is a
barcode on the plate, scan it in before proceeding. The Loading order is determined
by the spot cutter settings. Identify the first well you want to load. If you specify a
starting well other than the default of A1, the skipped wells will be noted in the dialog
box. Click Done when you have completed your selections.
A green circle will appear on the image, indicating the spot to be cut. Note that the
circle is the size of the actual cutting tip (1.0 mm or 1.5 mm, as specified in the spot
cutter Settings dialog box).
Continue clicking on spots in the image with Add Cut selected to make additional
cuts. The number of cuts will appear in the Cuts Selected field.
If you specify more cuts than there are wells in the microplate, you will be prompted
to name a new plate and starting well for subsequent cuts.
Moving a Cut Circle
To move a cut circle, make sure that Add Cut is selected, then position your cursor
inside the cut circle. The appearance of the cursor will change to a multidirectional
arrow. Drag the circle to a new location and release the mouse button.
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PDQuest User Guide
Multiple Cuts of the Same Spot
You can make multiple cuts of the same spot. The cuts can be placed in separate
microtiter plate wells or in the same well (to increase the volume of sample in that
well).
To place multiple cuts of the same spot in different wells, make sure the Multiple
Cuts/Spot checkbox is UNCHECKED, then click on several locations inside the spot
to create individual cuts of the same spot. Drag these circles so that they touch but do
not overlap.
Fig. 8-9. Multiple cuts to be placed in different wells.
Note:
Overlapping circles will result in incomplete cuts that may not be fully extracted
by the spot cutter.
To place multiple cuts of the same spot in the same microtiter plate well, select the
Multiple Cuts/Spot checkbox, and enter the maximum number of cuts per spot in the
Max Cuts/Spot field.
Click the Add Cut button, then drag a box over the spot. Multiple cutting circles will
appear grouped inside a red box. All these cuts will be placed in the same well.
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Basic Excision Tool
Fig. 8-10. Multiple cuts to be placed in the same well.
Note:
If the box you create contains too few cutting circles, simply drag over the area
again to create a bigger box.
To move a group of multiple cuts, with Add Cut selected, position your cursor inside
the center circle in the box. The appearance of the cursor will change to a
multidirectional arrow. Drag the box to a new location and release the mouse button.
Importing a Cut List
If you have a cut list saved to a text file, you can import it into the Basic Excision
Tool. This is useful if you have a large number of cuts for similar gels.
Note:
You can only import a cut list to an image that does not have any cuts added.
However, after you import cuts, you can add additional cuts to the image.
Click Import to open the Import Excision Coordinate List dialog box.
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PDQuest User Guide
Fig. 8-11. Importing a cut list
If the first line of your file is header information, select Skip first line of file. Next
enter which column the coordinates are located in the file. For example, if your file
lists the spot number in column one, the X coordinates in column two and Y in three,
then you would enter two for column X and three for column Y. Columns must be
separated by tabs or by columns.
Click the browse button to locate the file you want to import.
Units indicates the measurements of the coordinates, and Origin determines starting
point for the measurements.
The image in the Basic Excision Tool displays the placement of the spots as orange
circles before you complete the import. If the cuts are out of position, you can click
and drag all cuts at once to better position them. If you need to move cuts
individually, you can do so after import by clicking Add cut, then clicking and
dragging the cut you want to move.
When you are satisfied with the placement of the cuts, click Done to complete the
import.
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Basic Excision Tool
Removing Cuts
To remove a cut circle from the image, position your cursor on the spot and rightclick. You will be prompted to confirm the deletion. To remove multiple cut circles,
click on the Remove Cut button, then click on the cut circles, or drag a box around the
circles.
Display Options
Click the Well Number check box (below the Basic Excision image window) to
display the well number associated with each cut request.
Click the Cut Position checkbox to display the position of the cut (in millimeters) on
the gel.
8.2.c
Step 3. Set Cutter Options
Before beginning a cut run using the Basic Excision Tool, you must select the cutter
options for that run.
Select Spin When Cutting to make the cutting tip spin as it cuts. This is recommended
for all cutting, including gels and blots. Deselect this option if you want to test X,Y
positioning without cutting.
Spin When Ejecting will cause the cutting tip to spin as it ejects the cut into a
microplate well. This is only recommended for PVDF or nitrocellulose blots.
Cut Longer will cause the cutting tip to hold in the down position longer when
making a cut.
Double Eject will cause the eject pin in the cutting tip to eject twice into a microtiter
plate well. This is useful for membrane blots if the membrane seems to be sticking to
the cutting tip.
No spin when lifting stops the tip from spinning after the cut is made and the tip is
lifted from the gel. This option may be useful for some gel or blot applications.
Check the Backed gel setting if you are using gels that have a backing on them.
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The Tip motion range determines the side-to-side motion in millimeters of the tip as it
is cutting backed gels. The default range is 2.0, while the maximum range is 5.0 mm.
The user should determine the optimum setting for their gel.
Note:
The backed gel option is recommended for the 1.0 mm cutting tip only. Use of
the 1.5 mm cutting tip may result in decreased gel pickup efficiency.
The Gel Defaults button automatically selects the most useful options when cutting
gels. The PVDF Defaults button automatically selects the most useful options when
cutting PVDF membranes.
8.2.d
Step 4. Set Wash Options
Select the wash stations to use after each cut by clicking on one or more checkboxes.
8.2.e
Performing a Cut Run
After you have identified all the cuts you want to make and selected the appropriate
options, you are ready to begin the cut run. If you are using a fluorescent spot cutter,
close the door of the enclosure before proceeding.
Click the Begin/Resume button. You will be prompted to place a microtiter plate in
the plate holder.
As each cut is being made, it will be highlighted in yellow on the screen. After each
cut has been made, the cut circle appears in white on the screen.Cuts that have not yet
been made are circled in green.
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Basic Excision Tool
Fig. 8-12. Cuts being made.
Confirming Cuts
For visible spots, inspect the gel to confirm that each cut is being made correctly.
For fluorescent spots, you must take a picture of the gel using UV light to ensure that
cuts are being made correctly. After one or more cuts have been made, click Pause to
pause the cut run. Note that the cutter will pause only after the most recent cut has
been deposited in a well.
Confirm the pause, then click the Confirm Cuts button under the main acquisition
window.
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PDQuest User Guide
Cut 1,A01 has been
missed.
Cut 1,A02 was made
correctly.
Fig. 8-13. Confirmation image; note that the first cut made incorrectly.
When you have confirmed that the cuts are being made in the correct locations, click
Begin/Resume to proceed with the rest of the cuts. Otherwise, you can abort the run.
Ending the Cut Run
Click Pause to pause the cut run. You will be prompted to confirm the pause. Note
that the cutter will pause only after the most recent cut has been deposited in a well.
Any remaining cut requests will remain selected in the image. At this point, you can
click Begin/Resume to complete the remainder of the cuts, or abort the run.
When all cuts are complete, the cutting tip will return to home position and you will
receive an alert message on the screen. At this point, you can visually inspect the cut
holes for visible spots, or take a UV image of the MTP and confirm the cuts.
8.2.f
Recutting Spots
You can specify re-cuts by clicking the Recuts button below the image window, or
when using the UV mode, follow the on-screen directions for imaging the MTP.
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Basic Excision Tool
Fig. 8-14. Specify the wells to re-cut.
In the pop-up box, enter the numbers of your recut requests (microplate number, well
number), separated by semicolons. Click OK to close the recut request dialog, then
click Begin/Resume to perform the recuts.
Re-cuts in Basic Excision Tool
The specification for the ProteomeWorks spot cutter spot pick-up is greater than 95%
effective at picking up a spot that has been cut from a gel. There may be some spots
that have not been picked up and these can be re-cut. The efficiency of pick up on the
second cut is again >95%, so the spot is reliably picked up the second time. The
missed pick ups are detected by the user looking in the microtiter plate for empty
wells. However, with fluorescent gels, the clear gel spot (with no visible stain) is very
difficult to see in the microtiter plate wells. In order to assess the wells of the
microtiter plate, the re-cut message on the screen for the fluorescent light source will
differ from the white light message. Use the following procedure for re-cutting spots
in fluorescent mode:
When UV-Fluorescent imaging has been used on the spot cutter, at the end of the cut
run the software will prompt with, "Confirm cuts?"
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PDQuest User Guide
Fig. 8-15. Confirm cuts window.
Step 1:
Answer "Yes" to bring up the following on-screen instructions for imaging the
microtiter plate in UV mode:
Fig. 8-16. Image Microtiter plate window.
Step 2:
Do not move the gel or membrane on the cutting platform
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Basic Excision Tool
Step 3:
Place a clean plastic Gel Sheet on top of the gel
Step 4:
Place the microtiter plate on top of the plastic sheet. Note the orientation so that
you can recognize the A1 location.
Step 5:
Close the Enclosure door
Step 6:
Click on "Acquire image" when the plate is in place. The UV lights will turn on
and the camera will acquire an image with the same exposure time as the first
spot cutter image.
Step 7:
Assess the image on the screen for the wells that need to be recut.
Fig. 8-17. Microtiter plate with fluorescent spots in wells.
Use the Zoom and transform functions on the right side of the "Image Microtiter
Plate" window to get a better view of the microtiter plate wells.
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PDQuest User Guide
Fig. 8-18. Microtiter plate after zoom and transform functions applied.
Step 8:
If any of the wells are empty and you want to re-cut those spots, click on
"Specify re-cuts." Then follow these on-screen instructions:
Fig. 8-19. Message window before recuts are started.
Step 9:
Return the microtiter plate to the plate holder.
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Basic Excision Tool
Step 10: Remove the plastic sheet from the top of the gel.
Step 11: Do not move the gel. The image and spot selections that were already made still
apply to the gel as long as it does not move.
Step 12: After removing the microtiter plate, click "OK" on the message. The "Image
Microtiter Plate" window will close and an image of the microtiter plate will
open in front of the Basic Excision Tool screen. If you need to resize the window,
close the recuts box, resize the window, make any zoom or transform changes
using the icons on the top bar above the window, then click on the "Recuts" icon
to bring the recuts box back up.
Fig. 8-20. Basic Excision Tool fluorescent mode re-cuts.
Step 13: Enter the well numbers of the missed pick-up spots in the "Re-cuts" box on the
screen.
Step 14: Click on "Begin/Resume" after the selections are made.
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8.2.g
Saving the Image
To save the image taken by the spot cutter, click the Save button in the Basic Excision
Tool window. You will be prompted to enter a name and specify a location for the
image before saving.
8.3
Other Spot Cutter Controls
Click the Move Head buttons (under Setup in the Basic Excision Tool
window) to move the cutting tip to the front left corner of the spot
cutter or the back right corner.
You can simulate the action of the cutter and its camera by clicking on the Simulate
Cutter and Simulate Camera checkboxes. These checkboxes will cut off
communication with the cutter and camera if selected. In simulation mode, you will
be prompted to open a file image of a gel in place of the camera cutter image.
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9.
Integrated Excision Tool
The Integrated Excision Tool allows you to cut spots that have been matched in a
MatchSet and placed in an analysis set (see section 7.1, Analysis Sets). First, select
the gels from the MatchSet in the Excision Gel Selection (EGS) Tool that you want to
cut from; then use the Integrated Excision tool to perform the cuts.
PDQuest tries to locate the cutter. If you are not connected to a cutter you will be
prompted to use a simulation.
Fig. 9-1. Fluorescent spot cutter.
Finally, you can export the completed (i.e. after cuts are done) cut list to Micromass’s
MassLynx software for mass spectrometry analysis (see the next chapter).
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9.1
Excision Gel Selection
The Excision Gel Selection tool is used to select the gel(s) in a MatchSet from which
to cut spots using Bio-Rad’s ProteomeWorks™ spot cutter and the ProteomeWorks™
Plus spot cutter systems.
First, you must create an analysis set of spots (see section 7.1, Analysis Sets) in the
MatchSet that you want to cut. Then you open the Excision gel Selection tool, select
that analysis set, and identify which gels you want to cut from. This creates a cut list
of spots and gels. Finally, you use the Integrated Excision Tool to perform the cuts.
Note:
You must open the MatchSet containing the gels whose spots you want to cut and
create an analysis set out of those spots before you can open the Excision Gel
Selection tool.
With your MatchSet open, select Excision Gel Selection from the Identification
menu. The Excision Gel Selection tool will open, and you will be prompted to select
from the list of available analysis sets in the MatchSet.
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Integrated Excision Tool
Fig. 9-2. Excision Gel Selection.
9.1.a
Step 1. Select Analysis Set
Select the analysis set you want to cut from the pop-up box. The Excision Gel
Selection dialog will display a table of the spots in the analysis set and their quantities
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PDQuest User Guide
in the different gels in the MatchSet. The first spot in the set is magnified and
highlighted in each of the gels of the MatchSet.
To select a different analysis set, click the pull-down button next to the analysis set
name and select from the list. The number of spots in the set is displayed next to the
name.
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Integrated Excision Tool
Fig. 9-3. Excision Gel selection, Step 2.
9.1.b
Step 2. Set Selection Mode
In this step, you can select parameters for automatically selecting which gels to cut.
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Auto-select
Select the Auto-select option to automatically select the best gels to cut from based on
spot quantity and quality. When you choose this option, the auto-selection parameters
will become available, and the gels will be selected in the table based on these
parameters.
Spot quantity usually varies among the gels in a MatchSet. In general, you want to cut
from gels in which spot quantity is higher (to get more sample). The Tolerance ratio is
used to determine the range of spot quantity that is acceptable when selecting which
gel to cut a particular spot from. If the quantity of a spot in only one gel falls within
the tolerance ratio, that gel will be auto-selected for the spot cut. However, if the
quantity of a spot in two or more gels falls within the tolerance ratio, then the autoselect mechanism will consider which gel will best minimize the time and effort
required to switch gels on the spot cutter platform.
For example, if you have five gels in a MatchSet with a tolerance ratio of 1 (the
lowest setting) auto-selection specifies different cuts in all five gels, requiring to you
to change gels in the spot cutter five times. However, by increasing the tolerance ratio
slightly, auto-selection finds that three gels contain spots whose quantities fall within
the specified tolerance, and it adjusts the cuts so that only those three gels are
selected. This will save you considerable time and effort switching gels in the spot
cutter.
Check the Include Spot Quality box to consider spot quality when auto-selecting
spots. When this checkbox is selected, a well-resolved spot of sufficient quantity will
be favored over a less well-resolved spot of higher quantity (within the specified
tolerance ratio).
If after analyzing the cut selections you wish to add or remove cuts, click on the cells
to add or remove them. Note that this will automatically switch the selection mode to
Manual. To return to the Auto-select mode, click the option again. A dialog opens
asking you to confirm overwriting your manual edits.
Single gel
To make all the cuts from the same gel, select the Single Gel option button. Then
select the gel name from the pull-down box. All the cut requests listed in the table will
be located in that gel.
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Integrated Excision Tool
If you wish to add or remove cuts, click on the cells to add or remove them. Note that
this will automatically switch the selection mode to Manual. To return to the Single
gel mode, click the option again. A dialog opens asking you to confirm overwriting
your manual edits.
Single group
To excise all the occurrences of spots across all the gels of a specific replicate group,
select the Single group option button. Then select the group name from the pull-down
box. The cut requests listed in the table will be made from every gel belonging to that
group. Multiple gel cuts of the same spots are placed in the same well.
If you wish to add or remove cuts, click on the cells to add or remove them. Note that
this will automatically switch the selection mode to Manual. To return to the Single
group mode, click the option again. A dialog opens asking you to confirm overwriting
your manual edits.
Manual
To choose gels and spots manually, click the Manual option button. The spots to be
cut are selected in the table based on the prior selection mode. You can then click on
spots to add or remove cut requests. To select all the cuts in all the gels, click the
Select all button. Multiple gel cuts of the same spot are placed in the same well. To
clear the table of all the currently selected cuts, click the Deselect all button.
Table Options
The table in the Excision Gel Selection tool lists the spots in the analysis set (by
Standard Spot Number) and their quantities in the different gels. The columns are
gels, the rows are spots. You can use this table to make cut requests or modify the
auto-selection of cuts.
Under Table Options, click Show (Quality) to display the spot quality in the table
cells in parentheses following the spot quantity.
Click Show Ratio to display a column in the table showing the quantity tolerance ratio
across all gels for each spot. This value is used in conjunction with the Tolerance
Ratio setting, described above.
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PDQuest User Guide
Show All Gels will show columns for all the gels in the MatchSet. If this is
unselected, only those gels with cut requests will be listed in the table.
Image Options
Display Cuts marks each cut request with a red box in the MatchSet gel(s), and also
shows the actual cut request circles. These circles indicate the region of the spot that
will be cut. Note that if the Multiple Cuts/Spot checkbox is selected, multiple circles
may appear on some or all spots.
Auto-zoom on cut will automatically magnify each spot you select in the table.
Active Gels Only displays only those gels listed in the table. Note that if the Show All
Gels checkbox is not selected, only those gels with cut requests are listed in the table,
and consequently the MatchSet will reconfigure to show only those gels.
Under Cut options, the Multiple Cuts/Spot option automatically calculates the size of
each spot that has a cut request and, if the spot is sufficiently large, specifies multiple
cuts of that spot. Enter a maximum number of cuts per spot in the Max Cuts/Spot
field. The multiple cut circles will be displayed in the MatchSet. To customize the
way multiple cuts are made for a spot, click the Specify Cuts button. This allows you
to redraw a cut box around a large spot.
9.1.c
Step 3. Specify Cuts
In this step, you review the cut selection in the table and make changes as appropriate.
Note:
Only those gels with cut requests will be listed in the table, unless the Show All
Gels checkbox is selected.
The spots selected to be cut are highlighted in the table in blue. The table will initially
show one cut request per spot—that is, one highlighted blue table cell per row.
If you click the Standard Spot Number (SSP) at the beginning of each row, that spot
will appear highlighted in the actual MatchSet. See Figure 9-2 “Excision Gel
Selection.” for a pictoral representation. Study the size and quality of each spot in the
gels in the MatchSet when selecting spots to cut.
You can manually add or remove cut requests by clicking on the table cells. Click on
a blue cell to delete a cut request. Click on a white cell to make a cut request. To cut
the same spot in different gels, click on multiple white cells in the same row; they will
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Integrated Excision Tool
be highlighted in blue. You can add as many cut requests in as many gels in the
MatchSet as you want.
At the bottom of the table, the total number of cut requests per gel is shown.
9.1.d
Performing the Cuts
When you have made your selections, click the Next button to close the Excision Gel
Selection tool and open the Integrated Excision Tool.
9.2
Integrated Excision Tool
The Integrated Excision Tool is used to cut a set of spots from the gels in a MatchSet
using Bio-Rad’s ProteomeWorks spot cutter and ProteomeWorks Plus spot cutter
systems. It provides all the features of the Basic Excision Tool, plus the ability to cut
spots from PDQuest MatchSets.
Before using this tool, you must have selected the spots and the gels to cut using the
Excision Gel Selection tool, as described above. The MatchSet containing the gels
must be open and active. Also, make sure the spot cutter is connected with the
computer, is switched on and has warmed up, and make sure the camera is connected
and has been turned on.
Note:
See the spot cutter hardware manual for instructions on installing the hardware
and software.
Select Integrated Excision Tool from the Identify menu. The software will connect
with the spot cutter instrument and camera, and the Integrated Excision Tool dialog
box will open.
9.2.a
Spot Cutter Setup
Before you can use the spot cutter for the first time, you must focus the camera and
adjust the cutting tip height. You must also adjust the spot cutter settings and calibrate
the instrument.
Note:
If you have already focused and calibrated the spot cutter using the Basic
Excision Tool, you do not need to perform these steps again.
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PDQuest User Guide
Focus Cutter Camera
Note:
If you are using a lens filter, make sure that it is installed before focusing. If you
add or remove the lens filter after focusing, you will need to refocus.
The Focus Cutter Camera dialog box controls the camera exposure setting. Focusing
and aligning the camera are described in the spot cutter hardware manual.
Click the Focus Cutter Camera button to open the dialog. The platform
light on the spot cutter will turn on and the camera will focus on a small
region at the center of the gel or membrane and begin taking continuous
exposures. The exposures will appear in the Focus window at the rate of about 2 per
second.
At the top of the dialog, click Auto-Expose to reset the exposure time of the camera.
The Time field shows the time of each exposure in milliseconds. The Ceiling field
shows the highest pixel value in the focused region. The Levels field shows the levels
of gray scale in the image. All of these settings are automatically determined by the
software, based on the position and aperture of the camera lens.
Manually adjust the camera lens and position as described in the hardware manual to
optimize the image. When you are satisfied with the image and the levels of gray
scale, click Stop to close the Focus dialog.
Set Cutting Tip Height
Click the Set Cutting Tip Height button to adjust the distance the cutting tip
drops down to cut a spot. The adjustment dialog box will open, and the
cutting tip will move to the center of the gel.
Click Lower Tip to drop the cutting tip to its lowest position. In this position,
manually adjust the cutting tip height as described in the hardware manual until it
presses lightly against the surface of the cutting mat.
Raise Tip raises the cutting tip.
Test Cut performs a test cut in the center of the gel. Click Done to accept the settings
and return the cutting tip to home position.
The cutting tip height only needs to be set at installation and checked every few
months depending on use.
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Integrated Excision Tool
Spot Cutter Settings
Click the spot cutter Settings button to open the settings dialog box for the
cutter.
Fig. 9-4. Spot cutter settings dialog box.
Cutter Model
Select the spot cutter model you will be accessing for your cut run. This setting most
likely will not change unless you have regular access to both types of spot cutters.
There are slight differences between the two types. For instance, because the
ProteomeWorks Plus spot cutter has a larger platform and cutting tip range, it is not
necessary to crop the left margin. The ProteomeWorks Plus spot cutter also allows
you to cut backed gels. See “Step 2. Select the Cut Run Options” on page 9-17.
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PDQuest User Guide
Microtiter Plate
Specify the microtiter plate size you are using by clicking on 96 wells or 384 wells.
Specify the order in which you want to the load the wells on the microplate by
selecting A1, A2, A3, … or A1, B1, C1, … This function is useful for matching a
partial plate to the load order of a digest robot.
Well A1 position
The fields and buttons in this section are used to calibrate the position of the
microplate for proper loading of samples. The number fields contain the X and Y
coordinates (in millimeters) of well A1 on a plate in the spot cutter plate holder. Once
you have set the position of this well, the cutter robot will automatically calculate the
positions of the remaining wells.
To set the well A1 position, position your cursor in the number in the X or Y
coordinate field, click to place the cursor in the number field, and press Enter on your
keyboard. The tip will move to that position without attempting to lower into the well.
If the tip is not centered over the well A1 on the microplate, you will need to adjust
the numbers in each field.
You can either type a new value directly in each field, or (for small adjustments) use
the arrow buttons to adjust the tip by fractions of a millimeter. The cutting tip will
move after you press Enter or when you click the arrow buttons.
Note that the arrow buttons are oriented as if you were facing the front of the spot
cutter; also, if you are facing the cutter, higher numbers in the first (X) field will move
the cutting tip to the right, while higher numbers in the second (Y) field will move the
cutting tip toward the back of the instrument.
When you are satisfied with the position of the cutting tip, click Test to lower the tip
into the well. The tip should be centered inside the well.
Click Park to return the tip to its home position.
Wash Station 1 position
Calibrate the position of Wash Station 1 on the cutting platform by setting the cursor
in one of the Wash station fields and pressing enter. Then follow the steps described
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Integrated Excision Tool
above for Well A1. Once you have set the position of station 1, the cutter robot will
automatically calculate the positions of the remaining wash stations.
Miscellaneous Settings
Specify the diameter of the cutting tip by selecting 1.0 mm or 1.5 mm. Note that the
cut request circles that you make on the image reflect the approximate size of the
cutting tip, and will change based on the tip size selection.
If you are using the ProteomeWorks spot cutter, you will need to crop the left margin
of your image. Crop Left Margin repositions the image in the Excision Tool window
to the left. Enter a value (in screen pixels) in the field to reposition the image. Note
that you must click Done to close the settings dialog and then acquire a new image for
this change to take effect.
Note that cropping the left margin does not change the raw image, only the portion of
the displayed image. It is important to crop so that you can see the right edge of the
gel holding area of the platform in order to maximize the area available for cutting.
Serial Port
In the dialog box, select the communications port to the spot cutter by clicking on
COM 1 or COM 2.
9.2.b
Spot Cutter Calibration
Before you can begin excising spots, you must calibrate the spot cutter. This
procedure requires a coomassie blue stained PVDF membrane.
Note:
Before calibration, you should focus the cutter camera and align the wash station
position. You should make sure the spot cutter camera image is properly set using
the Crop Left Margin setting.
Note:
If you are using the lens filter for UV light, make sure that it is installed BEFORE
calibration. If you add or remove the lens filter after calibration, you must
recalibrate.
Click the Calibrate Cutter button. A series of dialog boxes will guide you
through the calibration procedure. The first dialog box will prompt you to
specify whether you have installed the lens filter or not. The lens filter must
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PDQuest User Guide
be INSTALLED if you are using the fluorescent spot cutter in fluorescent mode. The
lens filter is not required if you are using the spot cutter only in visible light mode.
