ChemiDoc™ MP Imaging

System with Image Lab™

Software
User Guide

Version 5.0
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.
Bio-Rad reserves the right to modify its products and services at any time. This
user guide is subject to change without notice. Although prepared to ensure
accuracy, Bio-Rad assumes no liability for errors or omissions, or for any
damage resulting from the application or use of this information.

Credits
1. Image Lab software is based in part on the work of the Qwt project
(http://qwt.sf.net).
2. Image Lab software is based in part on the work of the CImg project
(http://cimg.sourceforge.net/).
See license for details at
http://www.cecill.info/licences/Licence_CeCILL-C_V1-en.html
3. Image Lab software is based in part on the work of the Independent JPEG
Group (http://www.ijg.org/)
GelStar is a trademark of FMC Corporation. IRDye is a trademark of LI-COR
Biosciences. OliGreen, PicoGreen, and Pro-Q are trademarks of Invitrogen
Corp. Alexa Fluor, Coomassie Fluor, Qdot, and SYPRO are trademarks of
Invitrogen Corporation. Coomassie is a trademark of BASF Aktiengesellschaft.
Cy2 and Cy3 are trademarks of GE HealthCare. DyLight and Krypton are
trademarks of Thermo Fisher Scientific Inc. Excel, PowerPoint, and Windows
are trademarks of Microsoft Corporation. FireWire, iWork, Mac, Mac OS, and
Numbers are trademarks of Apple Inc. GelGreen and GelRed are trademarks of
Biotium, Inc. Intel Core and Pentium are trademarks of Intel Corporation.
Mitsubishi is a trademark of Mitsubishi Companies. PulseNet International is a
trademark of Centers for Disease Control and Prevention. Slo-Blo is a
trademark of Littelfuse, Inc.
SYBR is a trademark of Molecular Probes, Inc. Bio-Rad Laboratories, Inc. is
licensed by Life Technologies, Inc. to sell reagents containing SYBR Green I for
use in real-time PCR, for research purposes only.
CHEF (U.S. Patent Number 5,549,796, issued to Stanford University) is
exclusively licensed to Bio-Rad Laboratories, Inc.
Precision Plus Protein standards are sold under license from Life Technologies
Corporation, Carlsbad, CA, for use only by the buyer of the product. The buyer
is not authorized to sell or resell this product or its components.
Bio-Rad Laboratories, Inc. is licensed by Invitrogen Corporation to sell SYPRO
products for research use only under U.S. Patent Number 5,616,502.
Copyright ©2013 Bio-Rad Laboratories, Inc.
 Table of Contents

Safety and Regulatory Compliance . . . . . . . . . . . . . . . . . . . . . . . . . .11

Important Safety Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
General Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Regulatory Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Instrument Safety Warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Notice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Power Safety Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Voltage Setting Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Fuses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

ChemiDoc MP Imager. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
CCD Camera and Lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Universal Hood III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Image Lab Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Emission Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Optional Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Printer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Conversion Screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Optional Light Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
ChemiDoc MP Imager Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
ChemiDoc MP Technical Specifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
ChemiDoc MP Imaging System Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

User Guide | iii

Table of Contents

Chapter 2 Setting Up the Instrument with Image Lab Software . . . 27

System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Image Lab Security Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Computer Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Installing Image Lab Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Installing the Drivers on Windows 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Setting Up Image Lab Security Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Activating and Deactivating Security Edition. . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Enabling and Disabling Image Lab Secure Mode . . . . . . . . . . . . . . . . . . . . . . . 42
Setting Security Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Rename Security Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Using Groups on a Local Domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Changing Security Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Chapter 3 Image Lab Software Overview . . . . . . . . . . . . . . . . . . . . . . 51

Interface Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Main Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Main Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Results Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Display Toolbox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Start Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Analysis Tool Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Status Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Menu Commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Chapter 4 Acquiring Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

The Protocol Setup Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Single-Channel Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Multichannel Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Creating a Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Setting Up a New Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Step 1. Gel Imaging for Single-Channel Protocols . . . . . . . . . . . . . . . . . . . . . . 70
Step 1. Gel Imaging for Multichannel Protocols . . . . . . . . . . . . . . . . . . . . . . . . 72

iv | ChemiDoc MP Imaging System with Image Lab Software

 Table of Contents

Step 2. Detect Lanes and Bands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Step 3. Analyze Molecular Weight. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Step 4. Specify Output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Review Protocol Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Creating a Multichannel Image from Single Images . . . . . . . . . . . . . . . . . . . . . . . 85
Editing a Saved Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Positioning the Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Running a Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Running Signal Accumulation Mode (SAM) Protocols . . . . . . . . . . . . . . . . . . . . . 90
Saving Signal Accumulation Mode (SAM) Images . . . . . . . . . . . . . . . . . . . . . . 91
Setting Up a Custom Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Regression Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Application Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Chapter 5 Viewing Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Displaying Gel Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Display Gel Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Zoom Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Fit in Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Image Transform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Image Colors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
3-D Projection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Image Info . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Displaying Multichannel Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Multichannel View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Splitting Multichannel Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Displaying Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Analysis Table Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Lane and Band Table Measurement Definitions . . . . . . . . . . . . . . . . . . . . . . . 117
Volume Measurement Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Lane Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

User Guide | v
Table of Contents

Chapter 6 Analyzing Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Image Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Using Auto Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Analysis Tool Box Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Image Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Correcting a Slanted Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Cropping a Gel Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Inverting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Merging Images. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Lane and Bands Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Detecting Lanes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Using the All Lanes and Single Lane Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Copying Lanes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Detecting Bands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Editing the Detected Bands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Normalizing Volume Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Using the Normalization Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Optimizing Normalization of Volume Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Detect the Lanes in the Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Make Adjustments to the Lanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Detect the Bands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Subtract any Extraneous Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Remove Compromised Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
View the Data in the Analysis Table. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Adding a Channel to a Single Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Molecular Weight (MW) Analysis Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Quantity Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Annotation Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Add Annotations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Alignment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Text Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Rotate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Volume Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

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 Table of Contents

Chapter 7 Generating Reports. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Report Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Print Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Print Report to a PDF File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Adjust the Printer Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

Chapter 8 Exporting Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Exporting Gel Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Exporting Gel Images for Publication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Exporting Gel Images for Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Exporting Gel Images to PulseNet International . . . . . . . . . . . . . . . . . . . . . . . 185
Exporting Lane and Band Tables to Excel . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Exporting Volume Tables to File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Screenshot Tool Export. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Analysis Table Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

Chapter 9 System Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Recalibrating Your Imager. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

Chapter 10 Image Lab Logs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Image Lab Logs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Viewing the Instrument Log. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
Viewing the System Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Viewing the Document Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Displaying Log Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Displaying Data Columns in Logs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Filtering Data in Logs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Collapsing or Expanding Data Rows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Exporting Logs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Printing Logs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

User Guide | vii

Table of Contents

Chapter 11 Using the Security Edition . . . . . . . . . . . . . . . . . . . . . . . 203

21 CFR Part 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Standard Mode versus Secure Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
User Names, Groups, and Roles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Role Restrictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Starting Image Lab Security Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Electronic Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Unsecured Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Secure Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Modifying Secure Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Signing Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Document Logs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Viewing the Document Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

Appendix A Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

UV Transilluminator Lamp and Starter Replacement . . . . . . . . . . . . . . . . . . . . . 213
Fuse Replacement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

Appendix B Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

Appendix C Setting Up Users and Groups . . . . . . . . . . . . . . . . . . 219

Setting Up Image Lab Users and Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
User Accounts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
User Authentication and Group Membership . . . . . . . . . . . . . . . . . . . . . . . . . 219
Finding the name of your authentication domain . . . . . . . . . . . . . . . . . . . . . . 221
Configuring Users and Groups on a Local Computer . . . . . . . . . . . . . . . . . . . 222
Configuring Users and Groups on a Network Domain . . . . . . . . . . . . . . . . . . 226
Password Security. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
Password Policy Setting Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Account Lockout Policy Setting Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Auditing Windows Event Logs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Miscellaneous Security Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

viii | ChemiDoc MP Imaging System with Image Lab Software

 Table of Contents

Appendix D Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

Calibrating Accessories. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Installing Optional Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Epi Light Modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
UV/White Light Conversion Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
XcitaBlue™ Conversion Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Gel Alignment Template Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Orange Fluorescence Reference Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

Appendix E Using the Criterion Stain Free System . . . . . . . . . . . 245

Electrophoresis with Stain-Free Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Imaging Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Imaging Blots. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

Appendix F Mitsubishi P93/P95 Thermal Printer . . . . . . . . . . . . . 249

Setting up a Thermal Printer on Windows. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Setting up a Thermal Printer on a Mac . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

Appendix G Regression Calculation Methods . . . . . . . . . . . . . . . 251

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

User Guide | ix
Table of Contents

x | ChemiDoc MP Imaging System with Image Lab Software

 Safety and Regulatory
Compliance
Important Safety Information
Please read these instructions before attempting to operate the
ChemiDoc MP™ imaging system.

This instrument is suitable for research use only. It must be used, therefore, only by
specialized personnel who know the health risks associated with the reagents that
are normally used with this instrument.

Use of the ChemiDoc MP imaging system involves UV illumination. Proper

precautions must be taken to avoid eye and skin exposure to the UV radiation. This
instrument is meant for use only by trained personnel who know the health risks
associated with the UV radiation normally used with this instrument. The acrylic
shield provides some UV protection. However, it does not guarantee complete
protection, and it is designed to shield only the person working in front of the
imager.

WARNING! Use of the acrylic screen does not guarantee the user protection
from UV radiation. The use of protective eyeglasses, mask, and/or gloves is
strongly recommended.

User Guide | 11
 | Safety and Regulatory Compliance

Warranty
The ChemiDoc MP imaging system is warranted against defects in materials and
workmanship for one year. If any defect occurs in the instrument during this
warranty period, Bio-Rad Laboratories, Inc. will repair or replace the defective parts
at its discretion without charge. The following defects, however, are specifically
excluded:

 Defects caused by improper operation

 Repair or modification done by anyone other than Bio-Rad Laboratories,

Inc. or the company’s authorized agent

 Use of spare parts supplied by anyone other than Bio-Rad Laboratories,

Inc.

 Damage caused by accident or misuse

 Damage caused by disaster

 Corrosion caused by improper solvents or samples

General Precautions
 Read the user guide carefully.

 The instrument must be used only for the intended purpose of gel
documentation in research laboratories.

 The instrument must be connected to a grounded power source line and

protected by a circuit breaker.

 Do not pour liquids directly on or inside the instrument.

 Switch off all lights on the instrument immediately after use.

 Clean the transilluminator sample area after use.

12 | ChemiDoc MP Imaging System with Image Lab Software

 Regulatory Notices

Regulatory Notices
The ChemiDoc MP imaging system is designed and certified to meet EN 61010, the
internationally accepted electrical safety standard, EMC regulations, and TUV
requirements. Certified products are safe to use when operated in accordance with
this user guide. Do not modify or alter this instrument in any way. Modification or
alteration of this instrument will:

 Void the manufacturer’s warranty

 Void the regulatory certifications

 Create a potential safety hazard

WARNING! Bio-Rad Laboratories, Inc. is not responsible for any injury or

damage caused by use of this instrument for purposes other than those for
which it is intended or by modifications of the instrument not performed by
Bio-Rad Laboratories, Inc., or an authorized agent.

Instrument Safety Warnings

Notes, cautions, and warnings are used to highlight certain operating procedures
and recommendations. The table below describes how each is used in this
document.

Table 1. Notes, Cautions, and Warnings

Icon Meaning
Note: Note: A note indicates a special procedure, an exception to normal
operation, or something else of specific interest to the reader. Notes are
preceded by the word Note.
Caution: A caution precedes an operational step that could damage the
instrument or destroy data unless the operator takes certain precautions.
Cautions located in the main text are preceded by the word Caution and are
accompanied by the caution symbol in the left margin.

User Guide | 13
 | Safety and Regulatory Compliance

Table 1. Notes, Cautions, and Warnings, continued

Icon Meaning
Caution: With the exception of cleaning or replacing light bulbs, refer all
servicing to qualified Bio-Rad personnel or their agents. If you experience
technical difficulties with the instrument, contact Bio-Rad to schedule
service. The instrument should not be modified or altered in any way.
Alteration voids the manufacturer’s warranty and might create a potential
safety hazard for the user.
Caution: If the case interlock is defeated, there is a possibility of UV-B
radiation hazard due to UV-B light exposure. Exercise caution when
servicing the instrument.
Caution: Bio-Rad is not responsible for any injury or damage caused by the
use of this instrument for purposes other than that for which it is intended, or
by the modification of this instrument when not performed by qualified
Bio-Rad personnel or their agents.
Caution: Disconnect the AC power cord before removing the instrument
cover.
Warning: A warning precedes an operating procedure that could cause
injury to the operator if not followed correctly. Warnings located in the main
text are preceded by the word Warning and are accompanied by the
warning symbol in the left margin.
Warning: This instrument must be connected to an appropriate AC voltage
outlet that is properly grounded.

Notice
TheChemiDoc MP imager is intended for laboratory use only. This device is meant
for use by specialized personnel who know the health risks associated with
reagents normally used in electrophoresis. The UV light source is computer
controlled, and proper interlocks are implemented to avoid users’ accidental
exposure to UV radiation. Bio-Rad Laboratories, Inc. is not responsible for any injury
or damage caused by use of this instrument for purposes other than those for which
it is intended, or for instrument modifications not performed by
Bio-Rad Laboratories, Inc. or an authorized agent.

14 | ChemiDoc MP Imaging System with Image Lab Software

 Power Safety Information

Power Safety Information

Voltage Setting Information

The universal hood of the ChemiDoc MP imager has a power supply that
automatically chooses the correct voltage for your country or region.

Fuses
The universal hood of the ChemiDoc MP imager has two user-serviceable fuses, F1
and F2, which are located on the bottom rear panel and are a part of the power entry
module. See Fuse Replacement on page 215 for information about replacing the
fuses.

User Guide | 15
 | Safety and Regulatory Compliance

16 | ChemiDoc MP Imaging System with Image Lab Software

 1 Introduction

The ChemiDoc™ MP imaging system offers exceptional application flexibility, high

performance, and ease of use. The imager contains a charge-coupled device (CCD)
camera to capture images in real time and enable you to accurately position your
sample and generate optimized image data.

The ChemiDoc MP imager uses a new generation lighttight enclosure (the universal
hood III), which contains built-in UV and white light illumination as well as available
red, green, and blue epi LED light sources. The imager features dynamic flat fielding
technology for superior image uniformity and accurate quantification.

Image Lab™ software controls image capture and optimization for your selected
applications, analyzes results, and produces reports based on your specified
output, all in a single workflow.

ChemiDoc MP Imager
The ChemiDoc MP imager is a high-resolution gel documentation system that
allows fast, easy quantification of gels and blots. Position your sample inside the
imager and follow the onscreen steps to run a protocol with only one click. You can
customize your applications within an existing protocol or create a new protocol
using the many options presented in Image Lab software.

The ChemiDoc MP imager also offers sensitive chemiluminescent detection. The

system includes a supersensitive 16-bit CCD camera that is deeply cooled for
faint-sample detection and for accurate quantification of image data.

User Guide | 17
1 | Introduction

Features include:

 Smart, application-based protocol setup using Image Lab software, which

assists by presenting appropriate filter and illumination sources for imaging
applications that require excellent sensitivity

 Exceptional sensitivity and a dynamic range greater than four orders of

magnitude

 Flexibility to image chemiluminescent, fluorescent, and colorimetric

samples with dynamic flat fielding specific to each application

System Components

CCD Camera and Lenses

The ChemiDoc MP imager’s camera is placed on top of a lighttight enclosure (the
universal hood) for capturing images. The camera comes with a motorized zoom
lens (MZL) that allows remote adjustment of the lens control functions (zoom, focus,
and iris).

A patent-pending software algorithm controls the MZL, giving the user automatic
image focus once an initial calibration is performed during system installation. See
the ChemiDoc MP Technical Specifications on page 23 for complete specifications
of the system.

A +1 diopter lens is factory installed to allow the entire sample stage to be visible.
This lens should always remain on the MZL assembly.

18 | ChemiDoc MP Imaging System with Image Lab Software

 System Components

Universal Hood III

The universal hood III is designed to capture fluorescent and chemiluminescent
images without using a photographic darkroom. The enclosure has built-in white
light epi-illumination and UV transillumination. For easy sample loading, the UV
transilluminator is located in the drawer of the universal hood and can be accessed
from the front of the enclosure. When not imaging, the lights in the darkroom
enclosure turn off automatically.

The universal hood III has touchpad buttons to perform various functions; however,
Image Lab software controls all of these functions remotely, removing any
requirement for manual control of the lens and lights. Running a protocol overrides
touchpad input.

Image Lab Software

The imager ships with a full version of Image Lab software. In addition to controlling
the imager, image capture, and optimization, Image Lab software can be used to
annotate and document images, analyze molecular weights (or base pairs, when
imaging nucleic acid gels), and determine accurate quantification and purity of
samples.

You can print all or a subset of your data in a report. Alternatively, you can export
your data to other software, such as Microsoft Office programs, for further analysis
or presentation options.

Emission Filters
The universal hood III can hold up to six different emission filters for fluorescent
applications. No filter is required to image chemiluminescent samples.

A standard filter is used for colorimetric (white light) applications and is included in
the installation kit.

User Guide | 19
1 | Introduction

Optional Accessories
Bio-Rad Laboratories, Inc. offers a selection of optional filters and illumination
sources. See Ordering Information on page 242 for a complete listing of accessory
filters, UV light sources, optional parts, and replacement parts.

Printer
For your convenience, Bio-Rad offers an optional USB printer for use with the
ChemiDoc MP system: the Mitsubishi thermal printer (catalog #170-8089).

Conversion Screens
White Light Conversion Screen
The white light conversion screen is a phosphor screen that produces white light
transillumination when placed on top of the UV transilluminator.

XcitaBlue Conversion Screen

The optional XcitaBlue™ screen kit (catalog # 170-8182) converts UV to blue light,
which enables you to visualize DNA samples while protecting them from UV
damage.

Optional Light Sources

Red LED Module
The optional red LED module kit contains the emission filter and excitation source
for fluorescent applications. Instructions are also included.

Green LED Module

The optional green LED module kit contains the emission filter and excitation source
for fluorescent applications. Instructions are also included.

Blue LED Module

The optional blue LED module kit contains the emission filter and excitation source
for fluorescent applications. Instructions are also included.

20 | ChemiDoc MP Imaging System with Image Lab Software

 ChemiDoc MP Imager Applications

ChemiDoc MP Imager Applications

The ChemiDoc MP imager is capable of running protocols to image blots that use
various detection reagents for chemiluminescent, colorimetric, and fluorescent
applications. It can also image singleplex, multiplex, and stain-free gels and blots.
Contact Bio-Rad technical support to determine whether your gel or blot can be
imaged on this instrument.

See Chapter 4, Acquiring Images, for detailed instructions on designing protocols.

Nucleic Acid Gels

 Ethidium bromide  OliGreen

 Fluorescein  PicoGreen
 GelGreen  SYBR® Gold
 GelRed  SYBR® Green
 GelStar  SYBR® Safe

Protein Gels

 Stain-free gel  Coomassie Fluor Orange

 Oriole™ fluorescent stain  Pro-Q Diamond
 Flamingo™ fluorescent stain  Pro-Q Emerald 300
 SYPRO Ruby  Pro-Q Emerald 488
 Krypton

Blots

 Chemiluminescent reagent  Alexa 680

 Chemi hi sensitivity reagent  DyLight 488
 Chemi hi resolution reagent  DyLight 549
 Stain-free blot reagent  DyLight 649
 Colorimetric  DyLight 680

User Guide | 21
1 | Introduction

 Cy2  IRDye 680

 Cy3  Rhodamine
 Cy5  Fluorescein
 Cy5.5  Qdots 525
 Alexa 488  Qdots 605
 Alexa 546  Qdots 625
 Alexa 647  Qdots 705

22 | ChemiDoc MP Imaging System with Image Lab Software

 ChemiDoc MP Technical Specifications

ChemiDoc MP Technical Specifications

Applications
Chemiluminescence Yes
Fluorescence* Yes
Colorimetry/densitometry Yes
Gel documentation Yes
Hardware Specifications
Maximum sample size  Length: 28 cm
 Width: 36 cm
Maximum image area  Length: 26 cm
 Width: 35 cm
Maximum image area for  Length: 25 cm
standard, UV-excited gels  Width: 26 cm
Excitation source  Trans-UV and epi-white are standard (302 nm included,
with 254 and 365 nm available as options).
 Optional trans-white conversion screen.
 Optional XcitaBlue™ UV/blue conversion screen. Blue,
green, and red epis.
Detector Supercooled CCD
Pixel size (H x V in microns) 6.45 x 6.45
Cooling system Peltier cooled
Camera cooling –30°C controlled
temperature
Filter selector  6-position filter wheel
 1 without filter for chemiluminescence
Emission filters  1 included (standard)
 3 optional (530, 605, 695)
Dynamic range >4.0 orders of magnitude
Pixel density (gray levels) 65,535

User Guide | 23
1 | Introduction

Dynamic flat fielding Application-specific, for all applications

Instrument size  Length: 36 cm
 Width: 60 cm
 Height: 96 cm
Instrument weight 32 kg
Operating Ranges
Operating voltage AC 110/115/230 V nominal
Operating temperature 10–28°C (21°C recommended)
Operating humidity <70% noncondensing
Automation Capabilities
Workflow automated Application driven, user-selected or recalled by a protocol
selection
Workflow automated Controlled by a protocol via application-specific setup for
execution image area, illumination source, filter, analysis, focus, and
reporting
Workflow reproducibility 100% repeatability via recallable protocols; from image
capture to quantitative analysis and reports
Autofocus (patent pending) Precalibrated focus for any zoom setting
Image flat fielding Dynamic; precalibrated and optimized per application
(patent pending)
Autoexposure 2 user-defined modes (intense or faint bands)
* Using the optional XcitaBlue kit (catalog # 170-8182) is highly recommended if performing
preparative DNA applications with blue excitable stains. The UV to blue conversion screen allows
you to visualize DNA samples while protecting against UV damage.

