Quantity One® User Guide for Version 4.2.
Quantity One User Guide Bio-Rad Technical Service Department Phone: (800) 424-6723, option 2, option 3 (510) 741-6576 Fax: (510) 741-5802 E-mail: LSG.TechServ.US@Bio-Rad.com (U.S.) LSG.TechServ.Intl@Bio-Rad.com (International) 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.
Table of Contents 1. Introduction ........................................................................ 1-1 1.1. 1.2. 1.3. 1.4. 1.5. 1.6. 1.7. 1.8. 1.9. Overview of Quantity One ......................................................................... 1-1 Digital Data and Signal Intensity .............................................................. 1-2 About Gel Quality ........................................................................................ 1-3 Steps Involved in Using Quantity One ...
Quantity One User Guide 3.7. Display ......................................................................................................... 3-10 3.8. Options ........................................................................................................ 3-12 4. Chemi Doc .......................................................................... 4-1 4.1. 4.2. 4.3. 4.4. 4.5. 4.6. 4.7. 4.8. Chemi Doc Acquisition Window ............................................................... 4-2 Step I.
Contents 7. GS-800 Imaging Densitometer .......................................... 7-1 7.1. 7.2. 7.3. 7.4. 7.5. 7.6. 7.7. GS-800 Acquisition Window ...................................................................... 7-2 Step I. Select Application ............................................................................ 7-3 Step II. Select Scan Area .............................................................................. 7-6 Step III. Select Resolution ...........................................
Quantity One User Guide 10.4. Acquire the Image .................................................................................... 10-6 10.5. Options ...................................................................................................... 10-7 11. Molecular Imager FX ........................................................ 11-1 11.1. 11.2. 11.3. 11.4. 11.5. 11.6. FX Acquisition Window .......................................................................... 11-2 Step I.
Contents 14. Bands ................................................................................ 14-1 14.1. 14.2. 14.3. 14.4. 14.5. 14.6. 14.7. 14.8. How Bands Are Identified and Quantified .......................................... 14-2 Automatically Identifying All Bands .................................................... 14-3 Identifying and Editing Individual Bands ......................................... 14-10 Plotting Traces of Bands ..............................................................
Quantity One User Guide 18. Differential Display and VNTRs ...................................... 18-1 18.1. Differential Display .................................................................................. 18-1 18.2. Variable Number Tandem Repeats ....................................................... 18-4 19. Reports ............................................................................. 19-1 19.1. 19.2. 19.3. 19.4. 19.5. 19.6. 19.7. The Report Window .......................................
Preface 1. About This Document This user guide is designed to be used as a reference in your everyday use of Quantity One®. It provides detailed information about the tools and commands of Quantity One for the Windows and Macintosh platforms. Any platform differences in procedures and commands are noted in the text.
Quantity One User Guide Macintosh version. Both versions of a menu or dialog box will be shown only when there is a significant difference between the two. 2. Bio-Rad Listens The staff at Bio-Rad are receptive to your suggestions. Many of the new features and enhancements in this version of Quantity One are a direct result of conversations with our customers. Please let us know what you would like to see in the next version of Quantity One by faxing, calling, or e-mailing our Technical Services staff.
1. Introduction 1.1 Overview of Quantity One Quantity One® is a powerful, flexible software package for imaging and analyzing 1-D electrophoresis gels, dot blots and other arrays, and colonies. The software runs in a Windows or Macintosh environment and has a graphical interface with standard pull-down menus, toolbars, and keyboard commands.
Quantity One User Guide images can be easily converted into TIFF format for compatibility with other Macintosh and Windows applications. 1.2 Digital Data and Signal Intensity The Bio-Rad imaging devices supported by Quantity One are light and/or radiation detectors that convert signals from biological samples into digital data. Quantity One then displays the digital data on your computer screen, in the form of gray scale or color images.
Introduction light source, or the Fluor-S and Fluor-S MAX MultiImagers with white light illumination. Signal intensity is expressed in counts when using the Personal FX or FX, or in the case of the Gel Doc, Chemi Doc, Fluor-S, or Fluor-S MAX when using the UV light source. 1.3 About Gel Quality Quantity One is very tolerant of an assortment of electrophoretic artifacts. Lanes need not be perfectly straight or parallel. Bands need not be perfectly resolved.
Quantity One User Guide Acquire Image Optimize Image Profile Analysis Volume Analysis Colony Counting Report Results Fig. 1-2. Steps Involved in Using Quantity One. Acquire Image Before you can use Quantity One to analyze a biological image, you need to capture the image into your computer.
Introduction Analyze Image Once a “clean” image is available, you can use Quantity One to gather and analyze your data. In the case of 1-D gels, the software has tools for identifying lanes and defining, quantifying, and matching bands. Volume tools allow you to easily measure and compare the quantities of bands, spots, or arrays. The colony counting controls allow you to count the number of colonies in a Petri dish, as well as perform batch analysis.
Quantity One User Guide RAM: 64 MB for Gel Doc, Chemi Doc, and Fluor-S imaging systems. 128 MB for FX, Personal FX, and densitometers. Hard disk space: 3 GB. Recommended: Removable storage media (such as an Iomega Jaz drive) or a network file server. Monitor: 17" monitor, 1024 x 768 resolution, True Color (24- or 32-bit). SCSI: Required for all Bio-Rad imaging devices except the Gel Doc and Chemi Doc. Adaptec SCSI card with EZSCSI software. Printer: Optional.
Introduction SCSI: Required to run all Bio-Rad imaging devices except the Gel Doc and Chemi Doc. Macintosh has built-in SCSI. Printer: Optional. 1.6 Installing on a PC The software can be installed on a PC from a CD-ROM or you can download the installation program from the Internet. Make sure that Windows is up and running on your computer before attempting to install. Note: Windows NT and 2000: You must be a member of the Administrators group to install Discovery Series software on a computer.