Step 1:
The second dialog box prompt for flat field calibration requires that you ensure
the stage is completely empty and dry. This includes the cutting mat, which must
be removed. Press Ready to proceed. If you have previously performed this step
you can skip to Step 2, although this is not recommended.
Fig. 9-5. Spot cutter calibration step 2.
Step 2:
The third dialog requires the calibration template grid pattern supplied with the
PDQuest system. Place the grid on top of the cutting mat, which should have
been previously installed on the cutter. Make sure the grid is flat and aligned with
the stage edges. Click Ready to proceed. You may also skip this step, return to the
previous dialog, cancel the action, or ask for help.
Step 3:
The fourth dialog prompts you to place the coomassie stained PVDF membrane
on the mat. Click Ready to proceed with cutting, Back to return to the previous
screen, or Cancel to cancel the action.
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Integrated Excision Tool
At any point in the procedure you can move back to repeat a step.
Note:
Calibration creates several parameter and image files in the Install.prm directory
in the Discovery Series directory on your hard drive. These files should not be
moved, deleted or altered, or calibration will be affected.
When calibration is complete, you can proceed with normal use of the spot cutter.
Once the cutter is calibrated, you do not need to repeat the procedure during normal
day-to-day use. You must recalibrate if:
•
The instrument is moved.
•
You install or remove the lens filter after calibration.
9.2.c
Cutting Spots
After you have focused the camera and calibrated the spot cutter, you are ready to
begin cutting spots. The control panel is designed to guide you through the spot
cutting procedure.
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PDQuest User Guide
1
Cut list
2
3
Fig. 9-6. Integrated excision tool.
1.
Select the Cut List. In this step, you select the cut list created using the Excision
Gel Selection tool, and specify which cuts you want to perform for this run.
2.
Select Cutter Options. In this step, you select the options for the current cut run.
3.
Align the Images. Next, you click the Align button and align the first MatchSet
image and the spot cutter image.
4.
Select Microtiter Plate. In this step, you select a microtiter plate and specify the
starting well.
5.
Finally, you perform the cut run. When the run is complete you can recut spots if
necessary.
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Integrated Excision Tool
Step 1. Select the Cut List
At the top of the Integrated Excision Tool dialog, click the pull-down button to select
from the available cut lists you created using the Excision Gel Selection tool.
The cut list will be displayed in table format in the middle of the dialog box. The cut
list displays the microplate well, spot number, gel, spot quantity in that gel, cut run
number, and number of cuts for each spot.
The cuts are listed in the table in order of gel, so that all the cuts will be performed on
one gel before you are prompted to load the next one.
At the beginning of each row is a Cut checkbox, specifying the spots that will be cut
in the current cut run. Uncheck the box if you do not want to perform that cut.
After each cut is performed, its status will be listed as “completed” and its cut
checkbox will be deselected.
After a cut run is complete, you can reselect checkboxes in the list to recut any missed
spots or cut spots that were skipped in the previous run.
Step 2. Select the Cut Run Options
Before beginning a cut run using the Integrated Excision Tool, you must select the
appropriate cutter options for that run.
Select Spin When Cutting to make the cutting tip spin as it cuts. This is recommended
for all cutting, including gels and blots. Deselect this option if you want to test X,Y
positioning without cutting.
Spin When Ejecting will cause the cutting tip to spin as it ejects the cut into a
microplate well. This is only recommended for PVDF or nitrocellulose blots.
Cut Longer will cause the cutting tip to hold in the down position longer when
making a cut.
Double Eject will cause the eject pin in the cutting tip to eject twice into a microtiter
plate well. This is useful for membrane blots if the membrane seems to be sticking to
the cutting tip.
No spin when lifting stops the tip from spinning after the cut is made and the tip is
lifted from the gel. This option may be useful for some gel or blot applications.
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PDQuest User Guide
Check the Backed gel setting if you are using gels that have a backing on them.
The Tip motion range determines the side-to-side motion in millimeters of the tip as it
is cutting backed gels. The default range is 2.0, while the maximum range is 5.0 mm.
The user should determine the optimum setting for their gel.
Note:
The Backed gel option is only available if you are using the ProteomeWorks Plus
spot cutter.
Note:
The backed gel option is recommended for the 1.0 mm cutting tip only. Use of
the 1.5 mm cutting tip may result in decreased gel pickup efficiency.
The Gel Defaults button automatically selects the most useful options when cutting
gels. The PVDF Defaults button automatically selects the most useful options when
cutting PVDF membranes.
Select the wash stations to use after each cut by clicking on one or more checkboxes
(Station 1, Station 2, etc.). The wash solution is set by the user. Typically water is
enough for washing.
Under Misc. Options, select Multiple Cuts/Spot to specify multiple cuts of larger
spots. The number of cuts/spot is selected in the Excision Gel Selection window. Note
that the software will automatically calculate the size of each spot based on its
Gaussian profile in the MatchSet gel when determining whether to make multiple
cuts.
Save Validation Image will create and save an image of the gel on the spot cutter
platform when the cut run is complete. You will be prompted to name the validation
image.
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Integrated Excision Tool
Step 3. Align Analysis and Cutter Images
After you have selected the cut list and the cut run settings, click the Align button.
Fig. 9-7. Place gel on spot cutter dialog.
You will be prompted to place the first gel in the cut list on the spot cutter platform.
Click Gel Ready when you have positioned the gel as shown in the image.
The Align Analysis and Cutter Images dialog box opens. The process of autoalignment consists of the following steps:
Step 1:
Acquire the image of the gel with the spot cutter camera.
Step 2:
Position the data area box in correct position.
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Step 3:
Perform alignment.
In Step 3, you will have the option of skipping auto-alignment by making the software
work in totally manual mode. This could be useful if you have very few spots to be
cut, so you can do the alignment yourself.
Step 4:
Set the confidence threshold value. Visually confirm and, if needed, manually
correct the alignment of all those spots whose alignment is below the confidence
threshold.
Fig. 9-8. Aligning the images.
In the Align Images dialog, the left window displays the MatchSet image, and the
right window will contain the cutter camera image of the corresponding gel.
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Integrated Excision Tool
Note:
You always have the option of manually correcting spot positions.
Auto-alignment is not always correct and the manual collection allows you to
readjust your spot positions.
Flag the spot you want to correct in the cutter camera image and drag the spot to the
correct position. Red indicates the spot is below the confidence threshold, green spots
indicate the spot is above the confidence threshold, and the spot becomes white while
it is dragged.
You also have the option of returning to Data Box Positioning.
Step 1 Light Options
To acquire this image, first select the light option appropriate for your gel/membrane/
stain type:
•
Light on (gels).
•
Light off (PVDF membranes). Ambient light will produce a better image for
PVDF membranes.
•
UV on (fluorescent gels). UV light is only available for the Fluorescent spot
cutters.
Fig. 9-9. Light settings.
If UV light is selected, specify an exposure time in seconds in the field. Also, make
sure that the UV lamp switch on the back of the cutter box is on.
Note:
For Sypro Ruby stains, an exposure time of 1-30 seconds is recommended.
When you have selected a light option, click Acquire Image.
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Note:
For UV exposures, there will be a 15-second delay while the lamps heat up
before the exposure begins.
The camera will take a single image of the gel or membrane on the platform, then a
background image to adjust for image background. When the exposure is complete,
the image appears in the window.
To adjust the brightness and contrast of the image, open the Transform
window by clicking on the button to the right of the Image window.You can
also use the Transform button on the main toolbar.
Step 2 Positioning the Data Area Box
You will be prompted to position the green data area box that surrounds the camera
cutter image.
Fig. 9-10. Position the data area box.
If the orientation of the image is incorrect, use the flip and/or rotate buttons to fix the
orientation so that it matches the MatchSet image. Next, position your cursor over the
box and drag it so that it defines the same area as the left-hand (MatchSet) image.
Click OK. To reposition the data area box at a later time, click the Position Data Box
button and drag the box.
Step 3 Perform Alignment
In step three click Auto to have PDQuest automatically align the image. If you wish
to manually align the image, click Manual.
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If spots in the cutter image have not aligned correctly with corresponding spots in the
analysis image, make sure the scanning device has set the correct file size. For
example, the FX, Densitometer, and the GS series automatically set file sizes,
whereas camera scanners cannot. If your image was generated from a camera
acquisition device, (the Gel Doc EQ, ChemiDoc EQ, or VersaDoc), it will not have
specified file dimensions. For such images, you will need to enter the physical size
dimensions correctly at the time of acquisition. It is important to have correct physical
dimensions at the beginning, before you proceed with the spot detection and
MatchSet creation, otherwise auto-alignment will not work correctly.
Step 4 Alignment Confirmation
After the auto-alignment has been completed, set the confidence threshold to an
acceptable level. We recommend it to be a high value. The spots that meet this level
display in green. The spots below that confidence level display in red.
Fig. 9-11. Alignment Confirmation
Use the zoom box button to magnify the images for greater visibility.
Jump and Confirm
Click Jump and Confirm to jump to a view of the next spot in the
cut list, whose alignment is below the specified confidence
threshold. The spot being confirmed will be displayed in blue and
will be at the center of the window. If the spot is aligned correctly in the two images,
click Jump and Confirm again to jump to the next spot below the confidence
threshold. If the spot is not correctly aligned, drag it on the camera image to the
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correct position. Note that you can in fact drag any spot on the camera image any time
to manually correct its alignment. Repeat the above procedure for every spot to be cut
in this gel.
Matched
spots
Spot displayed
in step 4
Spots to be
matched
Fig. 9-12. Matching spots above and below confidence level.
Note:
Note that the green circle represents the actual cut made by the cutting tip in the
gel, and will vary in size depending on the cutting tip size selected under spot
cutter Settings.
The following display options are available in the Align Analysis and Cutter Images
dialog box:
Cut Marks will mark the spots to be cut with red Xs in the analysis image.
Matches will mark matched spots with green circles in both the MatchSet image and
cutter camera image.
Auto-zoom will automatically change the magnification and position of the (righthand) camera cutter image to imitate the display in the analysis image.
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Integrated Excision Tool
Cut Area displays a green box on the cutter camera image; this shows the boundary
of the cutting area. The cutting head cannot cut spots outside this area.
Multiple Cuts displays the multiple cuts of large spots containing more than one cut.
Data box displays the green data area box. Uncheck this box if the data area box is
obstructing the view of a spot on the periphery.
The Compute cut overlaps function automatically calculates
overlapping spot areas. You can manually adjust overlapping
spots by placing your cursor on the center of an overlapping
spot and moving it to the left or right.
Display Cut Overlaps marks overlapping spots with ellipses in the MatchSet image,
and the percent overlap will be displayed next to the overlapped cluster.
Beginning a Cut Run
When all the cut spots have been aligned and confirmed in the Align Images dialog
box, click the Begin/Resume Cutting button. If you have not manually and visually
confirmed the spot alignment for spots below the confidence threshold with Jump and
Confirm, you are prompted to either go back and do so, or proceed.
Fig. 9-13. Not all spots confirmed warning
If you proceed without confirmation it may result in reduced positioning accuracy.
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Step 4. Select the Microtiter Plate
Once you have completed alignment, you need to select a microtiter plate for your
cuts. If you have one or more existing microtiter plates associated with the current
analysis set, you can choose to use one of these plates.
Fig. 9-14. Use an existing plate.
The Use existing plate displays for each existing plate. If you want to use a new plate,
click no for each plate until the New plate dialog box displays.
Fig. 9-15. Enter the new plate information.
In the Enter Plate Information dialog box, a default plate name will appear in the Plate
Name field. Note that the loading order of the plates are named sequentially (Plate
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Integrated Excision Tool
001, Plate 002, etc.) by default. However, you can enter any name you want in the
field.
If you have a barcode scanner attached, and there is a barcode on the plate, scan it in
before proceeding.
The Loading order is determined by the spot cutter settings. See Section 9.2.a, Spot
Cutter Setup, for further information.
In the Specify Starting Well field, specify the first well you want to load in the current
cut run. If you specify a starting well other than the default of A1, the skipped wells
will be noted. Click Done to make your selection.
Note:
Specifying a different starting well is essential if you want to load spots from
different MatchSets or different analysis sets on the same microtiter plate. You
would load your spots from one MatchSet/analysis set on the plate. Then select a
different MatchSet/analysis set, and skip the previously loaded wells.
When you have made your selections, click OK to complete the microplate selection.
You will be prompted to place the microplate in the plate holder. Click OK, and the
cut run will begin. The Integrated Excision tool dialog box grays out while it is
working.
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Fig. 9-16. Cutting progress.
As each cut is being made, it will be highlighted in the table list. In progress or
Completed displays in the Status column. The checkbox in the Cut column next to
that spot will be automatically deselected. The spot being cut displays in the Member
Image window and in the Cutter image window. The spot in the Cutter image window
is filled with yellow while it is being cut, and surrounded by a green circle when the
cut is complete. This process takes a while so please be patient.
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Fig. 9-17. Progress completed on six cuts in the cut run.
Confirming Cuts
The Confirm button helps you check the cut progress of a
fluorescent stained gel. With visible stained gels it is easier to look
in the spot cutter to confirm that the cutting tip is cutting the spot
accurately. Since the spots are not visible without UV illumination in a fluorescent
gel, the Confirm function provides an on-screen method for monitoring the cut run.
After one or more cuts have been made, click Pause to pause the cut run. Note that the
cutter will pause only after the most recent cut has been deposited in a well.
Confirm the pause, then click the Confirm button under the main acquisition window.
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Switching Gels
When all the spots on one gel have been cut, you will be prompted to place the next
gel on the spot cutter platform and align that gel image with the MatchSet image (see
above). Repeat the steps described above until the cut run is complete for each gel.
Ending the Cut Run
Click Pause to pause the cut run. Note that the cutter will pause only after the most
recent cut has been deposited in a well. Any remaining cut requests will remain
selected in the image. At this point, you can click Begin/Resume to complete the
remainder of the cuts, or abort the run.
When the cut run is finished, the cutting tip will return to home position. If you have
selected the Save Validation Image checkbox, the validation image will be acquired
and you will be prompted to save it. The validation image is a cutter camera image
showing the cut spots.
Recutting Spots
When all cuts are complete, visually inspect the cut holes for visible spots, or take a
UV image of the MTP and confirm the cuts as follows.
You can then select Cut checkboxes in the cut list to recut any missed spots or cut
spots that were skipped in the previous run. At any time, you can reopen an old cut list
in the Integrated tool and perform recuts or cut previously skipped spots.
As before, click Align to begin the cut run.
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Fig. 9-18. Confirm cuts window.
Step 1:
Answer "Yes" to bring up the following on-screen instructions for imaging the
microtiter plate in UV mode:
Fig. 9-19. Image Microtiter plate window.
Step 2:
Do not move the gel or membrane on the cutting platform
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Step 3:
Place a clean plastic Gel Sheet on top of the gel
Step 4:
Place the microtiter plate on top of the plastic sheet. Note the orientation so that
you can recognize the A1 location.
Step 5:
Close the Enclosure door
Step 6:
Click on "Acquire image" when the plate is in place. The UV lights will turn on
and the camera will acquire an image with the same exposure time as the first
spot cutter image.
Step 7:
Assess the image on the screen for the wells that need to be recut.
Fig. 9-20. Microtiter plate with fluorescent spots in wells.
Use the Zoom and transform functions on the right side of the "Image Microtiter
Plate" window to get a better view of the microtiter plate wells.
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Fig. 9-21. Microtiter plate after zoom and transform functions applied.
Step 8:
If any of the wells are empty and you want to re-cut those spots, click on
"Specify re-cuts." Then follow these on-screen instructions:
Fig. 9-22. Message window before recuts are started.
Step 9:
Return the microtiter plate to the plate holder.
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Step 10: Remove the plastic sheet from the top of the gel.
Step 11: Do not move the gel. The image and spot selections that were already made still
apply to the gel as long as it does not move.
Step 12: After removing the microtiter plate, click "OK" on the message. The "Image
Microtiter Plate" window will close and an image of the microtiter plate will
open in front of the Basic Excision Tool screen. If you need to resize the window,
close the recuts box, resize the window, make any zoom or transform changes
using the icons on the top bar above the window, then click on the "Recuts" icon
to bring the recuts box back up.
Fig. 9-23. Integrated Excision Tool fluorescent mode re-cuts.
Step 13: Enter the well numbers of the missed pick-up spots in the "Re-cuts" box on the
screen.
Step 14: Click on "Begin/Resume" after the selections are made.
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9.2.d
Exporting the Cut List
You can export the information in the cut list as a spreadsheet file. Click the Export
button, enter a name for the file, and select the directory in which to create the file.
The export command will create a tab-delimited file that can be opened as a Microsoft
Excel worksheet or as a simple text file.
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10. Mass Spectrometry
Analysis
After you have cut spots from a gel using the Integrated Excision Tool and digested
them, you can select the parameters for processing them using Micromass’s MALDI
or MS/MS mass spec instruments. You can then import the mass spec data back into
PDQuest in the form of annotations.
10.1
Setting up a PLGS run
If you are using Proteinlynx Global Server 2 for your spot identifications, click Setup
PLGS Run in the Identify menu.
Note:
The IP address of the server must be correctly identified in the Paths tab of the
Preferences dialog box or you will not be able to open the Setup PLGS Run
dialog box.
Fig. 10-1. Setting up a PLGS run.
The name of the current MatchSet is displayed. the dialog box automatically lists
cutlist or microtiter plate. To select a different cutlist or plate, click the drop down
arrows next to the respective fields and select a cutlist or plate from the drop down
lists.
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When you click Done, a project is automatically created in the folder where the
Server is running. The Proteinlynx Global Server accesses this project and searches
the database to identify the spots. Once this is complete, you can import the data back
into the MatchSet as annotations. See Section 10.3, Importing PLGS Results, for
further information on importing PLGS results.
10.2
Exporting a MassLynx Worksheet
If you are not running PLGS2, but are using some other search engine such as Mascot
to identify your spots, then use the Export MassLynx Worksheet.
First set up a project file using MassLynx software, then export the processing
parameters from PDQuest into the project file. After you have identified the proteins,
you can import the results back into PDQuest.
With the MatchSet containing your cut spots open, select Export MassLynx
Worksheet from the Identify menu to set up the batch file.
Step 1
In the dialog box, the MatchSet name is listed next to MatchSet. Select the cut list you
want to analyze from the Cut List drop down list. If you have performed more than
one cut run on this cut list, select the number of the run from the Cut Run Serial
Number drop down list.
Next, select the plate used for the cut run using the Microtiter Plate drop down list. If
more than one plate was used for the run, select the first plate you want to process.
Step 2
Select the appropriate Micromass instrument using the MALDI or MS/MS option
button.
The Export to Masslynx Worksheet automatically formats the worksheet for use with
Mascot which allows you to include the MatchSet name in the sample identifier string
so that results from the specific MatchSet can be filtered out of the master search log.
The purpose of this is to include all necessary information in the "MSDataName" tag
in the file. This tag eventually is used by Mascot as the title in the "searches.log" file.
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Mass Spectrometry Analysis
See your MassLynx documentation for processing your raw data for use with the
Mascot search engine.
When you have made all your selections, click Done.
10.3
Importing PLGS Results
After you have completed a mass spec analysis and processed the data using PLGS2,
you can import the results back into PDQuest and include them in annotations for the
spots you have analyzed.
With the appropriate MatchSet open, select Import PLGS Results from the Identify
menu.
Fig. 10-2. Importing PLGS Results dialog box.
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In Step 1, the MatchSet name will appear in the MatchSet field. Select the cutlist and
microtiter plate name from the drop down lists.
Note:
The IP address of the server must be correctly identified in the Paths tab of the
Preferences dialog box or you will not be able to open the Import PLGS Results
dialog box.
Step 2 - Select Samples lists the gel name, run, and the spot number of each cut in the
cutlist. to view the primary hits associated with the sample in Step 3.
Step 3 - Select Primary Hit lists the primary hits for the selected sample in step 2.
Highlight a hit in the list to display its protein sequence in the field below. Sequences
highlighted in red indicate a high probability of match, while those highlighted in
blue indicate low probability. Click Open SwissProt to view the hit in the SwissProt
database.
Select the protein that you think is the most likely identification, and click Apply to
import all the information into spot annotations in PDQuest. To quickly apply the
highest probability hits for all the samples, click Apply all.
Note:
The protein you select will be used as the name for the spot. (All the hits will be
included in a special annotation category called Hit Table.) If you change your
mind about the protein name, you can change the information directly in the Spot
Annotations dialog box, or re-import the results with a different selection.
Annotation Categories Created
When you click Apply, the following annotation categories are created, and the mass
spec data for each spot is entered as an annotation in the appropriate category. (See
section 7.2.b, Annotation Categories for more information on annotation categories.)
•
Protein Name - The annotation entry in this category is the name of the protein
selected by you from the hit list.
•
Accession Number - The annotation entry in this category is the database number
keyed to the selected protein name. Note that some databases (e.g., Swissprot)
include accession numbers, while others do not. If the database used to identify
the protein does not have accession numbers, this category is not created.
•
Hit Table - The annotation entry in this category is the entire list of proteins
identified for the spot.
•
Mass Spec Score - Both the Joint Probability and MOWSE scores are listed.
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Mass Spectrometry Analysis
Fig. 10-3. Example of the Hit Table annotation category and entries.
In addition, the protein name is included in the standard protname category (which
will be created if it doesn’t already exist), so you can sort the spots by protein name in
the Select Spot list.
Note:
10.4
Each category is specific for the mass spec instrument used. For example, if you
import information from both MALDI and MS/MS, you would create both a Hit
Table (MALDI) category and a Hit Table (MS/MS) category.
Mass Spec Score Overlay
If you have imported mass spec data, you can display overlays indicating the
confidence level of the mass spec protein identification.
Select Mass Spec Score Overlay from the Identification menu.
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Fig. 10-4. Mass Spec Score Overlay dialog.
In the dialog box, indicate the instrument you want to report scores for by selecting
MALDI or MS/MS. Note that if there is no data for an instrument, no spot overlays
will be shown.
Enter the threshold value for the score in the Threshold field. Spots above this
threshold can be considered confident hits; spots below this threshold are not.
Click the Format buttons next to Above Threshold, Below Threshold, and No Hits to
select the appearance of the different overlays.
No Hits are spots with no hits or with an unknown protein identification. This means
that the mass spec tried and failed to recognize the sequence.
10.5
Show Identified Spots
Use the Show Identified Spots command in the Identify menu to display identified
spots in the MatchSet.
In the Show Identified Spots dialog box, select the box labeled Mark all excised spots
with (marker) to mark the spots on the gels in the MatchSet. Click the marker to
change the format of the marker.
Select Show protein name to display the protein name of each excised spot in the
MatchSet.
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Mass Spectrometry Analysis
When the Show Identified Spots dialog box opens, all identified spots in the
MatchSet are displayed. Select a cut list form the drop down list to display only the
spots from a specific cut list.
Select Show well to display the well number where the cut was placed.
10.6
Import Mascot Results
When importing results from Mascot, PDQuest searches the "searches.log" file to
find results that apply to the current MatchSet. Once the identifications have been
applied, the researcher can right-click on a spot, launching the Mascot browser with
the corresponding data file.
Before you can import Mascot results, you must map the directories where your
Mascot install folder is located and enter the URL for the Mascot browser. Go the
Edit menu, select preferences, and click on the Paths tab. Click browse to locate the
Mascot install folder and enter an address for the browser.
The Import Mascot Results dialog allows you to import data from a Mascot spot
identification run. To import Mascot results, select Identify>Import Mascot results....
This opens the Import Mascot Results dialog.
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Fig. 10-5. Import Mascot results
Set Search Filters
In this step of the Import Mascot Results... dialog, select your search filters. The
currently open MatchSet determines the MatchSet name. If you wish to use the
MatchSet name check the box to the left of the field. To search for a specific cut list or
plate, select one from the drop down list. You can also choose a user name and a run
since filter. When you enter data into any of these fields, the box is automatically
checked when you tab out of the field. If you no longer want to use one of these
filters, clear the box.
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Mass Spectrometry Analysis
The Replace Existing data without confirmation check box allows you the option
of overwriting current data. If this box is unchecked, you will receive confirmation
every time existing data is about to be overwritten. This may be time consuming if
you have a considerable amount of data to import, which overwrites existing data.
Select Search
The Select Search field lists all searches based on the filters selected in step one.
The search title must be formatted correctly. If the search title is incorrectly
formatted, you will get an error message whether or not you have the search title
checked in the options dialog. A correctly formatted search title consists of the
MatchSet name, cut list name, cut run serial number, plate name, MS run sequence
number, and the SSP number. The search title must be formatted with all spaces and
underscores removed. The only underscores that should remain are the item
separators.
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Fig. 10-6. Peptide Mass Fingerprint Search dialog
For example, a data file created in the Export MassLynx Worksheet dialog box might
look like this:
\\MS142002_spots_1_Plate1_1_5303_mass.txt
Where MS142002 is the MatchSet name, spots is the cut list, 1 is the cut serial
number, plate1 is the plate name, 1 is the run sequence, and 5303 is the spot number.