24 | ChemiDoc MP Imaging System with Image Lab Software

 ChemiDoc MP Imaging System Workflow

ChemiDoc MP Imaging System Workflow

Following are the basic steps to acquiring, analyzing, and archiving an image using
the ChemiDoc MP imaging system and Image Lab software:

1. Select an existing protocol or customize a new one.

2. Position the gel or blot to be imaged.

3. Run your selected protocol.

4. View the displayed results.

5. Optimize the analysis.

6. Generate a report.

7. Save or export the results.

For More Information

Refer to the ChemiDoc MP Installation Guide found in your ChemiDoc MP
installation kit for instructions about assembling and calibrating the ChemiDoc MP
imager. Refer to Chapter 2, Setting Up the Instrument with Image Lab Software for
information about installing Image Lab software.

To recalibrate your imager because you have acquired new accessories, refer to
Chapter 9, System Calibration.

User Guide | 25
1 | Introduction

26 | ChemiDoc MP Imaging System with Image Lab Software

 2 Setting Up the Instrument
with Image Lab Software
System Requirements
Image Lab™ software runs on Microsoft Windows XP Professional, Microsoft
Windows 7, and Mac OS X. Images scanned at high resolution can be quite large.
The amount of memory required for using the program is determined by the size of
the images you scan and analyze.

For this reason, we recommend that you archive images on a network file server or
on removable storage media. Bio-Rad can also provide an appropriate computer to
use with this system. Contact your local Bio-Rad representative for more details.

Image Lab Security Edition

Note: The system requirements for Image Lab Security Edition are the same as
those for the Standard Edition. The software must be installed on a computer
running the Windows XP Professional or Windows 7 operating system to take
advantage of the secure mode features.

Computer Specifications

Specifications Minimum Recommended

Operating system Windows XP SP3 Windows XP SP3 Professional

Windows 7, 32- and 64-bit Windows 7 Professional, 64-bit
Mac OS X 10.6 Mac OS X 10.6
Processor Pentium 4 or equivalent (Windows) Intel Core 2 Duo 2.0 GHz or
at 2.0 GHz higher

User Guide | 27
2 | Setting Up the Instrument with Image Lab Software

Specifications Minimum Recommended

Hard disk space 20 GB >100 GB

Memory (RAM) 1,024 MB >1,024 MB
Ports for connecting 1 free USB 2.0 port 1 free USB 2.0 port
instrument
Other software Microsoft Excel 2003 or later Microsoft Excel 2003 or later
(optional) (Windows) (Windows)
Office 2008 or iWork software Office 2008 or iWork software
(Mac) (Mac)

Installing Image Lab Software

The process of installing Image Lab differs depending on your operating system.
This section explains how to install Image Lab software on a Windows PC and on a
Mac.

Note: During the Windows installation process, you are prompted to install
Image Lab Standard Edition or Security Edition. In order to enable secure
mode, you must have Image Lab Administrator role privileges. Bio-Rad
recommends that you create the required groups and assign the Image Lab
roles to users within those groups before installing Image Lab software.

See User Names, Groups, and Roles on page 204 for additional information about
the required Image Lab roles, groups, and users.

See Setting Up Users and Groups on page 219 for information about setting up
groups, user names, and passwords.

To install Image Lab on a Windows PC

1. Insert the Image Lab software CD in your CD-ROM drive.

28 | ChemiDoc MP Imaging System with Image Lab Software

 Installing Image Lab Software

The Image Lab installer automatically launches.

2. On the Welcome screen, click Next.

3. Accept the license agreement and click Next.

4. On the Edition Selection screen, choose the edition to install.

User Guide | 29
2 | Setting Up the Instrument with Image Lab Software

Note the following.

 If you are licensed to install the Security Edition but choose to install the
Standard Edition, you will need to uninstall the Standard Edition and install
the Security Edition before you can use it.

 If you are licensed to install the Security Edition and choose to install it,
only a user assigned the Image Lab Administrator role (or group) privileges
can enable and disable secure mode.

Note: A user assigned the Image Lab Administrator role will not
necessarily also be the network or IT administrator. You must be
assigned the correct role within Image Lab to enable or disable secure
mode.

For more information, see Setting Up Image Lab Security Edition on

page 35.

 If you are not licensed to install the Security Edition but choose to install it,
you will be prompted for a license key when you start Image Lab.

5. Click Next.

30 | ChemiDoc MP Imaging System with Image Lab Software

 Installing Image Lab Software

6. A screen appears requesting your license code.

Note: Your 18-digit license code can be found in the Image Lab product
folder pocket.

Enter the 18–digit code in the three text boxes. The software verifies the code.

Tip: If you do not know or do not have access to the code, contact your
Bio-Rad customer service representative.

Note: Until you provide a license code, Image Lab will function only in
standard mode.

7. Click Next.

8. On the Install Location screen, accept the default location or click Change and
browse to another folder.

9. Click Next.

User Guide | 31
2 | Setting Up the Instrument with Image Lab Software

10. On the Ready to Install the Program screen, click Install.

The wizard installs Image Lab.

32 | ChemiDoc MP Imaging System with Image Lab Software

 Installing Image Lab Software

11. When the installation is complete, you are prompted to display the Release
Notes and/or the Windows Installer log.

The following screen shot is an example of the Windows Installer log.

12. Select or clear the checkboxes in the Install Wizard Completed dialog.

13. Click Finish to exit the wizard.

User Guide | 33
2 | Setting Up the Instrument with Image Lab Software

The Image Lab icon appears on your desktop. Follow the instructions in the next
section to connect your system.

To install Image Lab on a Mac

1. Insert the Image Lab software CD in your CD-ROM drive.

2. Double-click the CD icon on your desktop to see the folder contents.

3. Double-click the file Image Lab.dmg.

4. Drag the Image Lab application icon into the Applications folder.

Follow the instructions in the next section to connect your system.

34 | ChemiDoc MP Imaging System with Image Lab Software

 Installing the Drivers on Windows 7

Installing the Drivers on Windows 7

If you are running Windows 7, the device driver is installed during the Image Lab
installation process. After successful installation, you see a message similar to the
following.

Note: During the installation process you might see a warning similar to the
following. You can ignore this warning because it appears even when the driver
has been installed correctly.

Setting Up Image Lab Security Edition

Activating and Deactivating Security Edition

 Before you activate or deactivate Image Lab Security Edition, close any
open document files.

 After you change the active status of the Security Edition, you must restart
Image Lab.

Activate the Image Lab Security Edition

1. Double-click the Image Lab icon on your desktop to open Image Lab.

User Guide | 35
2 | Setting Up the Instrument with Image Lab Software

The License Code field is populated with the 18-digit license code that you
entered when you installed Image Lab.

The Security Edition Activation dialog box appears.

You can activate the Security Edition automatically via the Internet, or you can
activate it manually by creating an activation email.

To activate the Security Edition via the Internet

1. Select Activate Via Internet.

2. Click Activate.

36 | ChemiDoc MP Imaging System with Image Lab Software

 Setting Up Image Lab Security Edition

Within about 30 seconds you will receive a confirmation that your Image Lab
Security Edition has been activated.

To activate the Security Edition via email

1. Double-click the Image Lab icon on your desktop to open Image Lab.

The Security Edition Activation dialog box appears.

The License Code field is populated with the 18-digit license code that you
entered when you installed Image Lab.

2. Select Activate Via Create Activation Email.

3. Click Create Email.

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2 | Setting Up the Instrument with Image Lab Software

A Save File window appears with the File name field already filled in. Do not
change this file name.

4. Click Browse Folders to choose a location for the file, and click Save.

5. In your email program, create an email addressed to

[email protected], with the subject line: Request to Activate
Image Lab software Security Edition.

6. Attach the ActivationEmail.txt file to the email and click Send.

The Bio-Rad Technical Support Department will process your request and reply
with an email containing an attachment with your activation code.

7. When you receive your reply email, open it and save the attached
UnlockCode.txt file to the folder in which you saved the ActivationEmail.txt file.

8. On the Security menu, click Activate Security Edition to display the Security
Edition Activation dialog box.

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 Setting Up Image Lab Security Edition

9. Select Activate Via Receive Activation Email.

10. Click Receive Email.

An Open File window appears.

11. Navigate to the location where you saved the UnlockCode.txt file, select the
file, and click Open at the bottom of the window.

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2 | Setting Up the Instrument with Image Lab Software

The Security Edition activation complete window appears.

12. Click OK to close the window.

Deactivate Image Lab Security Edition

Image Lab can be installed on more than one computer. Bio-Rad Laboratories
recommends that you install the software on only one desktop computer and one
laptop. To load the Image Lab Security Edition on a second computer, the software
must be deactivated on the first computer before it can be activated on another. You
can deactivate the Security Edition automatically via the Internet, or you can
deactivate it manually by sending a deactivation email.

To deactivate the Security Edition automatically via the Internet

1. On the Security menu, click Deactivate Security Edition to display the
Deactivate Security Edition dialog box.

2. Select Deactivate Via Internet.

3. Click Deactivate.

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 Setting Up Image Lab Security Edition

The system sends a message to the Bio-Rad Technical Support Department, a

window stating that your deactivation was successful appears, and Image Lab
Security Edition is immediately deactivated.

To deactivate the Security Edition via email

1. From the Security menu, click Deactivate Security Edition to display the
Deactivate Security Edition dialog box.

2. Select Deactivate Via Create Deactivation Email.

3. Click Deactivate.

User Guide | 41
2 | Setting Up the Instrument with Image Lab Software

A Save File window appears.

4. Navigate to the folder in which you want to save the deactivation email and
click Save.

5. Create an email addressed to [email protected], with the

subject line: Request to Deactivate Image Lab software Security Edition.

6. Attach the DeactivationEmail.txt file to the email and click Send in your email
program.

The Bio-Rad Technical Support Department processes your request and

deactivates Image Lab Security Edition.

Enabling and Disabling Image Lab Secure Mode

You must have Image Lab Administrator role privileges to switch between
Image Lab secure mode and standard mode.

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 Setting Up Image Lab Security Edition

To enable secure mode

1. From the Security menu, click Security Preferences to display the Security
Preferences dialog box.

2. Select Enable secure mode.

3. Click OK to display the Enable Secure Mode authentication dialog box.

4. Enter your Image Lab Administrator user name and password.

5. If you are set up on a Windows network server, type the name of the Windows
domain in the Domain field.

Note: The default is the domain on which the current Windows user is
located, and this name will appear in the field

6. Click OK to save your changes.

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2 | Setting Up the Instrument with Image Lab Software

A message appears stating that you must restart Image Lab for the new secure
mode enable setting to take effect.

7. Click OK.

The application exits.

Important: For full details on why and how to set your security preferences, see
Setting Security Preferences on page 46.

To disable secure mode

1. From the Security menu, click Security Preferences to display the Security
Preferences dialog box.

2. Clear the Enable secure mode checkbox.

3. Click OK.

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 Setting Up Image Lab Security Edition

The Admin Authentication dialog appears.

4. Enter your Image Lab Administrator user name and password.

5. If you are set up on a Windows network server, type the name of the Windows
domain in the Domain field.

Note: The default is the domain on which the current Windows user is
located, and this name will appear in the field.

6. Click OK to save your changes.

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2 | Setting Up the Instrument with Image Lab Software

A message appears stating that you must restart Image Lab for the new
security settings to take effect.

7. Click OK.

The application exits.

Setting Security Preferences

Here are three useful definitions used in these instructions.

 Network domain — a remote domain-controlling computer or system

which ensures that only authorized users with valid credentials can access
and run Image Lab

 Local domain (or local computer) — the computer on which Image Lab is
running, and which ensures that only authorized users with valid user
credentials can access and run Image Lab

 Credentials — the valid user name and password that allows or prohibits
specific user actions

There are three possible combinations of settings in the Security Preferences dialog
box. Here are explanations of how to choose the settings and why you will want to
choose any of the three.

1. To set preferences so that only users who are set up on a network domain can
use Image Lab:

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 Setting Up Image Lab Security Edition

 In the Domain to be used in authentication field, enter the name of your

network domain. See To find the name of your network domain on
page 222 for instructions on how to determine this name.

Note: The default domain name that will appear in this field is the
domain on which the current user is logged in.

 Do not select the Use local groups for establishing user security levels
checkbox.

2. To set preferences so that only users who are domain users and who are also
valid members of specific local groups can run Image Lab:

 In the Domain to be used in authentication field, enter the name of your

network domain. See To find the name of your network domain on
page 222 for instructions on how to determine this name.

Note: The default domain name that will appear in this field is the
domain on which the current user is logged in.

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2 | Setting Up the Instrument with Image Lab Software

 Select the Use local groups for establishing user security levels checkbox.

3. To set preferences so that only local users can run Image Lab:

 In the Domain used in authentication field, enter your local computer name.
See To find the name of your local domain on page 221 for instructions on
how to determine this name.

Note: The default domain name that will appear in this field is the
domain on which the current user is logged in.

 The Use local groups for establishing user security levels checkbox is
grayed out (not accessible).

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 Setting Up Image Lab Security Edition

Rename Security Groups

To rename any of the four default Security Groups
1. From the main menu, select Security > Rename Security Groups.

Note: This menu option is visible only if the person logged on to the local
computer is logged on as a member of the Windows Administrators group.

2. Click in any of the four Group Name fields.

3. Enter a new name.

4. Click OK to save your changes.

Note: The new user group name must comply with standard Windows Local
Users and Groups user names rules.

For more information on setting up security groups, see Setting Up Users and
Groups on page 219.

Using Groups on a Local Domain

If you choose not to create or use groups on the network domain, set up local
groups. Add the authorized users to the groups on the local domain. In the Security
Preferences dialog box, select Use local groups for establishing user security levels.

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2 | Setting Up the Instrument with Image Lab Software

For information about setting up users and groups for Image Lab Security Edition,
see Appendix C, Setting Up Users and Groups on page 219.

Changing Security Preferences

Changing the domain that is used to authenticate users is a two-step process. You
first authenticate on the first domain, then authenticate on the second domain. This
change in domains can be performed in either of two ways. It can be performed by
one individual assigned the Image Lab Administrator role on both domains, or it can
be performed by two individuals, one with the administrator role on the first domain,
and the other with the administrator role on the second domain. See User
Authentication and Group Membership on page 219 for more information about
using this dialog box.

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 3 Image Lab Software
Overview
Image Lab™ image acquisition and analysis software works with the
ChemiDoc™ MP imaging system to create a reproducible, automated, and
time-saving workflow for imaging and analyzing gels.

In Image Lab, a protocol is any combination of imaging, analysis, and report settings
that has been saved to run as a single workflow. Researchers can run one protocol
repeatedly or easily design a wide range of protocols.

With Image Lab you can view analyzed data, edit the analysis, and produce
customized reports that show precisely the settings applied in order to ensure
repeatable results.

Image Lab generates two types of files:

 Protocol files describe the parameters for imaging and analyzing your gel
images.

 Image files contain the imaged gel, annotations, and analysis performed on
the gel. An imaged gel, run according to a protocol file, generates an image
file.

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3 | Image Lab Software Overview

Table 2 lists the extensions and icons for the type of files that Image Lab generates.

Table 2. Image Lab file extensions and icons

File type File Extension Icon
Unsigned Signed Unsigned Signed
Protocols .ptl .sptl

Images .scn .sscn

Multichannel .mptl .smptl

protocols

Multichannel .mscn .smscn

images

52 | ChemiDoc MP Imaging System with Image Lab Software

 Interface Overview

Interface Overview
The following illustration shows the Image Lab main window. This section explains
the main software elements.

Main Window
Image Lab displays a single main window. All image and protocol dialog boxes that
present choices open in the workspace, which is the gray area of the main window.

If many screens are open in the workspace, you can make one active by clicking the
title bar at the top of the selected screen. A list of open protocols and image files
also appears in the main window menu. Select one to make it active.

You can view complete analyses for images or protocols one at a time or compare
image results by arranging screens in the workspace.

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3 | Image Lab Software Overview

Main Toolbar
Many Image Lab tools can be selected by clicking toolbar buttons. The Screenshot
tool enables you to send a screen capture of your image to the clipboard or to save
it as a file. You can view demonstrations of various functions by clicking Tutorials.
The unlimited Undo and Redo buttons enable you to correct missteps easily.

File management View results data

Results Data
Results data associated with gel images can be viewed as an analysis table, a lane
profile, a standard curve, or in a report. Different tools for viewing the results data
are easily accessible from the main toolbar. These tools are described in Chapter 5,
Viewing Images.

The views display the analysis for the selected image. All of the views can be
displayed at the same time. See Displaying Data on page 114 for details.

Display Toolbox
The display toolbox at the top of every image enables you to display images in the
most useful ways. See Chapter 5, Viewing Images for a description of each option.

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 Interface Overview

Start Page
The Start Page guides you through creating, opening, and viewing protocols and
images.

Analysis Tool Box

The Auto-Analysis button quickly analyzes images. The remaining

tools customize the analyzed data.

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3 | Image Lab Software Overview

An image file must be selected to make analysis tools available.

Image Tools enables you to flip, rotate, and crop images and to transform the
image files.

Lane and Bands enables you to resize, adjust, and bend lanes and to detect,
adjust, add, or delete bands.

Normalization enables you to normalize volume data in multichannel images, so

you can correct for sample loading errors in your gels.

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 Interface Overview

MW Analysis Tools (Molecular Weight Analysis) enables you to choose standard

samples, assign standard lanes, and choose a regression method.

Quantity Tools enables you to automatically quantify bands, using either relative or
absolute values.

Annotation Tools enables you to add formatted text and arrows to any area of a
gel.

Volume Tools enables you to manually quantify an object inside a boundary that
you define.

These tools are described in Analysis Tool Box Tools on page 127.

Status Bar
The status bar at the bottom of the main window shows the imager in use and the X
and Y values for the cursor position on the image file.

Note: The status bar also displays the intensity (Int) values for the image at the
cursor position. The maximum data range is 0–65,535. However, the actual
range varies depending on the values contained within each image.

Tip: For multichannel images, move your cursor over a multichannel pane to
display color-coded intensities for all channels.

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3 | Image Lab Software Overview

Menu Commands
The following section describes all menu commands in the File, Edit, View, Window,
and Help menus. Many commands are also available on the toolbar or the Start
Page.

File Menu Commands

New Protocol enables you to create a new protocol that contains the necessary
steps and choices. See Chapter 4, Acquiring Images for detailed instructions.
Protocols can also be altered and stored for reuse.

Open browses the file system to retrieve a previously saved protocol file or image
file.

Recent Images enables you to open a recent image file.

Recent Protocols enables you to open a recent protocol.

Save enables you to save a protocol or image file after it is named.

Save As enables you to name and store a protocol or image. Protocols are stored
with a .ptl or .sptl extension. Image files are stored with an .scn or .sscn extension.

Multiplex protocols are stored with an .mptl or .smptl extension, and multiplex
image files are stored with an .mscn or .smscn extension.

Create Multichannel Image enables you to create a multichannel image from

single-channel images and from single channels in other multichannel images.

Split Multichannel Image enables you to split the multichannel image into
individual image files. Each file has the same name as the multichannel image; the
application name is appended in parentheses.

Close closes the active window.

Close All closes all the windows.

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 Interface Overview

Export enables you to export gel images or analysis tables with the following
options:

 Export for Publication — exports a displayed image to a file. You can

select from .bmp, .png, .jpg, and .tif formats. The gel displays with any
lanes, bands, and annotations that appear on the screen. See Exporting
Gel Images for Publication on page 182 for more information.

 Export for Analysis — creates a .tif file that retains all gel image data.
Analysis data are not included. Use this option to analyze the image in
other software such as Quantity One®,FPQuest™, or InfoQuest™FP. See
Exporting Gel Images for Publication on page 182 for more information.

 Export for PulseNet — reduces the image to an 8-bit .tif file. Resolution is
limited and file size is restricted to 300 dots per inch (dpi).

 Lane and Band Table to Excel — exports your lane and band table data
to an Excel (or Numbers on a Mac) spreadsheet.

Note: Excel or Numbers must be installed on your computer.

 Lane and Band Table to File — exports as a comma-separated values

(CSV) file so that your lane and band table can be opened in a database
application.

 Volume Table to Excel — exports your volume table data to an Excel (or
Numbers on a Mac) spreadsheet.

Note: Excel or Numbers must be installed on your computer.

 Volume Table to File — exports as a CSV file so your volume table can be
opened in a database application. See Exporting Volume Tables to File on
page 185 for detailed information about exporting files.

See Chapter 8, Exporting Results for more information about exporting files.

Image Info displays information about individual gel and blot images, such as
acquisition date and data range, and image capture detail, such as exposure time
and illumination source used. Click the Image Details, Analysis Settings, and Notes
tabs to display these properties. See Image Info on page 107 for more information.

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3 | Image Lab Software Overview

Page Setup contains print controls such as orientation (landscape or portrait),

margins, printer used, and paper size.

Print displays a print preview of the gel and the header information, which includes
the filename of the image, the user’s name, and the date and time it was printed.
The usual Windows Print screen is available as well, enabling you to select a printer
and the number of copies to print.

Exit closes Image Lab (after prompting you to save changes to your protocols or
images).

Edit Menu Commands

Undo undoes the last action.

Redo restores the last action after an Undo.

Screenshot enables you to take a screen shot of the Lane Profile Window, the
Standard Curve Window, or the default choice, Current Image View. The screen shot
can include the name of the image, and it can be placed on the clipboard or saved
in a file.

Default Imager enables users who own two or more imagers to switch between
them.

Instrument Setup displays information about the instrument, including its name,
serial number, camera serial number, illumination options, and last calibration. If you
add accessories to the instrument, you can reset the system calibration in this
dialog box.

Report Settings enables you to configure reports. This dialog box has three tabs.
All of the checkboxes are selected by default. Clear the boxes to exclude
information from reports. Your selections apply to all reports until you change them.

 The General tab has options for excluding or reporting information about
your gel image.

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 Interface Overview

 The Lane and Band Table tab enables the researcher to choose whether to
include all lanes or selected lanes, with appropriate identifiers. Lane
profiles can also be included.

