ChemiDoc™ MP Imaging System with Image Lab™ Software User Guide Version 5.
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.
Table of Contents Safety and Regulatory Compliance . . . . . . . . . . . . . . . . . . . . . . . . . .11 Important Safety Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 General Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Regulatory Notices . . . . . . . . . . . . . . . . . .
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table of Contents Step 2. Detect Lanes and Bands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Step 3. Analyze Molecular Weight. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Step 4. Specify Output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Review Protocol Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Creating a Multichannel Image from Single Images . . . . . .
Table of Contents Chapter 6 Analyzing Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Image Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Using Auto Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Analysis Tool Box Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Image Tools . . . . . . . . . . . . . . . . . . .
Table of Contents Chapter 7 Generating Reports. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Report Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 Print Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 Print Report to a PDF File . . . . . . . .
Table of Contents Chapter 11 Using the Security Edition . . . . . . . . . . . . . . . . . . . . . . . 211 21 CFR Part 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Standard Mode versus Secure Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 User Names, Groups, and Roles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 Role Restrictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table of Contents Appendix D Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 Calibrating Accessories. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 Installing Optional Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 Epi Light Modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 UV/White Light Conversion Screen . . . . . . . . . . . . . .
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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.
| 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.
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.
| 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.
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 223 for information about replacing the fuses.
| 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.
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 ChemiD
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.
1 | Introduction Optional Accessories Bio-Rad Laboratories, Inc. offers a selection of optional filters and illumination sources. See Ordering Information on page 250 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).
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.
1 | 22 Introduction Cy2 IRDye 680 Cy3 Rhodamine Cy5 Fluorescein Cy5.
ChemiDoc MP Technical Specifications ChemiDoc MP Technical Specifications Applications Chemiluminescence Yes Fluorescence* Yes Colorimetry/densitometry Yes Gel documentation Yes Hardware Specifications Length: 28 cm Width: 36 cm Length: 26 cm Width: 35 cm Maximum image area for standard, UV-excited gels Length: 25 cm Width: 26 cm Excitation source Trans-UV and epi-white are standard (302 nm included, with 254 and 365 nm available as options).
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 selection Application driven, user-selected or recalled by a protocol Workflow automated execution Controlled by a protocol via app
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.
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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 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.
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 instrument 1 free USB 2.0 port 1 free USB 2.
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.
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.
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.
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.
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.
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.
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.
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 36 1. Select Activate Via Internet. 2. Click Activate.
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.
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 LSG.TechServ.US@Bio-Rad.com, with the subject line: Request to Activate Image Lab software Security Edition. 6. Attach the ActivationEmail.txt file to the email and click Send.
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.
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.
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.
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 LSG.TechServ.US@Bio-Rad.com, 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.
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.
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 44 1. From the Security menu, click Security Preferences to display the Security Preferences dialog box. 2. Clear the Enable secure mode checkbox. 3.
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.
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.
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 230 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. 2. Do not select the Use local groups for establishing user security levels checkbox.
2 | Setting Up the Instrument with Image Lab Software 3. Select the Use local groups for establishing user security levels checkbox. 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 229 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.
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.
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 227. 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.
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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.
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 60 File Extension Icon Unsigned Signed Multichannel protocols .mptl .smptl Multichannel images .mscn .
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.
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.
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.
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.
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 135.
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.
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 190 for more information. Export for Analysis — creates a .tif file that retains all gel image data. Analysis data are not included.
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.
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.
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.
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.
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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.
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.
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.
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 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.
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.
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 100. Tip: For a list of applications with all required detection reagents, light sources, and any conversion screens or filters noted, see Application Tables on page 103. Note: If you select the Stain Free option, you may select the gel activation time.
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 115). Step 1.
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 100 for more information.
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 103. 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.
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.
4 | Acquiring Images 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. 5.
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 140 for details.
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 139.
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 149. Note: You must select an application for at least one channel in Step 1.
4 | Acquiring Images 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.
Creating a Protocol 3. 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. To create a new standard, click New in the Manage Standards dialog box and complete the fields in the Edit Standard dialog.
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. 4. d.
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.
4 | Acquiring Images 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 183. Review Protocol Settings Click Protocol Summary in the left pane of any Protocol Setup window to see a quick review of all protocol settings.
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.
4 | Acquiring Images 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.
Editing a Saved Protocol You see the same set of menus and choices described in Creating a Protocol on page 76. 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.
4 | Acquiring Images 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.
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.
4 | Acquiring Images 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.
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.
4 | Acquiring Images 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 100 | 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.
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.
4 | Acquiring Images 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.
Application Tables 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.
4 | Acquiring Images 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.
Application Tables Table 8. XcitaBlue conversion screen XcitaBlue™ (catalog #170-8182) Standard Filter* 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.
5 | Viewing Images 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.
Displaying Gel Images 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.
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.
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.
5 | Viewing Images If you change the color of a selected channel in Image Transform, the change is reflected in the merged image.
Displaying Gel Images 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.
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.
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.
5 | Viewing Images 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.
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.
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.
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.
5 | Viewing Images Vertical View 120 | ChemiDoc MP Imaging System with Image Lab Software
Displaying Multichannel Images Horizontal View Single View User Guide | 121
5 | Viewing Images 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.
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 125.
5 | Viewing Images 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.
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.
5 | Viewing Images 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.
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).
5 | Viewing Images The zoom tools work the same way they work elsewhere in the software. For more information, see Zoom Tools on page 109. 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.
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.
5 | Viewing Images 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.