Copy the portion of the name that coincides with the Search title and paste it into the
Search title field of the Peptide Mass Fingerprint Search dialog (e.g. the entire data
file name minus the _mass.txt).
If you use Mascot Daemon to run your search instead of the browser, it automatically
formats the search title for you based on the parameters set in the Daemon. See
Mascot documentation for using Mascot Daemon.
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Mass Spectrometry Analysis
Highlight the desired search and the list of hits automatically appear in the Search
results field. Click Mascot Search Results to open the Mascot browser and view the
Mascot results.
Search Results
The Search results field lists all the hits from the selected search or searches in step 2.
To reduce the number of hits, enter a maximum number of hits to return.
To apply a hit to a spot, highlight the desired hit and click Apply. Based on the
selections you have checked in the Options dialog, PDQuest automatically imports
the data as annotations for the selected spot.
When you have finished applying hits to spots, click Close. For information on
viewing imported data, see Section 7.2, Annotation tool.
10.6.a
Options
The top portion of the Mascot Import Options dialog allows you to select the columns
you wish to appear in the Select Search field.
Note:
Selecting too many columns to view may expand the dialog beyond the display
area. Select only those columns necessary for your purposes.
The lower portion of the Options dialog allows you to select the types of hit data you
wish to import.
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11. Graphs and Reports
This chapter describes the graphs and reports found in PDQuest. While most of the
graphs and reports described in this section are available from the Report menu, Spot
review is found in the Analyze menu, and Graph Partial Matches and Graph Erratic
Responses are both located in the Edit Matches submenu of the Match menu.
Spot Review Tool
Fig. 11-1. Reports menu and Graph Toolbar.
11.1 Configure Graphs
The Configure Graphs tool of PDQuest allows you to configure the way graphs are
displayed. The Configure Graphs tool includes two configurations (all gels and
replicate groups) with the ability for you to create more. Open the Configure Graphs
tool by selecting Report>Configure Graphs...
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Fig. 11-2. Configure Graphs tool
To select a graph configuration, highlight the desired configuration and click Apply.
The tool closes and the new configuration takes effect.
11.1.a
Adding and Editing Configurations
To add a new configuration click Add, or to edit an existing configuration, highlight
the desired configuration and click Edit. This opens the Configure Graph dialog.
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Graphs and Reports
Fig. 11-3. Editing a Configuration
Step 1:
Enter a name and description in the requisite fields.
Step 2:
Next, select whether you want the graphs to show quantifications for gels,
groups, or classes.
Note:
If you have not created any replicate groups or classes, these choices will be
inactive.
Step 3:
Select the display mode. This allows you to either view quantifications for
replicate groups and classes as an average of the spots in the group or class or as
individual graphs for each spot.
Selecting colorize colorizes the graph based on the groups or classes. This is very
useful when viewing individual spots for replicate groups.
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Individual
Group
Fig. 11-4. Colorized graphs showing replicate group by individual and group
To delete a configuration, highlight the item you wish to delete and click Delete.
11.2 Histogram Graphs
Histogram graphs can be used to quickly compare a spot’s quantity in each member of
a MatchSet. A brief look at a histogram can give you a sense of the general trends in
spot quantity.
Histograms are also useful as tools for detecting problems such as spots that are not
matched and spots whose quantitation is suspiciously off the average. Reviewing the
histograms of spots in an Analysis Set is a quick and easy way to confirm that the
members of the Analysis Set meet the criteria that was specified when the set was
created.
Fig. 11-5. Sample Histogram graph.
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Graphs and Reports
11.2.a
Data Displayed in the Histogram
Each bar in a histogram for a spot represents the spot’s quantity in a member of a
MatchSet. If replicate group quantitation is selected, each bar represents the spot’s
quantity in a replicate group.
The bars from left to right represent the gels in the order in which they were loaded.
As long as the spot is matched to the Master, you can display a histogram for it.
Maximum Quantity
Normalization Units
Fig. 11-6. Sample histogram.
The Standard Spot (SSP) number is displayed beneath the histogram.
The number at the upper right of the histogram is the quantitation of the maximum bar
in the graph. The other bars are drawn proportional to the highest bar.
Beneath the maximum quantitation, the normalization units are displayed.
Normalization is a process by which quantitative data from different gels are adjusted
so that you can compare different samples to one another (for more information on
normalization, see section 6.6).
If a spot is saturated, the bar representing it on the histogram will be shaded red
instead of black.
If replicate groups are defined for the MatchSet (see section 6.4), you can look at the
quantitation of individual spots or the average data of duplicate gels. You can specify
which way you want to review spot quantitation by selecting choosing a configuration
in the Configure Graphs dialog. See Section 11.1 for further information.
If you specify All Replicate Groups, the bars on the histograms will display not only
the maximum spot quantitation for each replicate group, but also the average spot
quantitation and the standard deviation. Each bar represents one replicate group. The
order of the bars corresponds to the order in which the replicate groups were made.
See Section 11.1, “Configure Graphs” for more information.
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Maximum Quantity
Standard Deviation
Minimum Quantity
Fig. 11-7. Sample histogram with Replicate Group Quantitation.
11.2.b
Types of Graphs
Spot Review Tool
Spot Review Tool on the Analyze menu (section 6.1) displays a page of graphs
alongside the MatchSet. You can use this tool interactively with the MatchSet to
review spots.
Pop-up Graph
Pop-up Graph (F10) shows a histogram next to a selected spot. Select Pop-up Graph
from the menu or toolbar, then click on a spot.
With Pop-up Graph, only one histogram can be displayed in a window at a time. If
you select another spot, the first histogram disappears and a new one displays.
Margin Graph
Margin Graph (SHIFT+F10) on the Reports menu and Graph toolbar will display
histograms for several spots along the left and the right margins of the screen. Select
Margin Graph, then click on several spots. Their histograms will be displayed in the
left and right margins. The Margin Graph only applies to the MatchSet Master.
Depending on the size of your window, you will be able to display up to 10 margin
graphs at once. If you keep clicking on spots after you’ve displayed 10 graphs, the
graphs will be displayed on a new page. Use the Graph Advance (shift+page down)
Graph Retreat (shift+page up) commands to flip through the pages.
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Graphs and Reports
Column Graphs
Column Graph on the Reports menu and toolbar will fill the right and left margins
with histograms of spots in the Master gel. Select Column Graph; the spots whose
graphs are displayed are all located in a narrow column of the image, centered around
the location of the cursor. The Column Graph only applies to the MatchSet Master. To
change the column of spots, use the Graph Advance/Graph Retreat tools (see below).
Graph Partial Matches
Graph Partial Matches (Match > Edit Matches submenu) displays histograms of each
partially matched spot in the Master. Select the command from the submenu and click
in the Master. Use Graph Advance/Graph Retreat to graph different regions of spots.
Graph Erratic Responses
Graph Erratic Responses (Match > Edit Matches submenu) displays histograms of
each erratic spot in the Master. Select the command from the submenu, click in the
Master, enter a fold change factor in the pop-up box, and click Apply to display the
graphs. Use Graph Advance/Graph Retreat to graph different regions of spots.
Graph Advance/Graph Retreat
If you are displaying Margin Graphs, Column Graphs or graphing spots using the
Graph Partial Matches or Graph Analysis Set commands, you will need to shift the
display to graph spots in different regions of the image. The commands for doing this
are located on the Reports menu or the Graph toolbar.
Graph Advance (SFT+PAGE DOWN) shifts the column of spots being graphed to the
right. Graph Retreat (SFT+PAGE UP) shifts the column of spots being graphed to the
left. For Margin Graphs, these commands flip through the graph pages.
11.3
A-B Overlay
The A-B Overlay function is used on MatchSet Masters to highlight and compare the
spots that appear in two different MatchSet members.
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Select A-B Overlay from the Reports menu and click the Master of the MatchSet in
which you want to perform the comparison. A pop-box will prompt you to select the
first member you want to compare (Member A). Click on the member name. A
second pop-up box will prompt you to select the second member you want to compare
(Member B).
When you select the second member, the box disappears and the spots in the Master
are highlighted.
Fig. 11-8. A-B Overlay example.
Spots that are marked in both members will be highlighted in green, spots that appear
only in Member A will appear highlighted in red, and spots that appear only in
Member B will appear highlighted in blue.
Spots that appear in neither member will not be highlighted.
A color key appears at the bottom of the Master.
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Graphs and Reports
11.4
Quantity Table Report
If you select Quantity Table Report from the Report menu, the Quantity Table Report
dialog box opens.
Fig. 11-9. Quantity Table Report dialog box.
You can type a report title, the name of the researcher, and the name of the research
institution into the appropriate fields.
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Specify the spots to include in your report: All Matched Spots or the spots in a
Specified Analysis Set drop-down list. If you click Analysis Set, click the drop down
arrow to display a list of available Analysis Sets. The name of the set you choose
appears in the field.
Under Configuration, choose how you want to view the spot quantitation. Individual
gels displays the quantitation for each gel. If you have replicate groups defined in
your MatchSet, you can view spot quantitation by Replicate group. If you have
associated gels to samples in the Sample Database and have added attributes to your
samples, you can view spot quantitation according to these attributes by selecting
Class. (See Section 6.6.b, Classes, for further information regarding sample
attributes.)
You can sort your spots by SSP number, DSN, trend, ratio, molecular weight, or
isoelectric point by clicking on the appropriate Sort by button. To sort in ascending
order, check the box labeled Increasing.
Options
The following options can be included in the Quantity Table Report:
•
If DSN numbers are assigned to the protein spots and you would like to see them
on the report, click the DSN button.
•
MrpI data will be reported if you click the MrpI button.
•
Check the box labeled Trend to view the trend of each spot.
•
You can view the group count and the group critical value by checking Group
Count and GroupCV respectively.
•
You can view the ratio of spots to a specific gel by clicking Ratio then selecting a
gel from the drop down list.
•
You can print spot annotations by clicking the Annotation button and selecting
from the list of annotation categories. The report will include annotation
information for the spots that have annotation data in the selected category.
Other Options
Data - Choose whether to round values off the closest integer or include normalized
values.
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Graphs and Reports
Missing Spots - You can choose to estimate missing spots, exclude them from the
report, or enter a minimum value to include in the report.
Saturated spots - You can choose to estimate saturated spots, exclude them from the
report, or enter a minimum value to include in the report.
Once you have selected your options, click Done to open the report viewer.
Reformat report
Export
Fig. 11-10. Viewing a Quantity Table Report
The report viewer allows you the option of printing or exporting the report for use in a
spreadsheet application. Click reformat report button to return to the Quantity Table
Report dialog. You can also click on a row to highlight the spot in the MatchSet.
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11.4.a
Quantity Graph Report
If you select Quantity Graph Report from the Report menu, a dialog box will open in
which you can specify the format of your quantitation histograms.
Fig. 11-11. Quantity Graph Report dialog box.
You can type a report title, the name of the researcher, and the name of the research
institution into the appropriate fields.
You can specify the spots that will be included in your report: All Matched Spots or
the spots in a Specified Analysis Set. If you click Analysis Set, click the drop down
arrow to display a list of available Analysis Sets. The name of the set you choose
appears in the field.
The quantitation mode for the graphs is determined by the configuration selected in
the Configure Graphs dialog. See Section 11.1, Configure Graphs for further
information on determining the quantitation mode.
You can sort your spots by SSP number, DSN, Trend, or quantity by clicking the
appropriate Sort by button.
Once you have selected your options, click Done to open the report viewer.
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Graphs and Reports
Reformat
Fig. 11-12. Quantity Graph Report Viewer
The report viewer allows you the option of printing or reformatting the report. Click
reformat report button to return to the Quantity Graph Report dialog. Click on a graph
to view the spot in the MatchSet.
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11.5
MatchSet Member Report
The MatchSet Member Report allows you to create a report of any member image of
the current MatchSet. Open the Report menu and click MatchSet Member Report.
This opens the MatchSet Member Report dialog box.
Fig. 11-13. MatchSet member Report dialog box.
If you want to enter a report title, researcher name, and/or institution in the fields
provided, they will appear in the report header.
Select the image for the report from the drop down list. Select Use zoom if you want
to create a report based on the current view of the image. If this box is not checked,
the report will contain the full view of the image regardless of the current view.
Tip:
The list of images is based on which images are currently displayed in the
MatchSet window. If you want to create a report of an image not displayed, use
the Interchange image command before opening the MatchSet Member Report
dialog box.
Next, choose which spots to highlight in the report: all matched spots or spots
associated with a specific analysis set. If you have spots that have been annotated, you
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Graphs and Reports
can choose to highlight only these spots. Check the box labeled Annotations Category
and select a category from the drop down list.
The display options allow you to determine how selected spots are highlighted in the
report. Check the box labeled Mark spots to mark the spots in the report. The radio
buttons determine whether to display the spot numbers (SSP), Annotations, None, or
SSP+Legend. The SSP+Legend option displays the spot numbers in the image with
the annotations in a legend to the right of the image in the report. Click Format next to
each option to customize the format of the option.
When ready, click Done to open the report viewer.
Fig. 11-14. MatchSet Member Report
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PDQuest User Guide
The MatchSet Member Report viewer allows you the option of printing or
reformatting the report. Click the Print Preview button to view how the report will
print, which may be different from the report view due to such things as paper size
and layout. In Print Preview mode, you can also choose to print the current page by
clicking the Print Current Page button. Click the Reformat report button to return to
the MatchSet Member Report dialog box.
11.6
Scatter Plot Report
The Scatter Plot Report command on the Report menu allows you to print out a
scatter plot report for all the member pairs of a MatchSet. A scatter plot shows the
relatedness of two different gels that have been matched in a MatchSet.
Note:
To display a scatter plot for any two gels in a MatchSet, use the Scatter Plot Tool
in the Analysis menu (section 6.3, Scatter Plot Tool).
Fig. 11-15. Scatter plot Report.
When you select Scatter Plot Report from the Report menu, a dialog opens in which
you can enter a report title, researcher name, and the name of the research institution.
Click Done to open the report viewer.
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Graphs and Reports
Reformat
Fig. 11-16. Scatter Plot Report Viewer
The report viewer allows you the option of printing or reformatting the report. Click
the reformat report button to return to the Scatter Plot Report dialog.
11.7
Analysis Set Report
The Analysis Set Report allows you to create a report of the analysis sets associated
with the current MatchSet. Open the Report menu and click Analysis Set Report. This
opens the Analysis Set Report dialog box.
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PDQuest User Guide
Fig. 11-17. Analysis Set Report Dialog box
If you want to enter a report title, researcher name, and/or institution in the fields
provided, they will appear in the report header. Next, select the information you want
to appear in your report. If an option is checked, a column will appear in the report
even if there is no information to report.
When ready, click Done to open the report viewer.
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Graphs and Reports
Fig. 11-18. Analysis Set Report
The Analysis Set Report viewer allows you the option of printing, exporting, or
reformatting the report. Click the Print Preview button to view how the report will
print, which may be different from the report view due to such things as paper size
and layout. In Print Preview mode, you can also choose to print the current page by
clicking the Print Current Page button. Click the Export button to export the report to
the clipboard or a file. Click the Reformat report button to return to the Analysis Set
Report dialog box.
11.8
Image Report
Image Report in the Report menu opens a report of a selected image. If you have a
single image visible in the active window, clicking Image Report opens the report
viewer for that image. If you have more than one image visible in the active window,
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PDQuest User Guide
such as a MatchSet or scan set, click Image Report then select the image you want to
view in the report.
Fig. 11-19. Image Report view
The Image Report displays all available information about the image and is the same
information displayed in the Image Info dialog box. (See Section 2.3.g, Image Info,
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Graphs and Reports
for further information.) Click Print to print the report. Click the Print Preview button
to view how the report will print, which may be different from the report view due to
such things as paper size and layout. In Print Preview mode, you can also choose to
print the current page by clicking the Print Current Page button.
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Appendix A
Gel Doc EQ
Fig. A-1. Gel Doc EQ.
Before you can begin acquiring images, the Gel Doc EQ system must be properly
installed and connected with the host computer. See the Gel Doc EQ hardware
manual for installation, startup, and operating instructions.
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PDQuest User Guide
To use the Gel Doc EQ, you will need to have the Bio-Rad-supplied acquisition board
installed in your PC or Macintosh. The drivers for this board will be installed when
you install the main software application.
Make sure that your Gel Doc EQ camera is turned on. If the camera is not turned on,
the Gel Doc EQ acquisition window will open and an image capture error will be
displayed.
Simulation Mode
Any of the imaging device acquisition windows can be opened in “simulation mode.”
In this mode, an acquisition window will open and the controls will appear active, but
instead of capturing real images, the window will create “dummy” images of
manufactured data.
You do not need to be connected to an imaging device to open a simulated acquisition
window. This is useful for demonstration purposes or practice scans.
To enter simulation mode, hold down the CTRL key and select the name of the device
from the File menu. The title of the acquisition window will indicate that it is
simulated.
A.1 Gel Doc EQ Acquisition Window
To acquire images using the Gel Doc EQ, go to the File menu and select Gel Doc
EQ.... The acquisition window for the instrument will open, displaying a control
panel and a video display window.
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Appendix A. Gel Doc
Fig. A-2. Gel Doc EQ acquisition window
The Gel Doc EQ video display window will open in “live” mode, giving you a live
video display of your sample. If no image is visible, make sure the camera is on,
check the cable connections, make sure the iris on the camera is not closed, and make
sure that the protective cap is off the camera lens. Also check to see that the
transilluminator is on and working.
The control panel has been arranged from top to bottom to guide you through the
acquisition procedure. There are six basic steps to acquiring an image using the Gel
Doc EQ:
1.
Position and focus the gel or other object to be imaged.
2.
Select Mode.
3.
Acquire the image.
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4.
Optimize the display.
5.
Analysis.
6.
Select the output.
A.2 Step I. Position Gel
The Gel Doc EQ window will open in “live” mode, giving you a live video display of
your sample. In this mode, the Live/Focus button will appear selected, and frames
will be captured and displayed at about 10 frames per second, depending on the speed
of your computer.
You can use live mode to zoom, focus, and adjust the aperture on the camera, while
positioning the sample within the area.
Note:
Newer versions of the Gel Doc EQ feature a motorized zoom lens that can be
controlled directly from the acquisition window using the Iris, Zoom, and Focus
arrow buttons. Click on the Up/Down buttons while viewing your sample in the
window to adjust the lens. These buttons will not be visible if you are connected
to older versions of the Gel Doc EQ without the motorized zoom lens.
Fig. A-3. Newer Gel Docs feature camera control buttons in the acquisition window.
You can also select the Show Alignment Grid checkbox to facilitate positioning.
Note:
After positioning your sample, you should check the Imaging Area dimensions
under Options (see section A.9, Options) to make sure that they conform to the
size of the area you are focusing on. To determine the size of the area you are
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Appendix A. Gel Doc
focusing on, you can place a ruler in the Gel Doc EQ cabinet so that it is visible
in the image.
A.3 Step II. Select Image Mode
The Image Mode option buttons allow you to set the type and scale of your data.
UV
Select this mode for fluorescent samples. With this mode selected, the data will be
measured in linear intensity units.
White Light
Select this mode for reflective and transmissive samples. With this mode selected, the
data will be measured in uncalibrated optical density (uOD) units.
A.4 Step III. Acquire Image
For many white light applications, you can skip this step and save and print images
directly from Live/Focus mode.
For UV light or faint samples, you can take an automatic exposure based on the
number of saturated pixels in the image or you can enter a specific exposure time.
Note:
“Exposure” refers to the integration of the image on the camera CCD over a
period of time. The effect is analogous to exposing photographic film to light
over a period of time.
Auto Expose
Auto Expose will take an exposure whose time length is determined by the number of
saturated pixels in the image. This is useful if you are uncertain of the optimal
exposure length.
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PDQuest User Guide
Note:
If you know the approximate exposure time you want (± 3 seconds), you can skip
this step and go directly to Manual Expose.
Click on the Auto Expose button to cancel Live/Focus mode and begin an automatic
exposure. The Auto Expose button will appear selected throughout the exposure.
During the auto exposure, the image is continuously integrated on the camera CCD
until it reaches a certain percentage of saturated pixels. This percentage is set in the
Options dialog box. (Default = 0.15 percent. See section A.9, Options.)
Click on Auto Expose;
button appears depressed
Can see Exposure Time
automatically changing
Fig. A-4. Auto Expose.
Once an image has reached the specified percent of saturated pixels, it is captured and
displayed in the video display window, Auto Expose is automatically deactivated, the
exposure time appears active in the Exposure Time field, and Manual Expose is
activated.
Note:
If you are having difficulty auto-exposing your sample, you can use Manual
Expose to adjust your exposure time directly. Most applications only require an
exposure time of a few seconds, which can be quickly adjusted using Manual
Expose.
Manual Expose
If you know the approximate exposure time you want, you can click on the Manual
Expose button. Manual Expose is automatically activated after Auto Expose has
deactivated.
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Appendix A. Gel Doc
Manual Expose active;
button appears selected
Exposure Time field active;
adjust number of seconds
Fig. A-5. Manual Expose.
With Manual Expose activated, you can adjust the exposure time directly by changing
the number of seconds in the Exposure Time field. Type in a number or use the arrow
buttons next to the field.
When the specified exposure time is reached, the last captured image will be
displayed in the Gel Doc EQ image window. The camera continues to integrate the
image on the CCD, updating the display whenever the specified number of seconds is
reached.
Once you are satisfied with the quality of the displayed image, click on the Freeze
button to stop the exposure process. The last full exposure will be displayed in the
image window.
Click on Freeze to stop
the exposure process
Fig. A-6. Freezing the exposure.
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PDQuest User Guide
Note:
Freeze is automatically activated if you adjust any of the subsequent controls
(e.g., Video Print, Image Mode, Display controls, etc.).
A.5 Step IV. Optimize Display
The Display controls are useful for quickly adjusting the appearance of your image
for output to a video printer. Adjusting these controls will automatically freeze the
video display and allow you to alter the image within the Gel Doc EQ window.
Fig. A-7. Display controls.
These controls are similar to those in the Transform dialog box.
Note:
The Display controls will only change the appearance of the image. They will not
change the underlying data.
High/Low Sliders
If Auto-scale doesn’t give you the appearance you want, you can use the High and
Low sliders to redraw the image yourself. In white light mode, dragging the High
slider handle to the left will make weak signals appear darker. In UV mode, dragging
the High slider handle to the left will make weak signals appear brighter. Dragging
the Low slider handle to the right will reduce background noise.
You can also type specific High and Low values in the text boxes next to the sliders.
Clicking anywhere on the slider bars will move the sliders incrementally.
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Appendix A. Gel Doc
Gamma Slider
Some images may be more effectively visualized if their data are mapped to the
computer screen in a nonlinear fashion. Adjusting the Gamma slider handle changes
the light and dark contrast nonlinearly.
Highlight Saturated Pixels
When this box is checked, any saturated pixels in the image will appear highlighted in
red in the scan window and in the pop-up image window. To view/hide saturated
pixels in the pop-up image window, use the Image > Transform command.
Invert Display
This checkbox will switch light spots on a dark background to dark spots on a light
background, and visa versa. This will only affect how the image is displayed on the
screen, not the actual image data.
Auto-scale
Clicking on Auto-scale will adjust your displayed image automatically. The lightest
part of the image will be set to the minimum intensity (e.g., white), and the darkest
will be set to the maximum intensity (e.g., black). You can then “fine tune” the
display using the High, Low, and Gamma sliders described below.
Reset
Reset will return the image to its original, unmodified appearance.
A.6 Step V. Analysis
The Analysis step of the Gel Doc EQ acquisition window allows you to add
annotations and analyze the newly acquired image.
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PDQuest User Guide
Annotate
Clicking on Annotate will open a separate image window displaying the captured
image. The default name for the image will include the date, time, and user (if
known).
The Text Overlay toolbar will also pop up to allow you to annotate your image.
The image will not be saved until you select Save or Save As from the File menu.
Analyze
Clicking on Analyze will open a separate image window displaying the captured
image. The default name for the image will include the date, time, and user (if
known).
You can then analyze the image using the other features in the main application.
The image will not be saved until you select Save or Save As from the File menu.
A.7 Step VI. Select Output
In Select Output you can select Video Print and Save as your output options.
Fig. A-8. Output options.
Video Print
Clicking on Video Print will automatically send the currently displayed frame (either
live or integrated) to a video printer. You can add information about your image to the
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Appendix A. Gel Doc
bottom of the printout by selecting the appropriate checkboxes in the Options dialog
box. (See section A.9, Options.)
Save
Clicking on Save will open a separate image window displaying the captured image.
A Save As dialog box will automatically open displaying the default file name for the
image, which will include the date, time, and user (if known). You can then change
the file name and storage directory.
You can also export your image as a TIFF image for viewing with other applications.
A.8 Exposure Status
The Exposure Status bar shows the progress of your exposure. If your exposure time
is greater than 1 second, the status bar display will give you a graphical representation
of the remaining time before exposure is complete.
If the exposure time is less than 1 second, the status bar will not refresh itself for each
exposure; it will remain at 100 percent.
A.9 Options
Click on the Options button to open the Options dialog box. Here you can specify
certain settings for your Gel Doc EQ system.