 The Volume Table tab enables the researcher to choose appropriate

identifiers for the volume table and provides the option of excluding the
table from reports.

Preferences enables you to set naming and color preferences for your image files.
This dialog box has two tabs.

 The Protocol tab shows presets for naming image files. You can choose to
include a designated Prefix, User Name, Date, and/or Time in the name of
your image files.

 The Colors tab enables you to choose colors for the graphic elements in
your gels, such as Lane Frame, Lane, Band, Band Attribute, and MW
Legend. This functionality ensures that these elements are visible,
regardless of the color of the gels.

View Menu Commands

Image Overview displays the gel image with a red rectangle outlining the area
visible in the larger main window. This is useful when you zoom in to a small section
of an image.

Image Transform displays a histogram that enables you to adjust the light and dark
values of a gel image. This adjustment does not change your data, only the way the
data display on your monitor.

Operations History displays the sequence of actions performed by both the user
and the software.

View System Log displays events related to running Image Lab software, including
enabling or disabling secure mode, and the users who log on to or log off of the
software.

View (Instrument) Log displays events related to the instrument, including

calibrating the instrument and the success or failure of the calibration. This log file is
visible only if Image Lab is connected to an instrument.

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3 | Image Lab Software Overview

View (Document) Log displays events related to creating and modifying secure
protocol and image files. This log file is created only when Security Edition is
enabled.

Security Menu Commands

Note: This menu is only visible in Image Lab Security Edition. For more
information about Security Edition, see Chapter 11, Using the Security Edition.

Security Preferences allows the Image Lab Administrator to enable and disable
secure mode. In this dialog box, the administrator chooses the domain to be used
for authentication and whether to use local groups for security levels.

Rename Security Groups allows a person logged on as a member the Windows

Administrators group to change any of the four default Image Lab security group
names (TDS_Administrator, TDS_User, TDS_Tech, and TDS_Guest).

Note: This menu option is visible only if the person logged on to the local
computer is logged on as a member of the Windows Administrators group.
Only users who are logged on as a member of that group maintain the authority
to change any of the four default Image Lab security group names. Any
changes made to these Security Group names must match the names your
Windows system administrator has given those groups.

Sign Document enables users to sign images and protocols. Users enter their user
name and password and provide a reason for signing. When the document is
signed, the reason is saved in the System Log file.

Window Menu Commands

The Window controls enable you to show and hide multiple open image files in your
workspace. A list of all currently open images and protocols appears in this menu.

Tile aligns all open image files so they are visible at the same time.

Tile Horizontal aligns all open image files from top to bottom.

Tile Vertical aligns all open image files from left to right.

Cascade stacks all open image files and protocols with overlapping title bars, so
each one can be easily chosen for viewing.

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 Interface Overview

Imitate Zoom changes the zoom setting of all open images to the same zoom
setting as the current image file.

Imitate Transform changes the brightness and contrast of all open images to the
same transform settings as the current image file.

Next cycles through all open image files from oldest to newest.

Previous cycles through all open image files from newest to oldest.

Help Menu Commands

Image Lab Help displays the help system.

User Guide displays the instruction manual in .pdf form.

About displays Image Lab software version and release date.

User Guide | 63
3 | Image Lab Software Overview

64 | ChemiDoc MP Imaging System with Image Lab Software

 4 Acquiring Images

Image Lab software runs specific applications with repeatable workflows using
configurable protocols that have a wide variety of settings. These protocols can be
retrieved, revised, and reused.

In Image Lab, a protocol is any combination of settings for imaging, analyzing, and
reporting that runs as a single workflow.

Image Lab supports two kinds of protocols: single-channel and multichannel. A

single-channel protocol enables you to choose one application for acquisition of a
single image from a gel or blot, with the exception of signal accumulation mode for
chemiluminescence. Multichannel protocols allow you to sequentially acquire up to
three separate images of a gel or blot, using different illumination, filter, and
exposure settings, to create a combined image. The multichannel image can be
viewed as an RGB (red, green, blue) color-composite image.

Note: The Image Lab software is designed to work with different imagers,
some of which can only produce single-channel images. The New Protocol
menu choices change depending on whether the imager supports multichannel
images or not. If you are using a ChemiDoc™ MP imager and you do not see
the multichannel option, check to see that your default imager selection is set
to ChemiDoc MP and not one of the single-channel imagers.

To create a new protocol, do one of the following:

 Click the New Protocol button on the toolbar, and then select Single
Channel or Multichannel in the menu that appears.

 In the Protocols box on the Start Page, click New Single Channel or New
Multichannel.

The appropriate Protocol Setup window appears for the type of protocol you
selected.

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4 | Acquiring Images

The Protocol Setup Window

Single-Channel Protocols
The left pane displays headings. Under the headings are numbered protocol steps.
You can enable or disable a step by selecting or clearing its checkbox. When you
select a step, the right pane of the window displays the detailed settings for that
step.

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 The Protocol Setup Window

You can review protocol settings by selecting Protocol Summary, which lists the
settings for each step in the right pane of the Protocol Setup window.

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4 | Acquiring Images

Multichannel Protocols
The Protocol Setup window for multichannels has some settings that differ from the
single-channel Protocol Setup window, but you work with it the same way as the
single-channel Protocol Setup window.

Select a step in the left pane and configure the settings for that step in the right
pane.

Click Protocol Summary in the left pane to view all your protocol settings in the right
pane.

Creating a Protocol
There are three categories of settings when setting up a protocol:

 Acquisition Settings – settings to acquire the image

 Analyze Image – settings to detect lanes and bands and to analyze the
molecular weight

 Generate Output – settings to generate the output

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 Creating a Protocol

The three categories are listed in the left pane of the Protocol Setup window.
Numbered steps in each category appear under these headings. To select an option
under a protocol step, select the accompanying checkbox. Options for that step
appear in the right pane of the window. To disable any step, clear its checkbox.

There are some differences in the gel imaging settings of single-channel protocols
and multichannel protocols. These settings are explained in Step 1. Gel Imaging for
Single-Channel Protocols on page 70 and Step 1. Gel Imaging for Multichannel
Protocols on page 72, in the Protocol Setup window. The remaining steps in the
protocol setup are virtually identical.

Setting Up a New Protocol

The following steps set up (or create) a protocol:

 Step 1. Gel Imaging for Single-Channel Protocols — use this step (on
page 70) if you are creating a single-channel protocol

 Step 1. Gel Imaging for Multichannel Protocols — use this step (on
page 72) if you are creating a multichannel protocol

 Step 2. Detect Lanes and Bands

 Step 3. Analyze Molecular Weight

 Step 4. Specify Output

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4 | Acquiring Images

Step 1. Gel Imaging for Single-Channel Protocols

To take an image of the gel or blot, you need to configure the acquisition settings for
the protocol.

To configure the acquisition settings

1. In the right pane, click Select and choose an application from the menu. The
detection reagents appear in submenus under each application type.

When you choose an application and detection reagent, any required filter or
illumination source displays in the Protocol Setup window.

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 Creating a Protocol

Choose Custom to save and run an existing application with a new name or to
create and run a new application. Previously saved custom applications display
here. To create a custom application, see Setting Up a Custom Application on
page 92.

Tip: For a list of applications with all required detection reagents, light
sources, and any conversion screens or filters noted, see on page 97.

Note: If you select the Stain Free option, you may select the gel activation
time. See Appendix E, Using the Criterion Stain Free System, on page 245
for more information.

2. In Imaging Area, select from the list of Bio-Rad gels or enter the image area
dimensions. The red box represents the imaging area for the selected gel, and
the gray rectangle represents the imager sample stage.

3. In Image Exposure, select from one of the following options:

 Auto Exposure — this setting estimates an optimal exposure time and

ensures the best use of the dynamic range. Intense Bands optimizes for all
bands and Faint Bands makes faint bands more visible but might
overexpose more prominent bands.

 Intense Bands — optimizes exposure for all bands

 Faint Bands — a longer exposure time is used making faint bands

more visible, but more prominent bands might be overexposed

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4 | Acquiring Images

 Manual Exposure — use this setting to manually override automated

imaging. Exposure time can range from 0.001 to 7,200 seconds with the
ChemiDoc MP imager.

 After imaging a gel with automatic exposure optimization, the display

provides a reference point from which to set your manual exposure time.

Note: You can view the exposure time of the image in the Image Info
window (see Image Info on page 107).

Step 1. Gel Imaging for Multichannel Protocols

You must configure acquisition settings for at least two channels to create a valid
multichannel protocol. Each channel can be assigned a color (red, green, or blue) so
you can easily identify each channel. Configure each channel separately.

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 Creating a Protocol

To configure the acquisition settings

1. In the right pane of the Protocol Setup window, click Configure in the Channel 1
box. The Configure Channel 1 dialog box appears.

2. Click Select and choose an application from the dropdown list that appears.

Note: Select Custom to run an existing application with a new name or to

create an application unlike existing applications. See Setting Up a Custom
Application on page 92 for more information.

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4 | Acquiring Images

For a list of applications with all required detection reagents, light sources, and
any conversion screens or filters noted, see Application Tables on page 95.

3. In Image Exposure, if you select the first radio button, Image Lab acquires an
image estimating an optimal exposure time. This option ensures the best use of
the dynamic range.

 If you choose Intense Bands from the list, exposure is optimized for all
bands.

 If you choose Faint Bands from the list, a longer exposure makes faint
bands more visible, but more prominent bands might be overexposed.

 If you select the second radio button to manually override automated

imaging, you can set the exposure time from 0.001–7,200 seconds.

After imaging a gel with automatic exposure optimization, the exposure time
displays in the protocol so you can manually adjust it if needed.

Note: You can also view the exposure time of the image later, in the Image
Info window (see Image Info on page 107).

Signal Accumulation Mode — if you are running a chemiluminescence

application on the ChemiDoc MP imager, you can also use signal accumulation
mode (SAM).

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 Creating a Protocol

SAM is used to simplify the capture of a good image from a chemiluminescent

sample. This sample type often requires long integration times to obtain an
image that represents the best range of signal.

Rather than manually acquiring a series of independent images with different

imaging times, SAM presents a series of cumulative images with progressively
greater signal in each image. To run SAM you must estimate the shortest and
longest times expected to generate an image with the appropriate signal
intensity. You then decide how many total images to acquire in this window of
time.

For example, if the minimum expected time to image the sample is 1 minute
and the maximum is 5 minutes, these values are entered (in sec) in the setup
window. By entering 5 in the Total number of images field, three images will be
acquired between the first and last images.

To use SAM, click Signal Accumulation Mode and select Setup to display the
Signal Accumulation Setup dialog box.

In this example, the bar in the Signal Accumulation Setup dialog box indicates
that images will be acquired at 1 minute intervals, starting at 1 minute and
ending at 5 minutes. The second 1 minute image is added to the first 1 minute
image, and the final image is the result of integrating these two images. The
third 1 minute image is added to the previous image, and so on, until the last
image is presented.

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Although SAM is useful for determining the optimum imaging time for a
chemiluminescent sample, it results in data that are not as accurate as data
from a single image. Signal that is near the intensity of background noise
becomes increasingly masked as the number of cumulative images grows. To
identify extremely faint signals in an image, reacquire it as a single image, using
the time the SAM tool found to be appropriate.

4. Set the following attributes in Display Options:

 Highlight saturated pixels — select this checkbox to see any saturated

pixels in red. This shows how much of the gel image is saturated. You can
change this option later by selecting View > Image Transform.

 Channel Color — select a color to display the sample image. Assigning

each channel a different color makes it easy to identify channels. After you
set up the first channel, the second channel box becomes active.

5. Repeat steps 1–4 to set up the second and third channels, if applicable. The
software determines which channels are available based on the previously
selected applications. Each Configure box displays the settings for that
channel.

You can reconfigure channel settings by clicking Configure in the channel box
and changing the settings.

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 Creating a Protocol

6. In Imaging Area, select from a list of Bio-Rad gels or enter the image
dimensions. The red line represents the imaging area for the selected gel, and the
gray rectangle represents the imager sample stage.

Step 2. Detect Lanes and Bands

To analyze the gel or blot, Image Lab must detect lanes and bands on the image.
Lanes are detected automatically, and then the background is subtracted
automatically. Refer to Using the All Lanes and Single Lane Tools on page 132 for
details. Customize band detection with the following options.

To configure the lane and band detection settings

1. Select the Lane and Band Detection checkbox in the left pane of the Protocol
Setup window.

2. In the right pane, select one of the following lane and band detection options:

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4 | Acquiring Images

 Low Band Detection Sensitivity — this option sets detection at a low

level (25) for images with more prominent bands. Faint bands are not
detected with this setting.

 High Band Detection Sensitivity — this option sets detection at a higher

level (75) for images that are fainter. Extraneous bands can be removed
later, using the Band tools in the Analysis Tool Box. See Lane and Bands
Tools on page 131.

 Custom — select a numeric value from 1 to 100 to choose the best

detection sensitivity for your sample.

Note: You cannot specify different sensitivity levels for individual channels
of a multichannel image. The same sensitivity level is applied to all
channels. After the image is generated, you can change the sensitivity level
for individual channels using the Lane and Bands tools.

The following is a screen shot of the Detect Lanes and Bands window for
multichannel protocols.

To configure normalization settings

1. (Multichannel protocols only) Click Normalization Channel and specify which
channel to use as the normalization channel.

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 Creating a Protocol

The selected channel is used as the normalization factor against which all other
lanes in the channels are compared to normalize the volume data.

You can defer making a selection during the protocol setup and normalize your
data after the image is generated. For more information on normalizing volume
data, see Normalizing Volume Data on page 141.

Note: You must select an application for at least one channel in Step 1.
Gel Imaging for Multichannel Protocols on page 72 in order to see a list of
choices.

2. Select the type of normalization to use:

 Total Lane Protein Normalization — one lane in the normalization

channel is used to calculate the normalization factor.

 Housekeeping Protein Bands Normalization — a single band of a

housekeeping protein is used to calculate the normalization factor.

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Step 3. Analyze Molecular Weight

Determining molecular weight depends on selecting the proper protein standards.

Many protein standards are available from Bio-Rad and many different DNA
standards are also available. See Appendix D, Accessories for all Bio-Rad standards
and their catalog numbers.

To specify how molecular weight is analyzed

1. Select the Analyze Molecular Weight checkbox in the left pane of the Protocol
Setup window.

The software calculates the molecular weight for each band based on the
specified standard.

2. To estimate the size of the molecules in the bands of your gel, enter the
standard you are using.

Alternatively, you can choose from available standards:

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 Creating a Protocol

a. Click Change to display the Manage Standards dialog box.

b. Use the Show dropdown list to show all, only the protein, or only the DNA
standards.

c. Choose the standard that you need and click OK to exit the dialog box.

3. To create a new standard, click New in the Manage Standards dialog box and
complete the fields in the Edit Standard dialog.

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4 | Acquiring Images

a. Enter a name for the new standard.

b. Choose a standard type: protein or nucleic acid

A protein standard can be expressed in either isoelectric point (pI) or

molecular weight (kDa) units. A nucleic acid standard can be expressed in
base pairs (bp), kilo base pairs (kb), or mega base pairs (Mb) units.

c. Click Add to display the Edit dialog box.

Note: At least two values must be added to create a new standard.

d. Enter the value and description of your new standard and click OK to
return to the Edit Standard dialog box.

e. Click Add to display the Edit dialog box again, and enter a second value
and description for your new standard.

f. Click OK to save your changes and close the Edit dialog box.

g. Click OK in the Edit Standard dialog box to save your new standard.

h. Click OK in the Manage Standards dialog box to save your changes.

4. Choose the lane(s) that contain your standards by typing lane numbers or the
words First and Last in the Standard Lanes field. The format is xx, xx, xx, and
so on, where xx is the lane number. For example, if you run an 18-well gel and
want your standards in lanes 1, 10, and 18, enter First, 10, Last.

Note: Lane detection works best when standards are placed in the first
and last lanes. For nucleic acid samples, use this step to determine the
size of the bands in base pairs.

For more information, see Molecular Weight (MW) Analysis Tools on page 152.

5. Select the appropriate regression method for the gel type:

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 Creating a Protocol

 Gradient gels — the linear (semilog) regression method works well for
these gels because the mobility of the bands is linear to the log of their
molecular weight. As an alternative, the point-to-point (semilog) method
can be used if the R2 value is not sufficient.

 Fixed percentage gels — these gels have a nonlinear relationship

between the gels’ mobility and their molecular weight. For these gels,
choose the logistic or cubic spline regression method.

For more information about regression methods, see Regression Methods on

page 94.

Step 4. Specify Output

To specify the output of the protocol

1. Select Specify Output in the left pane of the Protocol Setup window to display
output options.

2. In the right pane, you can choose to

 Automatically print the image

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 Automatically print a report

 Display the report

Image Lab prints to the default printer unless you select otherwise. For
information about customizing reporting options, see Report on page 175.

Review Protocol Settings

Click Protocol Summary in the left pane of any Protocol Setup window to see a
quick review of all protocol settings.

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 Creating a Multichannel Image from Single Images

Creating a Multichannel Image from Single Images

You can create a multichannel image from existing single images or from single
channels in other multichannel images. Only images with the same aspect ratio can
be combined in a multichannel image. These images are not linked to one another.
When you make a change to one image, it is not propagated to the other images.

To create a multichannel image from single images

1. Open the single images from which you want to create a multichannel image.

2. In the File menu, click Create Multichannel Image. The list of open images
appears in the left pane of the Create Multichannel Image dialog box.

3. Drag each image into one of the channel boxes in the right pane. After you
select the first file, the Available Open Images list displays only files with the
same aspect ratio.

4. (Optional) Specify a color for each channel in the accompanying dropdown

lists. The resulting multichannel image appears in the Image Preview section of
the dialog box.

5. Click OK to save the multichannel image.

To replace a channel in a multichannel image

1. Open the multichannel image and the new image you want to use.

2. In the File menu, select Create Multichannel Image. The open image files are
listed in the Compatible Open Images list of the Create Multichannel Image
dialog box.

3. Drag the images you want to keep from the Available Open Images list (left
pane) into the channels in the New Multichannel Image pane (right pane).

4. Drag the new image you want to use into one of the available channel boxes.

5. Click OK to save the new multichannel image.

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Editing a Saved Protocol

You can change the protocol settings and rename the protocol using Image Lab
tools.

To edit a saved protocol

1. To open a saved protocol, you can:

 Click Open on the Start Page

 Click File > Open on the menu bar

You can also choose a recently used protocol from the lists on the Start Page.

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 Editing a Saved Protocol

You see the same set of menus and choices described in Creating a Protocol
on page 68.

2. To update an existing protocol, edit and then save your changes without
renaming the protocol.

3. To create a new protocol:

a. Edit the protocol.

b. Choose File > Save As and type a different name.

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Positioning the Gel

To position a gel
1. Click the yellow Position Your Gel button in the Protocol Setup window.

2. Place a gel on the imager stage and view the gel in Image Lab.

3. Use the slider below the image to zoom the image into place. You can also
move the gel manually until it is centered properly on the stage.

Note: The Bio-Rad gel alignment template kit allows four sizes of standard
agarose gels to be centered quickly and easily and ensures the consistent
placement of each gel. See Appendix D, Accessories, on page 235 for
more information.

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 Running a Protocol

Running a Protocol
To run a protocol
 Click Run Protocol in the left pane.

The software runs through the steps in imaging mode, imaging each channel. A
message at the bottom of the screen indicates the channel being imaged and a
progress indicator tracks the process.

Note: If you run a chemiluminescence application using autoexposure, you

might see the following error message:

The sample is too faint to use the autoexposure setting. Change the Image
Exposure setting to use manual exposure or Signal Accumulation Mode (SAM), then
run the protocol again.

This error occurs if Image Lab, using the initial autoexposure algorithm, determines
that it would take too long (approximately 30 minutes or more) to acquire an image.
You can always use the manual Image Exposure setting and specify a longer
exposure time.

After the images are taken, Image Lab continues with the detection, analysis, and
reporting steps if these steps were selected in the Protocol Setup pane. When
Image Lab has completed running the protocol, it displays the images that were
acquired. You can then edit and save these images or do further analysis on them.

To end the protocol

 Click Cancel Run.

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Running Signal Accumulation Mode (SAM) Protocols

You can interrupt the acquisition of images for a SAM-enabled protocol at any time
by clicking Stop Acquire and Continue with Selected. The acquisition process stops
and continues the protocol with the selected image. Any other images that were
acquired are discarded.

At the end of the acquisition process, a thumbnail of each image appears at the
bottom of the window. The last image acquired appears, by default, in the main
window. You can view any of the SAM images in the main window by clicking on the
thumbnail image.

Review the images, select the image you want to use in your analysis, and click
Select Image and Continue. Image Lab continues to the next step in the protocol
using the selected image.

Note: After you click Select Image and Continue, only the selected image is
retained. All other images are deleted. Therefore, save any images you want to
keep before continuing with the protocol. See Saving Signal Accumulation
Mode (SAM) Images on page 91 for more information.

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 Running Signal Accumulation Mode (SAM) Protocols

Saving Signal Accumulation Mode (SAM) Images

You can save any individual image or all images at once by right-clicking on the
image and selecting an option from the shortcut menu.

To save a single SAM image

1. Right-click the thumbnail and click Save.

2. In the Save File dialog box, accept the default name for the file or enter another
name. Click Save.

To save all SAM images

1. Right-click any of the images and click Save All.

2. In the Select Directory dialog, enter the name of the folder and click Select
Folder.

The images are saved in the specified folder. The name of the file includes the
user name, timestamp, and exposure time. For example: John Doe 2012-05-01
15 hr 44 min_Exposure_5.0sec.

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Setting Up a Custom Application

Use the Manage Custom Application dialog box to save an existing application with
a new name or to create an application unlike existing applications.

To create a custom application

1. Select Custom on the Applications menu to display the Manage Custom
Applications dialog box. If you have stored custom applications, they display
here.

2. Click New.

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 Setting Up a Custom Application

The Create Custom Application dialog box appears.

