ChemiDoc™ MP System with Image Lab™ Software Instruction Manual
Copyright ©2011 Bio-Rad Laboratories, Inc. Reproduction in any form, either print or electronic, is prohibited without written permission of Bio-Rad Laboratories, Inc. 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 detail at http://www.cecill.info/licences/Licence_CeCILL-C_V1-en.html 3.
ChemiDoc MP 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 system involves UV illumination.
ChemiDoc MP with Image Lab Software | Safety and Regulatory Compliance General Precautions • • • • • • Please read the instruction manual 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 the lights immediately after use Clean the transilluminator platen after use Regulato
FUSES The universal hood of the ChemiDoc MP imaging system has two userserviceable fuses, F1 and F2, which are located on the bottom rear panel and are a part of the power entry module. Refer to the Maintenance chapter for fuse replacement instructions.
ChemiDoc MP with Image Lab Software | Safety and Regulatory Compliance vi
Contents Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ChemiDoc™ MP Imaging System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 System Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 CCD Camera and Lenses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Universal Hood III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ChemiDoc MP with Image Lab Software| Contents Output Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Review Protocol Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Creating a Multichannel Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Acquisition Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Output Settings . . . . . . . . . . . . . . . . . .
Volume Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Chapter 7 Generating Reports . . . . . . . . . . . . . . . . . . . . . . . . . 91 Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 General Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Lane and Band Table Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ChemiDoc MP with Image Lab Software| Contents XcitaBlue™ Conversion Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Gel Alignment Template Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Orange Fluorescence Reference Plate. . . . . . . . . . . . . . . . . . . . . . . . 115 Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Appendix D. Mitsubishi P93/P95 Thermal Printer Settings . 121 Windows . . .
ChemiDoc MP with Image Lab Software 1 Introduction The ChemiDoc™ MP imaging system offers exceptional application flexibility, high performance, and ease of use. The system 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.
ChemiDoc MP with Image Lab Software | Introduction • • • Smart, application-based protocol setup using Image Lab software, which assists you 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 L
System Components Image Lab Software The system ships with a full version of Image Lab software. In addition to controlling the imager system, 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 quantitation and purity of samples. You can print all or a subset of your data in a report.
ChemiDoc MP with Image Lab Software | Introduction Optional Accessories Bio-Rad Laboratories, Inc. offers a selection of optional filters and illumination sources. See Appendix C for ordering information and 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 P93DW Thermal Printer, (catalog #1708089).
System Requirements System Requirements Image Lab software runs under Windows XP Professional, 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 mainly 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.
ChemiDoc MP with Image Lab Software | Introduction NUCLEIC ACID GELS • Ethidium bromide • GelGreen • ® SYBR Green • GelRed • SYBR® Safe • Fluorescein • SYBR® Gold • OliGreen • PicoGreen • GelStar PROTEIN GELS • Stain-Free Gel • Coomassie Fluor Orange • Oriole™ fluorescent gel stain • Pro-Q Diamond • Flamingo™ fluorescent gel stain • Pro-Q Emerald 300 • SYPRO Ruby • Pro-Q Emerald 488 • Krypton BLOTS 6 • Chemi • Alexa 680 • Chemi Hi Senstivity • Dylight 488 •
ChemiDoc MP Technical Specifications ChemiDoc MP Technical Specifications Applications Chemiluminescence Yes Fluorescence* Yes Colorimetry/densitometry Yes Gel documentation Yes Hardware Specifications Maximum sample size (cm) 28 x 36 Maximum image area (cm) 25 x 26 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.
ChemiDoc MP with Image Lab Software | Introduction ChemiDoc MP Imager Workflow Following are the basic steps to acquiring, analyzing, and archiving an image using the ChemiDoc MP system and Image Lab software: 1. Select an existing protocol or customize a new one. 2. Position the sample 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.
ChemiDoc MP with Image Lab Software 2 System Calibration When your system is installed, system calibration is performed with a calibration wizard. See the Installation Guide in your installation kit for detailed instructions. The instrument calibration wizard performs several procedures required to automate the system and prevent focus problems.