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PDQuest User Guide
Fig. A-9. Available options in the Gel Doc EQ acquisition window.
Click on OK to implement any changes you make in this box. Clicking on Defaults
restores the settings to the factory defaults.
DAC Settings
Note:
The default DAC settings are highly recommended and should be changed with
caution.
These sliders may be used to adjust the minimum and maximum voltage settings of
your video capture board. The minimum slider defines the pixel value that will appear
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Appendix A. Gel Doc
as white in the image, while the maximum slider defines the pixel value that will
appear as black. The slider scale is 0–255, with the defaults set to 60 minimum and
130 maximum.
Imaging Area
These fields are used to specify the size of your imaging area in centimeters, which in
turns determines the size of the pixels in your image (i.e., resolution). When you
adjust one imaging area dimension, the other dimension will change to maintain the
aspect ratio of the camera lens.
Note:
Your imaging area settings must be correct if you want to do 1:1 printing. These
are also important if you are comparing the size of objects (e.g., using the
Volume Tools) between images.
Auto Exposure Threshold
When you click on Auto Expose, the exposure time is determined by the percentage
of saturated pixels you want in your image. This field allows you to specify that
percentage.
Typically, you will want less than 1 percent of the pixels in your image saturated.
Consequently, the default value for this field is 0.15 percent.
Reminder
When this checkbox is selected, the software will warn you to turn off your
transilluminator light when you exit the Gel Doc EQ acquisition window or when
your system is “idle” for more than 5 minutes.
Video Printing Footer Information
The checkboxes in this group allow you to specify the information that will appear at
the bottom of your video printer printouts.
Save Options
To automatically create a backup copy of any scan you create, select the Make
Backup Copy checkbox.
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PDQuest User Guide
With this checkbox selected, when you save a scan, a backup copy will be placed in
the same directory as the scanned image. Backup images are identified by "Backup
of" followed by the original image file name.
Note:
Backup files are read only files. If you attempt to save a backup file, you will be
promted to give it a new name. This protects the backup file and preserves it from
any changes.
Simple Acquisition Mode
The Simple Acquisition mode option allows you to reconfigure the Gel Doc EQ
acquisition window to simple mode. In simple mode, the acquisition window contains
the same steps as the advanced acquisition window with the exception of Step IV,
Optimize display, and Step V, Analysis. Manual exposure is also disabled in simple
mode.
Fig.A-10. Simple Acquisition Window
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Appendix A. Gel Doc
To change the acquisition window to Simple Acquisition Mode, check the box
marked Simple Acquisition Mode. The change will take effect the next time you open
the acquistion window.
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A-16
Appendix B
ChemiDoc EQ
Fig.B-1. ChemiDoc EQ.
Before you can begin acquiring images, the ChemiDoc EQ system must be properly
installed and connected with the host computer. See the ChemiDoc EQ hardware
manual for installation, startup, and operating instructions.
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PDQuest User Guide
To use the ChemiDoc EQ, you will need to have the Bio-Rad-supplied acquisition
board installed in your PC or Macintosh. The drivers for this board will be installed
when you install the main software application.
Make sure that your ChemiDoc EQ camera is turned on. If the camera is not turned
on, the ChemiDoc EQ acquisition window will open and an image capture error will
be displayed.
Simulation Mode
Any of the imaging device acquisition windows can be opened in “simulation mode.”
In this mode, an acquisition window will open and the controls will appear active, but
instead of capturing real images, the window will create “dummy” images of
manufactured data.
You do not need to be connected to an imaging device to open a simulated acquisition
window. This is useful for demonstration purposes or practice scans.
To enter simulation mode, hold down the CTRL key and select the name of the device
from the File menu. The title of the acquisition window will indicate that it is
simulated.
B.1 ChemiDoc EQ Acquisition Window
To acquire images using the ChemiDoc EQ, go to the File menu and select ChemiDoc
EQ.... The acquisition window for the instrument will open, displaying a control
panel and a video display window.
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Appendix B. ChemiDoc
Fig.B-2. ChemiDoc EQ acquisition window
The ChemiDoc EQ video display window will open in “live” mode, giving you a live
video display of your sample. If no image is visible, make sure the camera is on,
check the cable connections, make sure the iris on the camera is not closed, and make
sure that the protective cap is off the camera lens. Also check to see that the
transilluminator is on and working.
The control panel has been arranged from top to bottom to guide you through the
acquisition procedure. There are six basic steps to acquiring an image using the
ChemiDoc EQ:
1.
Position and focus the gel or other object to be imaged.
2.
Select Mode.
3.
Acquire the image.
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4.
Optimize the display.
5.
Analysis.
6.
Select the output.
B.2 Step I. Position Gel
The ChemiDoc EQ window will open in “live” mode, giving you a live video display
of your sample. In this mode, the Live/Focus button will appear selected, and frames
will be captured and displayed at about 10 frames per second, depending on the speed
of your computer.
You can use live mode to zoom, focus, and adjust the aperture on the camera, while
positioning the sample within the area.
Note:
Newer versions of the ChemiDoc EQ feature a motorized zoom lens that can be
controlled directly from the acquisition window using the Iris, Zoom, and Focus
arrow buttons. Click on the Up/Down buttons while viewing your sample in the
window to adjust the lens. These buttons will not be visible if you are connected
to older versions of the ChemiDoc EQ without the motorized zoom lens.
Fig.B-3. Live/Focus button and camera control buttons.
You can also select the Show Alignment Grid checkbox to facilitate positioning.
Note:
After positioning your sample, you should check the Imaging Area dimensions
under Options (see section B.9, Options) to make sure that they conform to the
size of the area you are focusing on. To determine the size of the area you are
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Appendix B. ChemiDoc
focusing on, you can place a ruler in the ChemiDoc EQ cabinet so that it is
visible in the image.
B.3 Step II. Image Mode
The Image Mode option buttons change the type and scale of the data, as well as the
behavior of the ChemiDoc EQ when acquiring an image.
UV
Select this mode for fluorescent samples. With this mode selected, the data will be
measured in linear intensity units.
White Light
Select this mode for reflective and transmissive samples. With this mode selected, the
data will be measured in uncalibrated optical density (uOD) units.
Chemi
This mode is designed for chemiluminescent samples. With this mode selected, the
data is measured in linear intensity units; however, the data is inverted, so that
samples will appear dark on a light background.
Also, Chemi mode changes the behavior of the Auto and Manual Expose functions, as
described above.
B.4 Step III. Acquire Image
For many white light applications, you can skip this step and save and print images
directly from Live/Focus mode.
For UV light, chemiluminescent applications, or faint samples, the ChemiDoc EQ
control panel has several features for creating image exposures. You can take an
automatic exposure based on the number of saturated pixels in the image, you can
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enter a specific exposure time, or you can take a series of exposures and select the
best one.
Note:
“Exposure” refers to the integration of the image on the camera CCD over a
period of time. The effect is analogous to exposing photographic film to light
over a period of time.
Auto Expose
Use Auto Expose if you want to take a single exposure but are uncertain of the
optimal exposure time.
Note:
If you know the approximate exposure time you want (± 3 seconds), you can skip
this step and go directly to Manual Expose.
Click on the Auto Expose button to cancel Live/Focus mode and begin an automatic
exposure. The Auto Expose button will appear selected throughout the exposure.
During the auto exposure, the image is continuously integrated on the camera CCD
until it reaches a certain percentage of saturated pixels. This percentage is set in the
Options dialog box. (Default = 0.15 percent. See section B.9, Options.)
Click on Auto Expose;
button appears selected
Can see Exposure Time
automatically changing
Fig.B-4. Selecting Auto Expose.
Once an image has reached the specified percentage of saturated pixels, it is captured
and displayed in the video display window, Auto Expose is automatically deactivated,
and the exposure time appears active in the Exposure Time field.
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Appendix B. ChemiDoc
At this point, if you are in UV or White image mode, Manual Expose will be
automatically activated. If you are in Chemi mode, the Freeze button will be
automatically activated.
Note:
If you are having difficulty auto-exposing your sample, you can use Manual
Expose to adjust your exposure time directly. Most non-chemi applications only
require an exposure time of a few seconds, which can be quickly adjusted using
Manual Expose.
Manual Expose
If you know the approximate exposure time you want, you can click on the Manual
Expose button. In UV or White image mode, Manual Expose is automatically
activated after Auto Expose is complete.
Manual Expose active;
button appears selected
Exposure Time field active;
adjust number of seconds
Fig.B-5. Setting a manual exposure.
With Manual Expose activated, you can adjust the exposure time directly by changing
the number of seconds in the Exposure Time field. Type in a number or use the arrow
buttons next to the field.
In UV or White image mode, when the specified exposure time is reached, the last
captured image will be displayed in the ChemiDoc EQ image window. The camera
will continue to integrate the image on the CCD, updating the display whenever the
specified number of seconds is reached.
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Once you are satisfied with the quality of the displayed image, click on the Freeze
button to stop the exposure process. The last full exposure will be displayed in the
image window.
Click on Freeze to stop
the exposure process
Fig.B-6. Freezing the manual exposure.
In Chemi mode, Manual Expose will expose an image over the specified exposure
time and then stop automatically.
Note:
Freeze is automatically activated if you adjust any of the subsequent controls
(e.g., Video Print, Image Mode, Display controls, etc.).
Live Acquire
Live Acquire mode allows you to specify an interval over which a series of
progressively longer exposures are taken. All exposures are then displayed on the
screen, and you can choose the one with the best image.
Click on the Live Acquire button. A settings dialog box will open in which you can
specify the total exposure time, starting exposure time, and number of exposures.
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Appendix B. ChemiDoc
Fig.B-7. Live Acquire settings.
Note:
You should specify no more than 10 exposures in the Live Acquire Settings
dialog, to avoid excessive build-up of image background in later exposures. The
fewer the exposures, the less background will be added to the image. See the
Release Notes for additional instructions on reducing background in images
captured using Live Acquire.
Select the Save Images checkbox if you want to automatically save each exposure as
it is taken.
Click on OK in the settings dialog to begin taking exposures. If you selected Save
Images, a Save dialog box will open in which you can specify the base file name and
location of the exposure files. When you click on Save, the exposures will be taken.
The specified number of exposures will be taken at equal intervals between the
starting exposure time and total exposure time. When each exposure is complete, an
image window containing that exposure will open behind the ChemiDoc EQ window.
When the full exposure time has lapsed, all the image windows will move in front of
the ChemiDoc EQ window.
Note that the first exposure will have the base file name (the default base file name is
the computer user name and a time stamp). Each subsequent exposure will have a
version number (v2, v3, v4, etc.) appended to the base file name. The highest version
number will be the final exposure. If you did not elect to auto-save the exposures as
they were created, then each image will be unsaved.
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To stop the Live Acquire, click on the Freeze button or adjust any of the subsequent
controls (e.g., Video Print, Image Mode, Display controls, etc.).
Note:
Exposures captured before freezing will be displayed in image windows.
Study the different images and select the best exposure(s) to keep. You can then
proceed to the next step.
B.5 Step IV. Optimize Display
The Display controls are useful for quickly adjusting the appearance of your image
for output to a video printer. Adjusting these controls will automatically freeze the
video display and allow you to alter the image within the ChemiDoc EQ window.
Fig.B-8. Display controls.
These controls are similar to those in the Transform dialog box.
Note:
The Display controls will only change the appearance of the image. They will not
change the underlying data.
High/Low Sliders
If Auto-scale doesn’t give you the appearance you want, you can use the High and
Low sliders to redraw the image yourself. In white light mode and chemi mode,
dragging the High slider handle to the left will make weak signals appear darker. In
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Appendix B. ChemiDoc
UV mode, dragging the High slider handle to the left will make weak signals appear
brighter. Dragging the Low slider handle to the right will reduce background noise.
You can also type specific High and Low values in the text boxes next to the sliders.
Clicking anywhere on the slider bars will move the sliders incrementally.
Gamma Slider
Some images may be more effectively visualized if their data are mapped to the
computer screen in a nonlinear fashion. Adjusting the Gamma slider handle changes
the light and dark contrast nonlinearly.
Highlight Saturated Pixels
When this box is checked, any saturated pixels in the image will appear highlighted in
red in the scan window and in the pop-up image window. To view/hide saturated
pixels in the pop-up image window, use the Image > Transform command.
Invert Display
This checkbox will switch light spots on a dark background to dark spots on a light
background, and visa versa. This will only affect how the image is displayed on the
screen, not the actual image data.
Auto-scale
Clicking on Auto-scale will adjust your displayed image automatically. The lightest
part of the image will be set to the minimum intensity (e.g., white), and the darkest
will be set to the maximum intensity (e.g., black). You can then “fine tune” the
display using the High, Low, and Gamma sliders described below.
Reset
Reset will return the image to its original, unmodified appearance.
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B.6 Step V. Analysis
The Analysis step of the ChemiDoc EQ acquisition window allows you to add
annotations and analyze the newly acquired image.
Annotate
Clicking on Annotate will open a separate image window displaying the captured
image. The default name for the image will include the date, time, and user (if
known).
The Text Overlay toolbar will also pop up to allow you to annotate your image.
The image will not be saved until you select Save or Save As from the File menu.
Analyze
Clicking on Analyze will open a separate image window displaying the captured
image. The default name for the image will include the date, time, and user (if
known).
You can then analyze the image using the other features in the main application.
The image will not be saved until you select Save or Save As from the File menu.
B.7 Step VI. Select Output
The ChemiDoc EQ window has several output options.
Fig.B-9. Output options.
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Appendix B. ChemiDoc
Video Print
Clicking on Video Print will automatically send the currently displayed frame (either
live or integrated) to a video printer. You can add information about your image to the
bottom of the printout by selecting the appropriate checkboxes in the Options dialog
box. (See section B.9, Options.)
Save
Clicking on Save will open a separate image window displaying the captured image.
A Save As dialog box will automatically open displaying the default file name for the
image, which will include the date, time, and user (if known). You can then change
the file name and storage directory.
You can also export your image as a TIFF image for viewing with other applications.
B.8 Exposure Status
The Exposure Status bar shows the progress of your exposure. If your exposure time
is greater than 1 second, the status bar display will give you a graphical representation
of the remaining time before exposure is complete.
If the exposure time is less than 1 second, the status bar will not refresh itself for each
exposure; it will remain at 100 percent.
B.9 Options
Click on the Options button to open the Options dialog box. Here you can specify
certain settings for your ChemiDoc EQ system.
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PDQuest User Guide
Fig.B-10. Available options in the ChemiDoc EQ acquisition window.
Click on OK to implement any changes you make in this box. Clicking on Defaults
restores the settings to the factory defaults.
DAC Settings
Note:
The default DAC settings are highly recommended and should be changed with
caution.
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Appendix B. ChemiDoc
These sliders may be used to adjust the minimum and maximum voltage settings of
your video capture board. The minimum slider defines the pixel value that will appear
as white in the image, while the maximum slider defines the pixel value that will
appear as black. The slider scale is 0–255, with the defaults set to 60 minimum and
130 maximum.
Imaging Area
These fields are used to specify the size of your imaging area in centimeters, which in
turns determines the size of the pixels in your image (i.e., resolution). When you
adjust one imaging area dimension, the other dimension will change to maintain the
aspect ratio of the camera lens.
Note:
Your imaging area settings must be correct if you want to do 1:1 printing. These
are also important if you are comparing the size of objects (e.g., using the
Volume Tools) between images.
Auto Exposure Threshold
When you click on Auto Expose, the exposure time is determined by the percentage
of saturated pixels you want in your image. This field allows you to specify that
percentage.
Typically, you will want less than 1 percent of the pixels in your image saturated.
Consequently, the default value for this field is 0.15 percent.
Reminder
When this checkbox is selected, the software will warn you to turn off your
transilluminator light when you exit the ChemiDoc EQ acquisition window or when
your system is “idle” for more than 5 minutes.
Note:
If you are performing experiments that are longer than 5 minutes (e.g.,
chemiluminescence), this should be deselected.
Video Printing Footer Information
The checkboxes in this group allow you to specify the information that will appear at
the bottom of your video printer printouts.
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Save Options
To automatically create a backup copy of any scan you create, select the Make
Backup Copy checkbox.
With this checkbox selected, when you save a scan, a backup copy will be placed in
the same directory as the scanned image. Backup images are identified by "Backup
of" followed by the original image file name.
Note:
Backup files are read only files. If you attempt to save a backup file, you will be
promted to give it a new name. This protects the backup file and preserves it from
any changes.
Simple Acquisition Mode
The Simple Acquisition mode option allows you to reconfigure the ChemiDoc EQ
acquisition window to simple mode. In simple mode, the acquisition window contains
the same steps as the advanced acquisition window with the exception of Step IV,
Optimize display, and Step V, Analysis. Manual exposure is also disabled in simple
mode.
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Appendix B. ChemiDoc
Fig.B-11. Simple Acquisition Window
To change the acquisition window to Simple Acquisition Mode, check the box
marked Simple Acquisition Mode. The change will take effect the next time you open
the acquistion window.
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Appendix C
ChemiDoc XRS
Fig.C-1. ChemiDoc XRS.
Before you can begin acquiring images, the ChemiDoc XRS system must be properly
installed and connected with the host computer. See the ChemiDoc XRS hardware
manual for installation, startup, and operating instructions.
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To use the ChemiDoc XRS, you will need to have the Bio-Rad-supplied acquisition
board installed in your PC or Macintosh. The drivers for this board will be installed
when you install the main software application.
Make sure that your ChemiDoc XRS camera is turned on. If the camera is not turned
on, the ChemiDoc XRS acquisition window will not open.
Simulation Mode
Any of the imaging device acquisition windows can be opened in “simulation mode.”
In this mode, an acquisition window will open and the controls will appear active, but
instead of capturing real images, the window will create “dummy” images of
manufactured data.
You do not need to be connected to an imaging device to open a simulated acquisition
window. This is useful for demonstration purposes or practice scans.
To enter simulation mode, hold down the CTRL key and select the name of the device
from the File menu. The title of the acquisition window will indicate that it is
simulated.
C.1 ChemiDoc XRS Acquisition Window
To acquire images using the ChemiDoc XRS, go to the File menu and select
ChemiDoc XRS.... The acquisition window for the instrument will open, displaying a
control panel and a video display window.
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Appendix C. ChemiDoc XRS
Fig.C-2. ChemiDoc XRS acquisition window
When the ChemiDoc XRS window first opens, no image will be displayed.
The control panel has been arranged from top to bottom to guide you through the
acquisition procedure. There are three basic steps to acquiring an image using the
ChemiDoc XRS:
1.
Select the application.
2.
Position and focus the gel or other object to be imaged.
3.
Acquire the image.
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C.2 Step I. Select Application
To set the appropriate parameters for the type of object you are imaging, click on the
Select button under Select Application. You have the option of selecting a UV, White,
or Chemiluminescence application. You can also select a custom setting. Once you
select an application, the name of the application and its settings appear in the Select
application step.
C.2.a. Custom Applications
The Bio-Rad installed applications have pre-set gain and bin settings. If the settings
for the available applications do not meet your needs, you have the option of creating
a custom application, which allows you to set your own binning settings.
To use a custom application, click Select and choose custom. Next, select a custom
application from the list, or select Create to create a new application. This opens the
custom dialog.
Fig.3-3. Creating a custom ChemiDoc XRS application.
Enter a name for the application. From the Illumination drop down list, select an
illumination mode. You can select UV, white or no illumination. Next select Gain and
Binning settings from the drop down list.
A higher Binning setting (2x2, 3x3) provides optimal sensitivity for low-light
applications such as chemiluminescence. In this mode, the pixels in the camera are
“binned” (e.g., four pixels are combined into one) to increase the amount of signal per
pixel without increasing noise. Note that combining the pixels results in a reduction in
the resolution of the image.
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Appendix C. ChemiDoc XRS
Click OK to save and add the new custom application to the custom list. The new
application is automatically selected as your application.
C.3 Step II. Position Gel
Click the Live/Focus button, and frames will be captured and displayed at about 10
frames per second, depending on the speed of your computer.
You can use live mode to zoom, focus, and adjust the aperture on the camera, while
positioning the sample within the area.
Note:
ChemiDoc XRS features a motorized zoom lens that can be controlled directly
from the acquisition window using the Iris, Zoom, and Focus arrow buttons.
Click on the Up/Down buttons while viewing your sample in the window to
adjust the lens.
Fig.C-4. Live/Focus button and camera control buttons.
You can also select the Show Alignment Grid checkbox to facilitate positioning.
Note:
After positioning your sample, you should check the Imaging Area dimensions
under Options (see C.5, Options) to make sure that they conform to the size of
the area you are focusing on. To determine the size of the area you are focusing
on, you can place a ruler in the ChemiDoc XRS hood so that it is visible in the
image.
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C.4 Step III. Acquire Image
The ChemiDoc XRS control panel has several features for creating image exposures.
You can take an automatic exposure based on the number of saturated pixels in the
image, you can enter a specific exposure time, or you can take a series of exposures
and select the best one.
Note:
“Exposure” refers to the integration of the image on the camera CCD over a
period of time. The effect is analogous to exposing photographic film to light
over a period of time.
Auto Expose
Use Auto Expose if you want to take a single exposure but are uncertain of the
optimal exposure time.
Note:
If you know the approximate exposure time you want (± 3 seconds), you can skip
this step and go directly to Manual Expose.
Click on the Auto Expose button to cancel Live/Focus mode and begin an automatic
exposure. The Auto Expose button will appear selected throughout the exposure.
During the auto exposure, the image is continuously integrated on the camera CCD
until it reaches a certain percentage of saturated pixels. This percentage is set in the
Options dialog box. (Default = 0.75 percent. See C.5, Options.)
Click on Auto Expose;
button appears selected
Can see Exposure Time
automatically changing
Fig.C-5. Selecting Auto Expose.
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Appendix C. ChemiDoc XRS
Once an image has reached the specified percentage of saturated pixels, it is captured
and displayed in the video display window, Auto Expose is automatically deactivated,
and the exposure time appears active in the Exposure Time field.
Note:
If you are having difficulty auto-exposing your sample, you can use Manual
Expose to adjust your exposure time directly. Most non-chemiluminescent
applications only require an exposure time of a few seconds, which can be
quickly adjusted using Manual Expose.
Manual Expose
If you know the approximate exposure time you want, you can click on the Manual
Expose button. Manual Expose is automatically activated after Auto Expose is
complete.
Manual Expose active;
button appears selected
Exposure Time field active;
adjust number of seconds
Fig.C-6. Setting a manual exposure.
With Manual Expose activated, you can adjust the exposure time directly by changing
the number of seconds in the Exposure Time field. Type in a number or use the arrow
buttons next to the field.
When the specified exposure time is reached, the last captured image will be
displayed in the ChemiDoc XRS image window.
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Click on Freeze to stop
the exposure process
Fig.C-7. Freezing the manual exposure.
Live Acquire
Live Acquire mode allows you to specify an interval over which a series of
progressively longer exposures are taken. All exposures are then displayed on the
screen, and you can choose the one with the best image.
Click on the Live Acquire button. A settings dialog box will open in which you can
specify the total exposure time, starting exposure time, and number of exposures.
Fig.C-8. Live Acquire settings.
Note:
You should specify no more than 10 exposures in the Live Acquire Settings
dialog, to avoid excessive build-up of image background in later exposures. The
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Appendix C. ChemiDoc XRS
fewer the exposures, the less background will be added to the image. See the
Release Notes for additional instructions on reducing background in images
captured using Live Acquire.
Select the Save Images checkbox if you want to automatically save each exposure as
it is taken.
Click on OK in the settings dialog to begin taking exposures. If you selected Save
Images, a Save dialog box will open in which you can specify the base file name and
location of the exposure files. When you click on Save, the exposures will be taken.
The specified number of exposures will be taken at equal intervals between the
starting exposure time and total exposure time. When each exposure is complete, an
image window containing that exposure will open in front of the ChemiDoc XRS
window.
Note that the first exposure will have the base file name (the default base file name is
the computer user name and a time stamp). Each subsequent exposure will have a
version number (v2, v3, v4, etc.) appended to the base file name. The highest version
number will be the final exposure. If you did not elect to auto-save the exposures as
they were created, then each image will be unsaved.
To stop the Live Acquire, click on the Freeze button.
Note:
Exposures captured before freezing will be displayed in image windows.
Study the different images and select the best exposure(s) to keep.
C.4.a. Exposure Status
The Exposure Status bar shows the progress of your exposure. If your exposure time
is greater than 1 second, the status bar display will give you a graphical representation
of the remaining time before exposure is complete.
Illumination Flat Fielding
For UV or white light transilluminatoin applications, you should use the appropriate
reference plate to ensure a uniform intensity in the image. This will compensate for
normal variations in image pixel intensity that occur with a transilluminating light
source.
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To enable this feature, select the Flat Fielding checkbox.
UV Illumination Flat Fielding: When you first select the Flat fielding checkbox, and
then acquire an image using the UV transilluminator, you will be prompted to remove
your sample and place the fluorescent reference plate on the ChemiDoc XRS sample
stage and turn on the UV transilluminator (see the ChemiDoc XRS User Manual).