3. Enter a unique application name.

4. Select a light source, filter, and image color from the lists.

5. Select a binning setting. Choosing a higher binning setting combines pixels to

increase the amount of signal without increasing noise. While a higher setting
provides optimal sensitivity for low-light applications such as
chemiluminescence, it also reduces image resolution.

6. Click OK to create the custom application.

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Regression Methods
A regression method is used to calculate the molecular weight of the unknown
bands. The software uses the relative front and molecular weight values of the
standard bands to calculate the standard curve. This standard curve is then used to
calculate the values of the unknown bands. The shape of the standard curve is
based on the selected regression method. Choose one of the four regression
methods listed in Table 3.

Table 3. Methods of generating a standard curve

Regression Method Minimum number
of standard bands
Linear (semilog) 2
Point-to-point (semilog) 2
Logistic 5
Cubic spline 5

If you do not have enough data points for the selected method, the molecular
weight of the unknown bands is not calculated.

You can check how well each regression method fits the data in the standard curve
window (see Standard Curve on page 122 for more information). The linear (semilog)
regression method provides a measurement that describes how well the standard
curve fits the data R2 value. The closer the R2 value is to 1.0, the better the data fit
the standard curve.

The molecular weight of each band is displayed in the Mol. Wt./Base Pair column in
the analysis table. See Molecular Weight (MW) Analysis Tools on page 152 for more
information about molecular weight.

For information about the calculations behind the regression methods, see
Appendix G, Regression Calculation Methods.

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Application Tables
The following tables list the light type and filter to be used with various stains for
each application for the ChemiDoc MP imager.

Table 4. UV, Standard filters

Base System — UV, Standard Filter
Ethidium bromide Krypton
SYBR® Green Coomassie Fluor Orange
SYBR® Safe Pro-Q Diamond
SYBR® Gold Pro-Q Emerald 300
GelGreen Chemiluminescent
reagent
GelRed Chemi Hi Res
Fluorescein Chemi Hi Sens
OliGreen Stain-free blot
PicoGreen Colorimetric reagent
GelStar Rhodamine
Stain-free Qdot 525
Oriole™ stain Qdot 605
Flamingo™ stain Qdot 625
SYPRO Ruby

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Table 5. White trans standard and blue Epi filters

White Trans, Standard Filter Blue Epi (catalog #170-8285)
530/30 Filter
Fast Blast™ stain Pro-Q Emerald 488
Coomassie Blue Cy2
Silver stain Alexa 488
Copper stain DyLight 488
Zinc stain

Table 6. Red epi filter

Red Epi (catalog #170-8283) 695/55 Filter
Cy5 DyLight 650
Cy5.5 DyLight 680
Alexa 647 IRDye 680
Alexa 680 Qdots 705

Table 7. Green epi filter

Green Epi (catalog #170-8284) 605/50 Filter
Cy3 DyLight 549
Alexa 546

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 Application Tables

Table 8. XcitaBlue conversion screen

XcitaBlue™ (catalog #170-8182) Standard Filter*
SYBR® Green (excision) Fluorescein (excision)
SYBR® Safe (excision) OliGreen (excision)
SYBR® Gold (excision) PicoGreen (excision)
GelGreen (excision) GelStar (excision)
* Use the Xcita Blue screen to visualize gels without causing UV damage
to the DNA. This is useful when you want to excise portions of the DNA.

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 5 Viewing Images

After a gel is imaged, the image appears in the workspace. Many controls are
available to optimize viewing and to analyze the image.

The following screen shot shows a gel image with band and lane detection as well
as annotations. The labels are overlays that you can display or hide.

There are many ways to view the data associated with the results. You can view
data as an analysis table, a standard curve, and a report.

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Displaying Gel Images

The display toolbar is located above the gel image. Each tool is described in the
following sections.

Display Gel Options

This section describes the settings in the Display Gel Options dialog box.

Annotations
You can choose whether to show text and arrow annotations that have been drawn
on the image.

Lanes and Bands

You can turn on or off any image overlays, such as lane frames, lanes, bands, lane
labels, molecular weight legends, and band edges.

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Band Attributes
You can show the following attributes for selected lanes or for all lanes.

 Band number

 Band label

 Molecular weight

 Relative front

 Volume

 Absolute Quantity

 Relative Quantity

 Band %

 Lane %

Volumes
If you drew volume boundaries on the gel, you can display the boundaries and their
volume labels.

Zoom Tools
The zoom tools resize the gel image. Click the magnifying glass with the plus sign to
make the image larger. Click the magnifying glass with the minus sign to make the
image smaller.

You can also zoom in on an area using the right mouse button. Right-click and drag
to select the area you want to magnify. You can also resize the image by right-
clicking and using the scroll wheel on your mouse.

Tip: You can return to the original view by right-clicking anywhere on the
image.

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5 | Viewing Images

Fit in Window
If you zoomed in on an area of an image, this button brings the entire image back
into view.

Image Transform
The Image Transform dialog box adjusts image brightness and contrast, optimizing
the image display so faint details can be seen.

The minimum to maximum range varies depending on the light and dark values
present in the image. These adjustments do not change the data. They change only
the way the data are displayed. The human eye cannot see as great a range as the
image contains.

The frequency distribution histogram shows the total data range in the image and
the amount of data at each point in the range.

Auto Scale determines an optimal setting for the image automatically. The lightest
part of the image is set to the minimum intensity, and the darkest is set to the
maximum.

 The High progress indicator determines which intensity value is shown at

the maximum gray scale (or other color) in the gel image.

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 Displaying Gel Images

 The Low progress indicator determines which intensity value is shown at

the minimum gray scale (or other color) in the gel image.

 The Gamma progress indicator changes the gray scale curve. A value of 1
is linear. A value <1 redistributes a greater proportion of the gray scale to
the first half of the intensity values. A value >1 redistributes a greater
proportion of the gray scale to the second half of the intensity values.

You can also type numerical values in the boxes next to the progress indicators.
Clicking anywhere on the progress indicator bars moves the progress indicator
incrementally.

Options:
 Invert image display — inverts dark bands on a light background to light
bands on a dark background. Light bands on a dark background are
inverted to dark bands on a light background.

 Highlight saturated pixels — when this checkbox is selected, areas of the

image with saturated signal intensity (higher than a measurable range) are
highlighted in red.

 Linear or logarithmic histogram — this adjustment changes the y-axis on

the histogram to display the number of pixels at each intensity value using
either a linear or a logarithmic scale.

In multichannel images, you can individually highlight the saturated pixels on a

channel in red. You cannot highlight the saturated pixels in a merged image.

For Multichannel Images

In the Image Transform dialog box, you can make changes to only one image at a
time. When you work with a multichannel image, you can select each channel, in
turn, at the bottom of the Image Transform dialog box and make any changes to the
image.

In addition to the adjustments described in Image Transform on page 102, you can
change the color of each channel. Changing the channel color in the Image
Transform dialog box automatically updates the title bar and the channel buttons on
the multichannel image.

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5 | Viewing Images

If you change the color of a selected channel in Image Transform, the change is
reflected in the merged image.

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Image Colors

You can choose a color map for your image results file. Viewing the image with a
different color scheme can make it easier to see all of the elements in the image, but
it does not change your data.

Note: In multichannel images, colors can be changed only for the individual
channels. You cannot change the colors of a merged image.

The first eight color choices imitate the colors of stained gels. The remaining
choices supply enough color variation to highlight small differences in the image
data. The available colors include:

 Gray

 EtBr (ethidium bromide)

 Coomassie

 Stain-free

 SYBR® Green

 SYPRO Ruby

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5 | Viewing Images

 Flamingo

 Silver

 False color

 Spectrum

 Gold-Silver

 Pseudo

3-D Projection

The 3-D view transforms the gel image into a solid three-dimensional model
spinning in space with x, y, and z dimensions. Accentuate or diminish the relative
heights of data points by pulling the slider at the bottom of the window to the right
or left.

Note: For multichannel images, you can view each channel separately in 3-D.
A merged image cannot be displayed in 3-D.

To view the intensity of various bands

1. Select the 3-D button in the display toolbar.

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 Displaying Gel Images

2. Click and drag the model to rotate it into your preferred view.

3. Bring the window into focus by clicking the image.

4. Press “C” to display an inverted green cone, which can be dragged around to
evaluate the intensity of various bands.

5. Press “C” again to hide the tool.

Image Info
The Image Info dialog box provides information about the active image.

Note: For multichannel images, select a channel to display details for that
image.

The dialog box has three tabs.

 Image Details — acquisition and image information appear in this tab.

 Analysis Settings — settings that were used when the gel was analyzed
are displayed here. For example, Band Detection and Molecular Weight
Analysis will appear if they were performed.

 Notes — on this tab you can add notes, point out the types of samples
used, and add any other information about the results. You can create
custom labels for the lanes in your image. In a multichannel image, the
custom labels are applied to all channels.

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Displaying Multichannel Images

The Multichannel View includes a pane that displays the merged channels and
panes for individual channels. Application names appear in the toolbar. A yellow
border surrounds the active pane.

In the display toolbox above the image, additional controls are available for viewing
the multichannel image.

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 Displaying Multichannel Images

Multichannel View
Click the Multichannel View button to display or hide the merged image panel in the
multichannel display.

You can show or hide each channel using the channel buttons.

You can merge the three channels into a single multichannel pane.

You can also specify the channels to include in the merged view using the dropdown
list.

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5 | Viewing Images

Splitting Multichannel Images

You can work separately on the images that make up the multichannel image by
splitting the multichannel image into individual image files. When you split a
multichannel image, a new file is created for each channel (except the RGB
channel). Each new file has the same name as the multichannel image; the
application name is appended in parentheses. All acquisition settings and overlays
are copied to the new files.

To split a multichannel image into separate files

1. Open a multichannel image.

2. Select Split Multichannel Image in the File menu. Each channel is displayed in
its own window (except the RGB channel).

3. Save each image in its own file.

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 Displaying Multichannel Images

Change Layout
You can choose a layout for the image panes. Clicking Change Layout shows a list
of display options for the image panes. You can select from one of the four views
that follow.

 Grid View

By default, multichannel images appear in grid view.

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5 | Viewing Images

Vertical View

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 Displaying Multichannel Images

Horizontal View

Single View

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Displaying Data
You can view results from analyzed data associated with a gel image using an
analysis table, a lane profile, a standard curve, or a report.

The buttons in the main toolbar turn these views on or off. You can view your data
with all views simultaneously.

Analysis Table Options

Numerical data associated with an analysis can be viewed in an analysis table. Data
from the Lane and Band analysis can be viewed in the Lane and Band Table tab. If
volume analysis was performed, these data can be viewed in the Volume Table tab.
The buttons above the table provide options for displaying and exporting analysis
table data.

To change the size of the Analysis Table window

 Move your cursor to the top of the window until it changes into a
double-headed arrow. Click and drag the edge of the window until you can see
all of the data.

Note: Resizing the Analysis Table window is restricted when a protocol window
is open.

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 Displaying Data

Display Data Options

The Display Data Options dialog box has three tabs.

 Measurements — enables you to choose the measurements to display in

the table. Use the arrows to move the columns between the Not Displayed
pane and the Displayed pane. By default, all measurements are displayed
in the Analysis table.

Note: For a description of each Lane and Band measurement type, see
Lane and Band Table Measurement Definitions on page 117. For a
description of each Volume measurement type, see Volume Measurement
Definitions on page 117.

 Display — enables you to set the display for the analysis table. The
following settings appear on the Display tab:

 Default display settings — The Move selected lane to top checkbox is

selected by default. When you click a lane on the image, the Analysis table
scrolls so that the data for the selected lane appears first in either the
vertical or horizontal view of the table.

 Per Measurement Precision — set the precision (decimal places) for the
measurements in the Lane and Band table and the Volume table.

 Example — shows an example of how measurements will display with the

selected measurement and precision settings.

 Export — enables you to choose how to export the analysis data. The
following settings appear on the Export tab:

 Export formatting — select checkboxes to include lane headers (Lane

and Band table tab only) and/or column headers in the exported file.

 Export delimiter — select a delimiter option for the exported file.

 Comma delimited

 Tab delimited

 Use other delimiter (user defined)

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Change Analysis Table Orientation

This button toggles between two table orientations.

Horizontal — displays the lanes/volumes beside each other, so you can scroll
through the table from left to right.

Vertical — displays the lanes/volumes on top of each other, so you can scroll
through the table from top to bottom.

Copy Analysis Table to the Clipboard

Copies the table data to the clipboard so that you can paste the data into word
processing or presentation applications.

Tip: It is best to use the vertical table orientation when copying to an

8.5 x 11-inch page, to give the columns enough room to display.

Export Analysis Table to a File

Exports the table data as a CSV file so you can open it in a database application.

Export Analysis Table to Excel

Exports the table data to a spreadsheet so you can use the sorting and formula
functions to manipulate your data. If you have Excel (PC or Mac) or Numbers (Mac)
installed on your computer, the program opens with your spreadsheet displayed.

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 Displaying Data

Lane and Band Table Measurement Definitions

This section defines the measurements that display in the Lane and Band tab in the
Analysis table. Use the Data display options button to choose the columns to
display.

Band Number — each band in a lane has a unique number, sorted from top to
bottom.

Band Label — you can assign a custom label to each band by clicking the Band
Label field of the Lane and Band table.

Molecular Weight — the molecular weight of the band is calculated based on the
user-defined standard and regression method. Italic values indicate extrapolated
values. When using nucleic acid gels, the size of the band is displayed in base pairs.

Relative Front — values between 0–1 indicate the relative movement of the band
from top to bottom.

Volume — the sum of all intensities within the band boundaries.

Abs. Quant. — absolute quantification of the band.

Rel. Quant. — relative quantification of the band compared to the reference band.

Band % — percentage of the band’s volume compared to all band volumes in the
lane.

Lane % — percentage of the band’s volume compared to the entire volume of the
lane.

Volume Measurement Definitions

This section defines the measurements that display in the Volume tab in the Analysis
table. Use the Display Data Options button to choose the columns to display.

Volume Number — a unique number is assigned to each volume.

Volume Label — software-generated labels for different types of volumes

(U – unknown, B – background, S – standard). Label can be changed in Volume
Properties.

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Volume — the sum of all the intensities within the band boundaries.

Adjusted Volume — the background-adjusted volume.

Mean Background — the mean value of the background.

Absolute Quantity Volume — the quantity of the volume based on the standard
volumes and the regression method.

Relative Quantity Volume — the ratio of the adjusted volume and the adjusted
volume of the reference volume.

# Pixels — number of pixels inside the volume boundary.

Minimum Value — intensity of the pixel with the minimum intensity inside the
volume.

Maximum Value — intensity of the pixel with the maximum intensity inside the
volume.

Mean Value — mean value of all pixels inside the volume boundary.

Standard Deviation — standard deviation of all pixels inside the volume boundary.

Area — area of the volume in mm2.

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 Displaying Data

Lane Profile

Background

Band
boundaries

The Lane Profile option shows a cross-section view of a single lane rotated 90°.

To view the lane profiles of the different channels in a multichannel image

1. Click on the channel. The Lane Profile window updates and displays the lane
profile of the selected lane in the channel.

2. Use the Next and Previous buttons to page through the lanes in the channel.

The title bar identifies which lane profile is in view (Lane 1, Lane 2, and so on). If the
image is a multichannel image, the title also includes the name of the channel, for
example, Lane 2 (DyLight 650).

There are several settings in the title bar.

 Scale to fit graph

 Include Background

 Identify Bands by

These settings, as well as the zoom tools, are global. They apply to all the profiles.

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5 | Viewing Images

The zoom tools work the same way they work elsewhere in the software. For more
information, see Zoom Tools on page 101.

In addition to the graph of the lane intensities, the Lane Profile tool also shows an
image of the selected lane below the graph. The multichannel channel of
multichannel images is always a gray scale image of the lane with the default
transform applied to that lane. For single-channel images and individual channels of
a multichannel image, the transform and color map are applied to the gray-scale
image.

As you move your cursor over the profile, the current relative front (Rf) value and the
average intensity (Int) value at the Rf value display in the lower-right corner of the
Lane Profile window.

Scale to Fit Graph

You can choose the highest point of the display to define the range of the graph.
This provides the best view of the lane profile.

You can clear the Scale to Fit Graph checkbox to display the entire range of
possible intensity values in the graph. Doing so allows valid comparisons between
different lanes.

Include Background
When the Include Background checkbox is selected, the Lane Profile window
shows the subtracted background under the blue line. The area used for band
quantification appears in green under the red line.

When you clear the Include Background checkbox, the area of the lane profile that
represents the background of the image does not display.

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 Displaying Data

Identify Bands by
You can change how the bands are labeled by choosing from the options in the
Identify Bands by dropdown list. By default, the bands are labeled by band number.

You can display one of the following attributes:

 Band Number (Band No.)

 Band Label

 Molecular Weight (Mol. Wt.)

 Relative Front

 Volume

 Absolute Quantity (Abs. Quant.)

 Relative Quantity (Rel. Quant.)

 Band %

 Lane %

 Normalization Factor (multichannel images only)

 Normalization Volume (multichannel images only)

Adjusting Band Boundaries

Below the profile of each lane there is a strip that displays the bands. Each band is
surrounded by a pair of vertical lines delimiting its boundaries. You can move the
vertical lines and change these boundaries.

To change the boundaries of a band

1. Hold your cursor over one of the boundary lines until a double arrow appears.

2. Click and drag the cursor to the new position.

Note: Boundary lines cannot overlap one another. Therefore, you will not
be able to move any boundary line beyond the next boundary line.

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Standard Curve

The Standard Curve dialog box displays the best curve fit for the defined standards
and the bands relative to this curve for the lane selected in the image. The tabs at
the bottom of the dialog box display the standard curves for three different
analyses.

Standards appear in green. Unknown bands appear in red. You can toggle the
molecular weight display on the y-axis between linear and log scale by clicking the
Log y-axis box at the upper left. The regression method you chose in Molecular
Weight Analysis Tools appears, as well as the formula (if applicable) and the R2 value
of the regression method.

Tabs in this window enable you to view the molecular weight standard curve, the
absolute quantity standard curve, or the volume standard curve.

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 Displaying Data

Report
See Generating Reports on page 175, for information about reports.

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 6 Analyzing Images

Analysis Tool Box tools are enabled when an image file is opened and in focus. An
active or “in focus” window has a darker blue menu bar on a Windows PC. On a
Macintosh, the window control icons display more brightly when a window is active.
This distinction helps you to identify the active window among many open image
files in your workspace.

Image Types
Image Lab™ creates three types of images:

 Single-channel images

 Linked multichannel images

 Unlinked multichannel images

The multichannel view in a multichannel image is a merged display of single

channels. You can have up to three images in the multichannel image. The linked
multichannel image is acquired using the multichannel protocol. Unlinked
multichannel images are generated by combining several images using the Create
Multichannel Image feature.

The Analysis Tool Box tools work differently on these images. Some tools, such as
lane detection, when applied to one channel in a linked multichannel image,
propagate the changes to all the other channels. With unlinked multichannel
images, lane detection must be applied separately to each channel.

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Using Auto Analysis

Auto analysis detects the lanes and bands and, optionally, calculates the molecular
weight of the bands in your image. Click Auto-Analysis in the Analysis Tool Box to
do the following:

 Analyze images obtained with protocols that did not include steps for auto
detection and analysis

 Change your analysis parameters to reanalyze your images

Note: If you change any settings for an analyzed gel, the initial analysis is
overwritten. To preserve both analyses, save each image file with a different
name.

Note: The Band Detection Sensitivity and Molecular Weight Analysis Settings
are applied to all channels in a multichannel image. If you want to use different
sensitivity levels to detect bands in each channel, use the Lane and Bands
tools.

Auto Detection Settings

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 Analysis Tool Box Tools

The band detection sensitivity options are as follows:

Low Band Detection Sensitivity — sets detection at a low level for images with
prominent bands. Faint bands are not detected with this setting.

High Band Detection Sensitivity — sets detection at a higher level for images that
are faint. Extraneous bands can be removed using the Band Tools in the Analysis
Tool Box. See Lane and Bands Tools on page 131.

Custom — enables you to set a value between 1 and 100 to select the best
detection sensitivity for your sample. You can also drag the sliding bar left or right to
set the value.

When Low Band Detection Sensitivity is used, the numerical value is set at 25; when
High Band Detection Sensitivity is used, the value is set at 75.

Molecular Weight Analysis Settings

Molecular Weight Standard — choose any of the many Bio-Rad standards or
another standard you added to your standards list.

Standard Lanes — choose or change the lanes in which the standards are placed.

Regression Method — four regression methods are available. For more

information, see Regression Calculation Methods on page 251.

Analysis Tool Box Tools

All Analysis Tool Box tools customize the analyzed data in image files. These tools
are available only when an image file is open. Click a specific image to select among
the many windows that might be open in your workspace.

Note: Some tools delete the existing analysis.

To access a tool
 Click any of the toolbox buttons.

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6 | Analyzing Images

To return to the Analysis Tool Box menu

 Click the green Up arrow to the right of the tool name.
Up arrow

Image Tools
The image tools enable you to manipulate your images.

To display the image tools menu

 Click Image Tools.

 Flip — you can flip the gel image horizontally or vertically.

 Rotate — you can rotate the gel image 90° using the Left or Right buttons.
Or you can set a custom rotation using the Custom button.

 Crop — you can trim the outer edges of your image to any shape or area.

 Invert Data — you can toggle the image data from positive to negative.

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 Image Tools

 Merge — you can merge a chemiluminescent blot image with a

colorimetric image of the same blot.

The sections that follow describe how to use these tools in greater detail.

Correcting a Slanted Gel

To correct a slanted gel
1. In the Rotate section, click Custom.

Red arrows appear over the gel image.

2. Rotate the red arrows to any degree between 0 and 360 by dragging them.

3. Right-click the gel image and click Rotate to set your gel in the new position.
Click Cancel if you do not want to set the rotation.

Cropping a Gel Image

You can save crop settings and use them to crop other images. This feature is useful
when you want to crop the same area in several images.