ChemiDoc MP with Image Lab Software | System Calibration The Instrument Setup dialog box available from the Edit menu displays these calibration results for reference. The calibration wizard needs to be run only once, when your system is installed. The calibrations remain valid over the life of the instrument. The calibration need not be changed unless you add equipment, such as a new light source.
ChemiDoc MP with Image Lab Software 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 software, 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.
ChemiDoc MP with Image Lab Software | Image Lab™ Software Overview Interface Overview The following illustration shows the Image Lab software main window. The paragraphs below describe the main software elements. Main Window Image Lab software 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.
Interface Overview Toolbar Many Image Lab software tools can be selected by clicking toolbar icons. 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. Additional tools for viewing protocols and results data presented in various forms are labeled below.
ChemiDoc MP with Image Lab Software | Image Lab™ Software Overview Display Toolbox The display toolbox near 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. Start Page The Start Page guides you through creating, opening, and viewing protocols and images.
Interface Overview Analysis Tool Box The Auto Analysis button quickly analyzes images. The remaining tools customize the analyzed data. Note: An image file must be selected (on a Windows computer, the title bar is dark blue) to make analysis tools available. Image Tools enable you to flip, rotate, and crop images and to transform the image files. Lane and Band Tools control the detection function, enabling you to resize, adjust, and bend lanes and to detect, adjust, add, or delete bands.
ChemiDoc MP with Image Lab Software | Image Lab™ Software Overview Quantity Tools enable you to automatically quantitate bands, using either relative or absolute values. Annotation Tools are useful for drawing attention to any area of a gel. Volume Tools enable you to manually quantitate an object inside a boundary that you define. These tools are describedon page 69.
Interface Overview Recent Images gives you the choice of opening a recent image file. Recent Protocols gives you the choice of opening a recent protocol. Save enables you to save a protocol or image file once the protocol or image is named. Save As enables you to name and store a protocol or image. Protocols are stored with a .ptl file extension. Image files are stored with an .scn extension. Multiplex protocols are stored with an .mptl file extension. Multiplex image files are stored with an .
ChemiDoc MP with Image Lab Software | Image Lab™ Software Overview • Volume Table to File—exports as a CSV file so your volume table can be opened in a database application. See Exporting Results on page 95 for detailed information about exporting files Image Info displays information about individual gel and blot images such as acquisition date, data range, and image capture details including the exposure time and illumination source used, in three tabs (Image Details, Analysis Settings, and Notes).
Interface Overview • 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 contains two tabs. • The Protocol tab shows presets for naming image files.
ChemiDoc MP with Image Lab Software | Image Lab™ Software Overview HELP 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.
ChemiDoc MP with Image Lab Software 4 Acquiring Images Image Lab software can run specific applications with repeatable workflows using custom protocols that have a wide variety of settings. These protocols can be retrieved, revised, and reused. In Image Lab software, a protocol is any combination of settings for imaging, analyzing, and reporting that has been saved to run as a single workflow. Image Lab software supports two kinds of protocol: single channel and multichannel.
ChemiDoc MP with Image Lab Software | Acquiring Images The Protocol Setup Window This is the Protocol Setup window for a single-channel protocol. 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 The Multichannel Protocol Setup window has some different settings, but you work with it the same way as the Single-Channel Protocol Setup window. Select a step in the left pane and configure that step’s detailed settings in the right pane. Select Protocol Summary in the left pane to view all your protocol settings in the right pane.
ChemiDoc MP with Image Lab Software | Acquiring Images Creating a Single-Channel Protocol Setting up a single-channel protocol consists of three processes: • Choosing acquisition settings • Analyzing the image • Generating output The three processes are listed in the left pane of the Protocol Setup window. Numbered steps in each process 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.
Creating a Single-Channel Protocol Note: For a list of applications with all required detection reagents, light sources, and any conversion screens or filters noted, see Application Tables on page 45. Note: If you select the Stain Free application you have the option of selecting the gel activation time. See Appendix A. Using the Criterion Stain Free™ System, for more information. Choose the Custom application to run an existing application with a new name or an application unlike existing applications.