Fig.C-9. Illumination flat fielding reminder
A reference image of the plate will be acquired and saved on your computer hard
drive. The reference image will be applied to the sample image to generate a flat field
corrected exposure.
White Light Illumination Flat Fielding: When you first select the checkbox, and then
acquire an image using the white light conversion screen or the white light
transilluminator, you will be prompted to remove your sample, turn on the white light
source, and collect an exposure of the white light screen/transilluminator (see the
ChemiDoc XRS User Manual). A reference image of the screen/transilluminator will
be acquired and saved on your computer hard drive. The reference image will be
applied to the sample image to generate a flat field corrected exposure.
For subsequent UV or white light trans exposures, you will be prompted to either use
the appropriate saved flat field image or acquire a new one.
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Appendix C. ChemiDoc XRS
Fig.C-10. Using the saved reference image.
Note:
Flat fielding is unavailable for White EPI illumination and chemiluminescence
applications.
C.5 Options
Click on the Options button to open the Options dialog box. Here you can specify
certain settings for your ChemiDoc XRS system.
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Fig.C-11. Available options in the ChemiDoc XRS acquisition window.
Click on OK to implement any changes you make in this box.
C.5.a. Imaging Area
These fields are used to specify the size of your imaging area in centimeters, which in
turn determines the size of the pixels in your image (i.e., resolution). When you adjust
one imaging area dimension, the other dimension will change to maintain the aspect
ratio of the camera lens.
Note:
Your imaging area settings must be correct if you want to do 1:1 printing. These
are also important if you are comparing the size of objects (e.g., using the
Volume Tools) between images.
C.5.b. Reminder
Show Light Warning - When this checkbox is selected, the software will warn you
to turn off your transilluminator light when you exit the ChemiDoc XRS acquisition
window or when your system is “idle” for more than 5 minutes.
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Appendix C. ChemiDoc XRS
Note:
If you are performing experiments that are longer than 5 minutes (e.g.,
chemiluminescence), this should be deselected.
Show Filter Warning - When this checkbox is selected, the software will warn you
to move the filter slider either to chemi or the UV/White position.
C.5.c. Dark Subtraction
At the time of installation a reference dark image file was generated and saved to your
hard drive. The purpose of this file is to reduce dark current noise generated from the
CCD. Dark current noise is typical of all CCDs and is a result of the accumulation of
charge in the absence of light
The default time of the reference dark image is ten minutes. The default reference file
is optimized for ChemiDoc XRS applications. If you select to change the reference
dark image, ensure that the time of the reference dark image is of equal or greater
duration than your typical sample image. When the sample image time is longer than
the reference dark image time the ability to detect very faint samples may be
compromised.
To reset the reference dark image time, select options in the ChemiDoc XRS
acquisition window. Select Reset Referenced.
Fig.C-12. Reset Reference Dark pop-up box
Enter the desired time in seconds. Select OK. You will be prompted to close and reopen the acquisition window. Place the lens cap on the camera and close the door of
the hood (See ChemiDoc XRS instruction manual). Select OK at the prompt and new
reference dark images of the entered time interval will be taken for each of the five
possible combinations of camera gain and bin settings. These will be retained as the
reference dark images until the time is reset again.
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C.5.d. Save Options
To automatically create a backup copy of any scan you create, select the Make
Backup Copy checkbox.
With this checkbox selected, when you save a scan, a backup copy will be placed in
the same directory as the scanned image. Backup images are identified by "Backup
of" followed by the original image file name.
Note:
Backup files are read only files. If you attempt to save a backup file, you will be
promted to give it a new name. This protects the backup file and preserves it from
any changes.
C.5.e. Auto-scale Transform
Auto-scale Transform after Acquisition allows the user the option of having the image
automatically perform the Auto-Scale transform function upon completion of image
acquisition. This eliminates the need to transform when the acquisition time was too
short or the iris not opened enough.
To enable the auto-scale transform function, check the box labeled, Auto-Scale
Transform after Acquisition in the Options dialog.
C.5.f. Auto Exposure Threshold
When you click on Auto Expose, the exposure time is determined by the percentage
of saturated pixels you want in your image. This field allows you to specify that
percentage.
Typically, you will want less than 1 percent of the pixels in your image saturated.
Consequently, the default value for this field is 0.75 percent.
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Appendix C. ChemiDoc XRS
C.6 Other Features
Fig.C-13. Other ChemiDoc XRS acquisition window features.
Highlight Saturated Pixels
When this box is checked, any saturated pixels in the image will appear highlighted in
red in the scan window and in the pop-up image window. To view/hide saturated
pixels in the pop-up image window, use the Image > Transform command.
File Size of Images
Image File Size shows the size of the image file you are about to create. This size is
determined by the bin setting used for the selected application.
If you do not have enough computer memory for the specified file size, an error
message will appear when you attempt to acquire an image. (Macintosh users can
increase the application memory partition. See your Macintosh computer
documentation for guidance.)
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Appendix D
GS-800 Imaging Densitometer
Fig.D-1. GS-800 Imaging Densitometer
Before you can begin scanning images with the GS-800 Imaging Densitometer®, your
instrument must be properly installed and connected with the host computer. See the
hardware manual for installation, startup, and operating instructions.
PC Only: A Note About SCSI Cards
The GS-800 is connected to your computer by a Small Computer System Interface
(SCSI) cable. To use the GS-800, you must have a SCSI card installed in your PC. If
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you have a PC with a Windows 98 or Windows ME operating system, you may also
need to load the SCSI and WinASPI drivers that came with the card.
Simulation Mode
Any of the imaging device acquisition windows can be opened in “simulation mode.”
In this mode, an acquisition window will open and the controls will appear active, but
instead of capturing real images, the window will create “dummy” images of
manufactured data.
You do not need to be connected to an imaging device to open a simulated acquisition
window. This is useful for demonstration purposes or practice scans.
To enter simulation mode, hold down the Ctrl key and select the name of the device
from the File menu. The title of the GS-800 acquisition window will indicate that it is
simulated.
Note:
There is no simulated calibration for densitometers.
D.1 GS-800 Acquisition Window
To begin acquiring images, go to the File menu and select GS-800. The acquisition
window for the densitometer will open, displaying a control panel and a scanning
window.
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Appendix D. GS-800
Fig. D-2. GS-800 acquisition window
The scanning window is marked by grid lines that divide the area into square
centimeters. These are numbered 1-40 top to bottom and 1-30 left to right if the light
source is reflective, and 1-40 top to bottom and 1-29 left to right if the light source is
transmissive.
To hide the gridlines, select the Hide Grid checkbox under Options.
Below the main scanning window is the calibration strip window. Every time the
densitometer calibrates, an image of the calibration strip will appear in this window.
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The control panel has been arranged from top to bottom to guide you through the
acquisition procedure. There are five basic steps to scanning an image using the GS800:
1.
Select the application.
2.
Select the scan area.
3.
Select the resolution of the scan.
4.
Calibrate the instrument. (This is automatic, after you enter the step tablet values
before you first scan after installation.)
5.
Acquire the image.
D.2 Step I. Select Application
To set the parameters for your particular scan, you can:
1.
Select from a list of possible applications, or
2.
Choose your own filter and light source settings.
Selecting from the List of Applications
When you select from the list of applications, the software automatically sets the
appropriate filter(s) and other parameters for that particular application.
To select from the list of applications, click on the Select button under Step I. Select
Application.
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Appendix D. GS-800
Fig. D-3. Example of the application tree in the GS-800 dialog box.
The applications are listed in a tree that expands from left to right. First you select the
category of your application, then you select your particular application.
To exit the tree without selecting, press the Esc key.
Gray file
X-ray film
Blue film
Linear
Coomassie Blue
Silver stain
Gel
Copper stain
Zinc stain
Stain all
Positive (dark bands, light background)
Photograph
Negative (light bands, dark background)
Blot
Coomassie stain
Colloidal gold stain
Amido black stain
AP-Substrate (BCIP/NBT)
HRP-Substrate (4CN)
HRP-Substrate (DAB)
Fig. D-4. Applications available in the GS-800 acquisition window.
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PDQuest User Guide
Choosing Your Own Settings
If you know the filter and light source settings you want, or want to experiment with
different settings, you can choose them yourself.
Fig. D-5. GS-800 Custom Application controls.
Next to Filter, click on either the Red, Green, Blue, or White checkbox, or a
combination of two of the first three (Red-Green, Green-Blue, Red-Blue).
Filter Color
Wavelength
Application Examples
Red
595–750 nm
Coomassie G-250, BCIP/NBT, Fast Green
FCF, Methylene Blue
Green
520–570 nm
Coomassie R-250, Basic Fuchsin
Blue
400–530 nm
Crocein Scarlet
White
400–750 nm
Silver Stains, Copper Stains, Film
Fig. D-6. Examples of filter colors and applications.
Next to Light, select the appropriate light source.
•
Select Reflective for scanning opaque mediums such as dried gels on filter paper,
arrays, or photographs. The scanning dimensions in this mode are 30 cm x 40
cm.
•
Select Transmissive for scanning transparent mediums such as films, gels, or
slides. The scanning dimensions in this mode are 29 cm x 40 cm.
D-6
Appendix D. GS-800
D.3 Step II. Select Scan Area
Preview Scan
Before selecting the particular area to scan, you can preview the entire scanning area
to determine the exact position of your sample.
Click on Preview Scan. A quick, low-resolution scan of the entire scanning area will
appear in the scanning window. Your sample should be visible within a portion of this
scan.
Selecting an Area
Using the preview scan as a guide, select your scan area by dragging your mouse
within the scanning window. The border of the scan area you are selecting will be
marked by a frame.
Note:
The scan area you select must be at least 1 cm wide.
When you release the mouse button, the border changes to a dashed blue line,
indicating a selected area.
•
To reposition the scanning area box you have created, position your cursor inside
the box and drag. The entire box will move.
•
To resize the box, position your cursor on a box side and drag. The side you have
selected will move.
•
To redo the box entirely, position your cursor outside the box and drag. The old
box will disappear and a new box will be created.
You can also select the scanning area by entering coordinates in the appropriate fields
(Top, Bottom, Left, Right). As you enter a coordinate, the position of the scanning
area box will change accordingly.
When selecting, be sure to include the entire area of interest, and be generous with
borders. You can always crop the image later.
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PDQuest User Guide
D.4 Step III. Select Resolution
To select from a list of possible scanning resolutions, click on the Select button under
Step III. Select Resolution. This will open the Select Scan Resolution dialog box.
Fig. D-7. Select Scan Resolution dialog box.
Available resolutions are listed from highest to lowest in terms of the dimensions of
the resulting pixels (in microns). Smaller pixels equal higher resolution. Each
resolution is listed with its typical use.
In general, the size of your pixels should be one-tenth the height of your smallest
object.
Specifying Your Own Resolution
If you select Oversample in the Options dialog box, you can specify your own
resolution within the range of 32–169 micrometers. With Oversample selected, enter
values directly in the fields next to X resolution and Y resolution in the main
acquisition window.
D-8
Appendix D. GS-800
Fig. D-8. Entering a custom resolution (with Oversample selected).
Image File Size
The size of the scan file for the selected resolution is listed next to Image File Size. If
you do not have enough computer memory for the specified file size, an error
message will appear when you attempt to scan. If this happens, select a lower
resolution or decrease the size of the area to be scanned. (Macintosh users can also
increase the application memory partition. See your Macintosh computer
documentation for guidance.)
D.5 Calibration
The GS-800 automatically calibrates the densities of scanned images to ensure
accuracy and reproducibility of results. The GS-800 has built-in step tablets for both
transmissive and reflective scanning.
•
The transmissive step tablet calibrates transmissive scans from 0 to 3.0 O.D.
•
The reflective step tablet targets reflective scans from 0 to 2.0 O.D.
The first time you use the GS-800, you must select some settings to ensure accurate
calibration. Click on the Options button in the main acquisition window. This will
open the Densitometer Options dialog box.
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PDQuest User Guide
Fig. D-9. Densitometer Options dialog box.
Note that calibration is always on for the GS-800.
D.5.a Step Tablet Values
The built-in transmissive and reflective step tablets on your densitometer have
specific density values. Before scanning for the first time, you must enter the values
for your transmissive step tablet into the software. You can use the default values in
the software for the reflective step tablet.
Transmissive Step Tablet
Before you scan in transmissive mode for the first time, you must specify the values
for your transmissive step tablet.
First, make sure that the Transmissive checkbox is selected in the main acquisition
window, then click on the Options button. In the Densitometer Options dialog, click
on the Edit Step Tablet button. The Step Tablet Values for GS-800 Transmissive
dialog box will open.
D-10
Appendix D. GS-800
Fig. D-10. Step Tablet dialog box.
Attached to the outside of your GS-800, you will find a copy of the manufacturer’s
printout of the diffuse density values for each segment of the transmissive step tablet,
as well as a serial number for the tablet. These exact density values must be entered
into the software to associate a correct density value with each step on the step tablet.
Note:
Scanning in transmissive mode with incorrect step tablet values can cause
significant errors in the reported densities of your scans.
First, type the serial number for the tablet into the Tablet Serial Number field.
The Quantity Offset field does not apply in the GS-800 in transmissive mode. This
value should remain at zero.
Next, enter the step tablet values into the appropriate fields under the Diffuse column.
After the step tablet is scanned, the software will associate each density value with its
corresponding segment on the step tablet. The density values do not need to be
reentered each time you calibrate.
When you are finished entering the transmissive step tablet values, click on OK.
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PDQuest User Guide
Reflective Step Tablet
For the reflective step tablet, it is recommended that you use the default target values
in the software. Although you can edit these values based on your own measurements
and testing, the default values have been preset based on careful testing and review.
To view or edit the default reflective values, make sure that the Reflective checkbox
is selected in the main acquisition window, then click on the Options button. In the
Densitometer Options dialog, click on the Edit Step Tablet button. The Step Tablet
Values for GS-800 Reflective dialog box will open.
When you are finished viewing/editing the reflective step tablet values, click on OK.
Diffuse Versus Specular O.D.
In the step tablet form, you enter O.D. as diffuse density, and then the software
automatically calculates the specular density.
Specular density is a measure of the light that passes directly through a medium.
Diffuse density includes light that is scattered as it passes through the medium. Step
tablet values are given in diffuse density, but are measured by the scanner in specular
density, and therefore must be converted according to the specular/diffuse O.D. ratio.
This conversion does not affect quantitation.
Specular Light (passes directly through medium)
Medium (gel, film, etc.)
Diffuse Light (is scattered by medium)
Fig. D-11. Specular and diffuse density
Diffuse density values are converted to specular optical density units according to the
following formula:
Specular OD = 1.4 ⋅ Diffuse OD
D-12
Appendix D. GS-800
D.5.b Other Calibration Settings
After you have entered the step tablet values, you can immediately calibrate by
clicking on the Calibrate Now button (in the Densitometer Options dialog box).
You can also specify how often you want the GS-800 to automatically recalibrate.
Either click on the Calibrate Before Every Scan checkbox or specify a period
between automatic recalibrations (in minutes) in the Recalibration Interval field.
Note:
The scanner will automatically recalibrate each time you change your filter or
your reflective/transmissive setting. (If you select a different application with the
same filter and light settings, it will not auto recalibrate.)
To print out a calibration report each time the densitometer calibrates, click on the
Calibration Report checkbox.
D.6 Acquire the Image
Note:
Before scanning in transmissive mode, make sure the white balance region of the
scanning area is not covered or obstructed in any way.
To begin to scan, click on the Acquire button. The scanned image will begin to
appear in the scanning window, line by line.
To interrupt a scan, click on the Stop button. A message will ask you to confirm the
interrupt, and then you will be asked if you want to keep the partial scan. This feature
is useful if you overestimated the size of the area you selected.
After the scan is complete, a window will open displaying the scan image, at which
point you can analyze and save it.
Note:
The image will open with a default file name that includes the date, time, and (if
applicable) user name. However, unless you have selected Auto Save After
Scan, the file will not be saved until you perform a Save or Save As operation.
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PDQuest User Guide
D.7 Other Options
Oversample
This feature allows you to scan at the maximum resolution of the scanner and then use
spatial averaging to create an image with lower resolution. This can result in better
images at lower resolution—however, it takes longer to scan.
To turn on oversampling, click on the Options button in the acquisition window and
select the Oversample checkbox in the options dialog.
With oversampling on, you can specify your own resolution within the range of the
densitometer by entering values directly in the fields next to X resolution and Y
resolution in the main acquisition window.
Auto Save After Scan
To automatically save any scan you create, select the Auto Save After Scan
checkbox.
With this checkbox selected, when you click on Acquire, a Save As dialog box will
open asking you to specify a file name and location for the image you are about to
create. The scan will begin when you click on the Save button.
Make Backup Copy
If you have selected Auto Save After Scan, you can also automatically create a
backup copy of any scan you create.
Click on the Make Backup Copy checkbox. With this checkbox selected, when you
save a scan, a backup copy will be placed in the same directory as the scanned image.
Backup images are identified by "Backup of" followed by the original image file
name.
Note:
Backup files are read only files. If you attempt to save a backup file, you will be
promted to give it a new name. This protects the backup file and preserves it from
any changes.
D-14
Appendix D. GS-800
Highlight Saturated Pixels
When this box is checked, any saturated pixels in the image will appear highlighted in
red in the scan window and in the pop-up image window. To view/hide saturated
pixels in the pop-up image window, use the Transform command on the main toolbar
or Image menu.
Hide Grid
To hide the gridlines in the scanning area window, click on the Hide Grid checkbox.
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PDQuest User Guide
D-16
Appendix E
Personal Molecular Imager FX
Fig.E-1. Personal Molecular Imager FX
Before you can begin acquiring images using the Personal Molecular Imager® FX,
the instrument must be properly installed and connected with the host computer. See
the Personal FX hardware manual for installation, startup, and operating instructions.
Note:
The Personal FX should be turned on and the initialization sequence completed
before the host computer is turned on (except in the case of certain Power
Macintosh configurations). See the hardware manual for more details.
E-1
PDQuest User Guide
PC Only: A Note About SCSI Cards
The Personal FX is connected to your computer by a Small Computer System
Interface (SCSI) cable. To use the Personal FX, you must have a SCSI card installed
in your PC. If you have a PC with a Windows 98 or Windows ME operating system,
you may also need to load the SCSI and WinASPI drivers that came with the card.
Simulation Mode
Any of the imaging device acquisition windows can be opened in “simulation mode.”
In this mode, an acquisition window will open and the controls will appear active, but
instead of capturing real images, the window will create “dummy” images of
manufactured data.
You do not need to be connected to an imaging device to open a simulated acquisition
window. This is useful for demonstration purposes or practice scans.
To enter simulation mode, hold down the CTRL key and select the name of the device
from the File menu. The title of the acquisition window will indicate that it is
simulated.
E.1 Personal FX Acquisition Window
To acquire images using the Personal FX, go to the File menu and select Personal
FX.... The acquisition window for the imager will open, displaying a control panel
and the scanning area window.
E-2
Appendix E. Personal FX
Fig.E-2. Personal FX acquisition window
The default scanning window is marked by grid lines that divide the area into
quadrants. There is also an outer box and inner box marked by thicker lines. The
quadrants are numbered 1–16 left to right and lettered A–U top to bottom.
If you prefer a scanning window measured in centimeters, deselect the Quadrant
Mode checkbox in the control panel by clicking on it. To hide the gridlines, click on
the Hide Grid checkbox under Options.
The control panel has been arranged from top to bottom to guide you through the
acquisition procedure. There are three basic steps to scanning an image using the
Personal FX:
1.
Select the scan area
E-3
PDQuest User Guide
2.
Select the resolution
3.
Acquire the image
E.2 Step I. Select Scan Area
To select a scan area, drag your mouse within the scanning window. (In the scanning
window, your cursor appearance will change to a cross.) The border of the scan area
you are selecting is marked by a frame.
Drag cursor to
define scan
area
Fig.E-3. Selecting a scan area.
If you are in quadrant mode, note that the frame “locks” onto the next quadrant as you
drag. When you release the mouse button, the border changes to a dashed blue line,
indicating a selected area.
•
To reposition the scanning box you have selected, position your cursor inside the
box and drag. The entire box will move.
•
To resize the box, position your cursor on a box side and drag. The side you have
selected will move.
•
To redo the box entirely, position your cursor outside the box and drag. The old
box will disappear and a new box will be created.
E-4
Appendix E. Personal FX
You can also select the scanning area by entering coordinates in the appropriate fields
(Top, Bottom, Left, Right). After you enter a coordinate, the position of the scanning
area box will change accordingly.
When selecting, be sure to include the entire area of interest, and be generous with
borders. You can always crop the image later.
E.3 Step II. Select Resolution
The Personal FX acquisition window allows you to scan at 50, 100, 200, or 800
micrometers. These resolutions are listed as option buttons in the control panel.
Fig.E-4. Resolution option buttons.
The resolution you select should be based on the size of the objects (e.g., bands,
spots) you are interested in. For example:
•
50 micrometer resolution should be reserved for images requiring the highest
level of detail, e.g., high density in situ samples, 1,536-well microplates, high
density arrays, samples with very closely spaced bands. Files scanned at 50
micrometers can be very large.
•
100 micrometer resolution should be used for typical gels and arrays.
•
200 micrometer resolution is useful for gels with large bands and dot blots.
•
800 micrometer resolution should be reserved for very large objects, such as CAT
assays.
E-5
PDQuest User Guide
File Size of Images
Image File Size (below Select Resolution) shows the size of the scan file you are
about to create. If you do not have enough computer memory for the specified file
size, an error message will appear when you attempt to scan. If this happens, select a
lower resolution or decrease the size of the area to be scanned. (Macintosh users can
also increase the application memory partition. See your Macintosh computer
documentation for guidance.)
E.4 Acquire the Image
Once you have selected your scan area and resolution, you ready to acquire an image.
Click on the Acquire button. There may be a short delay while the image laser warms
up; then the scanned image will begin to appear in the scanning window, line by line.
To interrupt a scan, click on the Stop button. A message will ask you to confirm the
interrupt, and then you will be asked if you want to keep the partial scan. This feature
is useful if you overestimated the size of the area you selected.
Note:
If the image you are scanning has more than 10 saturated pixels, you will receive
a warning message.
Saving the Image
After the scan is complete, a message will appear asking you if you want to keep the
scan. If you select Yes, a separate window will pop up containing the new image.
You can then save and analyze the image using the standard menu and toolbar
functions.
E-6
Appendix E. Personal FX
E.5 Options
Auto Save After Scan
To automatically save any scan you create, click on the Auto Save After Scan
checkbox.
Note:
In PDQuest, this option is preselected and cannot be turned off. All images must
be automatically saved when acquired.
With this checkbox selected, when you click on Acquire, a Save As dialog box will
open asking you to specify a file name and location for the image you are about to
create. The scan will begin when you click on the Save button.
Make Backup Copy
You can automatically create a backup copy of any scan you create. To do so, first
select Auto Save After Scan (see above), then select the Make Backup Copy
checkbox.
With this checkbox selected, when you save a scan, a backup copy will be placed in
the same directory as the scanned image. Backup images are identified by "Backup
of" followed by the original image file name.
Note:
Backup files are read only files. If you attempt to save a backup file, you will be
promted to give it a new name. This protects the backup file and preserves it from
any changes.
Highlight Saturated Pixels
When this box is checked, any saturated pixels in the image will appear highlighted in
red in the scan window and in the pop-up image window. To view/hide saturated
pixels in the pop-up image window, use the Image > Transform command.
Hide Grid
To hide the gridlines in the scanning area window, click on the Hide Grid checkbox.
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PDQuest User Guide
Warn on Saturated Pixels
The Warn on Saturated Pixels option is checked by default. Uncheck this box to
disable this warning.
E-8
Appendix F
Molecular Imager FX
Family (FX Pro, FX Pro
Plus and Molecular FX)
Fig. F-1. Molecular Imager FX
Before you can begin acquiring images using any of the Molecular Imager FX family
of products, the particular instrument must be properly installed and connected with
the host computer. See the corresponding FX hardware manual for installation,
startup, and operating instructions.
F-1
PDQuest User Guide
Note:
The FX should be turned on and the initialization sequence completed before the
host computer is turned on (except in the case of certain Power Macintosh
configurations). See the hardware manual for more details.
PC Only: A Note About SCSI Cards
The FX is connected to your computer by a Small Computer System Interface (SCSI)
cable. To use the FX, you must have a SCSI card installed in your PC. If you have a
PC with a Windows 98 or Windows ME operating system, you may also need to load
the SCSI and WinASPI drivers that came with the card.
Simulation Mode
Any of the imaging device acquisition windows can be opened in “simulation mode.”
In this mode, an acquisition window will open and the controls will appear active, but
instead of capturing real images, the window will create “dummy” images of
manufactured data.
You do not need to be connected to an imaging device to open a simulated acquisition
window. This is useful for demonstration purposes or practice scans.
To enter simulation mode, hold down the CTRL key and select the name of the device
from the File menu. The title of the acquisition window will indicate that it is
simulated.
F.1 FX Acquisition Window
To acquire images using the FX, go to the File menu and select FX.... The acquisition
window for the imager will open, displaying the control panel for the imager and the
scanning area window.