To crop a gel image

1. Click Crop. A red box outlines the image area.

2. Drag the red box to surround the image area you want to keep.

3. (Optional) Right-click the image to open the Crop menu and click Save Crop
Settings.

The Save Crop Settings dialog box appears.

4. (Optional) Type a name for the crop settings and click OK.

5. Right-click and select Crop or Cancel. Selecting Crop crops the image to the
area inside the red box.

To crop an image using saved crop settings

1. Click Crop. A red box outlines the image area.

2. Right-click the image to open the Crop menu and click Load Crop Settings.

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6 | Analyzing Images

3. Select the saved crop settings that you want to use and click Load. The red box
resizes and the crop specifications appear on the image.

4. Right-click and select Crop. The image is cropped to the area specified in the
crop settings you selected.

To delete crop settings

1. With an image open, click Crop.

2. Right-click the image inside the red box to open the Crop menu and click
Delete Crop Settings.

3. Select the crop settings in the dialog box that appears and click Delete.

Inverting Data
Use this button to change the image data from positive to negative. Invert Data is
used for negative stains and zymograms. Intensity values of bands must be greater
than background to perform analysis on the gel. View the gel as a 3-D projection to
determine if the data must be inverted.

Merging Images
Use this button to merge a chemiluminescent blot image with a colorimetric image
of the same blot. If you have used colorimetric prestained standards for a
chemiluminescent blot, you can acquire an epi-white light image of the blot to show
the standards and a chemiluminescent image to show immunodetection. These two
images can then be merged into a combined image with both signals.

Note: Merging images can have an adverse effect on quantification. If accurate

quantification is required, perform analysis on the original, separate images.
Only images of the same size can be merged.

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 Lane and Bands Tools

Lane and Bands Tools

The Lane and Bands tools enable you to identify the lanes and bands in your
images.

To open the Lane and Bands tool

 In the Analysis Tool Box, click Lane and Bands, then select the tab for the
Lanes or the Bands tool.

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6 | Analyzing Images

Detecting Lanes
To detect the lanes in the image, do one of the following:
 Click Automatic if the gel image is fairly typical.

 Click Manual to detect a specific number of lanes or if automatic lane

detection did not find all the lanes.

Detecting Lanes in Multichannel Images

To use automatic lane detection with multichannel images, select one of the
channels and click Automatic. Automatic lane detection works differently in
multichannel images depending on whether the channels are linked or unlinked.

 For linked multichannel images, the lane detection is based on the selected
channel, and the lanes are detected in all channels.

 For unlinked multichannel images, lane detection is applied to only the

selected channel. You must individually select each channel and apply
automatic lane detection.

The lanes in unlinked multichannel images might fall in slightly different places on
each channel, and you might need to have more control over where the lanes fall.
You can detect the lanes in one channel of an unlinked multichannel image using
either the automatic or manual lane detection tools. Then you can copy those lanes
into the other channels and position them so that the lanes in each channel align
with one another. For more information on copying lanes, see Copying Lanes on
page 136.

Using the All Lanes and Single Lane Tools

To use the All Lanes and Single Lane tools
 Click the lane tool first, then click the lane to which you want to apply the
change. Tool actions in linked multichannel images are applied across all
channels. Tool actions in unlinked multichannel images are applied in only the
selected channel.

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All Lanes Tools

You can modify all the lanes in your image using these tools.

 Resize — resizes the lanes in your image. Click Resize and then drag the
handles of the red squares to resize all lanes.

 Adjust — adjusts the orientation of all lanes. Click Adjust and then drag a
single corner of the lane frame. The Adjust All Lanes tool does not resize
lane width.

You can add anchor points to the top or bottom borders of the rectangle by
clicking the lane frame. Remove any unneeded anchor point by right-clicking it.
By dragging these additional anchor points, you can adjust for “smiling” gels.

Note: You can move the entire lane frame when resizing or adjusting the
lanes. Click Resize or Adjust and make the changes to your lanes. Then
click anywhere in the frame and move it to the desired location.

Tip: On the PC, you can press the Shift key or the Ctrl key and use the
arrow keys on your keypad to move the lane frame. On a Mac, press the
Shift or the Command key and use the arrow keys.

 Delete — deletes all lanes.

 Width — changes the width of all lanes at the same time. Click Width and
then drag one of the anchor points on any lane to change the width of all
lanes. In a multichannel image, the width of all the lanes in all channels are
changed.

Tip: You can use the plus key (+) on your keyboard to increase the lane
width, and the minus key (-) to decrease the width.

You can also change the lane width by specifying the size of the lanes. Click
Width, enter a number (in mm) in the box, and click Apply.

Note: You might see that the number in the box changes after you click
Apply. For example, you enter 5.75 and the number changes to 5.71. The
image is made up of pixels and the Image Lab™ software can only draw a
boundary for the lane between pixels. If the number you specify would

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6 | Analyzing Images

cause the boundary to fall anywhere on the pixels, Image Lab moves the
boundary so that it falls between two pixels and updates the number
accordingly.

Note: The All Lanes Width tool makes the width of all lanes uniform.
Therefore, if you used the Single Lane tool to change the width of an
individual lane, this change is overridden by the All Lanes Width tool.

Single Lane Tools

Specific information about how to use each lane tool is described in the following
section. You can modify an individual lane in your image using these tools.

 Add — adds a lane to a gel image. Click Add, then click within the lane
frame where you want to place the new lane. The lanes are automatically
renumbered.

Note: To add a lane outside the frame, add a lane inside the frame and
click Move to expand the lane outside the frame’s boundaries.

 Delete — deletes a lane. Click Delete, then click either the lane or its lane
number. The lanes are automatically renumbered.

 Bend — bends a lane to better fit the gel image. Click Bend, then drag
square anchor points to fit the lane to the image.

Note: To add anchor points, left-click within the lane. Drag these anchor
points to adjust the lane to fit the gel image. To remove an anchor point,
right-click on it.

 Move — moves a lane to a new position on a gel image. Click Move, then
click the lane you want to move. Drag it to a new location. The lanes are
renumbered according to their new positions.

 Width — changes the width of a lane. Click Width, then click within the
lane. Click on the anchor points to adjust the lane width.

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 Lane and Bands Tools

You can also change the lane width by specifying the size of the lanes. Click the
Width button, enter a number (in mm) in the box, and click Apply.

Note: You might notice that the number in the box changes after you click
Apply. For example, you enter 5.75 and the number changes to 5.71. The
image is composed of pixels and the software can draw a boundary only in
the lane between pixels. If the number you specify would cause the
boundary to fall anywhere on the pixels, Image Lab moves the boundary so
that it falls between two pixels and updates the number accordingly.

Lane Background Subtraction

To perform lane-based background subtraction
 Select Enable Subtraction in the Background Subtraction field. Use the Lane
Profile view to see the subtracted lane background.

Disk Size — specifies the size of a hypothetical rolling disk (between 0.5 and
99.5 mm in 0.5 mm increments) that removes background levels along the length of
the lane. The size of the disk determines how closely the background level follows
the intensity profile.

A large disk follows the profile trace less closely, touching fewer points along the
trace and removing less background. A smaller disk more closely follows the profile
trace, removing more background.

A disk radius that is too large will result in poor background removal. A disk radius
that is too small might subtract actual data. For most samples, a size of <10 mm is
usually appropriate. You can perform this task several times until you are satisfied
with the amount of background removed. Use the Lane Profile tool to evaluate the
appropriate disk size for background subtraction.

Apply to selected Lane — applies the specified level of background subtraction

only to the selected lane. This option enables you to set different background
subtraction levels for each lane.

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6 | Analyzing Images

Copying Lanes
You can copy the lanes from one image into any other image. This tool is useful if
you want to create a multichannel image from unlinked images, and you want to
identify and match the lanes in your channels. The entire frame and all the lanes are
copied. Individual lanes cannot be copied.

To copy lanes between channels

1. Open the Lane and Bands tool.

2. Select the channel that contains the lanes you want to copy.

3. From the Edit menu, select Copy.

The message Copying All Lanes appears.

4. Select the channel that you want to copy the lanes into.

5. From the Edit menu, select Paste.

A channel can contain only one lane frame at a time. Therefore, if you paste
lanes into a channel that already contains a lane frame, you are prompted to
confirm the deletion of the existing lanes. If you click Yes, the existing lanes are
deleted and replaced with the copied lanes.

Note: If you copy lanes into a channel of a linked multichannel image, the
lanes are copied into all channels of the image, including the Multichannel
channel.

After the lanes are copied into the channel, you can manipulate individual lanes
using the lane tools so that they are correctly positioned.

Detecting Bands
Note: Bands are detected for individual images. For multichannel images, you
must detect the bands for each channel, one at a time. This applies whether the
multichannel image is linked or unlinked. Band detection on the Multichannel
channel is disabled. The multichannel view is a composite of the other
channels. Detecting bands in this view is not particularly useful because
quantifying overlapping bands will result in combined values from multiple
channels.

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 Lane and Bands Tools

To detect bands in the image

 Click Detect Bands to open the Band Detection dialog box. Select band
detection sensitivity and the lanes to which it applies.

Note: With multichannel images, you can leave the Band Detection dialog
box open as you select each channel one by one and detect the bands in
each channel. You do not have to close and reopen the dialog box for each
channel.

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6 | Analyzing Images

Displaying Advanced Detection Options

You can also select Display Advanced Options to set specific parameters for band
detection. When initially expanded, the values in Advanced Options are set in
relation to the band detection sensitivity that you select in Detection Settings. You
can set specific parameters for the sensitivity level and apply the parameters to all
lanes or to a specific lane.

If later you change the band detection sensitivity in Detection Settings, the values in
Advanced Options change in relation to the new sensitivity levels. You might need to
reset these parameters to the original values.

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 Lane and Bands Tools

Advanced Detection Options

 Sensitivity — determines the minimum optical density that will be defined
as a band. The higher the sensitivity value, the lower the minimum signal
intensity, therefore the more bands will be detected.

If the sensitivity is set too high, background staining might be detected as

bands. If the setting is too low, bands of interest might not be detected.

The default sensitivity setting is 10.0. If the gel has faint bands (for example, if
the optical density is less than 0.05, and counts are less than 2,000), you may
want to increase this value to 20.0.

 Size Scale — distinguishes between trends in signal intensity and random

intensity fluctuations. It is the number of pixels in a vertical column that are
taken together to determine whether a band is present.

The Size Scale parameter uses the size of objects in the image to determine the
nature of those objects. If a gel image has high levels of background noise, a
larger size scale is appropriate. At low noise levels, a smaller value is preferable.
You can also increase the size scale if the gel has only a small number of thick
bands scanned at high resolution.

 Noise Filter — minimizes the number of small fluctuations (or noise) in the
image that are called bands while still recognizing larger features (the real
bands). This filter becomes especially important at higher sensitivity levels.

The noise filter value refers to the size of the filter in pixels (for example, a value
of 2.50 equals a filter size of 2.50 x 2.50 pixels). Features smaller than the filter
size will not be recognized as bands. Entering a noise filter size of zero turns it
off completely.

If band detection identifies doublets as single bands, decrease the noise filter
setting and/or increase the sensitivity level.

Tip: You can also decrease the Size Scale parameter instead of the noise
filter to improve the detection of closely spaced bands. However, if you
decrease both the noise filter and the size scale, the fuzziness around
bands might be mistakenly detected as separate bands.

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6 | Analyzing Images

 Shoulder — band detection tries to distinguish shoulders as separate

bands. When looking at a lane trace, these bands appear as flat or gently
sloping abutments to darker, better-defined bands (that is, there is no dip
on the trace between the two bands). Increasing the shoulder sensitivity
results in more shoulders being detected as bands. Changing this setting
to zero results in no shoulders being recognized as separate bands.

If band detection calls a doublet a single band, check the lane trace to see if
there is a dip between the peaks of the two bands. If there is no dip, increasing
the shoulder sensitivity value will help resolve the two bands.

 Normalize Sensitivity — compensates for differences in intensity between

lanes.

The intensity of each lane is determined by the darkest band in that lane. For
example, suppose that in all but one of the lanes the darkest band has an
intensity of 50,000 counts. In the light lane, the darkest band is only 25,000
counts. With normalization, band detection will be twice as sensitive when
processing the light lane, improving the detection of faint bands.

Note: It does not normalize for band quantitation.

 Band Limit — enables you to limit the number of bands that will be
detected in each lane, thus reducing the need for later editing.

Editing the Detected Bands

You can optimize the bands in your images using these tools.

 Add — manually add a band to a lane. Click Add, then click at the desired
location in a lane. Image Lab then locates a faint band close to where you
clicked.

Tip: You can darken your entire image to view faint bands more easily
using the sliders in the Image Transform dialog box. For instructions, see
Image Transform on page 102.

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 Normalizing Volume Data

 Delete — deletes a band from a lane. Use this feature to remove bands
that are not relevant to your analysis. Click Delete, and then click on the
band you want to remove.

 Adjust — adjusts the height of a band. Click Adjust. Boundary lines appear
on either side of each band. Move the cursor over a boundary line until you
see a double-headed arrow. Move the boundary line up or down. The
center is recalculated and the band appears there.

Note: You can also adjust band boundaries in the Lane Profile view.

Normalizing Volume Data

There are several reasons why you might want to normalize the volume data in
multichannel images:

 The lanes are loaded with the same volume, but you do not know the total
protein in each lane.

 Pipetting errors result in variations in the lane volumes.

 Differences occur in the transfer of protein from a gel to a membrane.

You can correct for these differences by normalizing the volume data. There are two
ways to normalize volume data:

 Total Lane Protein Normalization – one lane in the normalization channel

is used to calculate the normalization factor.

 Housekeeping Protein Bands Normalization – a single band of a

housekeeping protein is used to calculate the normalization factor. In order
to get accurate results, the housekeeping protein must be stable and
impervious to the treatments in your experiment. It must be the same in
pretreatment and post-treatment.

In both cases, the normalization factor is then used to calculate the normalized
volumes in the lanes for all channels.

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Using the Normalization Tool

Click Normalization on the Analysis Tool Box.

The Normalization Steps guide you

through the steps to normalize your
volume data.

The Normalization Steps outline the recommended steps to follow. You will need to
review your lanes and bands and possibly make adjustments to them.

Tip: From the Normalization tools pane, click Adjust Lanes/Bands to quickly
navigate to the Lane/Bands Tool.

See Optimizing Normalization of Volume Data on page 143 for an explanation of

each step in the process.

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 Optimizing Normalization of Volume Data

Optimizing Normalization of Volume Data

Follow these steps to get the best results when normalizing your volume data:

1. Detect the Lanes in the Channels

2. Make Adjustments to the Lanes

3. Detect the Bands

4. Subtract any Extraneous Background

5. Remove Compromised Data

6. View the Data in the Analysis Table

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6 | Analyzing Images

Detect the Lanes in the Channels

Start by detecting the lanes in your multichannel image. You can use either the
automatic or the manual lane detection method. For more information on when to
use each method, see Lane and Bands Tools on page 131. If your multichannel
image consists of unlinked images, you will need to pay particular attention to the
lanes that are detected to ensure that the corresponding lanes are correctly mapped
for each channel. For example, you can use the Copy Lanes tool to map your lanes.
For more information on that tool, see Copying Lanes on page 136.

Make Adjustments to the Lanes

Regardless of which method you use to detect your lanes, you should review the
lane borders to ensure that each lane encompasses all the protein in a particular
lane, and that the lane does not overlap into another lane. For example, in the
following figure, lanes were detected using the automatic lane finder. Some of the
lanes exclude material that should be included.

Note: Image Lab automatically excludes standard lanes from normalization.

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 Optimizing Normalization of Volume Data

Use the Lanes tools to make the necessary adjustments to your lanes. For more
information on these settings, see Using the All Lanes and Single Lane Tools on
page 132. In this example, the lanes were slightly widened to include the relevant
material. The corrected image follows.

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Detect the Bands

Use the band detection tool to detect the bands in the channels. If you are using the
total lane protein method to normalize your data, it is recommended that you do not
use the band detection tool on the normalization channel, because this will generate
large amounts of superfluous data in the analysis table. For more information, see
Detecting Bands on page 136.

If you are using a housekeeping protein to calculate the normalization factor, you
must isolate this protein in your image. Remove all bands other than the
housekeeping protein from your image. The normalization channel should have only
one detected band in each lane. Use the Delete Bands tool to exclude all other
bands from the calculation.

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 Optimizing Normalization of Volume Data

Subtract any Extraneous Background

The lane volume data is background-subtracted. Review the background
subtraction being applied to each lane by using the Lane Profile tool. Verify that an
equivalent background profile is being subtracted from each lane in the
normalization channel so the normalization factor is accurate. For more information
on the lane Background Subtraction tool, see Volume Background Subtraction on
page 170.

Remove Compromised Data

There are several reasons why lanes should be excluded from your analysis:

 Empty lanes — the first nonstandard lane in the selected channel is used
as the normalization factor against which all other lanes in all the channels
are compared. If the first nonstandard lane is not a valid lane (for example,
an empty lane), delete it from the channel.

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6 | Analyzing Images

 Lanes contain saturated pixels — check your images for saturated

pixels, indicated in red. These points cannot be read and cannot be used in
quantification.

Saturated Pixels

 Poor transfer quality — check the quality of the transfer. If the transfer is
poor with splotchy or blurred areas, delete these lanes.

View the Data in the Analysis Table

The first nonstandard lane in the selected channel is used as the normalization
factor against which all other lanes in all the channels are compared. The
normalization values are calculated based on the total background-corrected signal
in the selected channel.

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 Optimizing Normalization of Volume Data

The Analysis Table displays the uncorrected and normalized volumes, as well as the
normalization factor used to calculate the normalized volume.

Tip: Omit the normalization channel from the Analysis Table using the
Multichannel View button. (See Displaying Multichannel Images on page 108
for information on displaying and hiding channels.)

Adding a Channel to a Single Image

The Normalization tool can be used only with multichannel images. If you have a
single image, there is an Add Channel feature in the Normalization tools pane that
guides you through the process of creating a multichannel image. After the
multichannel image is created, you can use the tool to get normalized values.

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6 | Analyzing Images

To use the Normalization tools with a single image

1. Click Normalization in the Analysis Tool Box.

2. Click Add Channel to open the Add Normalization Channel window.

Note: The image that is selected when you click Add Channel is the first
image in your multichannel image.

The selected image appears as the first image in the Sample Data field.

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 Optimizing Normalization of Volume Data

3. To add a second image, double-click on an image from the Compatible Open

Images list.

If no images appear in the list or you want to use a different image, click Browse
to navigate and select the desired image.

Note: Image Lab checks to see if the image has the same aspect ratio as
the Sample Data image. If the aspect ratio is not compatible with the
Sample Data image, it will not appear in the list of Compatible Open
Images. The aspect ratios might not be compatible if they were created
using different instrument models (for example, Gel Doc™ EZ and
ChemiDoc™ MP) or if the images are cropped at different aspect ratios.

Tip: You can move an image to the Normalization Data box by dragging it
from the Compatible Open Images list, and you can remove it by dragging
it back to the list.

4. Click OK.

The Auto-Analysis window appears, prompting you to detect the lanes and
bands in your images.

5. In the Auto-Analysis window, select the level of sensitivity and click OK.

6. Follow the Normalization Steps listed in the pane to get the best results.

See Optimizing Normalization of Volume Data on page 143 for an explanation

of each step in the process.

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6 | Analyzing Images

Molecular Weight (MW) Analysis Tools

Molecular Weight Analysis tools enable you to determine molecular weight (or base
pairs, if using nucleic acid gels) by comparing a test sample with known standards.

Note: Before you can use the Molecular Weight Analysis tools, you must detect
the lanes and bands in your image.

You can view each band’s molecular weight in the molecular weight column of the
Lane and Band tab in the Analysis Table.

You can also display the molecular weight of the bands on the gel image by opening
the Display Gel Options window and selecting Mol. Wt. from the dropdown list in the
Band Attributes section. (See Displaying Gel Images on page 100 for information
about displaying band attributes.)

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 Molecular Weight (MW) Analysis Tools

Molecular Weight Standard

You can change the standards to ones that are relevant to your samples.

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6 | Analyzing Images

To change standards
 Click Change to access the Manage Standards dialog box. Choose another
standard or add your own standards.

Standard Lanes
Standard samples are placed in the first and last lanes by default. You can specify
other standard lanes by selecting the box below each lane or by entering the
standard lane numbers separated by commas in the MW Analysis Tools dialog box.
In the Lane and Bands view, Std appears below the lanes, identifying them as
standard lanes. In the Molecular Weight Analysis view, these lanes are indicated
with a check mark below the lane.

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 Molecular Weight (MW) Analysis Tools

Molecular Weight Analysis in Single-Channel Images

There are three standard lanes in the screen shot below (lanes 1, 16, and 17).
Standard lanes are identified with a check mark in the boxes below the lane. The
molecular weights of the bands in the standard lanes appear on either side of the
lane frame. The red lines running from one end of the lane frame to the other identify
the location of the bands in the standard lanes. You can use these lines to see where
the bands in the other lanes fall relative to the bands in the standard lanes.

Note: You can use the lane labels to identify your standard lanes.

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6 | Analyzing Images

The values for the molecular weight in any standard lane appear in bold. In this
example, the values for the first lane are in bold. (Lanes 16 and 17 are not included
in the screen shot, but their values would also be in bold.) The molecular weight of
the bands in the remaining lanes is calculated relative to these standards.

Molecular Weight Analysis in Multichannel Images

Molecular weight analysis is performed differently in multichannel images
depending on whether the channels of the image are linked or unlinked. If the
channels are linked, then the standard lanes are synchronized across all channels. If
the channels are unlinked, each channel has its own standard.

When you select the standard lane in one channel of a linked multichannel image,
the same lane is selected as the standard in all other channels. The combined data
in the multichannel image are used to detect the bands in this standard lane and
these bands are synchronized across all channels. All the bands in all the channels
are calculated using the same standard lane. If you deselect this lane as the
standard, the bands in the standard lane are deleted from all channels, including the
multichannel image.