ChemiDoc MP with Image Lab Software | Acquiring Images 2. Choose a unique application name. 3. Select a light source, filter, and image color on the drop-down menus. 4. 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. 5. Click OK to create your custom application. 6.
Creating a Single-Channel Protocol prominent bands. Exposure time can range from 0.001 to 7200 seconds with the ChemiDoc™ MP system After imaging a gel with automatic exposure optimization, the exposure time displays in the protocol (see below) so you can manually adjust the exposure time if needed. Note: You can also view the exposure time of the image later, in the Image Info window (see Image Info on page 55).
ChemiDoc MP with Image Lab Software | Acquiring Images In this example, the bar in the Signal Accumulation Setup dialog box shows that images will be acquired at one-minute intervals, beginning at 1 min and ending at 5 min. The second one-minute image is added to the first one-minute image, resulting in a two-minute total integration time image. The third one-minute image is added to the previous image, and so on, until the last image is presented.
Creating a Single-Channel Protocol Analyze Image STEP 2. DETECT LANES AND BANDS To analyze the gel or blot, Image Lab software must detect lanes and bands on the image. Lanes are detected automatically, and then the background is subtracted automatically. Refer to Lane and Band Tools for details. Customize band detection with the following options. 9. Select the Lane and Band checkbox in the left pane of the Protocol Setup window. The right pane displays lane and band detection options.
ChemiDoc MP with Image Lab Software | Acquiring Images STEP 3. ANALYZE MOLECULAR WEIGHT 10. To analyze molecular weight automatically, select Analyze Molecular Weight in the left pane of the Protocol Setup window. When this checkbox is selected, the software calculates the molecular weight for each band based on the specified standard. 11. To estimate the size of the molecules in the bands of your gel, enter the standards you are using and specify the lane(s) in which the standards are placed.
Creating a Single-Channel Protocol STANDARD LANES 12. Choose which lane contains your standards by typing lane numbers or the words First and Last in the Standard Lanes field. The format is xx, xx, xx, ... where xx is the lane number. For example, if you run an 18well gel and want your standards in lanes 1, 10, and 18, enter First, 10, and Last. Note: Lane detection works best when standards are placed in the first and last lanes.
ChemiDoc MP with Image Lab Software | 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 below. Table 1.
Creating a Single-Channel Protocol Output Settings Image Lab software prints to the default printer unless you select otherwise. You have three choices for specifying output: • You can automatically print the image; this is useful for gel documentation, especially if a thermal printer is connected. See Appendix D.
ChemiDoc MP with Image Lab Software | Acquiring Images Review Protocol Settings 16. Click Protocol Summary in the left pane of any Protocol Setup window to see a quick review of all protocol settings. 17. Click Save in the left pane to save the protocol.
Creating a Multichannel Protocol Creating a Multichannel Protocol Setting up a multichannel protocol consists of two parts: • Choosing acquisition settings • Generating output The two processes are listed in the left pane of the Protocol Setup window. A numbered step in each process appears under these headings. To select an option under a protocol step: • Select the accompanying checkbox.
ChemiDoc MP with Image Lab Software | Acquiring Images APPLICATION OPTIONS 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. Choose an application—click Select and choose an application from the drop-down list that appears. Note: Use the Custom application to run an existing application with a new name or an application unlike existing applications. See Setting Up a Custom Application on page 43 for more information.
Creating a Multichannel Protocol 3. Choose Image Exposure Time—if you select the top radio button, Image Lab software acquires an image estimating an optimal exposure time. This option ensures the best use of the dynamic range.
ChemiDoc MP with Image Lab Software | Acquiring Images 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. SAM requires you to 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.
Creating a Multichannel Protocol Channel Color—select a color to display the sample image. Assigning each channel a different color makes it easy to identify each channel.Once you set up the first channel, the second channel box becomes active. 5. Repeat steps 1 through 5 to set up the second channel as well as the third, if applicable. The software determines which applications are available based on your selection of other channels. Each Configure box displays the settings for that channel.