Quantity One User Guide Installed Files and Directories The installation program will guide you through a series of screens. The installer will create a default directory tree under Program Files on your hard disk called Bio-Rad/The Discovery Series/Bin (you can select your own directory if you wish). The main program will be placed in the Bin directory.
Introduction the instructions to download the installer and place it on your desktop. Then you can double-click on the installer icon to begin running the installer. Installed Files and Folders The installer will guide you through a series of screens. The installer will create a folder on your hard drive that contains the main application and associated sample images (you can select a different folder if you wish).
Quantity One User Guide 1.8.a Software License When the program first opens, you should see a “Welcome” dialog box that shows the current status of your software license. This program is protected by a software licensing system. You can have full use of the program for 30 days free of charge, after which you must purchase the software and obtain a password for continued use. Most users will be able to start the program and begin the free trial period immediately after installation.
Introduction PC Fig. 1-6. PC Hardware Security Key Before proceeding, please turn off your computer. If you have a printer attached to your computer’s parallel port, please turn that off as well. The HSK attaches to the parallel port on the back of your PC. If a printer cable is attached to this port, disconnect it. After you have attached the HSK, you can attach the printer cable to the key itself and restart your computer and printer. The code for the PC hardware security key is EYYCY.
Quantity One User Guide Macintosh Fig. 1-7. Macintosh Hardware Security Key Before proceeding, please turn off your Macintosh. The Macintosh HSK must be inserted in the Apple Desktop Bus (ADB) path. The ADB port is located on the back of your Macintosh. Fig. 1-8. Apple Desktop Bus icon on back of Macintosh. The HSK can be inserted at any point in the ADB path—between the computer and the keyboard, between the keyboard and the mouse, between the keyboard and the monitor, etc.
Introduction Fig. 1-9. Free Trial screen. Click on the Free Trial button. This will open the Software License Registration Form (see next section). Temporary License (With HSK or Network License) If you have an HSK attached or are using a Network License, the Software License screen will reflect the fact that you receive a 30-day temporary license (“Your license will expire on _______”).
Quantity One User Guide Fig. 1-10. Temporary License screen. Click on the Run button to begin using the software. If you are using an HSK, some time during the 30-day license period, click on the Registration Form button to register your software. If your 30-day period has expired, a Free Trial button will appear when you open the software. Click on this button to open the Software License Registration Form and register your software.
Introduction Fig. 1-11. Software License Registration Form. Note: If you do not yet have a Purchase Order Number or Software Serial Number, you may leave these fields blank to receive a trial license. Registering by Internet If you have Internet access on your computer (the same computer on which you loaded the software), you can register quickly and easily. In the Software License Registration Form, click on the Submit via Internet button.
Quantity One User Guide Software License screen to update your license. (To access the Software License screen from within the application, select Help > Register.) If you have not yet submitted your Software Serial Number, open the form again, enter the serial number, and resubmit it over the Internet. In 1–2 days, click on the Check License button to update your license.
Introduction Fig. 1-12. Enter Password screen. In the Enter Password screen, type in your password in the field. Once you have typed in the correct password, the OK light next to the password field will change to green and the Enter button will activate. Click on Enter to run the program. 1.9 Contacting Bio-Rad Bio-Rad technical service hours are from 8:00 a.m. to 4:00 p.m., Pacific Standard Time in the U.S. Phone: 800-424-6723, option 2, option 3 510-741-6576 Fax: 510-741-5802 E-mail: LSG.TechServ.
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2. General Operation 2.1 Graphical Interface 2.1.a Menu Bar Quantity One has a standard menu bar with pulldown menus that contain all the major features and functions available in the software. Fig. 2-1. Menu bar. • File—General file commands (Open, Save, Print), imaging device acquisition windows (Gel Doc, Chemi Doc, GS-700, GS-710, Fluor-S, Fluor-S MAX, Personal FX, FX). • Edit—Text Overlay tools, Preferences, miscellaneous editing tools.
Quantity One User Guide Below the menu bar is the main toolbar, containing some of the most commonly used commands. Next to the main toolbar are the status boxes, which provide information about cursor selection and toolbar buttons. 2.1.b Main Toolbar The main toolbar appears below the menu. It includes buttons for the main file commands (Open, Save, Print) and essential viewing tools (Zoom Box, Grab, etc.
General Operation The first box displays any function that is assigned to the mouse (see section 2.2, Mouse-assignable Tools). If you select a command such as Zoom Box, the name and icon of that command will appear in this status box and remain there until another mouse function is selected or the mouse is deassigned. The second status box is designed to supplement Tool Help (see above). It provides additional information about the toolbar buttons.
Quantity One User Guide Expanded format Horizontal format Vertical format Hold cursor over icon to reveal Tool Help Click on question marks for on-line help Click on resize button to toggle format Fig. 2-4. Secondary toolbar formats and features. The expanded toolbar format shows the name of each of the commands. Click on the “?” icon next to the name to display on-line Help for that command. 2.1.
General Operation Fig. 2-5. Quick Guides listed on Help menu and main toolbar. The Quick Guides are similar in design to the secondary toolbars, but are application-specific. Each Quick Guide contains all of the functions necessary for a particular application, from opening the image to outputting data.
Quantity One User Guide Note: You will find that as you become more familiar with the application, you will skip operations that are not needed for your particular images. In their expanded format, the Quick Guide commands are numbered as well as named, so that the order of operation is clear. Simply follow the steps and the Quick Guide will lead you through the analysis. As with the secondary toolbars, you can click on the “?” next to the name of a function to display the Help text. 2.