With unlinked multichannel images, each channel is treated independently as if it

were a single-channel image. You select a standard lane for each channel and the
molecular weight is calculated for only the bands in that channel.

In the following screen shot, lane 1 in the linked multichannel image is selected as
the standard lane for all channels. Because the images are linked, the same
molecular weights for the standard lane and the same red lines showing the location
of the bands in the standard lane are displayed in all channels.

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 Molecular Weight (MW) Analysis Tools

The molecular weights of the bands in the three channels are calculated based on
standards established in lane 1 and are displayed in the analysis table.

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6 | Analyzing Images

Regression Methods
There are four regression methods.

 Linear (semilog)

 Point-to-point (semilog)

 Logistic

 Cubic spline

See Regression Calculation Methods on page 251.

Quantity Tools
You can quantify bands in test samples automatically using either the Relative or
Absolute tab under Quantity Tools.

Relative Quantity Tab

To compare the relative quantities of bands
1. Select the Relative tab.

2. Click Select.

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 Quantity Tools

3. Click the band you want to use as a reference. A small R appears near the band
you selected.

To review the relative quantities of bands

 Go to the Rel. Quant. column of the Analysis table (Lane and Band tab). The
relative quantity is the ratio of the band volume divided by the reference band
volume.

All other bands now display numerical values that are relative to the reference band.
If the reference band value is 1.00, values higher than 1.00 indicate that the band
quantity is greater than the reference band. Values lower than 1.00 indicate the band
quantity is less than that of the reference band.

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6 | Analyzing Images

Absolute Quantity Tab

Absolute quantification is used to quantify bands based on known standard bands
using a calibration curve.

To calculate the absolute quantities of the bands

1. Select the Absolute tab.

2. Click Select.

3. Select at least two standard (known) bands and assign quantity values. Small
A’s appear (shown below circled in red) near the bands you selected.

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 Quantity Tools

The values display in the Standard Bands table. The greater the number of
known bands and the wider the range of their values, the more accurate the
absolute quantity calculation of the unknown bands will be.

Note: Any standard band selection can be deleted. To do so, select the
entry in the Standard Bands field and then click Delete.

4. Select a unit of measure from the Units dropdown list.

5. Select a regression method from the dropdown list.

Consider the following guidelines when making your choice.

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6 | Analyzing Images

Linear — generates a straight line that is the best fit of the values you provided
and is preferred in most cases.

Point-to-point — generates a curve in which each data point is connected

directly to the next, regardless of the shape of the resulting curve.

Cubic spline — generates a smooth curve that connects each data point. At
least four standard points are required to use this method of least-squares
polynomial fits.

Table 9. Regression Methods

Regression Minimum Number Minimum Number with Force
Method of Standard Bands Through Origin Option
Linear 2 1
Point-to-point 2 1
Cubic spline 5 4

6. Click Standard Curve on the toolbar and select the Absolute Quantity Standard
Curve tab.

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 Quantity Tools

The calibration curve displays. Standards are represented by green triangles.

Unknown values are represented by red triangles.

Note: Selecting the Force Through Origin checkbox always starts the
standard curve graph at 0,0, regardless of the best curve fit.

Note: Clicking the Standard Curve table generates a crosshair tool that
displays the numerical values associated with the placement of the cursor
in the graph.

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6 | Analyzing Images

Annotation Tools
You can annotate results with text and arrows to emphasize areas of interest.

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 Annotation Tools

Add Annotations
 Text — enables you to add text annotations to images to emphasize
important details. Click Text, then click an area on the image where you
want to insert your comment. A box appears with a dotted-line border.
Type your comment in the box.

Tip: To add a new line to the box, place your cursor where you want the
break and press Shift+Enter.

To move the box

 Click and drag the box to change its position.

 Arrow — enables you to add arrows to images to emphasize important

details. Click where you want the arrow to start and drag to stretch the
arrow point to the location you want to emphasize. To move the arrow on
the image, click the middle of the arrow and drag it to the new position.

To change where the arrow points

 Click either end of the arrow. Square boxes appear. Drag a box to change the
length or orientation of the arrow.

Note: In multichannel images, you can add annotations in all the channels.
Each annotation, including the merged channel, is channel specific.

Alignment
The alignment buttons enable you to align multiple annotations, such as lane
numbers, which you have manually added.

To select multiple annotations

 Press the Ctrl key (Command key on a Mac) and click each item or drag a
selection box around them.

Note: In multichannel images, you can also copy annotations from one channel
to another using the same method.

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6 | Analyzing Images

Text Properties
You can change the type size and font of your text annotations.

 Font — click the box you want to change. Open the dropdown Font menu
to show all fonts installed on your system. Select a new font for the text
annotation.

 Size — click the box whose font you want to resize. Open the dropdown
Size list to increase or decrease the size of the text. You can set the font
size from 6 to 72 points using the dropdown list.

Color
You can change the color of text annotations to make them visible with any color
scheme and emphasize them further by adding a color to the annotation’s
background, which is invisible by default.

To change the color of multiple items

 Press the Ctrl key and click each item.

 Foreground — click a text annotation or arrow. This activates the

Foreground field, so you can select a foreground color from the dropdown
list.

 Background — click a text annotation. This also activates the Background

field so you can select a background color from the dropdown list.

Rotate
You can rotate text annotations 90 to the left or right by clicking the Rotate buttons.

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 Volume Tools

Volume Tools
Volume tools enable you to manually quantify features on a sample image when
automated lane and band analysis is not appropriate or possible, such as in dot
blots.

Note: The analysis table displays the color-coded volume drawn for each
channel of a multichannel image. In multichannel view, you can draw a volume
on individual panes, but you cannot draw a volume on the Multichannel pane.

You can use Volume tools to quantify the signal intensity of bands, spots, arrays,
and other image data. Define an area of interest by surrounding it with a shape. You
can choose a rectangle, circle, freehand, or lane shape by clicking the appropriate
button under the Volumes field.

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6 | Analyzing Images

A default label appears within the shape drawn. The volume label is assigned a
sequential number and can be one of three types:

 U — unknown

 Std — standard

 B — background

Each new volume you create initially has a red border, which indicates that the
volume is selected. When you click elsewhere on the image, the border changes to
blue, indicating that the volume is no longer selected.

Note: Double-click a volume area to change its properties.

To review data for the volumes

 Open the analysis table and select the Volume tab. Volumes are listed based on
their number and/or the associated information per volume. See Volume
Measurement Definitions on page 117.

Note: In multichannel images additional column bars, channel numbers,

and volumes are color-coded based on their channel association.

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 Volume Tools

Volume Types
You can define the volume type (unknown, standard, or background), the quantity of
standard volumes, or enter a custom name to replace the default label.

Unknown volumes are volumes you want to quantify.

Standard volumes are used for absolute quantities. See Absolute Volume Quantity
on page 171.

Background volumes are used to remove the background from the calculation. The
result of volume background subtraction appears in the Adjusted Volume column of
the analysis table (Volume Table tab).

Note: This volume type needs to be assigned only when using Global
Background subtraction.

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6 | Analyzing Images

Volume Background Subtraction

When you draw a volume, some nondata background pixels might be included
inside the volume. These background pixels usually have an intensity value that you
do not want to include in your volume quantification. There are two methods of
calculating this background intensity for your volumes: local and global.

 Local — local background subtraction calculates a separate background

intensity for each unknown and standard volume you create. For each
volume, the intensities of the pixels in a 1-pixel border around the volume
are added together and divided by the total number of border pixels. This
gives an average intensity for the background around each, which is then
subtracted from the intensity of each pixel inside the volume. If the
background value is greater than the pixel value inside the volume, the
background-adjusted quantity of the volume could be <0. In this case,
redraw the border for this volume.

 Global — global background subtraction calculates a single background

intensity for the entire gel. This average background intensity is then
subtracted from all the volumes in the gel. The average intensity of the
pixels in the background volume is calculated and subtracted from each
pixel in all standard and unknown volumes. Therefore, it is not necessary
for the background volume area to be the same size as your unknown.

To calculate global background subtraction

1. Use one of the Volume Tools to create a volume in a representative background
region of your image (that is, a nondata region similar to the background
surrounding your data).

2. Double-click the volume. This opens the Volume Properties dialog box.

3. Select the Background option button.

Note:

 If you select Global in the Volume Tools toolbox but do not define a
background volume as described, no background subtraction is
performed.

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 Volume Tools

 If you create more than one background volume, all the pixels in those
background volumes are used to calculate the average background. Your
background volume(s) will have default names B1, B2, and so on based on
the sequence in which they were created.

 If the region you defined as background has a higher average intensity

value than your data object, you obtain a negative value for your adjusted
volume in the analysis table. If this happens, select a new background
region with less intensity than your data object.

Relative Volume Quantity

You can choose any one volume as a reference volume by selecting the Reference
Volume checkbox in the Volume Properties dialog box. The reference volume is
indicated by an asterisk on the volume label, for example, U1*.

Relative quantities are displayed in the Relative Quantity column in the analysis
table (Volume Table tab). The relative quantity is the ratio of the background-
adjusted volume divided by the background-adjusted reference volume.

All other volumes now display numerical values relative to your reference volume.
Values higher than 1.00 indicate that the volume is greater than the reference
volume. Values lower than 1.00 indicate the volume is less than the reference
volume.

Absolute Volume Quantity

Note: Absolute volume quantity analysis is not available for multichannel
images.

If you have drawn your volume around an object of known quantity, you can use it to
calculate the quantity of your unknown volumes. The quantities of your unknown
volumes are calculated based on the standard volumes and the selected regression
method.

To classify a particular volume as a standard

1. Double-click the volume. This opens the Volume Properties dialog box.

2. Select the Standard option button and enter the quantity in the Quantity box.

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6 | Analyzing Images

3. Click OK to close the dialog box.

Standard volumes will have the default names S1, S2, and so on, based on the
sequence of their creation.

To review the regression curve

 Open the Standard Curve window and select the Volume Standard Curve tab.

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 Volume Tools

Regression Methods
Three regression methods are available to generate the volume quantification curve
used for absolute quantity: linear, point-to-point, and cubic spline. To display the
standard curve, click the Standard Curve button on the toolbar and select the
Volume Standard Curve tab in the Standard Curve dialog box. See Regression
Calculation Methods on page 251 to learn how each of these methods is calculated.

The data for volume standards are found in the Absolute Quantity column of the
Volume Table.

Note: Selecting the Force Through Origin checkbox always starts the standard
curve graph at 0,0, regardless of the best curve fit.

Alignment
Align volumes by using the appropriate alignment button. To select several volumes,
Ctrl-click each one, then select one of the alignment buttons. Hover over any of the
six alignment buttons to display its function (Align Left, Align Right, etc.)

Copy and paste selected volumes by pressing Ctrl+C to copy. Press Ctrl+V to
paste.

When you click the Standard Curve button on the toolbar, a chart displays all
unknown and standard quantities.

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6 | Analyzing Images

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 7 Generating Reports

After viewing results, you can generate a report that displays the analyzed gel
images, all of the settings used in the protocol, and as much information about the
data as you want to include.

You can choose print settings within the Report Settings dialog box in the Edit menu
or by clicking Report in the main toolbar.

Report
To produce a preview of your report
 Click Report on the toolbar.

Use the following dialog boxes to customize the content in your reports. Doing so
does not modify the data from the analysis.

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7 | Generating Reports

Report Settings
Use this dialog box to customize the content of your report.

General Tab

The General tab includes the following settings:

 Include Gel Image — specify whether the image is included in the report.

If the image is included, the following options determine which overlays are
displayed on the gel image:

 Show Lanes and Bands

 Show Volumes

 Show Annotations

 Include Unannotated Image

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 Report

When you select this checkbox, both the annotated image (if available) and
unannotated images are printed in the report. The unannotated image
precedes the annotated image in the report.

Note: If you disable the other checkboxes in the Include Gel Image
group, the Include Unannotated Image checkbox is automatically
disabled.

 Image Info — specify what information is included in the report.

 Acquisition Information

 Analysis Settings

 Image Information

 Notes

 Signature History — details on when and why a secure document was

signed

If a secure document has been signed, the user name, the date and time of the
signature, and the reason for signing are all included in the Signature History
section. If a document has not been signed, this section is omitted from the
image report.

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7 | Generating Reports

Lane and Band Table Tab

The Lane and Band Table tab includes the following settings:

 Include Lane and Band Table — specify whether to include the Lane and
Band table in the report.

 Lanes to show — specify which lanes to display in the report.

 Show Lane Profile — specify whether to display the Lane Profile view.

 Print one lane per page — specify whether all lanes are printed on one
page or whether each lane is printed on a separate page (adds a page
break after each lane).

 Show Lane Profile — include the lane profile for each lane.

 Not Displayed/Displayed — remove columns that you do not want to

display in the report. By default, the report displays all columns from the
Lane and Bands table.

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 Report

Volume Table Tab

The Volume Table tab includes the following settings:

 Include Volume Table — clear to exclude this information from your

report.

 Not Displayed/Displayed — remove columns that you do not want to

display in the report. By default, the report displays all columns from the
Volume table.

Print Report
Click the Print Report button to print your report.

Print Report to a PDF File

The Print Report to .pdf File button opens a Save dialog box so the PDF file can be
saved on your system.

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7 | Generating Reports

Adjust the Printer Settings

The Printer Settings button accesses options for paper size, orientation, and page
margins.

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 8 Exporting Results

The most convenient way to archive complete information about experiments is to

produce reports. However, you might want to export only gel images or analysis
table data for analysis in different programs, such as Quantity One, FPQuest™, or
InfoQuest™FP software. Or you might need exported files for presentation or
publication.

Exporting Gel Images

Image Lab™ software includes features for exporting gel images several ways.

 You can export displayed image data to a publication (choose Export for
Publication).

 You can export raw image data as a 16-bit .tif file (choose Export for
Analysis).

 You can export image data to PulseNet. Doing so reduces the image to an
8-bit .tif file, limits its resolution, and restricts its file size to 300 Kb.

 You can export lane and band tables as well as volume tables to a
spreadsheet program or to a file.

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8 | Exporting Results

The options to export gel images are available using the Export option in the File
Menu.

Exporting Gel Images for Publication

Use this format only to export visual information to presentation or word processing
software, such as PowerPoint or Word.

To export a displayed image to a file

 Select File > Export > Export for Publication.

Note: You can select from .bmp, .png, .jpg, and .tif formats. The gel displays with
any lanes, bands, and annotations that appear on the screen. For a
multichannel image, select the image pane you want to publish before
exporting the image for publication. Image Lab software exports the active
pane.

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 Exporting Gel Images

In this dialog box you can:

 Select the entire image or the current view

 Select the resolution or specify a custom resolution

 Specify the publishing dimensions

 View the resulting published image size and dimensions

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8 | Exporting Results

You can zoom in on an area in a current view and export only that area, or you can
export the entire image. You can exclude annotations or overlays by clicking Display
Gel Options on the toolbar to access the appropriate settings.

Exporting Gel Images for Analysis

To export an image for analysis
Select File > Export > Export for Analysis.

This exports the raw data only as a 16-bit .tif file.

Note: 16-bit .tif images are not compatible with all image viewers.

The image might require contrast adjustment when it is imported into analysis
software. This option creates a file that can be analyzed in other programs such as
Quantity One, FPQuest, or InfoQuestFP software.

Note: For multichannel images, Image Lab software exports the separate
channel images, but not the multichannel image. Each exported channel image
is saved with its application name appended to the filename you selected.

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 Exporting Gel Images

Exporting Gel Images to PulseNet International

To export an image to PulseNet International
 Select File > Export > Export for PulseNet.

Image Lab reduces the image to an 8-bit .tif image file. Resolution is limited and file
size is restricted to 300 Kb.

Note: Export for PulseNet is not available for multichannel images.

Exporting Lane and Band Tables to Excel

If you have Excel (or Numbers on a Mac) installed on your computer, you can export
the data to the spreadsheet application.

To export the data to Excel (or Numbers)

 Select File > Export > Lane and Band Table to Excel.

This opens a table directly in the spreadsheet program. You can then use the Save
As option to produce other formats.

Exporting Volume Tables to File

To export an image as a CSV file
 Select Export > Volume Table to File.

Image Lab exports the image as a comma-separated values (CSV) file so the data
file can be opened in a database application.

Screenshot Tool Export

Use the Screenshot tool, available on the toolbar, to capture a displayed image to
the clipboard or to save it to a file (.bmp, .gif, .jpg, or .png).

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8 | Exporting Results

Analysis Table Export

You can export table analysis data from the File menu or by using the export buttons
at the top of the Analysis Table window.

The Analysis Table window has several buttons to export data to different formats,
depending on how the data are to be presented.

Copy Analysis Table to the Clipboard

Copies the analysis table to the clipboard and then pastes the analysis table into
word processing or presentation applications. It is best to use the vertical table
orientation when copying to an 8.5 x 11-inch page to accommodate the columns.

Export Analysis Table to a File

Exports an analysis table as a CSV file, so your data file can be opened in a
database application.

Export Analysis Table to a Spreadsheet

Enables you to use sort and formula functions of a spreadsheet program with your
data. If you have Excel (or Numbers on a Mac) installed on your computer, the data
open in the spreadsheet program.

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 9 System Calibration

When your imager is installed, instrument calibration is performed with a calibration

wizard. See the installation guide in your Image Lab™ upgrade kit for detailed
instructions.

The instrument calibration wizard performs several procedures required to automate

the system and prevent focus problems. Each of these calibrations affects your
system as follows:

 Focus Calibration — this calibration allows automated focus settings at

any zoom point, using a (patent pending) software algorithm. Therefore,
your focus remains correct whether you view an entire sample or an area of
interest.

 Focus Calibration with Height Offset — this calibration takes the tallest
of the available conversion screens into account, and extrapolates values
for the others, so that focus remains optimal, for whichever screen is used.

 Dark Reference Image — this calibration determines and corrects any

background signal present in your imager

 UV Flat Field Calibration — this calibration generates the flat field correction
profiles needed for the UV light source. Because of this calibration, your
images have more accurate quantity reporting and backgrounds of even
intensity.

 Lens Flat Field Calibration — this calibration corrects for the intensity roll-
off inherent in any lens.

 White Conversion Screen Calibration — this calibration generates a flat

field correction profile needed for the white light conversion screen.

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9 | System Calibration

Recalibrating Your Imager

When you add light sources or filters to a ChemiDoc™ MP imager, recalibrate your
imager using the instrument calibration dialog box.

To recalibrate your imager

1. In Image Lab, click Edit > Instrument Setup to open the Instrument Setup dialog
box.

2. If you have a new illumination source, select the appropriate box in the
Illumination Options field.

3. If you are adding new filters, use the dropdown list to match what is installed in
your instrument.

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 Recalibrating Your Imager

4. The software prompts you to restart the calibrations needed for the new
illumination sources.

5. Click OK to exit the dialog box. Your settings persist until you make further
changes.

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9 | System Calibration

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 10 Image Lab Logs

Image Lab Logs

Image Lab™ software provides three types of logs.

 Instrument log — records events related to the instrument, including

calibrating the instrument and the success or failure of the calibration. This
log file is visible only if the computer running Image Lab is connected to an
instrument.

 System log — records events related to running Image Lab, including

enabling or disabling of secure mode and the users who log on to or log off
of Image Lab.

 Document log — (Security Edition only) records events related to the

creating and modifying of secure protocol and image files.

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10 | Image Lab Logs

Viewing the Instrument Log

Instrument operations that you perform with Image Lab are recorded in the
instrument log.

To open the Instrument Log Viewer

 From the View menu, click the log for your instrument.

The menu displays the specific instrument to which you are connected. The
GS-900™ densitometer is shown as an example, above.

Note: You can view an instrument log only if Image Lab is connected to the
specified instrument.

The instrument log includes the following information.

 date and time of the action

 the user name of the user logged into the computer at the time of the
action

 the user’s security level

 the domain where the current user is logged in

 the type of action performed

 the result of the action taken, including complete records of the calibration

 the user’s reason for the action, if provided

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 Image Lab Logs

Viewing the System Log

Events related to running Image Lab, including enabling or disabling of secure mode
and users logging on to or logging off from Image Lab, are recorded in the system
log.

To open the System Log Viewer

 From the View menu, click View System Log.

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10 | Image Lab Logs

Viewing the Document Log

The View menu displays a list of logs for each open document. The document log
captures information about creating and editing the Image Lab protocol and image
files.

Note: Document logs are viewable only in Image Lab Security Edition, and only
on Windows-based computers. Document logs are not viewable on the Mac.

The document log captures changes from:

 Image tools

 Lane and bands tools

 Normalization tools

 Molecular weight tools

 Quantity tools

 Volume tools

You can view the document log for any open file. This file can be a previously saved
file, or it can be a newly created protocol or image file that is open on your desktop
but which has not yet been saved.

If the document was previously saved, the log is identified with this name. If the
document has not been saved, the log is identified with the time stamp, showing
when the document was created.

Log file for saved image files

Log file for open but unsaved image file

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 Image Lab Logs

To open the Document Log Viewer

 From the View menu, click the document log that you want to view. A typical
document log is shown below.

Displaying Log Data

The display toolbar is located above the log.

Displaying Data Columns in Logs

By default, the logs display the following columns:

 Date and time

 User

 Level — the security role of the user

 Domain — domain where the current user is logged in

 Type — the type of event

 Description — the actual event captured

 Reason (For Secure Mode only) — the reason for signing a document

You can change your view of any log by displaying or hiding data columns.

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10 | Image Lab Logs

To display or hide columns

1. From the View menu, open the log file.

2. Click the Display log viewer options icon.

3. In the Display Column Options dialog box, use the arrow keys to move columns
between the Not Displayed and Displayed lists.

4. Click OK.

Filtering Data in Logs

For all Image Lab logs, you can filter the entries in the following columns:

 Date and time

 User

 Type

For example, you can set the filter in the Type column to File and Image Lab will
display only the rows where Type is equal to File.

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 Image Lab Logs

Setting filters in logs

To filter the entries of a column
1. In the log, click the heading of the column that you want to filter. The Display
Filter options icon in the Actions tool bar is enabled. (By default, the log opens
with all event types displayed.)