F-2
Appendix F. FX
Fig. F-2. FX acquisition window
The default scanning window is marked by grid lines that divide the area into
quadrants. There is also an outer box and inner box marked by thicker lines. The
quadrants are numbered 1–16 left to right and lettered A–U top to bottom.
If you prefer a scanning window measured in centimeters, deselect the Quadrant
Mode checkbox in the control panel by clicking on it. To hide the gridlines, click on
the Hide Grid checkbox under Options.
The control panel has been arranged from top to bottom to guide you through the
acquisition procedure. There are four basic steps to scanning an image using the FX:
1.
Select the application(s).
2.
Select the scan area.
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PDQuest User Guide
3.
Select the resolution.
4.
Acquire the image.
F.2 Step I. Select Application
In Step 1, you select the appropriate filters and other scanning parameters for the type
of gel, blot, plate, or other object that you are imaging. Each set of parameters you
select is called an “application.”
The FX now supports multi-channel sequential scanning. This allows you to
automatically scan the same object using up to four different applications (e.g., to
detect different types of stains on the same gel).
First you select a channel, then you select the application under that channel.
F.2.a Selecting a Channel
The four channels are accessed using the tabs under Step 1. Channel 1 is always
enabled—that is, the FX will always scan using the application settings selected
under Channel 1 first.
To enable any of the remaining channels, click on a channel tab, then select the
Enable Channel checkbox and select the application for that channel as described in
the following section.
F-4
Appendix F. FX
Click on the tab to
select the channel
Select the checkbox
to enable the channel
Click on the Select
button to select the
application
Fig. F-3. Enabling Channel 2.
Enabled channels have a green check mark on their tabs.
You do not need to enable Channels 2–4 in sequence. For example, you can set up
your four most common applications using the different channels, but only enable
Channel 4 for a particular gel. Channel 1 would be scanned first, Channel 4 second.
Enabled channels are scanned sequentially. The separate scans are displayed and
saved as separate images. The total scanning time depends on the number and type of
enabled applications.
If you have selected Auto Save After Scan, the image created using Channel 1 will
have the base file name, and images created using subsequent channels will have the
base file name plus a version number. (v. 2, v. 3, v. 4). Note that the image version
number does not necessarily correspond to the channel number. For example, if you
scanned an image using only Channels 1 and 4, the image created using Channel 4
will be saved as version 2 (v. 2).
F.2.b Selecting an Application
To select an application (i.e., the appropriate filter and other parameters for the type of
object you are imaging), click on the Select button under a channel tab.
F-5
PDQuest User Guide
Fig. F-4. Example of an application tree: Ethidium Bromide gel.
Standard Applications
The standard applications and associated settings are listed in a tree that expands from
left to right. When you select a standard application, the software automatically
selects the appropriate filter(s) in the FX for that particular application. Some
applications are inactive in the FX Pro and FX Pro Plus systems.
Standard FX Applications
Category
Application
Radioisotopes
CS- or BI-Screen (Bio-Rad)1
K-Screen (Kodak)
Fuji-Screen
Chemiluminescence
Chemi-Screen (Bio-Rad)1
Chemifluorescence
ECL-Plus
Attophos
DNA Stain Gel
Ethidium Bromide
Sybr Green I & II
Sybr Gold
F-6
Appendix F. FX
Standard FX Applications
Protein Stain Gel
Sypro Orange
Sypro Red
Nile Red
Sypro Ruby
Fluorophores
Alexa 488
Alexa 532
Alexa 546
FITC
FAM
CY3
HEX
R6G
Texas Red
Microtiter Plate
DNA (Sybr Green I)
Protein (Nano Orange)
ssDNA (Oligreen)
DNA (Picogreen)
B-Gal (FDG)
GUS (FDG)
Densitometry
Coomassie Blue Gel/Blot
Copper Stain Gel/Blot
Silver Stain Gel/Blot
X-Ray Film (Grey Type)
1
Not supported in the FX Pro and FX Pro Plus systems.
First select your general application, next select the particular stain or medium you
are using, and finally (if appropriate) select the intensity of your samples.
Note:
Some applications require an external laser. If you choose one of these without
having an external laser attached, you will receive a warning.
To exit the tree without selecting, press the ESC key.
Your selection and settings will be displayed below the Select button.
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PDQuest User Guide
Sample Intensity
Many FX applications require that you select a sample intensity (High, Medium, or
Low) from the application tree. This is simply a rough estimate of how much sample
is visible in your gel or other object.
If you are unsure of the level of intensity of your sample, you can always select a
level, capture an image, then adjust the level and capture another image.
For example, if you select Low Sample Intensity and the resulting image has too
many saturated pixels, you will receive a warning message. Simply change the setting
to Medium Sample Intensity and rescan. If you select High Sample Intensity and the
resulting image is too faint, select Medium or Low and rescan.
Custom Applications
If your application is not listed, if you want to use user-installed filters, or if you want
to use an external laser, you can create and save your own custom application.
From the application tree, select Custom, then Create. This will open a dialog box in
which you can name your application and select your settings.
Fig. F-5. Creating a custom application.
F-8
Appendix F. FX
To select a filter (including user-defined) or filter combination, click on the buttons
for Filters A, B, and C, and make your choice from each pop-up list.
Note:
Filter wheel C is not available in the FX Pro or the FX Pro Plus systems.
Note:
The user-defined filters (User1, User2, etc.) cannot be renamed in the pop-up list,
so be sure to remember which filter you insert into each position in the FX (i.e.
690 nm filter, 605 nm BPfilter).
To use an external laser, click on the Laser button and select it from the pop-up list
(i.e. 488 nm or 635 nm laser). Otherwise, use the default internal laser (532/1064nm).
Note:
The dual laser (532/1064 nm) is not available in the FX Pro or the FX Pro Plus
systems.
Click on the PMT Voltage button to select a standard voltage for your custom
application or create a custom PMT voltage.
To select a custom voltage, click on the Custom option. In the dialog box, adjust the
slider to select a PMT voltage as a percentage of the maximum.
Fig. F-6. Selecting a custom PMT voltage.
Note:
For voltages above 80% of maximum, you will receive a warning message that
the high voltage could damage the PMT.
If you select Choose Later from the list of PMT voltages, the choices of sample
intensity will be displayed when you select your custom application.
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PDQuest User Guide
Finally, enter a name for your application in the Name field and click on OK to
implement your changes.
After you have created an application, you can select it from the application tree by
selecting Custom and the name you created. You can delete the application by
selecting Custom, Delete, and the name of the application.
Fig. F-7. Selecting a custom application.
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Š™™•’ŒŠ’˜— ›˜– Š— Ž¡’œ’— ˜—Ž
Fig. F-8. Application selection and settings.
F-10
Appendix F. FX
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F.3 Step II. Select Scan Area
To select a scan area, drag your mouse within the scanning window. (In the scanning
window, your cursor appearance will change to a cross.) The border of the scan area
you are selecting is marked by a frame.
Drag cursor to
define scan
area
Fig. F-9. Selecting a scan area.
If you are in quadrant mode, note that the frame “locks” onto the next quadrant as you
drag. When you release the mouse button, the border changes to a dashed blue line,
indicating a selected area.
•
To reposition the scanning box you have selected, position your cursor inside the
box and drag. The entire box will move.
•
To resize the box, position your cursor on a box side and drag. The side you have
selected will move.
•
To redo the box entirely, position your cursor outside the box and drag. The old
box will disappear and a new box will be created.
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PDQuest User Guide
You can also select the scanning area by entering coordinates in the appropriate fields
(Top, Bottom, Left, Right). After you enter a coordinate, the position of the scanning
area box will change accordingly.
When selecting, be sure to include the entire area of interest, and be generous with
borders. You can always crop the image later.
F.4 Step III. Select Resolution
The FX acquisition window allows you to scan at 50, 100, 200, or 800 micrometers.
These resolutions are listed as option buttons in the control panel.
Fig. F-10. Resolution option buttons.
The resolution you select should be based on the size of the objects (e.g., bands,
spots) you are interested in. For example:
•
50 micrometer resolution should be reserved for images requiring the highest
level of detail, e.g., high density in situ samples, 1,536-well microplates, high
density arrays, samples with very closely spaced bands. Files scanned at 50
micrometers can be very large.
•
100 micrometer resolution is useful for typical gels and arrays.
•
200 micrometer resolution is useful for gels with large bands and dot blots.
•
800 micrometer resolution should be reserved for very large objects, such as CAT
assays.
F-12
Appendix F. FX
File Size of Images
Image File Size (below Select Resolution) shows the size of the scan file you are
about to create. If you do not have enough computer memory for the specified file
size, an error message will appear when you attempt to scan. If this happens, select a
lower resolution or decrease the size of the area to be scanned. (Macintosh users can
also increase the application memory partition. See your Macintosh computer
documentation for guidance.)
F.5 Acquire the Image
Once you have selected your application, scan area, and resolution, you are ready to
acquire an image.
Click on the Acquire button. There may be a short delay while the image laser warms
up; then the scanned image will begin to appear in the scanning window, line by line.
To interrupt a scan, click on the Stop button. A message will ask you to confirm the
interrupt, and then you will be asked if you want to keep the partial scan. This feature
is useful if you overestimated the size of the area you selected.
Note:
If the image you are scanning has more than 10 saturated pixels, you will receive
a warning message. If this happens, you can go back and select a higher sample
intensity in the application tree.
Saving the Image
After the scan is complete, a message will appear asking you if you want to keep the
scan. If you select Yes, a separate window will pop up containing the new image.
You can then save and analyze the image using the standard menu and toolbar
functions.
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PDQuest User Guide
F.6 Options
Auto Save After Scan
To automatically save any scan you create, click on the Auto Save After Scan
checkbox.
With this checkbox selected, when you click on Acquire, a Save As dialog box will
open asking you to specify a file name and location for the image you are about to
create. The scan will begin when you click on the Save button.
If you are scanning using multiple channels, the image created using Channel 1 will
be saved using the base file name, and images created using subsequent channels will
have the base file name plus a version number. (v. 2, v. 3, v. 4). Note that the image
version number does not necessarily correspond to the channel number. For example,
if you scanned an image using only Channels 1 and 4, the image created using
Channel 4 will still be saved as version 2 (v. 2).
Make Backup Copy
You can automatically create a backup copy of any scan you create. To do so, first
select Auto Save After Scan (see above), then select the Make Backup Copy
checkbox.
With this checkbox selected, when you save a scan, a backup copy will be placed in
the same directory as the scanned image. Backup images are identified by "Backup
of" followed by the original image file name.
Note:
Backup files are read only files. If you attempt to save a backup file, you will be
promted to give it a new name. This protects the backup file and preserves it from
any changes.
Highlight Saturated Pixels
When this box is checked, any saturated pixels in the image will appear highlighted in
red in the scan window and in the pop-up image window. To view/hide saturated
pixels in the pop-up image window, use the Image > Transform command.
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Appendix F. FX
Hide Grid
To hide the gridlines in the scanning area window, click on the Hide Grid checkbox.
Warn on Saturated Pixels
The Warn on Saturated Pixels option is checked by default. Uncheck this box to
disable this warning.
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PDQuest User Guide
F-16
Appendix G
VersaDoc
Fig.G-1. VersaDoc.
Before you can acquire images using the VersaDoc Imaging System, you need to
install the Roper Scientific interface adapter and its associated device drivers in your
computer. The driver installation procedures for Windows operating systems inlcude
pre-installation and use of the Windows Hardware Wizards.
Note:
Important! Please read and follow the driver installation procedures contained in
the README documents before you install the VersaDoc camera interface
adapter. You can find the README documents in the VersaDoc Drivers folders
on The Discovery Series (TDS) CD.
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PDQuest User Guide
The CD also contains Roper Scientific camera interface driver installation
instructions for Macintosh computers.
See the VersaDoc hardware manual for further installation, startup, and operating
instructions for the VersaDoc Imaging System.
G.1 VersaDoc Acquisition Window
To acquire images using the VersaDoc, go to the File menu and select VersaDoc....
The acquisition window for the instrument will open, The acquisition window opens,
displaying a control panel and an image display window.
Fig.G-2. VersaDoc acquisition window.
When the VersaDoc window first opens, no image will be displayed.
The control panel has been arranged from top to bottom to guide you through the
acquisition procedure. There are four basic steps to acquiring an image using the
VersaDoc:
G-2
Appendix G. VersaDoc
1.
Select the application.
2.
Position and focus the object to be imaged.
3.
Set the exposure time.
4.
Acquire the image.
G.2 Step I. Select Application
In Step 1, you select the appropriate filters and other imaging parameters for the type
of gel, blot, plate, or other object that you are imaging. Each set of parameters you
select is called an application.
The VersaDoc supports multichannel sequential imaging. This allows you to
automatically image the same object using up to four different applications (e.g., to
detect different types of stains on the same gel).
First you select a channel, then you select the application under that channel.
G.2.a. Selecting a Channel
Note:
Illumination Flat Fielding will be disabled if you use multiple channels.
The four channels are accessed using the tabs under Step 1. Channel 1 is always
enabled—that is, the VersaDoc will always capture an image using the application
settings selected under Channel 1 first.
To enable any of the remaining channels, click on a channel tab, then select the
Enable Channel checkbox and select the application for that channel as described
below.
Enabled channels have a green check mark on their tabs.
Note:
If you use the Optimize Exposure feature, only the application under the selected
channel tab will be imaged; other channels, even if enabled, will be ignored.
You do not need to enable Channels 2–4 in sequence. For example, you can set up
your four most common applications using the different channels, but only enable
Channel 4 for a particular gel. Channel 1 would be scanned first, Channel 4 second.
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PDQuest User Guide
Enabled channels are imaged sequentially. The separate scans are displayed and saved
as separate images. The total imaging time depends on the number and type of
enabled applications.
If you have selected Auto Save After Scan, the image created using Channel 1 will
have the base file name, and images created using subsequent channels will have the
base file name plus a version number. (v. 2, v. 3, v. 4). Note that the image version
number does not necessarily correspond to the channel number. For example, if you
captured an image using only Channels 1 and 4, the image created using Channel 4
will be saved as version 2 (v. 2).
G.2.b. Selecting an Application
To select an application (i.e., the appropriate filter and illumination source for the type
of object you are imaging), click on the Select button under a channel tab.
Standard Applications
The standard applications and associated settings are listed in a tree that expands from
left to right. When you select an application, the software automatically sets the
appropriate filter in the VersaDoc for that particular application.
First select your general application, then select the particular stain or medium you
are using. When you select the stain or medium, the software automatically sets the
appropriate filter (520LP, 530DF60, 610LP, clear, or none), light type (UV, white, or
none), and light source (Trans, Epi, or neither) in the VersaDoc for that particular
application.
G-4
Appendix G. VersaDoc
Fig.G-3. The application tree in the VersaDoc acquisition window.
Note:
If you select an application that requires trans white illumination, you will need
to place the white light conversion screen on the sample stage area.
To exit the tree without selecting, press the Esc key or click outside the dialog box.
Your selection will be displayed below the Select button.
Custom Applications
If your application is not listed, if you want to use a user-installed filter, or if you want
to set the gain and bin settings manually, you can create and save your own custom
applications.
From the application tree, select Custom, then Create. This will open a dialog box in
which you can name your application and select your settings.
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PDQuest User Guide
Fig.G-4. Creating a new custom application.
Enter a name for your application in the Name field.
Next, click on the buttons next to the Filter, Illumination, Gain and Binning fields to
change these settings.
Note:
Note that if you select trans white illumination, you will need to place the white
light conversion screen on the sample stage.
A higher Binning setting (2x2, 3x3) provides optimal sensitivity for low-light
applications such as chemiluminescence. In this mode, the pixels in the camera are
“binned” (e.g., four pixels are combined into one) to increase the amount of signal per
pixel without increasing noise. Note that combining the pixels results in a reduction in
the resolution of the image.
Selecting a higher Gain (4x) provides higher sensitivity without reduced resolution;
however, noise will also increase. This is useful for faint signals (bright spots will
saturate). Selecting a lower gain (0.5x) is useful for brighter images that tend to
saturate. A gain setting of 1x provides the greatest dynamic range.
Click OK to implement your changes.
After you have created an application, you can select it from the application tree by
selecting Custom and the name you created.
G-6
Appendix G. VersaDoc
You can edit a custom application by selecting Custom, Edit, and the name of the
application. You can also use this feature to create a new custom application from an
existing one.
You can delete a custom application by selecting Custom, Delete, and the name of the
application.
G.3 Step II. Position/Focus
Before positioning and focusing the image, select the application that you will be
using under Step I.
Note that the optimal position and focus will be different for a filtered image versus
an unfiltered one. (All standard applications except Chemiluminescence use a filter.)
Position
After you have selected your application, you are ready to center your gel or other
object within the camera frame. To do so, click on the Position button. The VersaDoc
will begin capturing a “live” image and updating it every second.
With the Position button selected, study the image in the acquisition window while
you position your object in the center of the sample stage. If you have a zoom lens on
the camera, you can adjust the magnification while you position. (See the VersaDoc
User Manual for details on positioning.)
While you are positioning, you can select the Show Alignment Grid checkbox to
display a target grid overlay on the image.
When you click on Position, the light inside the camera box automatically switches
on. To turn the light off while positioning, deselect the Light On checkbox.
When you are finished positioning, click on the Stop button.
Focus
Note:
Before focusing, you should adjust the f-stop on the camera to the lowest setting
(i.e., the maximum aperture). This reduces the depth of field, allowing you to
more accurately focus the camera. Then, after focusing, increase the f-stop to the
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PDQuest User Guide
desired setting. See Table 1, “Recommended Exposure Times and Lenses,” on
page 9.
After you have positioned your sample, click on the Focus button and look at the
image in the acquisition window while adjusting the focus on the camera lens. While
focusing, the camera will limit its focus to a small portion of the sample (this will not
affect any zoom lens adjustments you may have made.) (See the VersaDoc User
Manual for details on focusing.)
When you click on Focus, the light inside the camera box automatically switches on.
To turn the light off while positioning, deselect the Light On checkbox.
When you are finished focusing, click on the Stop button.
G.4 Step III. Set Exposure Time
The “exposure time” is the period of time an image is integrated on the CCD. The
effect is analogous to exposing photographic film to light.
Setting an Exposure Time
Different applications have different optimal exposure times. If you are imaging using
multiple channels, you can select a different exposure time for each channel. First
select the channel using the tabs, then select the appropriate exposure time for that
channel.
See Table 1, “Recommended Exposure Times and Lenses,” on page 9.
You can enter an exposure time (in seconds) directly in the field, or use the Arrow
buttons to adjust the exposure time in 10 percent increments.
Fig.G-5. Selecting an exposure time.
G-8
Appendix G. VersaDoc
The following table provides recommended exposure times for various applications
Table 1: Recommended Exposure Times and Lenses
Recommended
Exposure
Lens & Filter
Accessories Used
Fluorescent Stain Gel
3–30 sec.
Zoom/IR
None
Fluorescence EndLabel Gel
30 sec.–5 min.
Zoom/IR
None
Fluorescent Blot
0.5–5 sec.
Zoom/IR
Sample/Chemi
Tray (if sample is
small)
Chemifluorescent
Blot
0.5–5 sec.
Zoom/IR
None
Colorimetric Gel
0.1–1 sec.
Zoom/IR
White light conversion screen
Colorimetric Blot
0.1–1 sec.
Zoom/IR
Sample/Chemi
Tray (if sample is
small)
X-ray film
0.1–1 sec.
Zoom/IR
White light conversion screen
Weak
Chemiluminescence1
5–10 min.
50 mm
Sample/Chemi
Tray
(if sample is
small)
Strong
Chemiluminescence1
10 sec.–2 min.
50 mm
Sample/Chemi
Tray
(if sample is
small)
Sample
1
For chemi applications, the 50mm lens is recommended. Always remove the 660 filter.
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PDQuest User Guide
Note:
For most applications, you can select an exposure time, capture an image, study
it, and then adjust the exposure time accordingly. Repeat this procedure as many
times as necessary to obtain a good image. For chemiluminescent samples, which
degrade over time and emit low levels of light, select a high exposure time
initially or use the Optimize Exposure command described on page 12.
Preview
For shorter exposures, you can use Preview to test different exposure times. Click on
the Preview button to create a preview exposure and display it in the acquisition
window.
Note:
The camera on the VersaDoc must be at the correct operating temperature before
capturing images. The temperature adjustment can take several minutes after the
camera is turned on, depending on your VersaDoc model and the ambient room
temperature. See your hardware manual for details.
A preview scan takes only half as long to create as a real scan, because the preview
scan does not capture a “dark” image. The progress of the exposure will be displayed
in the Exposure Status bar at the bottom of the dialog box.
You cannot save preview scans.
If you want to stop a preview scan that is in progress, click on the Stop button.
G.5 Acquire the Image
You can acquire a single exposure for each channel based on the time selected in Step
III (Set Exposure Time), or take a series of exposures for a particular channel over a
specified interval (Optimize Exposure).
Illumination Flat Fielding
For applications using the UV or white light transillumination, you should use the
appropriate reference plate to ensure a uniform intensity in the image. This will
compensate for normal variations in image pixel intensity that occur with a
transilluminating light source.
G-10
Appendix G. VersaDoc
To enable this feature, select the Flat Fielding checkbox.
Note:
For applications using the UV or white light transillumination, you should use
the appropriate reference plate to ensure a uniform intensity in the image. This
will compensate for normal variations in image pixel intensity that occur with a
transilluminating light source.
UV Illumination Flat Fielding: When you first select the Flat fielding checkbox, and
then acquire an image using the UV transilluminator, you will be prompted to remove
your sample and place the fluorescent reference plate on the VersaDoc sample stage
(see the VersaDoc User Manual). A reference image of the plate will be acquired and
saved on your computer hard drive. The reference image will be applied to the sample
image to generate a Flat Field corrected image.
White Illumination Flat Fielding: When you first select the checkbox, and then
acquire an image using the white light conversion screen, you will be prompted to
remove your sample and collect an exposure of the conversion screen. A reference
image of the screen will be acquired and saved on your computer hard drive. The
reference image will be applied to the sample image to generate a Flat Field corrected
image.
For subsequent UV or white light trans exposures, you will be prompted to either use
the appropriate saved Flat Fielding image or acquire a new one. Any changes in light
source, filter, or lens setting will require the acquisition of a new Flat Fielding
reference image.
Acquire
Click on the Acquire button to capture a single image for each enabled channel. An
exposure will be taken for each enabled channel based on the time selected for that
channel in Step III.
The progress of each exposure will be displayed in the Exposure Status bar at the
bottom of the acquisition window.
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PDQuest User Guide
Fig.G-6. Exposure Status bar when acquiring an image.
Depending on which dark subtraction type you have selected under Options (see
section G.6, Options), a dark image may be acquired immediately following image
acquisition.
If you want to stop an acquisition that is in progress, click on the Stop button. The
current acquisition will be terminated. If multiple channels are selected, you must
click on the Stop button once per channel to stop all acquisitions.
After an image has been acquired, a separate window will pop up containing the new
image. You can then analyze the image using the analysis functions.
Optimize Exposure
Optimize Exposure allows you to specify an interval over which a series of
progressively longer exposures are taken. All exposures are then displayed on the
screen, and you can choose the one that provides the best image.
Note:
Multiple exposures will be taken for only the selected multichannel tab. Other
channels, even if enabled, will not be used. Illumination Flat Fielding will be
disabled if you are using Optimize Exposure.
Click on the Optimize Exposure button. A settings dialog box will open in which you
can specify the total exposure time, starting exposure time, and number of exposures.
(The specified number of exposures will be taken at regular intervals between the
starting exposure time and the total exposure time.)
G-12
Appendix G. VersaDoc
Fig.G-7. Optimize Exposure dialog.
Note:
You should specify no more than 10 exposures in the Optimize Exposure dialog,
to avoid excessive build up of image background in later exposures. The fewer
the exposures, the less background will be added to the image. See the Release
Notes for additional instructions on reducing background in images captured
using Optimize Exposure.
Select the Save Images checkbox if you want to automatically save each exposure as
it is taken.
Click on OK in the settings dialog to begin taking exposures. If you selected Save
Images, a Save dialog box will open in which you can specify the base file name and
location of the exposure files. When you click on Save, the exposures will be taken.
The specified number of exposures will be taken at equal intervals between the
starting exposure time and total exposure time. The exposure status bar will show the
progress of each exposure.
Depending on which dark subtraction type you have selected under Options, a dark
image may be acquired immediately following each exposure.
When each exposure is complete, an image window containing that exposure will
open in front of the VersaDoc window. Subsequent exposures are tiled in front of the
VersaDoc window.
Note that the first exposure will have the base file name; the default base file name is
the computer user name and a time stamp. Each subsequent exposure will have a
version number (v2, v3, v4, etc.) appended to the base file name. (If you are using the
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PDQuest User Guide
default base file name, the time stamp may change in the course of the series of
exposures; in this case, the base file name will change and the version numbering will
reset for subsequent exposures.)
The highest version number will be the final exposure. If you did not elect to autosave the exposures as they were created, then each image will be unsaved.
To stop the image acquisitions, click on the Stop button.
Note:
Exposures captured before stopping will be displayed in image windows.