General Operation 2.3 Keyboard Commands Many commands and functions may be executed using keyboard commands (e.g., pressing the F1 key will activate the View Entire Image command). A list of key combinations and their associated commands will be displayed if you select Keyboard Layout from the Help menu. The pulldown menus also list the shortcut keys for the menu commands.
Quantity One User Guide Fig. 2-8. File menu. Open To open a previously saved image, select Open from the File menu or main toolbar. This opens the Open dialog box.
General Operation Macintosh version: Windows version: Fig. 2-9. Open dialog box. In the dialog box, open a file by selecting the file name and clicking on the Open button. To look for files in other directories, use the directory pulldown menu at the top of the dialog box. An image created in the Windows version of Quantity One can be opened in the Macintosh version, and visa versa. However, you must add a .1sc extension to your Macintosh files to open them in Windows.
Quantity One User Guide You can also open images captured by other Discovery Series software and Multi-Analyst™ software. The application comes with a selection of sample images. In Windows, these may be found in the Discovery Series/Sample Images/1D directory. On the Macintosh, they are stored in the Sample Images folder in the Quantity One folder. Opening TIFF Images The Open command can also be used to import TIFF images from other software applications.
General Operation Discovery Series, although most scanners that are capable of producing 24-bit and indexed color images will be able to produce grayscale scans as well. 3. Compressed Files. The software does not read compressed TIFF images. Since most programs offer compression as a selectable option, files intended for compatibility with the Discovery Series should be formatted with the compression option turned off. Close To close an image or scan, select File > Close.
Quantity One User Guide Save All Save All on the main toolbar or File menu saves all images that are currently open. In Windows, new images will be given a .1sc extension when they are first saved. Revert to Saved File > Revert to Saved will reload the last saved version of the image you are working on. This is a quick way to undo any alterations you may have made to an image since you last saved it. Any changes you have made since last saving the file will be lost.
General Operation Fig. 2-10. Image Info box. To print the file info, click on the Print button in the dialog box. Change the Image Dimensions You can change the dimensions of certain images using the Image Info dialog box. Note: This feature is only available for images captured by a camera (such as the Gel Doc or Fluor-S) or imported TIF images in which the dimensions are not already specified.
Quantity One User Guide Fields for changing the image dimensions Fig. 2-11. Image Info dialog box with fields for changing the image dimensions. Enter the new image dimensions (in millimeters) in the appropriate fields. Note that the pixel size in the image ( in micrometers) will change to retain the same number of pixels in the image. Reduce File Size Image files can be quite large, and computer systems do not have unlimited memory or storage space.
General Operation Note: Reducing the file size of an image will result in some loss of resolution. In most cases this will not affect quantitation. In general, as long as the pixel size remains less than 10 percent of the size of the objects in your image, changing the pixel size will not affect quantitation. Select File > Reduce File Size to open the Reduce File Size dialog box.
Quantity One User Guide Note: Since with most 1-D gels you are more concerned with resolving bands in the vertical direction than the horizontal direction, you may want to reduce the file size by making rectangular pixels. That is, keeping the pixel size in the “y” dimension the same, while increasing the size in the “x” dimension. This lets you decrease the total number of pixels and therefore file size without sacrificing detail. When you are finished, click on the OK button.
General Operation 1. Gel Doc 1000/2000 2. Chemi Doc 3. GS-700 Imaging Densitometer 4. GS-710 Imaging Densitometer 5. Fluor-S MultiImager 6. Fluor-S MAX MultiImager 7. Personal Molecular Imager FX 8. Molecular Imager FX Selecting a name in the File menu will open the acquisition window that allows you to scan using that instrument. See the individual chapters on the imaging devices for more details. Exit File > Exit quits the application.
Quantity One User Guide Fig. 2-14. Preferences dialog box. Click on the appropriate tab to access groups of related preferences. After you have selected your preferences, click on OK to implement them. 2.5.a Misc. Click on the Misc tab to access the following preferences. Memory Allowance The Memory Allowance field allows you to specify the amount of virtual memory allocated for the application at start up. The default value of 512 megabytes is recommended.
General Operation GLP/GMP Mode The GLP/GMP Mode checkbox allows you to prevent changes to an image that would change the raw image data.
Quantity One User Guide Show Volumes Quick Guide If this checkbox is selected, the Volumes Quick Guide will open automatically when you open the program. Align Quick Guide with Document If this checkbox is selected, the Quick Guides will open flush with the edge of your documents. Otherwise, they will appear flush with the edge of the screen. Quick Guide Placement and Toolbar Placement These checkboxes determine which side of the screen the Quick Guides and toolbars will first open—left or right.
General Operation functions, while experienced users can specify a longer delay once they are familiar with the icons. Tool Help Persistence determines how long the Tool Help will linger on the screen after you move the cursor off a toolbar icon. 2.5.c Application Click on the Application tab to access the following preferences.
Quantity One User Guide 1. Dividing the distance a band has traveled down a lane by the length of the lane (Follow Lane). This is useful if your gel image is curved or slanted. 2. Dividing the vertical distance a band has traveled from the top of a lane by the vertical distance from the top of the lane to the bottom (Vertical Projection). Note: “Lane” and “band” refer here to lanes and bands as you have defined them on your image.
General Operation Band Style Bands in your gel image can be marked with brackets that define the top and bottom boundaries of the band, or they can be marked with a dash at the center of the band. Indicate your preference by clicking on the Brackets or Lines button next to the Band Style prompt. (This setting can be temporarily changed in the Band Attributes dialog box. However, all newly opened images will use the preferences setting.) 2.5.e Paths Click on the Paths tab to access the following preference.