ChemiDoc MP with Image Lab Software | Acquiring Images STEP 4. SPECIFY OUTPUT 7. Select Specify Output in the left pane of the Protocol Setup window to display output options. 8. In the right pane, you can choose whether to automatically display or to print an image or a report after a protocol runs. The report can be customized with the options in Edit > Report Settings. For information about reporting options, see Report on page 91.
Choosing a Protocol You can also choose recently used protocols or image files from the lists on the Start Page. Editing Protocols Protocols can be changed and/or renamed using the many tools in Image Lab software. Open any saved protocol. You are presented with the same set of menus and choices described in Creating a Single-Channel Protocol on page 24. Make and save your changes without renaming the protocol to replace an older one. Choose Save As and type a different name to make a second protocol.
ChemiDoc MP with Image Lab Software | Acquiring Images Position Gel To position a gel: 1. Click the yellow Position Gel button in the Protocol Setup window shown on page 41. 2. Place a gel on the imager stage and view the gel in Image Lab software. 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.
Setting Up a Custom Application 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. If you enabled SAM in the protocol, the most recent image appears by default in the main window. To end the protocol: • Click Cancel Run To save and analyze an image at any time: 1. Right-click the thumbnail and select Save on the pop-up menu. 2.
ChemiDoc MP with Image Lab Software | Acquiring Images To create a custom application: 1. Select New in the Manage Custom Applications dialog box. The Create Custom Applications dialog box appears. 2. Choose a unique application name. 3. Select a light source, filter, and image color on the drop-down lists. 4. Select a binning setting. Choosing a higher binning setting combines pixels to increase the amount of signal without increasing noise.
Application Tables Application Tables The following tables list the applications and primary filters for each light source.
ChemiDoc MP with Image Lab Software | Acquiring Images Red Epi (catalog #170-8283) 695/55 Filter CY5 DyLight 650 CY5.5 DyLight 680 Alexa 647 IRDye 680 Alexa 680 Qdots 705 Green Epi (catalog #170-8284) 605/50 Filter CY3 DyLight 549 Alexa 546 Xcita Blue™ (catalog #170-8182) Standard Filtera SYBR® Green (excision) Fluorescein (excision) SYBR® Safe (excision) OliGreen (excision) SYBR® Gold (excision) PicoGreen (excision) GelGreen (excision) GelStar (excision) a.
Creating a Multichannel Image from Single Images Creating a Multichannel Image from Single Images You can create a multichannel image from existing single images and from single channels in other multichannel images. Only images with the same aspect ratio can be combined in a multichannel image. 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, select Create Multichannel Image.
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ChemiDoc MP with Image Lab Software 5 Viewing Images Once a gel has been imaged, the image appears in the workspace. Many controls are available to optimize viewing and to analyze the image. Results Overview The screen shows a gel image with band and lane detection as well as annotations. The labels represent overlays that you can show or hide. There are many ways to view the data associated with the results. You can view data as an analysis table, a lane profile, a standard curve, and a report.
ChemiDoc MP with Image Lab Software | Viewing Images Displaying Gel Images Notice the display toolbar buttons above the gel image. Each of these tools is described in the following sections. Display Gel Options 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, and molecular weight legends.
Displaying Gel Images • • • • • Volume Absolute Quantity Relative Quantity Band % Lane % VOLUMES If you have drawn volume boundaries on the gel, you can display the volume 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; the one with the minus sign to make the image smaller. You can also zoom with the right mouse button. Click and drag to select an area you want to magnify.
ChemiDoc MP with Image Lab Software | Viewing Images Image Transform Use the Image Transform dialog box to adjust image brightness and contrast, optimizing the image display so faint detail 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.
Displaying Gel Images • • Highlight saturated pixels—when this checkbox is selected, areas of the image with saturated signal intensity (above 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 Note: In multichannel images, you can individually highlight the saturated pixels on a channel in red.
ChemiDoc MP with Image Lab Software | Viewing Images Image Colors You can choose a colormap 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.
Displaying Gel Images 3-D Projection Clicking the 3-D button transforms the gel image into a solid 3-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.