Study the different images and select the best exposure(s) to keep.
G.6 Options
Click on the Options button to open the Options dialog box.
Fig.G-8. Options dialog box.
G.6.a. Dark Subtraction Type
All CCD cameras accumulate electrons that produce a signal that is indistinguishable
from light. This “dark current” adds to the noise in your images, particularly for long
G-14
Appendix G. VersaDoc
exposures. In most cases, you will want to subtract this dark current from your
images.
The settings for subtracting the dark current are in the Options dialog box. Click on
the Options button in the acquisition window to open this dialog.
Normal
The Normal option button selects the default dark subtraction type. In this mode, after
you acquire an image, a “dark” image of the same exposure length will be taken, and
this will be subtracted from your image.
The progress of the dark exposure will be displayed in the Exposure Status bar
following the regular image exposure.
In Normal mode, a dark image is only acquired the first time you perform a scan with
particular application and exposure settings. If you perform subsequent scans with the
same settings, no dark exposure will be taken.
Reference
If you do not want to perform a dark exposure with each acquisition, you can take a
“reference” dark exposure that will be saved and subtracted from all subsequent
acquisitions. Click on the Referenced button to activate this feature.
The first time you acquire an image after selecting this option, the VersaDoc will take
a dark exposure that will be saved and used to subtract the dark current from all
subsequent acquisitions.
Note:
The VersaDoc 1000 will take a 60-second reference dark exposure; the VersaDoc
3000 and 5000 will take a 180-second reference dark exposure.
For image exposures that are longer or shorter than the reference dark, the reference
dark will be scaled accordingly and then subtracted. It is recommended that the
reference dark exposure time be equal to or greater than the sample exposure time.
You can change the default reference dark exposure time using the Reset Reference
button (see below).
If you deselect the Referenced button and then reselect it, the old reference dark
exposure will still be available.
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PDQuest User Guide
Separate reference dark exposures will be taken for images that have different levels
of binning or gain. Once you have created a reference dark for each level of binning
or gain, the appropriate reference dark will be used according to the settings of your
selected application.
Reset Reference
If you would like a reference dark with an exposure time that more closely matches
that of your typical scans, click on the Reset Reference button.
A pop-up box will prompt you to enter a new dark exposure time in seconds.
Fig.G-9. Reset Reference Dark pop-up box.
Click on OK to implement your change. The new reference dark will be created when
you acquire your next image.
Because of the high sensitivity of the CCD, fluctuations in background radiation and/
or temperature in the room can affect the level of dark count. If you feel that radiation/
temperature conditions have changed in the room since your last reference dark was
created, use the Reset Reference button to delete your old reference and create a new
one under current conditions.
None
If you do not want to perform dark subtraction, select None. No dark exposure will be
acquired or subtracted.
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Appendix G. VersaDoc
G.6.b. Save
Auto Save After Scan
To automatically save any image you acquire using the Acquire button, click on the
Auto Save After Scan checkbox in the Options dialog box.
With this checkbox selected, when you click on Acquire, a Save As dialog box will
open asking you to specify a file name and location for the image you are about to
create. The scan will begin when you click on the Save button.
Make Backup Copy
You can automatically create a backup copy of any scan you create. To do so, first
select Auto Save After Scan, then select the Make Backup Copy checkbox.
With this checkbox selected, when you save a scan, a backup copy will be placed in
the same directory as the scanned image. Backup images are identified by "Backup
of" followed by the original image file name.
Note:
Backup files are read only files. If you attempt to save a backup file, you will be
promted to give it a new name. This protects the backup file and preserves it from
any changes.
G.6.c. Imaging Area Size
The imaging area is the area of the sample (in centimeters) that is captured by the
camera and displayed in the scan window. To specify the size of this area, enter a
dimension in the appropriate field under Imaging Area in the Options dialog box.
Click on the Options button in the acquisition window to open this dialog.
When you change one imaging area dimension, the other will change to maintain the
aspect ratio of the camera lens.
The imaging area will change depending on your zoom factor. For example, if you
have zoomed in on a area that is 4.5 x 3.5 cm, then you would enter 4.5 for the width
(3.5 for the height would be calculated automatically).
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PDQuest User Guide
Note:
Your imaging area settings must be correct if you want to do 1:1 printing. They
must also be correct if you want to compare the quantities of objects (e.g., using
the Volume Tools) in different images.
The imaging area dimensions also determine the size of the pixels in your image (i.e.,
resolution). A smaller imaging area will result in a higher resolution.
G.6.d. Auto-scale Transform
Auto-scale Transform after Acquisition allows the user the option of having the image
automatically perform the Auto-Scale transform function upon completion of image
acquisition. This eliminates the need to transform an image or re-scan an image when
the acquisition time was too short or the iris not opened enough.
To enable the auto-scale transform function, check the box labeled, Auto-Scale
Transform after Acquisition in the Options dialog.
G.7 Other Features
Fig.G-10. Other VersaDoc acquisition window features.
Highlight Saturated Pixels
When this box is checked, any saturated pixels in the image will appear highlighted in
red in the scan window and in the pop-up image window. To view/hide saturated
pixels in the pop-up image window, use the Transform command.
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Appendix G. VersaDoc
File Size of Images
Image File Size shows the size of the image file you are about to create. This size is
determined by the resolution of the camera and any binning you perform when
capturing the image.
If you do not have enough computer memory for the specified file size, an error
message will appear when you attempt to acquire an image.
Macintosh users can increase the application memory partition. See your Macintosh
computer documentation for guidance.
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G-20
Appendix H
Calibration and Merging
You can use calibration strips (calstrips) to calibrate your scans for more accurate
quantitation. Calstrips are required for both gel calibration and merging multiple
exposures.
H.1 Calibrating Gels
The commands for calibrating gels are located on the Edit > Calibrate Gels submenu.
Fig. H-1. Edit > Calibrate Gels submenu.
Creating a Calstrip File
If calstrip images are currently part of your gel images, you will have to crop them
and save them as separate files for use in PDQuest. To do so, select Advanced Crop >
Define Calstrip Area from the Image menu, draw a box around your calstrip, then
crop it and save it as a calstrip file. See section 3.13.a for instructions.
H-1
PDQuest User Guide
Configuring Calstrip Subwindows
If you are displaying multiple calstrips linked to multiple exposures (see section H.2,
Merging Multiple Exposures), Configure Calstrip Windows on the Edit > Calibrate
Gels submenu will automatically arrange the calstrip images in subwindows.
H.1.a Calibration Strip Data
Values and units for each segment of your calstrip must be entered into a file called a
Calstrip Record prior to calibration.
To do this, select Edit Calstrip Record from the Edit > Calibrate Gels submenu. The
Calstrip Record dialog box will open.
Fig. H-2. Calstrip Record Editor dialog box.
H-2
Appendix H. Techniques
Enter a title for the data record next to the Name prompt. You can enter descriptive
information about the calstrip in the Description box.
Next, select the Detection method/stain that was used to make the calstrip.
For Fluorography and Autoradiography detection methods, select the Isotope used.
Also enter the date the calstrip was made next to the Counting date prompt. The
format in which the date should be entered is dd-mmm-yyyy (e.g., 01-Nov-1993 or
01-11-1993). The first three letters of the name of the month or the number of the
month may be entered.
Specify the units for the quantitative values by clicking on one of the buttons next to
the Units prompt or typing the units in the text box next to the Other button.
Finally, enter the Segment values for each segment of the calstrip. These values
should correspond to the calstrip segments as they appear from left to right (i.e., if the
darkest segments are on the left, the values will decrease).
Range of Segment Values and Spot Quantities
The range of segment values should be approximately 2.0 to 2 x 104. The minimum
value the Calstrip Record will accept is 2.0. A typical maximum is 2 x 104, but this
value may vary. The range should span four orders of magnitude.
Typical spot quantities will range from 10-2 to 5 x 103, depending on the size of the
spot.
An example of segment values (DPM/cm2) for an 35S calstrip are:
Segment #:
1.
2.
3.
4.
5.
6.
278579.0
159376.0
100299.0
67078.0
40895.0
24686.0
7.
8.
9.
10.
11.
12.
14892.0
9207.0
5791.0
3670.0
2220.0
1396.0
13.
14.
15.
16.
17.
18.
827.0
516.0
315.0
134.0
93.0
44.0
Such a calstrip will produce spot quantitation values that typically range from 2.4
DPM to 502.6 DPM.
If you are working with Coomassie gels and you want to make a calstrip, you should
begin with a stock solution and make approximately 10 serial dilutions, each of which
will be used to make one segment of the calstrip.
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PDQuest User Guide
For example, you might begin with a 50 µg/ml stock solution of BSA and make
segments of the calstrip starting at 50 µg and progressing down to 5.0 ng in constant
ratio steps.
In order to enter numerical values in the Calstrip Record that will produce
interpretable spot quantitation values, convert all the numbers to ng (i.e., multiply all
µg values by 103).
For the example mentioned above, the values would range from 50,000 ng to 5.0 ng.
Saving/loading Calstrip Records
When you have completed the information in the dialog box, click on the Save button
at the bottom of the box.
To access the information in an existing Calstrip Record, click on the Load button. A
list of saved Calstrip Records will be displayed. Select the one whose data you wish
to see by clicking on it. The data associated with that record will be displayed on the
form.
To delete a saved Calstrip Record, click on the Delete button.
Click on the Cancel button to close the Calstrip Record Editor form.
H.1.b Scan Information
After the calibration data has been entered, you will also need to enter some
information about the gel that goes with it.
Start by loading the 2-D and calstrip scans. Display the calstrip scans by using the
Configure Calstrip Windows function (Edit > Calibrate Gels > Configure Calstrip
Windows).
Click on Gel Record in the Calibrate Gels submenu and click on the calstrip scan. The
Gel Record dialog box will open, in which you can enter information about the
calstrip.
H-4
Appendix H. Techniques
Fig. H-3. Gel Record Editor.
Enter the date on which the gel was run in the text box next to the Gel run date
prompt.
Next to the Calstrip prompt, click on the button that corresponds to the kind of
calibration associated with this gel. In most cases, you or someone in your lab has
made the calstrips, so the button labeled User Batch is the appropriate choice.
Next to the User Batch prompt, click on the arrow. From the list displayed, select the
calibration record name for the calstrip. The Detection and Isotope fields are
automatically obtained from the specified Calstrip Record.
Click on the appropriate button to indicate the type of detection used for your gel, if
not already defined from the batch file.
Click on the Isotope button to indicate the isotope used in the gel. This is usually,
though not necessarily, the same isotope as the one used in the calstrip.
Next, enter the number of counts loaded on the gel.
Enter the day on which you began exposing the gel to film in the text box next to the
Exposure start prompt.
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PDQuest User Guide
Note:
Most calibration problems arise from incorrectly entering the dates used to
calculate radioactive decay factors for the isotopes. Wrong values can make the
calstrip appear to have decayed to nothing.
Next to Duration (days), enter the number of days that the film was exposed.
Once this data has been entered, you will want to apply it to the 2-D scan and the
calstrip scan. To do so, click on the Apply button.
If you want to apply different calibration data to your 2-D scan than to your calstrip
scan (if, for example, your 2-D scan and calstrip scans use different isotopes), use the
Apply Here button to apply the gel record to the currently displayed image only (2-D
or calstrip scan).
Copying Gel Record Information
If you have scanned several exposures of the same gel, most of the calibration data
will be the same for all of them (except for the exposure start date and length). Instead
of entering all the information for each exposure, you can enter it once, and then copy
it between exposures using the Copy button.
Clicking on Copy will open a list of available gel records. Select the record that you
would like to apply to the current image and click the mouse.
Click on the Cancel button to exit the operation.
H.1.c Sampling the Calibration Strip Segments
The next step in calibrating your gels involves sampling the densities in the segments
of the calstrip to establish a correlation between the sampled intensities recorded by
the scanner and the counts entered into the Calstrip Record.
If you have not already done so, display the calstrips. If you have gels with multiple
exposures, use the Configure Calstrip Windows function to automatically divide the
screen into enough windows to display all the calstrips for a given gel.
Flip Calstrip (if necessary)
If the orientation of the scanned calstrip does not correspond to the order in which the
segment activities are entered in the Calstrip Record, a message will be displayed,
H-6
Appendix H. Techniques
indicating that the calibration density decreases while the counts increase (or vice
versa).
To change the orientation of the calstrip so it corresponds to the Calstrip Record
segment activities, use the Horizontal Flip function (under the Image menu). If the
dark segments were on the left, they will now be on the right, and vice versa. Again,
we recommend that you enter your data from highest to lowest activity and display
the calibration strip with the darkest segment on the left.
Box Segments in a Calstrip
Once the calstrip is in the correct orientation, go back to the Edit > Calibrate Gels
submenu and select Box Segment.
Go to the first (leftmost) segment of your calstrip. Sampling boxes must be drawn
beginning with the leftmost segment of the calstrip and moving right.
With Box Segment selected, drag a box on the calstrip. When you release the mouse
button, the area will be sampled.
Note:
When defining your box, be sure to avoid sampling regions that are very close to
the borders of the segment or any irregular areas (i.e., areas that are overly dark
or light).
The number of each segment will be displayed in the upper left corner of each
sampling box.
Above the box will be a number between 0.00 and 2.00. This represents the O.D.
value for the segment.
Beneath each box is the number of counts associated with that segment from the
Calstrip Record.
If you make a mistake, simply redraw a new box over the old one. Repeat this
procedure for all the segments of the calstrip.
Note:
All segments entered in the calstrip record must be boxed starting from the left.
The leftmost segment corresponds to Segment 1 on the Calstrip Record. Your
calstrips and calstrip data should be designed so that either (1) the darkest
segments are on the left and the count values decrease, or (2) the lightest values
are on the left and the count values increase.
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PDQuest User Guide
You can draw more boxes than the number of segments defined in the Calstrip
Record. However, they are automatically and permanently deactivated.
H.1.d Plot the Calibration Curve
Once you have created the sampling boxes, select Plot Calib. Curve to display the
calibration curve.
A graph will be displayed that correlates the optical density (usually between 0.00
and 2.00 O.D.) with DPMs or other specified units.
The Y axis represents the optical density (0.00–2.00) in each sampled segment, while
the X axis represents the corresponding DPM or other unit per image unit area
calculated from the calibration record data.
Fig. H-4. Example of a calibration curve.
H-8
Appendix H. Techniques
Calibration Curve Problems
Find errant data points and their corresponding segments using the function Display
Segment Quan.
Above each sampling box will be displayed the average O.D. value within the box,
and just below the box will be displayed the corresponding DPM (or other specified
unit). This should tell you which sampling boxes correspond to errant data points on
the graph.
Usually, problems arise when a box straddles the boundary between two adjacent
segments or the density is very uneven within the segment due to bleeding from an
adjacent segment. These sorts of problems should be remedied by redoing the
sampling boxes.
Use the function Remove Segment to remove an errant sampling box.
Draw a new sampling box on the segment using Box Segment, as described above.
Place the new box carefully to avoid repeating the problem. For example, keep the
box away from segment boundaries. If the segment shows bleeding, place the
smallest box possible in an area of the segment with representative O.D. Try not to
sample blotchy areas.
Replot the calibration curve using the function Plot Calib. Curve.
If there is still a problem, you can opt not to include that particular segment in the
calculation of the calibration curve. Select De/Activate Segment and click on the
problem segment. The segment will still exist, so its counts in the corresponding
Calstrip Record do not need to be changed or deleted. However, the segment will not
be included in the calculation of the calibration curve.
If you change your mind and want to reinclude the segment, select De/Activate
Segment again and click on the segment. The segment will be reactivated and
included in calibration curve calculations.
H.1.e Repeat for Each Calstrip
If you have multiple film exposures of the same gel, repeat the steps outlined above
for each exposure. The calibration strip batch number for each film exposure of a
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PDQuest User Guide
single gel should be the same since the same physical calibration strip was used to
make each exposure.
H.2 Merging Multiple Exposures
If you are working with 8-bit images and are using calstrips on your scans, you can
merge multiple exposures of the same gel into a single image with a greater dynamic
range.
Note:
You can only merge 8-bit images with calstrips. If you are not working with 8-bit
images or are not using calstrips, you cannot merge exposures.
To merge multiple exposures, make sure all your exposures and calstrips are loaded
into the same image window, then simply auto-detect spots as you would normally
(see the following chapter).
Creating Different Exposure Files
To identify different gel scans as different exposures of the same gel, open the scan
files, select Change Version/Exposure from the File menu, and enter consecutive
exposure numbers for the different scans. Make sure that the root names of the scan
files are the same.
Note:
You can give different exposures the same root name when you are scanning
them, or you can change them later using the Save As command. After you
change the exposure numbers, you will end up with files named RootName x1,
RootName x2, RootName x3, etc.
Why Merge Exposures?
Because 8-bit scanners have a limited dynamic range, dark spots may saturate on long
exposures, while faint spots may be missed on short exposures, and either extreme
can be missed on medium duration exposures. Therefore, it is frequently necessary to
combine the quantitative data from a number of 8-bit exposures of the same gel
exposed for different lengths of time into a “merged” synthetic image. Merging
creates such an image automatically as you spot detect.
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Appendix H. Techniques
Fig. H-5. Diagrammatic representation of merging.
Cropping and Aligning Exposures of the Same Gel for Merging
Before you can merge multiple exposures, you must crop and align them so that they
are exactly the same size and position. To do this, you will use the Advanced Crop
commands on the Image menu. See section 3.13.a for instructions.
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PDQuest User Guide
H-12
Appendix I
Cross-Platform File Exchange
It is possible to move image data between Discovery Series software applications on
different platforms. There are different protocols depending on the platform (PC or
Macintosh) you are transferring from and to.
I.1 Macintosh to PC
To transfer a file from a Discovery Series application running on a Macintosh to a
Discovery Series application running on a PC, you need to tag the file name with the
suffix appropriate to the image file type (e.g., 2-D scan).
For 2-D scans, use the suffix: .gsc
For Filtered images, use the suffix: .gim
For Gaussian images, use the suffix: .gsp
For calstrip scans, use the suffix: .csc
For MatchSets, use the suffix: .ms
For high-level MatchSets, use the suffix: .hms
I.2 PC to Macintosh
PC versions of Discovery Series files can be read directly by Macintosh applications,
with no required modifications.
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PDQuest User Guide
I-2
Appendix J. CFR Module
Appendix J
21 CFR Part 11
J.1 Introduction
Effective August 20, 1997 the United States Food and Drug Administration (FDA)
released Part 11 “Electronic Records; Electronic Signatures” of title 21 of the Code of
Federal Regulations. This rule states the conditions under which the FDA considers
electronic signatures and electronic records to be trustworthy, reliable, and equivalent
to traditional handwritten signatures. In this manner it defines the conditions under
which an organization must operate to meet its record keeping and record submission
requirements when using electronic signatures and records rather than handwritten
records and signatures.
J.1.a. CFR and The Discovery Series
The purchase of a separate license allows the use of the 21 CFR Part 11 System
Security features of the The Discovery Series. The Discovery Series software utilizes
these security features in concert with the operating system to assist the laboratory,
research institution, etc. in becoming CFR compliant. These features include audit
trail, electronic signature, and secure user login.
Note:
Bio-Rad makes no claim that The Discover Series is CFR compliant in and of
itself, nor does it guarantee compliance for the user. The organization must
establish policies and standard operating procedures that work in conjunction
with the tools provided by Bio-Rad to ensure compliance with 21 CFR Part 11.
Audit Trail
Section 11.10 (e) of 21 CFR Part 11 requires the use of “secure, computer-generated,
time-stamped audit trails to independently record the date and time” of activities
within the system. In System Security mode, The Discovery Series software
automatically records any events that affect data and analysis in the Audit Report. For
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PDQuest User Guide
instance, such things as cropping and spot detection parameters are recorded in the
Audit Report, while transform will not because it does not affect the data.
Although there are software controls to detect modification of data, protection of the
data from deletion must happen at the level of the operating system. As a result, data
must be stored only on an NTFS partition. Access to directories where data is stored
should be granted only to those who need to access that data. Ideally, access for a
single user will prevent the possibility of two users accessing the same file. Also, a
regular backup procedure should be in place.
Electronic Signature
Section 11.3 (b) (7) of 21 CFR Part 11 defines an electronic signature as “a computer
data compilation of any symbol or series of symbols executed, adopted, or authorized
by an individual to be the legally binding equivalent of the individual's handwritten
signature.” The Discovery Series software allows the user to “sign” images or
MatchSets thereby locking the file against future changes. If a user changes a
“signed” file, and attempts to save the file, the file automatically receives a new
version number, and the signed tag is removed from the window title.
Secure User Login
Section 11.3 (b)(4) defines a closed system as “an environment in which system
access is controlled by the persons who are responsible for the content of electronic
records that are on the system.” The Discovery Series software utilizes the Microsoft
Windows 2000 and XP Professional security model as the secure user login.
J.2 Using The Discovery Series in CFR Mode
Access Levels
Before CFR mode can be utilized, the system must be correctly configured by the
network administrator. There are four levels of access to The Discovery Series in
CFR Mode:
Guest - A user with guest access has the ability to open and view files, but a guest
cannot change data, nor can a guest save a file.
J-2
Appendix J. CFR Module
Tech - The tech level user has the ability to acquire images, transform images, crop,
flip, rotate, use the text and line tools, perform basic excision, print, and export. The
tech level user also has the ability to save and sign files.
User - At this level, all functions are available to the user. However, the user cannot
change the CFR settings.
Administrator - The administrator level has all the capabilities of the full user as well
as the ability to make changes to the CFR settings.
CFR Image Files
For files to be opened in CFR mode, the files must have been created on a CFR
system, either through acquisition or importing a TIFF file. Any files created on a
non-CFR system cannot be opened. To open a file created on a non-CFR system, open
the file on a non-CFR system and export it to TIFF. You can then import it in CFR
mode. To ensure accuracy, remember to select the analysis option as your export
mode when you export your non-CFR images.
Note:
Due to operating system limitations, it is possible to have image files open in two
or more locations over a network simultaneously. To safeguard your image files
keep them in a protected folder or on your local machine.
J.2.a Setting Security Preferences
Setting the Security preferences for CFR mode requires the user have administrator
privileges. To locate the Security Preferences, select Preferences from the Edit menu
and click the Security tab.
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PDQuest User Guide
Fig. J-1. Security Preferences
The following options are available on the Security tab of the Preferences dialog box:
Enable 21 CFR Part 11 mode - If this box is unchecked, all other options are inactive.
To activate the other options and CFR mode check this box.
Program times out if inactive - When this box is checked, you must enter your
password to resume using PDQuest. The minutes field indicates the amount of time
that must elapse before the application times out.
Require login on startup - If this box is checked, you must enter your password when
you start PDQuest. This password is the same password as the one used for logging
on to Windows. This is an added security measure to prevent unauthorized users from
opening PDQuest on a machine where they are not logged in.
Automatically create backup for new images - This option, when selected, creates a
backup file when you save a newly acquired or imported image. Locate the backup
file in the same folder as the original. The backup file is identified by “backup of”
preceding the filename of the original.
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Appendix J. CFR Module
J.2.b Secure User Login
PDQuest utilizes the Windows security model. When you open PDQuest, the
application determines the user name by the ID of the user currently logged on to that
system. If you have Require Login on startup checked on the Security tab of the
Preferences dialog box, then you must enter your Windows password to open
PDQuest.
To view the current user information, click Show User Information on the CFR menu.
The message displays the username, full name, and group membership for the
currently logged in user.
Note:
Although Windows XP allows you to switch users without logging off, you must
first close PDQuest before switching users. Windows 2000 does not allow the
ability to switch users.
J.2.c Audit Report
The Audit Report included with CFR mode tracks all changes to actual data of images
as well as any events associated with analysis.
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PDQuest User Guide
Fig. J-2. Audit Report
Each entry in the Audit report contains the date and time of the event as well as the
name of the user who performed the event. The Audit Report also contains a
description of the event with details, the file name, and the version of PDQuest used
when the event occurred. The comment column allows you to enter comments about
particular events. To enter a comment, click the cell under the comments heading for
the event to which you want to add the comment. This opens the comment editor
dialog box. Enter your comment and click Done.
J-6
Appendix J. CFR Module
Fig. J-3. Comment Editor
Audit Report Options
The Audit Report Options dialog box allows you to customize the Audit Report view.
Click the Options button to open the Audit Report Options Dialog box.
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PDQuest User Guide
Fig. J-4. Audit Report Options
The left hand column of the Audit Report dialog box lists the category filters. If a box
is checked, all events of that type display in the Audit Report. To hide a category in
the report viewer, clear the check box next to the corresponding category.
The right hand column lists the columns the report viewer displays. To hide a column,
clear the check box next to the corresponding column name.
Note:
These options only affect what is displayed in the report viewer, not what is
recorded. As previously stated, all changes to image data and events associated
with analysis are recorded.
If the Expand Details in Display option is checked, then the all details for each event
are displayed in the viewer. Otherwise, only the first row of each event is displayed.
Similarly, the Expand comments in Display functions the same way.