Note: The Display Filter options icon is enabled only when you click on a
column that can be filtered. The Display Filter options icon remains
disabled if you click a column that cannot be filtered.

2. Click the Display Filter options icon to display the Select filter values dialog box.

Tip: Alternatively, right-click the heading of a filterable column to display

the Select filter values dialog box.

3. Select a value and click OK.

Tip: You can select multiple values to filter.

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10 | Image Lab Logs

Removing filters in logs

The Remove all filters icon is enabled only after you set a filter on a column. This
icon clears all filters on all columns.

Each filterable column has a Remove filters icon as well. This icon removes the filter
for that column only.

Collapsing or Expanding Data Rows

You can expand the size of rows in any log to display the full content of the row, or
you can collapse the row size to display more rows in the table. The icon toggles
between collapse and expand.

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 Image Lab Logs

To display the full content of a row

 Click the Collapse or expand rows icon to expand the row height. The row
adjusts to fit the text of the longest entry.

This action expands all rows in the log where an entry is longer than the width
of the column. Rows that do not need to wrap are not affected.

To display more rows in the table

 Click the Collapse or expand rows icon to collapse the row height to the default
height setting.

This action collapses all rows in the log to display data on a single line.

Exporting Logs
From the log viewer, you can:

 Copy log entries to the clipboard — copies the log entries to the
clipboard, enabling you to paste them into a word processing or
presentation application.

Tip: You can also open a log in the log viewer and press Ctrl+C to copy
the log entries to the clipboard.

 Export log entries to a file — exports the log entries as a CSV file that can
be opened in a database application.

 Export log entries to Excel — exports the log entries to an Excel file
where you can use Excel’s sorting and formula functions to manipulate
your data. If Excel is not installed on your computer, this feature is
disabled.

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10 | Image Lab Logs

To export a log file

1. From the View menu, click the log you want to view and export.

2. Click one of the icons to export the data in one of the supported formats.
Copy log entries to clipboard
Export log entries to File
Export log entries to Excel

Printing Logs
Log data can be sent to a printer or saved to a PDF file.

To print a log
1. From the View menu, click the log you want to view and print.

2. In the log viewer, click Print log.

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 Image Lab Logs

The Log Print Preview window displays the contents of the log file.

From the Log Print Preview window, you can:

 Click Print log to print the log to a printer.

 Click Print log to PDF to save the log to a PDF file.

 Click Adjust printer settings to prepare the file for printing.

User Guide | 201

10 | Image Lab Logs

202 | ChemiDoc MP Imaging System with Image Lab Software

 11 Using the Security Edition

21 CFR Part 11
Image Lab™ Security Edition is a module within the Bio-Rad Image Lab software
that assists users in meeting the Food and Drug Administration’s regulations on
good lab practices in the pharmaceutical and biotechnology industries. The Security
Edition enables system administrators to ensure that Image Lab operates in
compliance with Title 21 of the Code of Federal Regulations (CFR) Part 11 within a
closed system. A closed system is defined 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” (Section 11.3 (b) (4).

Note:

 Image Lab Security Edition is not supported by the Mac.

 The security controls built into Image Lab Security Edition must be properly
configured and administered by the system administrator(s) in your
organization in order to be secure and in compliance with 21 CFR Part 11.

 Bio-Rad makes no claim that Image Lab Security Edition software is

CFR-compliant in and of itself, nor does the company guarantee
compliance for the user. Your 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.

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11 | Using the Security Edition

Standard Mode versus Secure Mode

Image Lab Security Edition can be run in either of the following modes:

 Standard Mode — in this mode, there are no restrictions on controlling the

instrument, operating the software, or changing the documents.

 Secure Mode — in this mode, in addition to the features available in

standard mode, extensive security features are enabled, including
document signing and log creation.

When Image Lab is in secure mode, a padlock symbol appears in the left corner
of the status bar. If no padlock is present, the software is running in standard
mode.

Lock indicates secure No lock indicates standard

mode is enabled mode is enabled

When Image Lab is installed, by default it is set to run in standard mode. It continues
to run in this mode until a user with Image Lab Administrator privileges enables
secure mode.

This chapter assumes you are running the application in secure mode unless
otherwise noted.

User Names, Groups, and Roles

When Image Lab Security Edition is run in secure mode, you must log in with a user
name and password in order to run the application. The Microsoft Windows system
administrator generally creates the user names and passwords. The Image Lab
administrator defines the groups to which the Security Edition roles will be
associated.

Note: See Setting Up Users and Groups on page 219 for additional information
about setting up groups, user names, and passwords.

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 User Names, Groups, and Roles

There are four default roles in Image Lab Security Edition. Each role is associated
with one of the four default Image Lab user groups, and each user is assigned a role
that provides the user access to specific features in the software. Table 10 lists the
default Image Lab Security Edition user groups and the corresponding roles. It also
provides a brief description of the permissions for each role.

Note: System administrators can change the user group names, if necessary,
to meet their local company standards.

Table 10. Image Lab Security Edition groups and roles

User Group Image Lab Role Description
TDS_Administrator Administrator Users with this role can enable or
disable secure mode.
Administrator users can also
view log files, but they do not
have access to any other
features.
TDS_User Supervisor Users with this role have full
access to all features and
functions of the application. They
can also sign files. Supervisors
cannot enable or disable secure
mode.
TDS_Tech Clinician Users with this role can perform
instrument operations, run
existing protocols, and view
protocol files, results files, and
log files. They can also sign files.
All other access is restricted. For
example, they cannot create or
edit protocols.
TDS_Guest Reviewer Users with this role can open
and view protocol files, results
files, and log files. They can also
sign files. Users with this role
cannot create new protocols and
cannot change protocols. All
other access is restricted.

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11 | Using the Security Edition

Role Restrictions
Your role determines which features of the security edition you have permission to
use. If you attempt to perform an action that is not permitted for a user in your role,
you will see an error message. In some instances the user’s role determines which
Security Edition features are visible and/or enabled. Therefore, you might not see all
of the features described in this chapter.

Table 11 lists the Image Lab Security Edition functions that each role has permission
to perform.

Table 11. User access to features by role

Function Administrator Supervisor Clinician Reviewer
Enable/disable secure mode X
View log files X X X X
Set up and recalibrate the X
instrument
Create new protocols X
Open existing protocols X X X
Run protocols X X
Edit protocols X
Create new images X X
Open existing images X X X
Edit images X X
Analyze images X X
Sign documents X X X

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 Starting Image Lab Security Edition

Starting Image Lab Security Edition

When secure mode is enabled, you are prompted to log on when you start the
Image Lab application.

To start Image Lab in secure mode

1. Click the Image Lab icon to start the application.

2. In the Log on to Image Lab dialog box, enter your user name and password.

3. Click OK.

Note: If you have any questions or problems logging on, see your system
administrator.

Electronic Records
Image Lab Security Edition enables you to create secure electronic records as
defined by 21 CFR Part 11. In Image Lab, the following are electronic records:

 Protocol files

 Image files

 Document log files

 Instrument log files

 System log files

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11 | Using the Security Edition

Unsecured Documents
Protocol and image files created in standard mode or created with previous versions
of Image Lab are unsigned, unsecure documents. Unsecure documents remain
unsecure. When you open these files in secure mode, you can make changes to
these files and save them without restrictions. A document log is generated for
changes to these files, but the log is not viewable in standard mode. You must log in
to secure mode to see the log.

You can also open unsecure documents in secure mode and sign them. You can
make changes to these files and save them as secure documents. The original
unsecure document remains unsigned and unsecure. The new document is saved
as read-only with an incremental revision number and Image Lab generates an audit
log. Image Lab will not be able to overwrite the secure file, but it can overwrite the
original unsecure file.

Secure Documents
Protocol and image files created in secure mode are known as secure documents
and generate a log. However, until they are signed, such documents save as
standard files. Only after they are signed are they saved with the secure
extension(.smptl, .smscn, .sptl, or .sscn). Image files generated from signed
protocols automatically open the signing dialog box when the scan completes.

If you create a default protocol based on an existing signed protocol, the resulting
new default protocol cannot be edited.

Note: A secure document can be saved unsigned. Likewise, a signed

document can be saved as a new, unsigned file. In this case, the saved file is
still a secure document. All changes to the document are captured in the
document log.

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 Electronic Records

Documents created in secure mode can be signed at any time. When a secure
document is signed, it is saved as read-only. Image Lab cannot overwrite the
document. You can open signed documents and sign them again. The newly signed
file is saved as a second revision, and the new signatures are captured in the
document log.

Note: Image Lab can never overwrite any signed file.

You can open secure documents in standard mode of Image Lab. Selecting Save As
from the File menu creates an unsecure file. The original file is preserved. No log is
generated for the new, unsecure document. However, an entry is added to the
original log that the file has been reverted to unsecure.

Secure Documents in SAM mode

Images saved in signal accumulation mode (SAM) follow the same rules as the main
image. The rules apply when the image is saved from within a protocol. If you create
an image from a signed protocol, all resultant images must also be signed. If you try
to save a file more than once, a warning message appears asking you to confirm the
re-save. And if you confirm this re-save and click OK, Image Lab creates a new file
with an incremental revision number.

Modifying Secure Documents

You can open and change a signed document in Image Lab. The original document
is read-only and cannot be overwritten. Saving the revisions opens the Save As
dialog box. The changes are saved into a new revision with an incremental revision
number.

Each time a secure document is modified, you must provide a reason for each
change before you can sign the document. The modifications are logged in the
document log. The new signed document takes with it the entire history of the
original document in its log.

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11 | Using the Security Edition

Signing Documents
To sign a document
1. Select the protocol or results file.

2. From the Security menu, click Sign Document. The Signing Document dialog
box appears.

3. Enter the user name and password of a user authorized to sign documents.

Note: The user name and password can be for a user other than the
current user.

4. Enter a reason for signing the document. Typical reasons include review,
approval, responsibility, or authorship.

Note: You must provide a reason in order to sign the document.

The user name, date and time of the signature, and reason for signing are
always included in the Signature History section of the image report (see
page 177).

5. Click OK.

A Save File dialog box appears.

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 Document Logs

6. Enter a new name for the file and click Save.

Note: Signed protocols are saved with an .smptl extension, signed images
are saved with an .smscn extention.

Document Logs
Any changes made or actions performed on a protocol or image file generate a
document log documenting each change or action. This log is created as soon as
the protocol or scan file is created. Protocols are updated when the protocol is
saved or run. If you make changes to a file and save it as a new file, regardless of
whether you are in secure mode, the document log is preserved in the new file. If
you are in secure mode, the signature of the previous file is noted as part of the
document log.

Note: When a protocol is run, its log entries are copied to the resulting image
file.

All major actions and changes are audited (generate a document log). Examples of
auditable actions include:

 Signing a file

 Changing protocol settings

 Changing the image, for example, by cropping or rotating it

 Modifying the analysis, for example, changing the lanes and bands, or
adding or editing annotation to the images

Minor changes that affect only the display are not audited, such as:

 Selecting different columns in the Analysis Table

 Changing the display options in Lane Profile

 Annotating or labeling the image using Annotation Tools

Each change you make to a signed protocol or image file must be documented in
the Reason for Change dialog box.

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11 | Using the Security Edition

Viewing the Document Log

The View menu displays a list of logs for each open document. The document log
captures information about creating and editing Image Lab protocol and image files.
You can view the document log for any open file. This file can be a previously saved
file, or it can be a newly created protocol or image file that is open on your desktop
but which has not yet been saved.

If the document was previously saved, the log is identified with this name. If the
document has not been saved, the log is identified with the time stamp of when the
document was created.

Log file for saved image file

Log file for open but unsaved image file

The document log includes:

 Date and time

 User

 Level — the security role of the user

 Type — the type of event

 Description — the actual event captured

 Reason — the reason for signing a document

For more information about logs, see Chapter 10, Image Lab Logs on page 191.

212 | ChemiDoc MP Imaging System with Image Lab Software

 A Maintenance

This chapter includes instructions for maintaining the universal hood in proper
working condition by replacing parts.

UV Transilluminator Lamp and Starter Replacement

Note: The UV filter surface should always be kept clean from the chemical
agents used as gel dyes. Use protective gloves when touching the UV
transilluminator cover.

Depending on usage, the UV bulbs and starters last for many years. Replace bulbs
when you notice them flickering. If a bulb does not turn on when it is new or moved,
replace the bulb starter and test the bulb again.

Three types of bulbs are available. The catalog numbers are listed in Ordering
Information on page 242. The standard bulb is 302 nm. Optionally, the 254 nm bulb
is used for cross-linking of protein, and the 365 nm bulb is used to minimize
denaturing of DNA.

To replace the lamps

1. Turn off the power.

2. Disconnect the power cord from the universal hood.

3. Remove the four screws located on the left/right sides of the transilluminator
cover.

4. Remove the cover with the UV glass by sliding it forward, then lifting up.

User Guide | 213

A | Maintenance

5. Place it on a nonabrasive surface so that the glass does not get scratched or
damaged.

Note: Do not put the UV cover directly on the bench. Wear gloves when
touching the lamps.

6. Rotate the lamp until it becomes loose and the pins come to a vertical position.

7. Remove the lamp. Install the new lamp by rotating so that the pins are
horizontal and the lamp is tight.

8. Remove the starter by rotating it counterclockwise, and then pull it out.

9. Insert a new starter into the holder and rotate clockwise.

10. .Reassemble the cover and retighten the screws on both sides.

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 Fuse Replacement

Fuse Replacement
Always unplug the instrument before changing or checking the fuses.

Fuse holders

This unit is protected by two fuses (5 x 20 mm, 2 A Slo-Blo). The fuses are located in
fuse holders housed in the power entry module. This module is located on the right
side of the back of the universal hood.

To replace the fuses

1. Unplug the main power cable from the power outlet.

2. Use a flat screwdriver to turn the slotted front of each fuse holder
counterclockwise; the holder pops out so you can extract the fuse.

3. Remove the blown fuses and replace them with two new fuses
(catalog #900-8935).

4. Slide each fuse holder into the power entry module until it snaps in place.

User Guide | 215

A | Maintenance

216 | ChemiDoc MP Imaging System with Image Lab Software

 B Troubleshooting

Follow these suggestions to troubleshoot yourChemiDoc MP systems.

Problem Possible Cause Solution

Camera does not  Power to the camera  Turn on the power to the camera.
respond/camera may be turned off.
not found  The camera cables may  Make sure that all cables are
not be seated properly. connected as shown in the
 The software driver for Installation Guide.
the camera is missing.  If the camera driver is not present,
reload the camera driver from the
 Computer power-saving Image Lab™ software CD.
modes may be  Disable the power-saving modes
interfering with the on the computer.
camera driver.
 The cables may be
defective.  Replace the cables.
 The camera may be
defective.  Replace the camera.
Horizontal stripes  The emission filter may  Move the filter lever so that the
in image when not be positioned filter slider positions the filter under
using the UV mode properly. the camera lens.

Image is not visible  The monitor settings are  See your computer manual for the
on the monitor incorrect. proper settings.
 The lens cap is attached.  Remove the lens cap.
Image is not bright  The incorrect emission  Verify and use the correct filter for
enough filter is in use. the application.
 For chemiluminescence,  Remove the emission filter from
the emission filter is in the front of the lens.
front of the lens.

User Guide | 217

B | Troubleshooting

Problem Possible Cause Solution

Printout does not  The monitor settings are  See your monitor manual for the
look like the wrong. appropriate settings.
monitor image
 The printer settings are  See your printer manual for the
wrong. appropriate settings.
Light leakage into  The lens body is not  Loosen the thumbscrew and seat
the darkroom seated properly against the lens properly against the
the gasket on the hood’s gasket on the hood’s adapter
adapter plate. plate.
Unable to focus on  Focus is not calibrated  Select Edit > Instrument Setup to
the sample using for samples using this recalibrate the focus for use with
white light light source. this accessory.
transilluminator or
conversion screen
Lens limits seem  The camera lens is not  Reseat the camera on the lens
artificially restricted seated properly on the mounting plate.
lens mounting plate.

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 C Setting Up Users and
Groups
Setting Up Image Lab Users and Groups
This appendix explains how to set up users and groups to run Image Lab™ Security
Edition software in secure mode.

Note: This task requires System Administrator privileges on the client

computers (and possibly their domain) on which Image Lab is installed.

User Accounts
To give users access to Image Lab Security Edition, you can create new Windows
user accounts, add existing user accounts to the four default user groups specified
in Table 10 on page 205, or rename any of the four default user groups.

 A user account can have any name, but must have a password defined for
the account. See the section on Password Security, on page 230 for
information about setting passwords for maximum security.

 Each user can belong to the Image Lab Administrator group and one other
Image Lab user group.

For example, a user can belong to the Administrator group and the Supervisor
group, but a user cannot belong to both the Clinician group and the Supervisor
group.

User Authentication and Group Membership

Authentication in Image Lab is made up of two processes: user authentication and
group membership evaluation.

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C | Setting Up Users and Groups

User Authentication
In the user authentication process, Image Lab matches (authenticates) a user name
with permissions assigned to that user name on the authentication domain. That
domain can reside on your local computer (a local domain) or on a network server (a
network domain). You set the location of this domain in the Domain to be used for
authentication field of the Security Preferences dialog box. This name must be the
exact name of your local computer or network server. For details on how to find this
name see Finding the name of your authentication domain, on page 221.

Enter the name of your local

computer, or the name of the
network server used for your
authentication domain.

In the Security Preferences dialog box, if you (or your network administrator) choose
a local domain to be used for authentication, you are considered a local user. If you
or your network administrator choose a network domain, you are considered a
domain user.

Group Membership Evaluation

In the group membership evaluation process, Image Lab verifies that a user is a
member of one or more of the four default Image Lab user groups
(TDS_Administrator, TDS_User, TDS_Tech, or TDS_Guest). The valid members for
each of these groups can be specified in one of two places, as defined by the Use
local groups for establishing user security levels checkbox on the Security
Preferences dialog box.

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 Setting Up Image Lab Users and Groups

This checkbox specifies whether

valid users and groups are defined
on (1) the local Image Lab
computer, or (2) on the network
domain.

If this checkbox is selected, only users and groups that are defined on the local
computer (on which Image Lab is installed) are recognized. If this checkbox is not
selected, only users and groups that are defined on the network domain are
recognized.

Finding the name of your authentication domain

Your authentication domain can be hosted on your local computer (a local domain)
or on a network server (a network domain).

To find the name of your local domain

1. From your Start menu, open the Control Panel.

2. Click System.

The System window appears. In the Computer name, domain, and workgroup
settings section, the name of your local computer is shown as Computer name.

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C | Setting Up Users and Groups

To find the name of your network domain

1. From your Start menu, open the Control Panel.

2. Click System.

The System window appears. In the Computer name, domain, and workgroup
settings section, the name of your network domain is shown as Domain.

Configuring Users and Groups on a Local Computer

To set up users and groups on a local computer
1. In the Windows Control Panel, select Administrative Tools.

2. Select Computer Management.

3. In the Computer Management window, expand the System Tools folder, and
then expand the Local Users and Groups folder.

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 Setting Up Image Lab Users and Groups

To create a new user on a local computer

1. Click on the Users folder to open it and select Action > New User. Alternatively,
use the right-click context menu.

The New User dialog box appears.

2. Fill out all the fields:

 User name — the user name must be unique.

 Full name — the full name must be unique.

Bio-Rad recommends using the user’s actual full name, as this name will
be shown in the document log and all the log reports. This is a requirement
of 21 CFR 11.50a.

 Description — this field must also be filled out.

Bio-Rad recommends entering the user’s title as the description.

 Password — enter and confirm a password for the user.

Tip: Select the User must change password at next logon checkbox.
This prevents the Windows system administrator from knowing the
passwords of the users.

Note: If you select the User must change password at next logon
checkbox, the user must actually log on to Windows and change the

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C | Setting Up Users and Groups

password before using Image Lab Security Edition. Otherwise,

Security Edition will not recognize the user.

To create a new group on a local computer

1. In the Computer Management window, click on the Groups folder to open it and
select Action > New Group. Alternatively, use the right-click context menu.

The New Group dialog box appears.

2. In the Group name field, enter the name of one of the groups specified in
Table 10 on page 205 (TDS_Administrator, TDS_User, TDS_Tech, TDS_Guest).
You can also enter a description in the Description field.

The group does not need any special operating-system level privileges.

3. Click Create to create and save the new group.

4. Repeat this task for the remaining groups specified in Table 10 on page 205.

To add a user to a group on a local computer

1. In the New Group dialog box, click Add. Alternatively, double-click an existing
group in the Groups folder to open its Properties dialog box, and click Add.

You see the Select Users dialog box.

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 Setting Up Image Lab Users and Groups

2. Click Advanced to expand the dialog box.

3. In the expanded dialog box, click Find Now to populate the bottom field with all
the users on the local computer.

4. Click a user name in the list to select it, or press the Ctrl key and click multiple
users to select them.

5. When you have selected all the users to add to the group, click OK, and click
OK again to close the Select Users dialog box.

6. Click Create to close the New Group dialog box and create the group.

Alternatively, you can click OK to close the existing group’s Properties dialog
box and accept the changes.

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C | Setting Up Users and Groups

Configuring Users and Groups on a Network Domain

Note: The network administrator must know how users and groups are set up
using the Windows server software at your site. The following example is used
to illustrate the choices.

To locate the users and groups on a Windows server

 Open Administrative Tools and select Active Directory.

Note that in the Active Directory window, the Users folder lists groups as well.

To create a new user on a Windows server

1. With the Users folder open, select Action > New User. Alternatively, use the
right-click context menu.

You see the New User dialog box.

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 Setting Up Image Lab Users and Groups

2. Fill out all the fields:

 User name — The user name must be unique.

 Full name — The full name must be unique.

Bio-Rad recommends using the user’s actual full name, as this name will be
shown in the document log and all the log reports. This is a requirement of
21 CFR 11.50a.

 Description — This field must also be filled out.

Bio-Rad recommends that you enter the user’s title as the description.

 Password — Enter and confirm a password for the user.