ChemiDoc MP with Image Lab Software | Viewing Images IMAGE DETAILS Acquisition and image information appear in this tab. Note: For multichannel images, channel settings also appear in this tab. Select a channel to display its details. ANALYSIS SETTINGS The 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.
Displaying Multichannel Images In the display toolbox above the image, additional controls are available for viewing the multichannel image. Multichannel View Click the Multichannel View button to display or hide the merged image panel in the multichannel display. You can show or hide channels individually using the three channel buttons. You can merge the three channels into a single multichannel pane.
ChemiDoc MP with Image Lab Software | Viewing Images You can also specify the channels to include in the merged view using the drop-down list. 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).
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: Grid View—by default, multichannel images appear in grid view.
ChemiDoc MP with Image Lab Software | Viewing Images Horizontal View Single View Displaying Data The results from analyzed data associated with the gel images can be visualized using an analysis table, a lane profile, a standard curve, or a report. Buttons to toggle these views on and off are located in the main toolbar. All of the views can be seen at one time.
Displaying Data 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 tab. If volume analysis was performed, these data can be viewed in the Volume Table tab. Clicking Analysis Table opens a tabular display of the data extracted from the results. The buttons above the table offer several ways of displaying and exporting analysis table data.
ChemiDoc MP with Image Lab Software | Viewing Images 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) CHANGE ANALYSIS TABLE ORIENTATION This button toggles between two table orientations.
Displaying Data Abs. Quant.—absolute quantification of the band. Rel. Quant.—relative quantification of the band compared to the reference band. Band %—percentage of the band 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 Definitions Volume Number—a unique number is assigned to each volume. Volume Label—software-generated labels for different types of volumes (U for unknown, B for background, S for standard).
ChemiDoc MP with Image Lab Software | Viewing Images Lane Profile Background The Lane Profile option shows a cross-section of the selected lane, rotated 90°. You can select other lanes by clicking them while the Lane Profile window is open. 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.
Displaying Data IDENTIFY BANDS BY You can display callouts on the Lane Profile to indicate these attributes: • Band Number • Band Label • Molecular Weight • Relative Front • Volume • Absolute Quantity • Relative Quantity • Band % • Lane % 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.
ChemiDoc MP with Image Lab Software | Viewing Images 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 Mol. Weight Standard Curve, the Abs.
ChemiDoc MP with Image Lab Software 6 Analyzing Images Analysis Toolbox tools are enabled once 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 Mac, 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.
ChemiDoc MP with Image Lab Software | Analyzing Images DETECTION SETTINGS 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 Band Tools on page 72.
Analysis Toolbox Tools MOLECULAR WEIGHT ANALYSIS SETTINGS Molecular Weight Standard—choose any of the many Bio-Rad standards or other standards you have placed in your standards list. For more information, refer to Standards on page 30. 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 Methods on page 32.
ChemiDoc MP with Image Lab Software | Analyzing Images Image Tools To show the image tools menu, click Image Tools. Click to return to the Analysis Toolbox menu FLIP You can flip the gel image horizontally or vertically. ROTATE You can rotate the gel image 90° using the Left or Right buttons. To correct a slanted gel: 1. Click Custom. 2. Rotate the red arrows that appear over the gel to any degree between 0 and 360° by dragging them. 3.
Analysis Toolbox Tools 4. (Optional) Type a name for the crop settings in the dialog box that appears 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. 3. Select the saved crop settings in the dialog box that appears and click Load.
ChemiDoc MP with Image Lab Software | Analyzing Images Lane and Band Tools Click Lane and Band Tools to select a tab, one for the lane tools and one for band tools. LANE TAB You can choose how lanes are detected by selecting Automatic or Manual Lane Finder. • • 72 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.
Analysis Toolbox Tools ALL LANES Resize—you can resize all lanes by dragging the handles on the corners of the red square to fit the gel image. Adjust—if the gel image is irregular, you can adjust the orientation of all lanes by dragging a single corner of the lane frame. The Adjust tool does not resize lane width. You can also add more anchor points on the top or bottom borders of the rectangle by clicking the lane frame. Remove any unneeded anchor point by right-clicking it.