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3. Gel Doc Fig. 3-1. Gel Doc. Before you can begin acquiring images, the Gel Doc system must be properly installed and connected with the host computer. See the Gel Doc hardware manual for installation, startup, and operating instructions. To use the Gel Doc, you will need to have the Bio-Rad-supplied acquisition board installed in your PC or Macintosh. The drivers for this board will be installed when you install the main software application.
Quantity One User Guide Make sure that your Gel Doc camera is turned on. If the camera is not turned on, the Gel Doc acquisition window will open but the screen will be black, and you will be unable to Video Print. Simulation Mode Any of the imaging device acquisition windows can be opened in “simulation mode.” In this mode, an acquisition window will open and the controls will appear active, but instead of capturing real images, the window will create “dummy” images of manufactured data.
Gel Doc Fig. 3-2. Gel Doc acquisition window The Gel Doc video display window will open in “live” mode, giving you a live video display of your sample. If no image is visible, make sure the camera is on, check the cable connections, make sure the f-stop on the camera is not closed, and make sure that the protective cap is off the camera lens. Also check to see that the transilluminator is on and working. The control panel has been arranged from top to bottom to guide you through the acquisition procedure.
Quantity One User Guide 3. Select the output. 3.2 Step I. Position Gel The Gel Doc window will open in “live” mode, giving you a live video display of your sample. In this mode, the Live/Focus button will appear selected, and frames will be captured and displayed at about 10 frames per second, depending on the speed of your computer. You can use live mode to zoom, focus, and adjust the aperture on the camera, while positioning the sample within the area.
Gel Doc the size of the area you are focusing on. To determine the size of the area you are focusing on, you can place a ruler in the Gel Doc box so that it is visible by the camera. 3.3 Step II. Acquire Image For many white light applications, you can skip this step and save and print images directly from Live/Focus mode.
Quantity One User Guide Click on Auto Expose; button appears depressed Can see Exposure Time automatically changing Fig. 3-4. Auto Expose. Once an image has reached the specified percent of saturated pixels, it is captured and displayed in the video display window, Auto Expose is automatically deactivated, the exposure time appears active in the Exposure Time field, and Manual Expose is activated.
Gel Doc Manual Expose active; button appears selected Exposure Time field active; adjust number of seconds Fig. 3-5. Manual Expose. With Manual Expose activated, you can adjust the exposure time directly by changing the number of seconds in the Exposure Time field. Type in a number or use the arrow buttons next to the field. When the specified exposure time is reached, the last captured image will be displayed in the Gel Doc image window.
Quantity One User Guide Note: Freeze is automatically activated if you adjust any of the subsequent controls (e.g., Video Print, Image Mode, Display controls, etc.). 3.4 Step III. Select Output The Gel Doc window has several output options. Fig. 3-7. Output options. Video Print Clicking on Video Print will automatically send the currently displayed frame (either live or integrated) to a video printer.
Gel Doc The image will not be saved until you select Save or Save As from the File menu. Analyze Clicking on Analyze will open a separate image window displaying the captured image. The default name for the image will include the date, time, and user (if known). You can then analyze the image using the menu and toolbar functions. The image will not be saved until you select Save or Save As from the File menu. Save Clicking on Save will open a separate image window displaying the captured image.
Quantity One User Guide White Light Select this mode for reflective and transmissive samples. With this mode selected, the data will be measured in uncalibrated optical density (uOD) units. 3.6 Exposure Status The Exposure Status bar shows the progress of your exposure. If your exposure time is greater than 1 second, the status bar display will give you a graphical representation of the remaining time before exposure is complete.
Gel Doc Highlight Saturated Pixels When this box is checked, any saturated pixels in the image will appear highlighted in red in the scan window and in the pop-up image window. To view/hide saturated pixels in the pop-up image window, use the Image > Transform command. Invert Display This checkbox will switch light spots on a dark background to dark spots on a light background, and visa versa. This will only affect how the image is displayed on the screen, not the actual image data.
Quantity One User Guide Reset Reset will return the image to its original, unmodified appearance. 3.8 Options Click on the Options button to open the Options dialog box. Here you can specify certain settings for your Gel Doc system. Fig. 3-9. Available options in the Gel Doc acquisition window. Click on OK to implement any changes you make in this box. Clicking on Defaults restores the settings to the factory defaults.
Gel Doc DAC Settings Note: The default DAC settings are highly recommended and should be changed with caution. These sliders may be used to adjust the minimum and maximum voltage settings of your video capture board. The minimum slider defines the pixel value that will appear as white in the image, while the maximum slider defines the pixel value that will appear as black. The slider scale is 0–255, with the defaults set to 60 minimum and 130 maximum.
Quantity One User Guide Video Printing Footer Information The checkboxes in this group allow you to specify the information that will appear at the bottom of your video printer printouts. Save Options To automatically create a backup copy of any scan you create, select the Make Backup Copy checkbox. With this checkbox selected, when you save a scan, a backup copy will be placed in the same directory as the scanned image. Windows backup files will have an “.sbk” extension.
4. Chemi Doc Fig. 4-1. Chemi Doc. Before you can begin acquiring images, the Chemi Doc system must be properly installed and connected with the host computer. See the Chemi Doc hardware manual for installation, startup, and operating instructions. To use the Chemi Doc, you will need to have the Bio-Rad-supplied acquisition board installed in your PC or Macintosh. The drivers for this board will be installed when you install the main software application.
Quantity One User Guide Make sure that your Chemi Doc camera is turned on. If the camera is not turned on, the Chemi Doc acquisition window will open but the screen will be black, and you will be unable to Video Print. Simulation Mode Any of the imaging device acquisition windows can be opened in “simulation mode.” In this mode, an acquisition window will open and the controls will appear active, but instead of capturing real images, the window will create “dummy” images of manufactured data.