Tip: Select the User must change password at next logon checkbox. This
prevents the Windows system administrator from knowing the users’
passwords.

Note: If you select the User must change password at next logon
checkbox, the user must actually log on to Windows and change their
password before using Image Lab Security Edition. Otherwise, Security
Edition will not recognize the user.

User Guide | 227

C | Setting Up Users and Groups

To create a new group on a Windows server

1. With the Users folder open, select Action > New Group. Alternatively, you can
use the right-click menu.

You see the New Object - Group dialog box.

2. In the Group name field, enter one of the group names specified in Table 10 on
page 205 (TDS_Administrator, TDS_User, TDS_Tech, TDS_Guest).

Type the name exactly as specified. You can also enter a description for the
group in the Description field.

Note: The group does not need to have any special operating-system
level privileges.

To add a user to a group on a Windows server

1. In the New Group dialog box, click Add. Alternatively, double-click an existing
group in the User Manager folder to open its Properties dialog box and click
Add.

You see the Select Users dialog box.

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 Setting Up Image Lab Users and Groups

2. Click Advanced to expand the dialog box.

3. In the expanded dialog box, click Find Now to populate the bottom field with all
the users.

4. Click a user name in the list to select it, or press the Ctrl key and click multiple
users to select them.

5. When you have selected all the users to add to the group, click OK, and click
OK again to close the Select Users dialog box.

6. Click Create to close the New Group dialog box and create the group, or click
OK to close the existing group’s Properties dialog box and accept the changes.

User Guide | 229

C | Setting Up Users and Groups

Password Security
21 CFR 11.300 (b) requires that passwords be “periodically checked, recalled, or
revised.” Password policies are therefore recommended, although the password
duration and rules are up to the system administrator and the organization. For
instance, the exact duration between password changes is flexible.

To set password policies on a local computer

1. Open the Control Panel and select Administrative Tools > Local Security Policy.

2. In the left pane of the Local Security Policy window, expand Account Policies
and then select Password Policy.

You see the password policies listed in the right pane of the window.

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 Password Security

3. To change password policy settings:

a. Right-click the policy and select Properties to open its properties dialog
box.

b. Modify the default setting to meet your company policy.

c. Click Apply.

d. Click OK to close the Properties dialog box.

4. Close the Local Security Policy window.

To set password policies in Active Directory

1. Open the Control Panel and select Administrative Tools > Domain Controller
Security Policy.

2. In the left pane of the Domain Controller Security Policy window, expand
Security Settings > Account Policies and select Password Policy.

3. To change password policy settings:

a. Right-click the policy and select Properties to open its properties dialog
box.

b. Modify the default setting to meet your company policy.

c. Click Apply.

d. Click OK to close the Properties dialog box.

4. Close the Domain Controller Security Policy window.

User Guide | 231

C | Setting Up Users and Groups

Password Policy Setting Examples

The following examples are only suggestions. Your organization needs to establish
its own password policy.

 Enforce password history: 12 passwords remembered

 Minimum password age: 5 days

 Maximum password age: 30 days

 Minimum password length: 8 characters

 Password must meet complexity requirements: Enabled

 Store passwords using reverse encryption: Enabled

Account Lockout Policy Setting Examples

 Account lockout duration: 0 (The account is locked out until the
administrator unlocks it.)

 Account lockout threshold: 3 logon attempts

 Reset account lockout counter after: 30 minutes

Auditing Windows Event Logs

Some global auditing information is stored in the Windows Event logs. It is a
requirement of 21 CFR Part 11 that these logs be archived. However, by default,
Windows systems automatically remove these data without warning.

Note: It is therefore critical that the event log is reconfigured to generate and
preserve all necessary log data. Regular manual intervention is also required to
preserve these data.

To open the Event Properties Log

1. Open Administrative Tools and click Event Viewer.

2. Right-click on each log and select Properties.

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 Miscellaneous Security Measures

3. In the section When maximum event log size is reached: select Do not
overwrite events (Clear logs manually).

4. Increase the maximum size of the event log to cover any possible messages.
The smaller the maximum size of the event log, the more often the system
administrator must manually view, archive, and clear the system log.

Auditing information generated by the operating system is recorded in the Security

Log. Logon failures in Image Lab Security Edition are recorded in this log.

During the review process, the log should be examined for attempted breaches of
security, such as a series of failed logon attempts. To avoid the risk of losing data,
the size should be very large and this inspection/archive process should occur daily.
The Audit Policy should be set as follows:

 Audit account logon events — Failure should be checked at a minimum

 Audit account management — both Success and Failure should be

checked

 Audit logon events — Failure should be checked at a minimum

 Audit policy change — both Success and Failure should be checked

Miscellaneous Security Measures

Bio-Rad recommends that you use the built-in protections that Windows Server
offers in order to protect the computer while the user is absent.

Note: Microsoft is continually updating its operating systems in response to

security issues. It is critical to keep all components of the Windows operating
system, especially any domain controllers, up to date.

User Guide | 233

C | Setting Up Users and Groups

234 | ChemiDoc MP Imaging System with Image Lab Software

 D Accessories

Calibrating Accessories
If you are installing accessories along with your original system installation, calibrate
your system with a one-time Instrument Calibration wizard. Complete instructions
are in the installation guide that arrives with your imager. If you acquire new
conversion screens, light sources, or filters for a ChemiDoc™ MP imaging system
after your original system installation, you will have to recalibrate your imager to use
them.

See Chapter 9, System Calibration for instructions on how to calibrate newly

acquired accessories.

Installing Optional Accessories

Epi Light Modules

Epi light modules are available for the ChemiDoc MP imager in three colors: red
(catalog #170-8283), blue (catalog #170-8285), and green (catalog #170-8284). For
installation instructions, see Installing an Epi Light, an instruction sheet that
accompanies each module.

User Guide | 235

D | Accessories

UV/White Light Conversion Screen

This optional white light conversion screen (catalog #170-8289) converts the UV
light generated in the universal hood to white light. Your imager must be calibrated
to use the white light conversion screen.

To use the white light conversion screen

1. Center the conversion screen on the imager stage.

2. Center your samples on top of the conversion screen.

3. Image the gel using your preferred application..

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 Installing Optional Accessories

XcitaBlue™ Conversion Screen

The optional XcitaBlue conversion screen kit (catalog #170-8182) converts UV to
blue light, which enables you to visualize DNA samples while protecting them
against UV damage.

The XcitaBlue conversion screen is held in place by adhesive-backed edge guides.

After the edge guides are installed, the conversion screen remains centered and will
not slide, even if you close the drawer rapidly.

Adhesive-backed
edge guide

Transilluminator
drawer front

Transilluminator
border edge

User Guide | 237

D | Accessories

To install the XcitaBlue conversion screen

1. Perform a trial placement first, without removing the paper tape. Place the edge
guides in each corner of your transilluminator, as shown. The edge guides
should touch the inside of the drawer front and fit over the edge of the metal
transilluminator border (shown in red).

2. Remove the paper tape from the bottom surface of each edge guide.

3. Press each edge guide into position carefully. After the adhesive surfaces
touch, it is difficult to reposition the guide.

4. Calibrate your imager to use this accessory by going to Edit > Instrument
Setup. Select the XcitaBlue Conversion Screen checkbox under Illumination
Options. The software prompts you to calibrate the focus with height offset.

5. To visualize a sample using the XcitaBlue conversion screen, place the screen
between the edge guides.

6. Center the gel on top of the XcitaBlue conversion screen, and proceed with
normal image capture. Use the gel alignment template kit to center your gels
easily and consistently.

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 Installing Optional Accessories

Gel Alignment Template Kit

The Bio-Rad gel alignment template kit (catalog #170-8184) allows four sizes of
standard agarose gels to be centered quickly and easily and ensures the consistent
placement of each gel.

Magnetic
locator
frame

7 x 7 cm 15 x 7 cm 7 x 10 cm
15 x 10 cm

The kit contains:

 Magnetic locator frame

 Instruction sheet

 Alignment guides for the following gel trays:

 Sub-Cell® GT UV-transparent mini-gel tray, 7 x 7 cm

 Sub-Cell GT UV-transparent wide mini-gel tray, 15 x 7 cm

 Sub-Cell GT UV-transparent mini-gel tray, 7 x 10 cm

 Sub-Cell GT UV-transparent gel tray, 15 x 10 cm

The gel alignment templates fit exactly into the XcitaBlue conversion screen frame
(catalog #170-8182).

User Guide | 239

D | Accessories

To install and use the gel alignment template kit

1. Place the locator frame over the transilluminator with the magnetic side down.
Match the corners of the magnetic locator frame with the edges of the
transilluminator. The UV symbol on the frame will be in the same orientation as
the UV symbol on the imager.

2. Place the gel alignment template that matches the size of your sample tray or
agarose gel into the magnetic locator frame.

3. Place your gel or gel tray into the open area of the template.

Note: No recalibration is necessary to use the gel alignment template kit.

Orange Fluorescence Reference Plate

The orange fluorescence reference plate enables you to apply UV flat fielding
corrections to your™ imager. Corrections are made for all UV illumination sources,
filters, and the camera lens.

The orange fluorescence reference plate can be used to correct for image
nonuniformities for red and orange gels, including:

 Ethidium bromide

 GelRed

 Flamingo™

 Coomassie Fluor Orange

 SYPRO Ruby

 Krypton

 Qdot 625

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 Installing Optional Accessories

The orange fluorescence reference plate (catalog #170-8008) can be used with
several of the Bio-Rad® Molecular Imager® series of products, including:

 Gel Doc™ XR+ imaging system with Image Lab™ software

 ChemiDoc™ XRS+ imaging system with Image Lab software

 ChemiDoc XRS+ imaging system with Quantity One® software

When you order the reference plate, you receive:

 Orange fluorescence reference plate, overall dimensions: 29.5 x 29.5 cm;

viewing surface: 27 x 27 cm

 Instruction sheet (10017296)

Calibrating the Imager to Use the Orange Fluorescence Reference

Plate
Your imager must be calibrated to use the orange fluorescence reference plate.

To calibrate the imager to use the orange fluorescence reference plate

 Choose Edit > Instrument Setup and select the appropriate checkbox in the
dialog box. Image Lab software guides you through the calibration.

User Guide | 241

D | Accessories

Ordering Information
The following table contains catalog numbers and descriptions for all parts available
for the ChemiDoc MP imager, plus all optional accessories and replacement parts.
For more information, see the Bio-Rad catalog.

Table 12. Ordering information

Catalog # Description
Molecular Imager Series of Products (includes Universal Hood III, Camera, Cables, and
Accessories)
170-8280 ChemiDoc MP imaging system
Installation Kits
170-8282 ChemiDoc MP installation kit
Universal Hood
170-8281 Universal Hood III
Imaging Cameras
170-8255 ChemiDoc MP camera with motorized zoom lens
Image Lab Software
170-9690 Image Lab software, Windows/Mac
Optional Accessories
170-8001 UV/White light conversion screen (UV to white light)
170-8182 XcitaBlue (UV to blue light) conversion screen kit, without standard detection filter
170-8283 Kit, Red LED Module
170-8284 Kit, Green LED Module
170-8285 Kit, Blue LED Module
170-8183 XcitaBlue (UV to blue light) conversion screen kit, with standard detection filter
170-8008 Orange fluorescent reference plate
170-8089 Mitsubishi P93W Printer, 100/240 V, USB
170-3759 Bio-Rad fluorescent ruler
170-3760 Gel cutter ruler
170-8184 Gel alignment template kit
Replacement Parts
170-8026 Image Lab focus calibration target
170-8027 Image Lab flat fielding disc
170-8185 XcitaBlue viewing goggles
170-7581 Mitsubishi thermal printer paper, 4 rolls
170-7813 Sample holders for gels

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 Ordering Information

Table 12. Ordering information, continued.

Catalog # Description
100-2784 UV transilluminator lid (includes UV filter glass)
1001-4106 Thumbscrew for camera
170-8081 Filter, standard emission, 62 mm
100-1370 UV bulb starter, quantity 3
930-2242 Multicolor target
Lamps
100-1361 UVB lamp, 302 nm (1 each)
170-8097 302 nm lamp kit, (6 lamps)
170-8098 254 nm lamp kit, (6 lamps)
170-6887 365 nm lamp kit, (6 lamps)
Fuses
900-8935 Fuse T 2 A, 250 V, quantity 10
900-0234 Fuse T 4 A, 250 V, quantity 10
Universal Hood III
100-2787 Universal Hood feet, quantity 4
170-8068 UV Shield for Universal Hood
Connection Cables
931-0071 Cable, USB, Type A to B, 10 ft
901-0064 Cable, USB, Type A to B, 6 ft
Optional Analysis Software
170-9600 Quantity One 1-D Analysis software
170-9300 FPQuest™ software
170-9310 InfoQuest™FP Basic Fingerprint software
Protein Standards
161-0363 Precision Plus Protein™ Unstained Standards. 1 ml
161-0373 Precision Plus Protein™ All Blue Standards, 500 pl
161-0374 Precision Plus Protein™ Dual Color Standards, 500 pl
161-0375 Precision Plus Protein™ Kaleidoscope™ Standards, 500 pl
161-0385 Precision Plus Protein™ WesternC™ pack, 50 applications
161-0318 Prestained SDS-PAGE standards, broad range, 500 pl
161-0317 Unstained SDS-PAGE standards, broad range, 200 pl
Nucleic Acid Standards
170-8351 EZ Load™ 20 base pairs molecular ruler
170-8352 EZ Load 100 base pairs molecular ruler
170-8353 EZ Load 100 base pairs PCR molecular ruler

User Guide | 243

D | Accessories

Table 12. Ordering information, continued.

Catalog # Description
170-8354 EZ Load 500 base pairs molecular ruler
170-8355 EZ Load 1 kb molecular ruler
170-8205 2.5 kb molecular ruler
170-8200 AmpliSize® molecular ruler
170-8356 EZ Load precision molecular mass ruler (base pairs/ng of sample)
Pulsed Field Standards and Markers
170-3624 CHEF DNA size standard, 5 kb ladder
170-3707 CHEF DNA size standard, 8 - 48 kb
170-3635 CHEF DNA size standard, lambda ladder
170-3605 CHEF DNA size marker, 0.2 - 2.2 Mb
170-3667 CHEF DNA size marker, 1 -3.1 Mb
170-3633 CHEF DNA size marker, 3.5 - 5.7 Mb

244 | ChemiDoc MP Imaging System with Image Lab Software

 E Using the Criterion Stain
Free System
The Criterion Stain Free™ system comprises the ChemiDoc™ MP
imager, Image Lab™ software, and three types of precast gels:

 Criterion™ TGX Stain-Free™

 Criterion Stain Free™

 Mini-PROTEAN® TGX Stain-Free™

The stain-free system eliminates the time-consuming staining and destaining steps
required by other protein detection methods. Stain-free gels include unique trihalo
compounds that allow rapid fluorescent detection of proteins with the
ChemiDoc MP imager — without staining.

The trihalo compounds in the gels react with tryptophan residues in a

UV light-induced reaction to produce fluorescence, which can be easily detected by
the ChemiDoc MP imager within gels or on low fluorescence PVDF membranes.
Activation of the trihalo compounds in the gels adds 58 Da moieties to available
tryptophan residues and is required for protein visualization. Proteins that do not
contain tryptophan residues cannot be detected using this system. The sensitivity of
the stain-free system is comparable to staining with Coomassie Brilliant Blue for
proteins with a tryptophan content >1.5%; sensitivity superior to Coomassie
staining is possible for proteins with a tryptophan content >3%.

The benefits of the Criterion Stain Free system include:

 Elimination of staining and destaining steps for faster time to results

 No background variability within a gel or between gels (as is often seen

with standard Coomassie staining)

User Guide | 245

E | Using the Criterion Stain Free System

 Elimination of the need for acetic acid and methanol in staining and
destaining, which reduces organic waste

 Visualization of transferred or blotted proteins on low fluorescence PVDF

membranes Stain-Free Workflow

For detailed information about the Activate/image gels step, refer to Chapter 4,
Acquiring Images, on page 65. For all other workflow steps, refer to the Criterion™
Precast Gels Instruction Manual and Application Guide or to the Mini-PROTEAN®
Precast Gels Instruction Manual and Application Guide.

Prepare buffers

Prepare gels and assemble

electrophoresis cell

Prepare and load samples

Perform electrophoresis

Activate/image gels

Analyze the separation

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 Electrophoresis with Stain-Free Gels

Electrophoresis with Stain-Free Gels

Stain-free gels are made and packaged without SDS so they can be used for both
SDS and native PAGE applications.

To perform electrophoresis with stain-free gels

1. Prepare the sample and running buffers.

2. Set up the electrophoresis cell

3. Perform the run.

Imaging Gels
Use unstained standards with stain-free gels, as some prestained standards are not
detected by the stain-free system. To monitor electrophoresis, use a 1:1 mixture of
unstained and prestained standards.

Setting up a protocol for stain-free gels is very similar to setting up protocols for
other applications. Follow the instructions in Setting Up a Custom Protocol on
page 84. Choose one of the following activation times based on your sample and
the purpose of your experiment:

 Gels used in blotting — use 1 min activation for optimal results when
using western blotting followed by immunodetection.

 Good sensitivity – use 2.5 min activation when samples are abundant and
when a fully optimized signal-to-noise ratio is not necessary.

 Best sensitivity – use 5.0 min activation for detection of proteins that are
in low concentration and for the best quantification of the maximum
number of bands. Because the reaction is near completion after 5 min, this
method offers an optimal signal-to-noise ratio.

Note: If the gel has already been activated for 2.5 min, activating it for another
2.5 min might improve it. But activating an image for more than 5 min will not.

User Guide | 247

E | Using the Criterion Stain Free System

Imaging Blots
To blot stain-free gels, use standard blotting procedures as described in the
instruction manual you are using. Use only PDVF membranes with low background
fluorescence, as membranes other than low fluorescence PDVF can result in high
background or low sensitivity with the ChemiDoc MP imager.

To assess transfer efficiency, be sure to activate and visualize the gel using the
imager before transfer.

248 | ChemiDoc MP Imaging System with Image Lab Software

 F Mitsubishi P93/P95
Thermal Printer
Setting up a Thermal Printer on Windows
The printer driver is on the Image Lab™ software installation CD in the Misc folder.

To set up a thermal printer on a Windows system

1. Install the printer driver.

2. Open the printer section in Control Panel.

3. Click the thermal printer icon and select Printing Preferences.

4. Configure the correct paper size. Select 1280 x 1280 from the dropdown list.

5. Click OK to apply your changes.

User Guide | 249

F | Mitsubishi P93/P95 Thermal Printer

Setting up a Thermal Printer on a Mac

The printer driver can be found on the Image Lab software installation CD in the
Misc directory.

To set up a thermal printer on a Mac system

1. Install the printer driver.

2. Connect the printer to the computer.

To configure the correct paper size

1. Start Image Lab software.

2. Select File > Page Setup.

3. In the Settings dropdown list, select Page Attributes.

4. In the Format For dropdown list, select the Mitsubishi printer.

5. In the Paper Size dropdown list, select 1280 x 1280.

6. In the Settings dropdown list, select Save as Default.

7. Click OK to save the settings.

250 | ChemiDoc MP Imaging System with Image Lab Software

 G Regression Calculation
Methods
Each regression method calculates a standard curve. Some of the methods provide
the formula for the standard curve. In this case, the molecular weight can be
calculated by:

x = relative front of the band of interest

y = molecular weight of the band of interest

Linear (semilog): The linear equation is y = a + bx, where a is the intercept and b is
the slope of the line.

Note: The linear equation is calculated on the log of the molecular weight
values.

The R2 value can be used to determine the overall quality of the linear fit. A linear
regression with an R2 value of >0.99 is considered a very good fit. The primary
advantage of this method is that it is extremely simple. The primary disadvantage is
that it will deliver incorrect results if the data are not very linear.

Point-to-point (semilog): No single equation is available for the point-to-point

method. The slope of each segment of the curve between data points is calculated
independently.

Note: The log of the molecular weight values is used to calculate the slope for
each segment of the curve.

Logistic: The logistic-4PL equation is

User Guide | 251

G | Regression Calculation Methods

where:

x = mobility

y = molecular weight

a = estimated molecular weight at infinity

b = slope of the tangent at midpoint

c = midpoint

d = estimated molecular weight at zero mobility

Since the curve generated by the logistic-4PL regression method represents a

perfectly shaped S, it might not fit the data very well in all cases.

Cubic spline: Cubic spline curves are smooth curves that go through every data
point. The model is a cubic polynomial on each interval between data points. In
some cases, a spline curve can work well as a standard curve for interpolation.
However, because the curve is calculated individually for every pair of points, it does
not correspond to any single equation.

252 | ChemiDoc MP Imaging System with Image Lab Software

 Glossary

Aspect ratio The ratio of the width to the height of an image.

CCD (Charge-coupled device) A light-sensitive silicon chip used as a
photodetector in ChemiDoc™ MP and ChemiDoc XRS+ camera
systems.
Colormaps Different color representations of a gel image.
Electrophoresis A technique for separating molecules based on the differential
movement of charged particles through a matrix when subjected to an
electric field.
Example precision The number of decimal places chosen for displaying a measurement.
Flat fielding An average intensity computation that compensates for
nonuniformities generated by an instrument.
Histogram A graphed representation of the brightness, or gray value, of an image.
Native charge The inherent electrical charge of a protein without the addition of SDS.
density
pl Isoelectric point; the pH at which a protein molecule carries no net
charge.
Rf Relative front value of the band. In Image Lab™ software, Rf has a
value between 0 and 1 and indicates the relative movement of the
band from top to bottom.
Quantitative Determines the quantity of a protein’s components through analysis of
imaging the pixel values in a digital image of the sample.
UV-B The range of ultraviolet light used by the system.
UV The part of the imager that transmits UV light through a sample.
transilluminator

User Guide | 253

 | Glossary

254 | ChemiDoc MP Imaging System with Image Lab Software

 Bio-Rad
Laboratories, Inc.

Life Science Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 5044 5699
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10022469 Rev D US/EG 13-0584 0213 Sig 1212