ChemiDoc MP with Image Lab Software | Analyzing Images 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 may subtract actual data. For most samples, a size of <10 mm is usually appropriate.
Analysis Toolbox Tools Detect Bands—this option opens a dialog box in which you can select band detection sensitivity and the lanes to which it applies. Add—use this option to add faint bands. First click Add, and then add a new band by clicking anywhere inside the lane. Note: You can darken your entire image to view faint bands more easily using the Gamma slider in the Image Transform dialog box. For instructions, see Image Transform on page 52.
ChemiDoc MP with Image Lab Software | Analyzing Images Molecular Weight 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. You can view each band’s molecular weight in the molecular weight column of the Lane and Band tab in the Analysis Table view or in the gel image by selecting Mol. Wt.
Analysis Toolbox Tools Click Change to access the Manage Standards dialog box, where you can choose another standard or add third-party 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. Standard lanes are labeled Std below the lane. REGRESSION METHODS There are four regression methods. See Regression Methods on page 32.
ChemiDoc MP with Image Lab Software | Analyzing Images To select a reference band from an image and quantitate all other bands relative to it: 1. Click the Select button. 2. Click the band you want to use as a reference. A small R appears near the band you selected. To review the relative band quantities: • Go to the Rel. Quant. column of the Analysis table (Lane and Band tab).
Analysis Toolbox Tools ABSOLUTE QUANTITY TAB Absolute quantification is used to quantify bands based on known standard bands using a calibration curve. To determine the absolute quantities of bands, first select the Absolute tab. To calculate the absolute quantities of the bands: 1. Click Select. 2. Select at least two standard (known) bands and assign quantity values. The values display in the Standard Bands table.
ChemiDoc MP with Image Lab Software | Analyzing Images 3. Select a unit of measure from the choices listed in the Units drop-down list. 4. Select a regression method from the drop-down list. Keep the following guidelines in mind while making your choice. 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.
Analysis Toolbox Tools 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.
ChemiDoc MP with Image Lab Software | Analyzing Images Annotation Tools You can annotate results with text and arrows to draw attention to areas of interest. ADD ANNOTATIONS Text—you can add text annotations to gel images to draw attention to important details. First click Text, and then click an area you want to emphasize. A text box appears with a dotted-line border. Type your comment in the text box. Drag the box to change its position. Note: While editing text, press SHIFT+ENTER to add text.
Analysis Toolbox Tools Arrow—to add an arrow, first click Arrow. Click the area where you want the arrow to start and then drag to stretch the arrow point to the location you want to emphasize. To move the arrow, click the middle and drag it. 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.
ChemiDoc MP with Image Lab Software | Analyzing Images Foreground—click a text annotation or arrow. This activates the Foreground field, so you can select a foreground color from the drop-down list. Background—click a text annotation. This also activates the Background field so you can select a background color from the drop-down list. ROTATE You can rotate text annotations 90° to the left or right by clicking the Rotate buttons.
Analysis Toolbox Tools Volume Tools Volume tools enable you to manually quantitate 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.
ChemiDoc MP with Image Lab Software | Analyzing Images A default label appears within the shape drawn. The volume label can be one of three types—U for unknown, Std for standard, or B for background—along with a number that is assigned sequentially. 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.
Analysis Toolbox 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 89. Background volumes are used to remove the background from the calculation.
ChemiDoc MP with Image Lab Software | Analyzing Images 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.
Analysis Toolbox Tools 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 above 1.0 indicate that the volume is greater than the reference volume. Values below 1.0 indicate the volume is less than the reference volume.
ChemiDoc MP with Image Lab Software | Analyzing Images ALIGNMENT Align volumes by using the appropriate alignment button. To select several volumes, CTRL-click each one or drag them to use a selection box. Copy and paste selected volumes by pressing CTRL+C to copy. Press CTRL+V to paste. When you click the Standard Curve button in the toolbar, a chart displays all unknown and standard quantities.
ChemiDoc MP with Image Lab Software 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. The print report settings can be chosen with the Report Settings dialog box in the Edit menu or by clicking Report in the main toolbar. Report Click Report to produce a Print Preview of your report.