Chemi Doc Fig. 4-2. Chemi Doc acquisition window The Chemi Doc video display window will open in “live” mode, giving you a live video display of your sample. If no image is visible, make sure the camera is on, check the cable connections, make sure the f-stop on the camera is not closed, and make sure that the protective cap is off the camera lens. Also check to see that the transilluminator is on and working.
Quantity One User Guide 3. Select the output. 4.2 Step I. Position Gel The Chemi Doc window will open in “live” mode, giving you a live video display of your sample. In this mode, the Live/Focus button will appear selected, and frames will be captured and displayed at about 10 frames per second, depending on the speed of your computer. You can use live mode to zoom, focus, and adjust the aperture on the camera, while positioning the sample within the area.
Chemi Doc 4.3 Step II. Acquire Image For many white light applications, you can skip this step and save and print images directly from Live/Focus mode. For UV light, chemiluminescent applications, or faint samples, the Chemi Doc control panel has several methods of creating image exposures. You can take an automatic exposure based on the number of saturated pixels in the image, you can enter a specific exposure time, or you can take a series of exposures and select the best one.
Quantity One User Guide Click on Auto Expose; button appears selected Can see Exposure Time automatically changing Fig. 4-4. Selecting Auto Expose. Once an image has reached the specified percentage of saturated pixels, it is captured and displayed in the video display window, Auto Expose is automatically deactivated, and the exposure time appears active in the Exposure Time field. At this point, if you are in UV or White image mode, Manual Expose will be automatically activated.
Chemi Doc Manual Expose active; button appears selected Exposure Time field active; adjust number of seconds Fig. 4-5. Setting a manual exposure. With Manual Expose activated, you can adjust the exposure time directly by changing the number of seconds in the Exposure Time field. Type in a number or use the arrow buttons next to the field. In UV or White image mode, when the specified exposure time is reached, the last captured image will be displayed in the Chemi Doc image window.
Quantity One User Guide In Chemi mode, Manual Expose will expose an image over the specified exposure time and then stop automatically. Note: Freeze is automatically activated if you adjust any of the subsequent controls (e.g., Video Print, Image Mode, Display controls, etc.). Live Acquire Live Acquire mode allows you to specify an interval over which a series of progressively longer exposures are taken.
Chemi Doc Click on OK in the settings dialog to begin taking exposures. If you selected Save Images, a Save dialog box will open in which you can specify the base file name and location of the exposure files. When you click on Save, the exposures will be taken. The specified number of exposures will be taken at equal intervals between the starting exposure time and total exposure time. When each exposure is complete, an image window containing that exposure will open behind the Chemi Doc window.
Quantity One User Guide Fig. 4-8. Output options. Video Print Clicking on Video Print will automatically send the currently displayed frame (either live or integrated) to a video printer. You can add information about your image to the bottom of the printout by selecting the appropriate checkboxes in the Options dialog box. (See Options, below.) Annotate Clicking on Annotate will open a separate image window displaying the captured image.
Chemi Doc You can then analyze the image using the other features in the main application. The image will not be saved until you select Save or Save As from the File menu. Save Clicking on Save will open a separate image window displaying the captured image. A Save As dialog box will automatically open displaying the default file name for the image, which will include the date, time, and user (if known). You can then change the file name and storage directory.
Quantity One User Guide Note: This setting will invert the data in your image, so that a pixel with an intensity of 255 will be changed to 0, and visa versa. Also, Chemi mode changes the behavior of the Auto and Manual Expose functions, as described above. 4.6 Exposure Status The Exposure Status bar shows the progress of your exposure. If your exposure time is greater than 1 second, the status bar display will give you a graphical representation of the remaining time before exposure is complete.
Chemi Doc Highlight Saturated Pixels When this box is checked, any saturated pixels in the image will appear highlighted in red in the scan window and in the pop-up image window. To view/hide saturated pixels in the pop-up image window, use the Image > Transform command. Invert Display This checkbox will switch light spots on a dark background to dark spots on a light background, and visa versa. Note: This will only affect how the image appears on your screen; it will not change the image data.
Quantity One User Guide Reset Reset will return the image to its original, unmodified appearance. 4.8 Options Click on the Options button to open the Options dialog box. Here you can specify certain settings for your Chemi Doc system. Fig. 4-10. Available options in the Chemi Doc acquisition window. Click on OK to implement any changes you make in this box. Clicking on Defaults restores the settings to the factory defaults.
Chemi Doc DAC Settings Note: The default DAC settings are highly recommended and should be changed with caution. These sliders may be used to adjust the minimum and maximum voltage settings of your video capture board. The minimum slider defines the pixel value that will appear as white in the image, while the maximum slider defines the pixel value that will appear as black. The slider scale is 0–255, with the defaults set to 60 minimum and 130 maximum.
Quantity One User Guide Video Printing Footer Information The checkboxes in this group allow you to specify the information that will appear at the bottom of your video printer printouts. Save Options To automatically create a backup copy of any scan you create, select the Make Backup Copy checkbox. With this checkbox selected, when you save a scan, a backup copy will be placed in the same directory as the scanned image. Windows backup files will have an “.sbk” extension.
5. GS-700 Imaging Densitometer Fig. 5-1. GS-700 Imaging Densitometer Before you can begin scanning images with the GS-700 Imaging Densitometer®, your instrument must be properly installed and connected with the host computer. See the hardware manual for installation, startup, and operating instructions. PC Only: A Note About SCSI Cards The GS-700 is connected to your computer by a Small Computer System Interface (SCSI) cable. To use the GS-700, you must have a SCSI card installed in your PC.