The AutoZoom on Detail Image Coordinates option is a convenient way to pinpoint
the location an event took place in the image. If the box is checked, click the
coordinates in the report viewer and the image will automatically zoom to the location
of the event. Note that this tool is only available when a specific location has been
recorded in the report.
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Appendix J. CFR Module
Printing and Exporting the Audit Report
The Audit Report Viewer allows you to print or export the report for use in a
spreadsheet application.
To print the report, click either the Print Current Page button or the Print button. Print
Current Page is only available in print preview mode and allows you to print the
currently viewed page. The print all pages button opens the Pint dialog box where you
can either print all pages or specify a print range. Click Page Setup to change the print
settings.
To export the report click the Export button. This opens the Export Options dialog
box where you can determine the delimiter (tab or comma) and the export destination
(save as a file or copy to the clipboard).
J.2.d Electronic Signature
As was previously stated, Section 11.3 (b) (7) of 21 CFR Part 11 defines an electronic
signature as “a computer data compilation of any symbol or series of symbols
executed, adopted, or authorized by an individual to be the legally binding equivalent
of the individual's handwritten signature.” PDQuest allows the user to “sign” an
image or MatchSet thereby locking the file against future changes. If a user changes a
“signed” file, and attempts to save the file, the file automatically receives a new
version number, and the signed tag is removed from the window title.
To “sign” a file in PDQuest, with the file open, select Sign from the CFR menu. A
modified version of the Comments dialog box opens. Enter any comments you may
have, and click Sign. This opens the Login User dialog box, which requires you to
enter your user name and password for signature validation.
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PDQuest User Guide
Fig. J-5. Electronic Signature dialog box
Once you have “signed” your file, the image window title bar indicates the file has
been signed, and the event is recorded in the Audit report.
Fig. J-6. A signed MatchSet
If your policies require signature verification by more than one person, such as a lead
researcher, a signed file can be signed again without changing the file version
provided the file has not been modified between signatures.
J-10
Index
Numerics
3D Viewer ............................................................................................................. 3-18
A
A-B Comparison ................................................................................................... 11-7
Acid-Base ............................................................................................................. 2-45
Add spot ................................................................................................................ 4-17
Add Spot to Standard ............................................................................................ 5-26
Analysis Set Manager ............................................................................................. 7-2
Analysis Sets .......................................................................... 7-13, 7-18, 7-20, 7-22
Analysis sets ........................................................................................................... 7-1
Arbitrary ........................................................................................................ 7-18
Boolean .......................................................................................................... 7-20
Creating annotations from ............................................................................. 7-36
Cutting spots from ........................................................................................... 9-2
Quantitative ..................................................................................................... 7-9
Statistical ....................................................................................................... 7-13
Analysis Tools ........................................................................................................ 6-1
Annotation categories ........................................................................................... 7-26
File name ....................................................................................................... 7-28
Protein name .................................................................................................. 7-29
URL ............................................................................................................... 7-27
Annotation Select Tool ......................................................................................... 7-25
Annotation Tool .................................................................................................... 7-25
Entering an annotation ................................................................................... 7-30
Image options ................................................................................................ 7-33
Selecting a category ....................................................................................... 7-29
Selecting spots ............................................................................................... 7-29
Index-1
PDQuest User Guide
Annotations ........................................................................................................... 7-24
Browsing ........................................................................................................ 7-35
Creating from analysis sets ............................................................................ 7-36
Displaying ...................................................................................................... 7-33
Exporting ....................................................................................................... 2-35
Importing ....................................................................................................... 7-30
Printing ........................................................................................................... 7-38
Transferring in a high-level matchset ............................................................ 7-39
Arrow keys .............................................................................................................. 3-8
Assigning images to windows ................................................................................. 3-3
Auto-add Spots to Standard .................................................................................. 5-24
B
Base-Acid .............................................................................................................. 2-45
Basic Excision Tool ................................................................................................ 8-1
Acquiring the image ......................................................................................... 8-9
Cut run options ............................................................................................... 8-17
Light options .................................................................................................... 8-9
Multiple cuts of the same spot ....................................................................... 8-14
Operation ......................................................................................................... 8-9
Pausing a run .................................................................................................. 8-18
Recutting spots ............................................................................................... 8-20
Specifying cuts ............................................................................................... 8-12
Stopping a run ................................................................................................ 8-18
Basic Export Matchset .......................................................................................... 2-33
Bio-Rad Technical Service ................................................................................... 1-16
Boolean Analysis Sets ........................................................................................... 7-20
Browse Annotations .............................................................................................. 7-35
C
Calibrating gel scans .............................................................................................. H-1
Calstrips
Index-2
Index
Boxing segments ............................................................................................ H-7
Calibration curve ............................................................................................ H-8
Configuring in subwindows ........................................................................... H-2
Cropping out of images .................................................................................. H-1
Deactivating a segment ................................................................................... H-9
Entering data ................................................................................................... H-2
Gel information .............................................................................................. H-4
Range of values .............................................................................................. H-3
Records ........................................................................................................... H-2
Removing segments ........................................................................................ H-9
Sampling segments ......................................................................................... H-6
Cancelling spots .................................................................................................... 4-22
Categories of annotations ..................................................................................... 7-26
Centering images .................................................................................................... 3-9
Chemi Doc
Acquiring the image ............................................................................... B-5, C-6
Adjusting the display ..................................................................................... B-11
Annotating images ............................................................................. A-10, B-12
Auto Expose ........................................................................................... B-6, C-6
Auto exposure settings ....................................................................... B-15, C-14
Backup images .................................................................................... B-16, C-14
Chemi mode ..................................................................................................... B-5
DAC settings ................................................................................................. B-14
Exposure Status bar .............................................................................. B-13, C-9
Exposure time ............................................................................... A-7, B-7, C-7
Freeze .............................................................................................................. B-8
Image mode ..................................................................................................... B-5
Imaging area ....................................................................................... B-15, C-12
Invert display ................................................................................................. B-11
Live Acquire ........................................................................................... B-8, C-8
Live image display .......................................................................................... B-4
Manual Expose ....................................................................................... B-7, C-7
Options ............................................................................................... B-13, C-11
Positioning the sample ............................................................................ B-4, C-5
Saturated pixels, highlighting ........................................................................ B-11
Saving images ................................................................................................ B-13
Simulation mode ..................................................................................... B-2, C-2
UV mode ......................................................................................................... B-5
Video card .............................................................................................. B-2, C-2
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PDQuest User Guide
Video display window .................................................................................... B-3
Video printing ............................................................................................... B-13
Video printing, footer info ............................................................................ B-15
White light mode ............................................................................................ B-5
ChemiDoc XRS ...................................................................................................... C-1
Classes ................................................................................................................... 6-20
Closing an image ................................................................................................... 2-15
Colony matching ................................................................................................... 5-31
Colors, setting ....................................................................................................... 3-13
Column Graph ....................................................................................................... 11-7
Combine spots ....................................................................................................... 4-23
Computer Requirements ......................................................................................... 1-7
Computer requirements
Macintosh ......................................................................................................... 1-7
PC ..................................................................................................................... 1-7
Configure Subwindows ........................................................................................... 3-1
Contrast, see Transform.
Crop
Saving crop settings ....................................................................................... 3-28
Cropping ................................................................................................................ 3-26
Advanced crop commands ............................................................................. 3-28
Custom ................................................................................................................... C-4
Custom Rotation ................................................................................................... 3-30
Cybergels
Landmarking .................................................................................................. 5-24
D
Database Node ID .................................................................................................
Database spot numbers. See DSNs
Delete
Group .............................................................................................................
Density Tools ........................................................................................................
Density at Cursor ...........................................................................................
Density in Box ...............................................................................................
Plot Cross-section ..........................................................................................
Index-4
2-45
6-10
3-11
3-11
3-11
3-12
Index
Plot Density Distribution ...............................................................................
Plot Vertical Trace .........................................................................................
Display settings .....................................................................................................
Downloading from Internet
Windows ........................................................................................................
DSNs .....................................................................................................................
Assigning .......................................................................................................
Displaying ......................................................................................................
Transferring in high-level matchsets .............................................................
3-12
3-13
2-41
1-15
6-34
6-35
6-35
6-36
E
Enter MrpI Data .................................................................................................... 6-30
Excision Gel Selection ............................................................................................ 9-2
Exit ........................................................................................................................ 2-20
Exporting
Annotations .................................................................................................... 2-35
Images ............................................................................................................ 2-27
Matchset data ................................................................................................. 2-30
Exposures
Merging ........................................................................................................ H-10
F
Faint spots ............................................................................................................. 4-24
File
Unloading ...................................................................................................... 2-15
File locations in Windows ...................................................................................... 1-9
File transfer between Windows and Macintosh .................................................... 2-12
File types ................................................................................................................. 2-7
Filter Wizard ......................................................................................................... 3-33
Filtered images ...................................................................................................... 4-12
Filtering image noise ............................................................................................ 3-32
Find File for .......................................................................................................... 7-28
Index-5
PDQuest User Guide
Finding spots ......................................................................................................... 4-29
Flipping images ..................................................................................................... 3-29
FX, see Molecular Imager FX
G
Gaussian images .................................................................................................... 4-12
Gaussian spots ....................................................................................................... 4-13
Gel Doc
Acquiring the image ........................................................................................ A-5
Auto expose .................................................................................................... A-5
Auto exposure settings .................................................................................. A-13
Backup images .............................................................................................. A-13
DAC settings ................................................................................................. A-12
Display settings ............................................................................................... A-8
Exposure Status bar ...................................................................................... A-11
Freeze .............................................................................................................. A-7
Image mode ..................................................................................................... A-5
Imaging area ................................................................................................. A-13
Invert display .................................................................................................. A-9
Live image display .......................................................................................... A-4
Manual expose ................................................................................................ A-6
Options .......................................................................................................... A-11
Positioning the sample .................................................................................... A-4
Saturated pixels, highlighting ......................................................................... A-9
Saving images ............................................................................................... A-11
Simulation mode ............................................................................................. A-2
UV mode ......................................................................................................... A-5
Video card ....................................................................................................... A-2
Video display window .................................................................................... A-3
Video printing, footer info ............................................................................ A-13
White light mode ............................................................................................ A-5
Gel Record ............................................................................................................. H-4
Grab ......................................................................................................................... 3-8
Graph
Advance ......................................................................................................... 11-7
Index-6
Index
Column Graph ............................................................................................... 11-7
Margin Graph ................................................................................................ 11-6
Partial Matches .............................................................................................. 5-30
Pop-up Graph ................................................................................................. 11-6
Retreat ............................................................................................................ 11-7
Graphs ................................................................................................................... 11-4
Display order of histogram bars .................................................................... 11-5
Group Consensus .................................................................................................. 6-13
GS-800 Imaging Densitometer
Applications, selecting ................................................................................... D-4
Calibration ...................................................................................................... D-9
Calibration settings ....................................................................................... D-13
Calibration step tablets ................................................................................. D-10
Filters and light source, selecting ................................................................... D-6
Options ......................................................................................................... D-14
Oversampling ............................................................................................... D-14
Preview scan ................................................................................................... D-7
Resolution, selecting ....................................................................................... D-8
Saturated pixels, highlighting ....................................................................... D-15
Scan area, selecting ........................................................................................ D-7
Scanning an image ........................................................................................ D-13
Scanning window ........................................................................................... D-3
SCSI card ........................................................................................................ D-1
Simulation mode ............................................................................................. D-2
H
Hide Overlays .......................................................................................................
High-level matchsets ............................................................................................
Creating .........................................................................................................
Transferring annotations ................................................................................
Transferring DSNs .........................................................................................
Transferring MrpI data ..................................................................................
Transferring SSPs ..........................................................................................
Histograms ............................................................................................................
Index-7
3-10
5-33
5-34
7-39
6-36
6-31
6-34
11-4
PDQuest User Guide
I
Image information ................................................................................................. 2-16
Image report, printing ........................................................................................... 2-24
Image Stack Tool .................................................................................................... 6-5
Image stack tool ...................................................................................................... 6-4
Images
Assigning to windows ...................................................................................... 3-3
Closing ........................................................................................................... 2-15
Exporting ....................................................................................................... 2-27
Interchanging ................................................................................................... 3-3
Opening .......................................................................................................... 2-10
Saving ............................................................................................................ 2-16
Stacking ........................................................................................................... 6-5
Types ................................................................................................................ 2-7
Imitate Zoom ........................................................................................................... 3-7
Importing TIFF files .............................................................................................. 2-12
Installation
Windows .......................................................................................................... 1-9
Integrated Excision Tool ......................................................................................... 9-9
Aligning images ............................................................................................. 9-19
Beginning a run .............................................................................................. 9-25
Confirming cuts ............................................................................................. 9-29
Cut run options ............................................................................................... 9-17
Exporting cut list ............................................................................................ 9-35
Microtiter plate selection ............................................................................... 9-26
Pausing a cut run ............................................................................................ 9-30
Recutting spots ............................................................................................... 9-30
Selecting gels ................................................................................................... 9-2
Selecting the cut list ....................................................................................... 9-17
Setup ................................................................................................................ 9-9
Validation image ............................................................................................ 9-30
Interchanging images .............................................................................................. 3-3
Invert
Image data ...................................................................................................... 3-38
Image display ................................................................................................. 3-26
Index-8
Index
K
Keyboard shortcuts ................................................................................................. 2-5
L
Landmarking
Auto-match after ............................................................................................
Landmarks ............................................................................................................
Marking .........................................................................................................
Unlandmarking ..............................................................................................
License ..................................................................................................................
Registration ....................................................................................................
Line tool ................................................................................................................
Location in Gel .....................................................................................................
Low quality spots ..................................................................................................
Low quantity spots ................................................................................................
2-45
5-17
5-19
5-19
1-11
1-12
3-40
3-10
4-28
4-25
M
Macintosh ............................................................................................................. 1-10
Macintosh, memory assigned to PDQuest .............................................................. 1-8
Magnifying images ................................................................................................. 3-4
Margin Graph ........................................................................................................ 11-6
Mark
MrpI Spots ..................................................................................................... 6-30
Partial Matches .............................................................................................. 5-29
Match
Offsets ............................................................................................................ 5-20
Overview ......................................................................................................... 1-6
Matching ............................................................................................................... 7-22
Automatic ...................................................................................................... 5-12
Colonies ......................................................................................................... 5-31
Index-9
PDQuest User Guide
Landmarking .................................................................................................. 5-17
Unmatching .................................................................................................... 5-31
Unmatching individual spots ......................................................................... 5-29
Matchset
Partial Matches .............................................................................................. 5-29
Show Match ................................................................................................... 5-20
Standard ........................................................................................................... 5-8
Matchsets
Adding spots .................................................................................................. 5-24
Closing ........................................................................................................... 2-15
Creating ............................................................................................................ 5-3
Exporting data ................................................................................................ 2-27
High-level ...................................................................................................... 5-33
Matching ........................................................................................................ 5-12
Opening .......................................................................................................... 2-10
Removing all matches .................................................................................... 5-31
Saving ............................................................................................................ 2-16
Undoing matches ........................................................................................... 5-28
Member sharing ...................................................................................................... 5-8
Memory allowance ................................................................................................ 2-37
Menus ...................................................................................................................... 2-1
Merging multiple exposures ................................................................................. H-10
Molecular Imager FX
Acquiring the image ....................................................................................... F-13
Control panel .................................................................................................... F-2
File size of images ......................................................................................... F-13
Options ........................................................................................................... F-14
Saturated pixels, highlighting ........................................................................ F-14
Saving the image ............................................................................................ F-13
Scanning window ............................................................................................. F-3
Selecting an application ................................................................................... F-4
Selecting resolution ........................................................................................ F-12
Selecting scan area ......................................................................................... F-11
Simulation mode .............................................................................................. F-2
Mouse-assignable tools ......................................................................................... 2-48
Behavior with keyboard commands ................................................................ 2-6
MrpI Data .............................................................................................................. 6-29
Displaying ...................................................................................................... 6-30
Removing ....................................................................................................... 6-30
Index-10
Index
Transferring in high-level matchsets ............................................................. 6-31
Multi-Channel Viewer .......................................................................................... 3-16
N
Normalization ....................................................................................................... 6-23
O
Opening files ......................................................................................................... 2-10
Optimizing images, see Transform.
Overlays, hiding .................................................................................................... 3-10
P
Password, entering ................................................................................................ 1-14
Personal Molecular Imager FX
Acquiring an image ......................................................................................... E-6
Control panel ................................................................................................... E-2
File size of images ........................................................................................... E-6
Options ............................................................................................................ E-7
Saturated pixels, highlighting .......................................................................... E-7
Saving the image ............................................................................................. E-6
Scanning window ............................................................................................ E-3
Selecting resolution ......................................................................................... E-5
Selecting scan area .......................................................................................... E-4
Simulation mode .............................................................................................. E-2
pH Gradient .......................................................................................................... 2-45
Plot
DSN Grid ....................................................................................................... 6-35
SSP Grid ........................................................................................................ 6-33
Pop-up Graph ........................................................................................................ 11-6
Index-11
PDQuest User Guide
Preferences
Display ........................................................................................................... 2-41
File paths ........................................................................................................ 2-38
General ........................................................................................................... 2-37
Toolbars ......................................................................................................... 2-43
Printing
Annotations .................................................................................................... 7-38
Image reports ................................................................................................. 2-24
Video printouts .............................................................................................. 2-26
ProteomeWorks Spot Cutter
Basic Tool ........................................................................................................ 8-1
Q
Qualitative Analysis Sets ........................................................................................ 7-6
Quality of spots ..................................................................................................... 4-27
Quantitation
Replicate Groups .............................................................................................. 6-9
Single Gels ....................................................................................................... 6-9
Quantitative Analysis Sets ...................................................................................... 7-9
Quantity Graphs .................................................................................................. 11-12
Quantity of spots ................................................................................................... 4-24
Quantity table ........................................................................................................ 4-26
R
Reduce File Size .................................................................................................... 2-18
Registration
By fax/e-mail ................................................................................................. 1-14
By Internet ..................................................................................................... 1-13
Registration form .................................................................................................. 1-12
Remove MrpI Data ................................................................................................ 6-30
Remove Segment ................................................................................................... H-9
Removing spots ..................................................................................................... 4-18
Index-12
Index
Replicate Group
Edit Group ..................................................................................................... 6-10
Rename .......................................................................................................... 6-10
Reports
A-B Comparison ............................................................................................ 11-7
Normalization ................................................................................................ 6-23
Quantitation Table ......................................................................................... 11-9
Quantity ......................................................................................................... 11-9
Quantity Graphs ........................................................................................... 11-12
Scatter Plots ................................................................................................. 11-16
Revert to Saved ..................................................................................................... 2-16
Rotating images .................................................................................................... 3-29
S
Sample Database ................................................................................................... 6-17
Sample images ...................................................................................................... 2-12
Sample Pairs ......................................................................................................... 6-21
Saturated pixels, highlighting ............................................................................... 3-26
See also individual imaging devices
Saturated spots ...................................................................................................... 4-24
Saving a file .......................................................................................................... 2-16
Scanset .................................................................................................................. 4-12
Scansets ................................................................................................................... 2-8
Scatter plot tool ....................................................................................................... 6-7
Show Match .......................................................................................................... 5-20
Simulation mode, see individual imaging devices
Software license .................................................................................................... 1-11
Spot boundaries .................................................................................................... 4-19
Contour tool ................................................................................................... 4-20
Copying and pasting ...................................................................................... 4-22
Deleting ......................................................................................................... 4-22
Freehand tool ................................................................................................. 4-21
Moving .......................................................................................................... 4-22
Spot Crosshairs ..................................................................................................... 4-14
Spot cutter
Index-13
PDQuest User Guide
Basic tool ......................................................................................................... 8-1
Calibration ............................................................................................. 8-6, 9-13
Cut run options ............................................................................................... 8-17
Cutting tip height ................................................................................... 8-3, 9-10
Focussing the camera ............................................................................. 8-3, 9-10
Settings ................................................................................................... 8-4, 9-11
Setup ........................................................................................................ 8-2, 9-9
Spot Detection Wizard ............................................................................................ 4-1
Background subtraction ................................................................................... 4-7
Finding spot centers ......................................................................................... 4-4
Identifying faint, small, and large spots ........................................................... 4-3
Noise removal .................................................................................................. 4-7
Parameters, loading .......................................................................................... 4-9
Processing gels ................................................................................................. 4-9
Saving parameters ............................................................................................ 4-9
Sensitivity and size scale ................................................................................. 4-5
Streak removal ................................................................................................. 4-7
Spot Ellipses .......................................................................................................... 4-14
Spot Review Tool .................................................................................................... 6-1
Spots
Adding ........................................................................................................... 4-17
Boundary tools ............................................................................................... 4-19
Cancelling ...................................................................................................... 4-22
Combining ..................................................................................................... 4-23
Contour tool ................................................................................................... 4-20
Crosshairs ....................................................................................................... 4-14
Detecting .......................................................................................................... 4-9
Detection parameters, selecting ....................................................................... 4-1
Detection report ............................................................................................. 4-12
Detection Wizard ............................................................................................. 4-1
Ellipses ........................................................................................................... 4-14
Faint ............................................................................................................... 4-24
Finding ........................................................................................................... 4-29
Freehand tool ................................................................................................. 4-21
Low quality sets ............................................................................................. 4-28
Low quantity sets ........................................................................................... 4-25
MrpI value ...................................................................................................... 4-28
Parameters ...................................................................................................... 4-28
Peak value ...................................................................................................... 4-28
Index-14
Index
Quality ................................................................................................. 4-27, 4-28
Quantity ............................................................................................... 4-24, 4-28
Quantity table ................................................................................................ 4-26
Removing ...................................................................................................... 4-18
Review tool ...................................................................................................... 6-1
Saturated ........................................................................................................ 4-24
SSP numbers ......................................................................................................... 6-32
Displaying ...................................................................................................... 6-32
Transferring in high-level matchsets ............................................................. 6-34
Standard image ....................................................................................................... 5-8
Selecting .......................................................................................................... 5-8
Standard image in a matchset
Adding spots .................................................................................................. 5-24
Standard spot numbers. See SSP numbers
Statistical Analysis Sets ........................................................................................ 7-13
Status boxes ............................................................................................................ 2-3
Step ......................................................................................................................... C-4
Subwindows
Configuring ...................................................................................................... 3-1
Definition ......................................................................................................... 3-1
T
Technical Service, contacting ............................................................................... 1-16
Text Overlays
Creating ......................................................................................................... 3-38
Editing ........................................................................................................... 3-40
Line tool ......................................................................................................... 3-40
Viewing ......................................................................................................... 3-41
Three by three (3x3) sections, viewing ................................................................. 3-10
TIFF files
Importing ....................................................................................................... 2-12
Tile Windows .......................................................................................................... 3-4
Tool Help ................................................................................................................ 2-2
Toolbars
Main ................................................................................................................. 2-2
Index-15
PDQuest User Guide
Secondary ......................................................................................................... 2-3
Tools, mouse-assignable ....................................................................................... 2-48
Transfer
DSN’s ............................................................................................................. 6-36
SSP’s .............................................................................................................. 6-34
Transform .............................................................................................................. 3-21
Auto-scale ...................................................................................................... 3-23
Gamma slider ................................................................................................. 3-24
High/Low Sliders ........................................................................................... 3-23
Highlight Saturated Pixels ............................................................................. 3-26
Histogram ....................................................................................................... 3-22
Invert Display ................................................................................................ 3-26
U
URL annotations ................................................................................................... 7-27
V
VersaDoc
Acquire .......................................................................................................... G-11
Acquiring the image ...................................................................................... G-10
Binning ................................................................................................... C-4, G-6
Control panel ................................................................................................... G-2
Custom settings ............................................................................................... G-5
Dark subtraction ................................................................................. C-13, G-14
Exposure time ................................................................................................. G-8
Exposure times, recommended ....................................................................... G-9
File size of images ........................................................................................ G-19
Gain ................................................................................................................. G-6
Illumination Flat Fielding ............................................................................. G-10
Imaging area size .......................................................................................... G-17
Optimize exposure ........................................................................................ G-12
Optimize uniformity ....................................................................................... C-9
Index-16
Index
Options ......................................................................................................... G-14
Positioning ...................................................................................................... G-7
Preview scan ................................................................................................. G-10
Saturated pixels, highlighting ....................................................................... G-18
Save options .................................................................................................. G-17
Selecting an application .................................................................................. G-3
Video printing ....................................................................................................... 2-26
View Entire Image .................................................................................................. 3-6
Viewing images
Centering on the cursor ................................................................................... 3-9
Undoing view commands ........................................................................ 3-4, 3-7
W
Windows
Assigning/interchanging images .....................................................................
Imitate Zoom ...................................................................................................
Tiling ...............................................................................................................
Windows and subwindows, overview ....................................................................
Windows file locations ...........................................................................................
3-3
3-7
3-4
3-1
1-9
Z
Zoom Box ............................................................................................................... 3-5
Zoom In/Zoom Out ................................................................................................. 3-6
Zoom tools
Changing behavior ......................................................................................... 2-41
Index-17
PDQuest User Guide
Index-18
The Discovery Series™ Software
Software License Agreement
READ THIS! THIS IS A LEGAL AGREEMENT BETWEEN YOU, THE CUSTOMER,
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