ChemiDoc MP with Image Lab Software | Generating Reports Use the following dialog boxes to remove information from reports. Doing so does not delete the data from the analysis. General Tab By default all information is included, but you can customize reports here. Clear any of the following settings to exclude information from a report. Custom Report Title—you can provide a custom report title for the report. Include Gel Image—the following options determine which overlays display on the gel image.
Report Image Info • Acquisition Information • Analysis Settings • Image Information • Notes Lane and Band Table Tab Clear any of the following settings to exclude information from the report. Include Lane and Band Table Lanes to show: • All lanes or only user-defined lanes • All lanes on one page, or one lane per page (add page break after each lane) Show Lane Profile—includes the lane profile for each lane.
ChemiDoc MP with Image Lab Software | Generating Reports Volume Table Tab Include Volume Table—clear to exclude this information from your report. Not Displayed/Displayed—move items that you do not want to include in the report to the left column. Print Report Click the Print Report icon to print your report. Print Report to .pdf File The Print Report to .pdf File icon brings up a Save dialog box so the .pdf file can be saved on your system.
ChemiDoc MP with Image Lab Software 8 Exporting Results The most convenient way to archive complete information about experiments is to produce reports. However, you may 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 may need exported files for presentation or publication. Exporting Gel Images Image Lab™ software includes features for exporting gel images several ways.
ChemiDoc MP with Image Lab Software | Exporting Results The available export options are described in the following sections. Exporting Gel Images for Publication Use this format only to export visual information to presentation or word processing software, such as PowerPoint or Word. When you select File > Export > Export for Publication, you can export a displayed image to a file. You can select from .bmp, .png, .jpg, and TIFF formats.
Exporting Gel Images Note that you can select the entire image or the current view, select the resolution or specify a custom resolution, specify the publishing dimensions, and view the resulting published image size and dimensions. You can zoom in on an area in a current view to export only that area, or you can export the entire image. You can exclude annotations or overlays by clicking Display Gel Options in the toolbar to access the appropriate settings.
ChemiDoc MP with Image Lab Software | Exporting Results Exporting Gel Images for Analysis When you select File > Export > Export for Analysis, you can export full image data (raw data only) for analysis as a 16-bit TIFF file. Note: 16-bit TIFF images are not compatible with all image viewers. The image may 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.
Exporting Gel Images 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 Click this button to copy the analysis table to the clipboard and then paste the analysis table into word processing or presentation applications.
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ChemiDoc MP with Image Lab Software 9 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.
ChemiDoc MP with Image Lab Software | Maintenance 4. Remove the cover with the UV glass by sliding it forward, then lifting up. 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.
Fuse Replacement Fuse Replacement Always unplug the instrument before changing or checking the fuses. Fuse holders This unit is protected by 2 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.
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ChemiDoc MP System with Image Lab Software Appendix A. 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.
ChemiDoc MP System with Image Lab Software • • 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 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, except where otherwise noted.
Imaging Gels 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 in many ways the same as setting up protocols for other applications. Follow the instructions in Creating a Single-Channel Protocol, on page 24, but after step 3, set the activation options for the gel as follows.
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ChemiDoc MP with Image Lab Software Appendix B. Troubleshooting Follow these suggestions to troubleshoot your ChemiDoc™ MP system.
ChemiDoc MP with Image Lab Software | Appendix B.
ChemiDoc MP with Image Lab Software Appendix C. Accessories Calibrating Accessories If you are installing accessories along with your original system installation, you calibrate your system with a one-time Instrument Calibration wizard. Complete instructions are in the installation guide that arrives with your system.
ChemiDoc MP with Image Lab Software | Appendix C. Accessories UV/White Light Conversion Screen This optional white light conversion screen (catalog #170-8001) converts the UV light generated in the universal hood to white light. Your system must be calibrated to use the white light conversion screen. To calibrate, go to the Edit > Instrument Setup dialog box and select the appropriate checkbox. The Image Lab™ software guides you through the calibration. To use the white light conversion screen: 1.