Quantity One User Guide appear active, but instead of capturing real images, the window will create “dummy” images of manufactured data. You do not need to be connected to an imaging device to open a simulated acquisition window. This is useful for demonstration purposes or practice scans. To enter simulation mode, hold down the CTRL key and select the name of the device from the File menu. The title of the acquisition window will indicate that it is simulated.
GS-700 Imaging Densitometer Fig. 5-2. GS-700 acquisition window The scanning window is marked by grid lines that divide the area into square centimeters. These are numbered 1–35 top to bottom and 1–21 left to right if the light source is uncalibrated reflective, and 1–25 top to bottom and 1–20 left to right if the light source is uncalibrated transmissive (see below for details). To hide the gridlines, click on the Hide Grid checkbox under Options.
Quantity One User Guide The control panel has been arranged from top to bottom to guide you through the acquisition procedure. There are four basic steps to scanning an image using the GS-700: 1. Select the application. 2. Select the scan area. 3. Select the resolution of the scan. 4. Acquire the image. 5.2 Step I. Select Application To set the parameters for your particular scan, you can: 1. Select from a list of possible applications, or 2. Choose your own filter and light source settings.
GS-700 Imaging Densitometer The applications are listed in a tree that expands from left to right. First you select the category of your application, then you select your particular application. To exit the tree without selecting, press the ESC key.
Quantity One User Guide Next to Filter, click on either the Red, Green, Blue, or White checkbox, or a combination of two of the first three (Red-Green, Green-Blue, Red-Blue). Filter Color Wavelength Application Examples Red 595–750 nm Coomassie G-250, BCIP/NBT, Fast Green FCF, Methylene Blue Green 520–570 nm Coomassie R-250, Basic Fuchsin Blue 400–530 nm Crocein Scarlet Gray Scale 400–750 nm Silver Stains, Copper Stains, Film Fig. 5-6. Examples of filter colors and applications.
GS-700 Imaging Densitometer Selecting an Area Using the preview scan as a guide, select your scan area by dragging your mouse within the scanning window. The border of the scan area you are selecting will be marked by a frame. Note: The scan area you select must be at least 1 cm wide. When you release the mouse button, the border changes to a dashed blue line, indicating a selected area. • To reposition the scanning area box you have created, position your cursor inside the box and drag.
Quantity One User Guide Fig. 5-7. Select Scan Resolution dialog box. Available resolutions are listed from highest to lowest in terms of the dimensions of the resulting pixels (in microns). Smaller pixels equal higher resolution. Each resolution is listed with its typical use. In general, the size of your pixels should be one-tenth the height of your smallest object. Some of the resolutions are asymmetrical, meaning that the resolution is higher in the vertical dimension (i.e.
GS-700 Imaging Densitometer Fig. 5-8. Entering a custom resolution (with Oversample selected). Image File Size Image File Size (under Select Resolution) shows the size of the scan file you are about to create. If you do not have enough computer memory for the specified file size, an error message will appear when you attempt to scan. If this happens, select a lower resolution or decrease the size of the area to be scanned. (Macintosh users can also increase the application memory partition.
Quantity One User Guide Save After Scan, the file will not be saved until you select Save or Save As from the File menu. 5.6 Calibration If you have installed a calibration overlay, you can automatically calibrate your transmissive and reflective scans. (Calibration overlays for the GS-700 can be ordered from Bio-Rad.) To set the automatic calibration settings, click on the More Options button in the GS-700 acquisition window. This will open the Densitometer Options dialog box. Fig. 5-9.
GS-700 Imaging Densitometer described below. You do not need to change the values in the reflective step tablet form; you can use the default values. With Calibration On selected, the other calibration settings become active. Calibration Strip Window When calibration is turned on, a calibration strip window will appear below the main scanning window and the length of the main scanning window will be reduced to 29 cm reflective and 23 cm transmissive.
Quantity One User Guide Fig. 5-10. Step Tablet dialog box. The dialog box will indicate whether the displayed form is for the transmissive or reflective step tablet, depending on whether you are in transmissive or reflective mode. Note: In transmissive scanning, the scanner is calibrated against a transmissive step tablet. In reflective scanning, the scanner is calibrated against a reflective step tablet, unless the reflective calibration strip is uncalibrated.
GS-700 Imaging Densitometer Finally, enter the values in the appropriate fields under the Diffuse column. After the step tablet is scanned, the software will associate each density value with its corresponding segment on the step tablet. The step tablet density values do not need to be entered each time you calibrate. Once they have been entered and saved, they will be automatically recalled when the calibration strip is scanned. You only need to enter new values if you use a new step tablet.
Quantity One User Guide 5.6.b Calibration Settings After you have entered the step tablet values, you can immediately calibrate by clicking on the Calibrate Now button (in the Densitometer Options dialog box). You can also specify how often you want the densitometer to automatically recalibrate. Either click on the Calibrate Before Every Scan checkbox or enter a Recalibration Interval (in minutes) in the appropriate field.
GS-700 Imaging Densitometer Auto Save After Scan To automatically save any scan you create, click on the Auto Save After Scan checkbox. With this checkbox selected, when you click on Acquire, a Save As dialog box will open asking you to specify a file name and location for the image you are about to create. The scan will begin when you click on the Save button. Make Backup Copy If you have checked Auto Save After Scan, you can also automatically create a backup copy of any scan you create.
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6. GS-710 Imaging Densitometer Fig. 6-1. GS-710 Imaging Densitometer. Before you can begin scanning images with the GS-710 Imaging Densitometer®, your instrument must be properly installed and connected with the host computer. See the hardware manual for installation, startup, and operating instructions. PC Only: A Note About SCSI Cards The GS-710 is connected to your computer by a Small Computer System Interface (SCSI) cable. To use the GS-710, you must have a SCSI card installed in your PC.