Installing Optional Accessories 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 To install the XcitaBlue conversion screen: 1. Perform a trial placement first, without removing the paper tape.
ChemiDoc MP with Image Lab Software | Appendix C. Accessories 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. 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.
Installing Optional Accessories 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 allows you to apply UV flat fielding corrections to your ChemiDoc MP imager.
ChemiDoc MP with Image Lab Software | Appendix C. Accessories CALIBRATING THE SYSTEM TO USE THE ORANGE FLUORESCENCE REFERENCE PLATE Your system must be calibrated to use the orange fluorescence reference plate. To calibrate, go to the Edit > Instrument Setup dialog box and select the appropriate checkbox. Image Lab software guides you through the calibration.
Ordering Information Ordering Information The following table contains catalog numbers and descriptions for all parts available for the ChemiDoc MP system, plus all optional accessories and replacement parts. For more information, see the Bio-Rad catalog.
ChemiDoc MP with Image Lab Software | Appendix C.
Ordering Information 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.
ChemiDoc MP with Image Lab Software | Appendix C.
ChemiDoc MP with Image Lab Software Appendix D. Mitsubishi P93/ P95 Thermal Printer Settings Windows 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 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.
ChemiDoc MP with Image Lab Software 4. Configure the correct paper size: select 1280 x 1280. 5. Click OK to apply your changes. 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.
Mac 3. In the Settings drop-down list, select Page Attributes. 4. In the Format For drop-down list, select the Mitsubishi printer. 5. In the Paper Size drop-down list, select 1280 x 1280. 6. In the Settings drop-down list, select Save as Default. 7. Click OK to save the settings.
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Appendix E: Regression Calculation Methods Appendix E. 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.
ChemiDoc MP with Image Lab Software | Appendix E. Regression Calculation Logistic: The logistic-4PL equation is 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.
ChemiDoc MP with Image Lab Software Glossary 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.
ChemiDoc MP with Image Lab Software | Glossary Quantitative imaging: Determines the quantity of a protein’s components through analysis of the pixel values in a digital image of the sample. UV-B: The range of ultraviolet light used by the system. UV transilluminator: through a sample.
ChemiDoc MP with Image Lab Software Index lane-based 73 local 88 volume 87 Blots 6 merging images 70, 71 Blotting stain-free gels 107 A Absolute quantity 79 Absolute volume 89 Accessories 111 Activation options Stain Free gel 107 Adding annotations to gel images Analysis table changing orientation 62 copying to clipboard 99 exporting to spreadsheet 99 Analyzing molecular weight 29, 76 Annotation tools 82 Application custom 25, 43 Application tables 45 Applications, list of 36 B Background subtraction glo
ChemiDoc MP with Image Lab Software | Index Crop tool 70 Cubic spline defined 126 Cubic spline regression method Custom application 25, 43 creating 25 80 resizing Results files Flat fielding 127 Fuse v replacing 103 51 G Gel D Dark reference image 9 Detect lanes and bands Display gel options 50 29 E Editing a protocol 41 Electrophoresis defined 127 Emission filters 3 Epi light modules 111 Exporting analysis tables 99 lane and band tables 98 results 95 volume tables 98 Exporting gel images 95 for pub
ChemiDoc MP with Image Lab Software Image Lab software 3 Image tools 70 Image Transform set image contrast 52 Instrument calibration wizard installing 111 Optional conversion screen 4 Optional light sources 4 Optional printer 4 Orange fluorescence reference plate 9 115 Ordering Information 117 L Lane background subtraction 73 detecting 72 Lane and band tools 72 Lanes and bands detecting 29 Light sources optional 4 Linear regression method 80 Local background subtraction 88 M Maintenance 101 Merging b
ChemiDoc MP with Image Lab Software | Index Relative volume 88 Relative volume quantity 89 Replacing fuse 103 UV starter 101 UV transilluminator 101 Report 91 adjusting printer settings 94 generating 91 printing 94 printing to .
ChemiDoc MP with Image Lab Software Z Zoom tools resizing images 51 133
Bio-Rad Laboratories, Inc. Life Science Group 10022469 Rev B Web site www.bio-rad.