Quantity One User Guide appear active, but instead of capturing real images, the window will create “dummy” images of manufactured data. You do not need to be connected to an imaging device to open a simulated acquisition window. This is useful for demonstration purposes or practice scans. To enter simulation mode, hold down the CTRL key and select the name of the device from the File menu. The title of the acquisition window will indicate that it is simulated.
GS-710 Imaging Densitometer Fig. 6-2. GS-710 acquisition window The scanning window is marked by grid lines that divide the area into square centimeters. These are numbered 1–29 top to bottom and 1–21 left to right if the light source is reflective, and 1–23 top to bottom and 1–20 left to right if the light source is transmissive (see below). To hide the gridlines, click on the Hide Grid checkbox under Options. Below the main scanning window is the calibration strip window.
Quantity One User Guide The control panel has been arranged from top to bottom to guide you through the acquisition procedure. There are five basic steps to scanning an image using the GS-710: 1. Select the application. 2. Select the scan area. 3. Select the resolution of the scan. 4. Calibrate the instrument. (This is automatic, after you enter the step tablet values before you first scan after installation.) 5. Acquire the image. 6.2 Step I.
GS-710 Imaging Densitometer Fig. 6-3. Example of the application tree in the GS-710 dialog box. The applications are listed in a tree that expands from left to right. First you select the category of your application, then you select your particular application. To exit the tree without selecting, press the ESC key.
Quantity One User Guide Choosing Your Own Settings If you know the filter and light source settings you want, or want to experiment with different settings, you can choose them yourself. Fig. 6-5. GS-710 Custom Application controls. Next to Filter, click on either the Red, Green, Blue, or White checkbox, or a combination of two of the first three (Red-Green, Green-Blue, Red-Blue).
GS-710 Imaging Densitometer 6.3 Step II. Select Scan Area Preview Scan Before selecting the particular area to scan, you can preview the entire scanning area to determine the exact position of your sample. Click on Preview Scan. A quick, low-resolution scan of the entire scanning area will appear in the scanning window. Your sample should be visible within a portion of this scan. Selecting an Area Using the preview scan as a guide, select your scan area by dragging your mouse within the scanning window.
Quantity One User Guide 6.4 Step III. Select Resolution To select from a list of possible scanner resolutions, click on the Select button under Select Resolution. This will open the Select Scan Resolution dialog box. Fig. 6-7. Select Scan Resolution dialog box. Available resolutions are listed from highest to lowest in terms of the dimensions of the resulting pixels (in microns). Smaller pixels equal higher resolution. Each resolution is listed with its typical use.
GS-710 Imaging Densitometer Specifying Your Own Resolution If you select Oversample under More Options (see section 6.7, Other Options, for details), you can specify your own resolution within the range of 43–169 microns (micrometers). With Oversample selected, enter values directly in the fields next to X resolution and Y resolution in the main acquisition window. Fig. 6-8. Entering a custom resolution (with Oversample selected).
Quantity One User Guide The first time you use the GS-710, you must select some calibration settings to ensure that your calibration is accurate. To set the automatic calibration settings, click on the More Options button in the GS-710 acquisition window. This will open the Densitometer Options dialog box. Fig. 6-9. Densitometer Options dialog box. 6.5.
GS-710 Imaging Densitometer step tablet to which those density values belong. Those exact density values must be entered into the computer for the software to associate a correct density value with each step on the step tablet. Note: Scanning in transmissive mode with incorrect step tablet values entered into the computer can cause significant errors in the reported densities of your scans.
Quantity One User Guide If you are in transmissive mode, you do need to enter the correct step tablet values that were shipped with your GS-710. You can type the serial number for these values into the Tablet Serial Number field. The Quantity Offset field does not apply in the GS-710. This value should be set to zero. Next, enter the values for the transmissive step tablet in the appropriate fields under the Diffuse column.
GS-710 Imaging Densitometer Diffuse density values are converted to specular optical density units according to the following formula: Specular OD = 1.4 ⋅ Diffuse OD 6.5.b Calibration Settings After you have entered the step tablet values, you can immediately calibrate by clicking on the Calibrate Now button (in the Densitometer Options dialog box). You can also specify how often you want the GS-710 to automatically recalibrate.
Quantity One User Guide After the scan is complete, a window will open displaying the scan image, at which point you can analyze and save it. Note: The image will open with a default file name that includes the date, time, and (if applicable) user name. However, unless you have selected Auto Save After Scan, the file will not be saved until you select Save or Save As from the File menu. 6.7 Other Options Oversample This feature allows you to scan at the maximum resolution of the GS-710 (42.3 x 42.
GS-710 Imaging Densitometer Click on the Make Backup Copy checkbox. With this checkbox selected, when a scan is created and saved, a backup copy will be placed in the same directory as the scanned image. Windows backup files will have an “.sbk” extension. Macintosh backup files will have the word “backup” after the file name. This backup copy will be read-only, which means that you cannot make changes to it.
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7. GS-800 Imaging Densitometer Before you can begin scanning images with the GS-800 Imaging Densitometer®, your instrument must be properly installed and connected with the host computer. See the hardware manual for installation, startup, and operating instructions. PC Only: A Note About SCSI Cards The GS-800 is connected to your computer by a Small Computer System Interface (SCSI) cable. To use the GS-800, you must have a SCSI card installed in your PC.
Quantity One User Guide 7.1 GS-800 Acquisition Window To begin acquiring images, go to the File menu and select GS-800.... The acquisition window for the densitometer will open, displaying a control panel and a scanning window. Fig. 7-1. GS-800 acquisition window The scanning window is marked by grid lines that divide the area into square centimeters.
GS-800 Imaging Densitometer To hide the gridlines, click on the Hide Grid checkbox under Options. Below the main scanning window is the calibration strip window. Every time the densitometer calibrates, an image of the calibration strip will appear in this window. The control panel has been arranged from top to bottom to guide you through the acquisition procedure. There are five basic steps to scanning an image using the GS-800: 1. Select the application. 2. Select the scan area. 3.
Quantity One User Guide Fig. 7-2. Example of the application tree in the GS-800 dialog box. The applications are listed in a tree that expands from left to right. First you select the category of your application, then you select your particular application. To exit the tree without selecting, press the ESC key.
GS-800 Imaging Densitometer Choosing Your Own Settings If you know the filter and light source settings you want, or want to experiment with different settings, you can choose them yourself. Fig. 7-4. GS-800 Custom Application controls. Next to Filter, click on either the Red, Green, Blue, or White checkbox, or a combination of two of the first three (Red-Green, Green-Blue, Red-Blue).
Quantity One User Guide 7.3 Step II. Select Scan Area Preview Scan Before selecting the particular area to scan, you can preview the entire scanning area to determine the exact position of your sample. Click on Preview Scan. A quick, low-resolution scan of the entire scanning area will appear in the scanning window. Your sample should be visible within a portion of this scan. Selecting an Area Using the preview scan as a guide, select your scan area by dragging your mouse within the scanning window.
GS-800 Imaging Densitometer 7.4 Step III. Select Resolution To select from a list of possible scanner resolutions, click on the Select button under Select Resolution. This will open the Select Scan Resolution dialog box. Fig. 7-6. Select Scan Resolution dialog box. Available resolutions are listed from highest to lowest in terms of the dimensions of the resulting pixels (in microns). Smaller pixels equal higher resolution. Each resolution is listed with its typical use.
Quantity One User Guide Fig. 7-7. Entering a custom resolution (with Oversample selected). Image File Size Image File Size (under Select Resolution) shows the size of the scan file you are about to create. If you do not have enough computer memory for the specified file size, an error message will appear when you attempt to scan. If this happens, select a lower resolution or decrease the size of the area to be scanned. (Macintosh users can also increase the application memory partition.
GS-800 Imaging Densitometer Fig. 7-8. Densitometer Options dialog box. 7.5.a Editing the Step Tablet Before you scan for the first time, you must enter the step tablet values for your transmissive calibration strip into the Step Tablet Values form.
Quantity One User Guide Fig. 7-9. Step Tablet dialog box. In the step tablet dialog box, type the serial number for the printout into the Tablet Serial Number field. The Quantity Offset field does not apply in the GS-800. This value should be set to zero. Next, enter the values for the transmissive step tablet in the appropriate fields under the Diffuse column. After the step tablet is scanned, the software will associate each density value with its corresponding segment on the step tablet.
GS-800 Imaging Densitometer Diffuse Versus Specular O.D. In the step tablet form, you enter O.D. as diffuse density, and then the software automatically calculates the specular density. Specular density is a measure of the light that passes directly through a medium. Diffuse density includes light that is scattered as it passes through the medium.
Quantity One User Guide application with the same filter and light settings, it will not auto recalibrate.) Calibration Report To print out a calibration report each time the densitometer calibrates, click on the Calibration Report checkbox. 7.6 Acquire the Image Note: Before scanning in transmissive mode, make sure the white balance region of the scanning area is not covered or obstructed in any way. To begin to scan, click on the Acquire button.
GS-800 Imaging Densitometer To turn on oversampling, click on the More Options button in the acquisition window and click on the Oversample checkbox. With oversampling on, you can specify your own resolution within the range of the densitometer by entering values directly in the fields next to X resolution and Y resolution in the main acquisition window. Auto Save After Scan To automatically save any scan you create, click on the Auto Save After Scan checkbox.
Quantity One User Guide Hide Grid To hide the gridlines in the scanning area window, click on the Hide Grid checkbox.
8. Fluor-S MultiImager Fig. 8-1. Fluor-S MultiImager. Before you can begin acquiring images using the Fluor-S® MultiImager, the imaging system must be properly installed and connected with the host computer. See the Fluor-S hardware manual for installation, startup, and operating instructions. Note: Make sure that the temperature light on the Fluor-S is green before attempting to capture an image.
Quantity One User Guide PC Only: A Note About SCSI Cards The Fluor-S is connected to your computer by a Small Computer System Interface (SCSI) cable. To use the Fluor-S, you must have a SCSI card installed in your PC. If you have an older PC, you may also need to load the SCSI and WinASPI drivers that came with your card. Simulation Mode Any of the imaging device acquisition windows can be opened in “simulation mode.
Fluor-S MultiImager Fig. 8-2. Fluor-S acquisition window When the Fluor-S window first opens, no image will be displayed. The control panel has been arranged from top to bottom to guide you through the acquisition procedure. There are four basic steps to acquiring an image using the Fluor-S: 1. Select the application. 2. Position and focus the gel or other object to be imaged. 3. Set the exposure time. 4. Acquire the image.
Quantity One User Guide 8.2 Step I. Select Application To set the appropriate filter and other parameters for the type of object you are imaging, click on the Select button under Select Application. The available applications and their associated settings are listed in a tree that expands from left to right. Fig. 8-3. The application tree in the Fluor-S acquisition window. First select your general application, then select the particular stain or medium you are using.
Fluor-S MultiImager Custom Applications If your application is not listed, if you want to use a user-installed filter, or if you want to access High Sensitivity mode (see below), you can create and save your own custom application. From the application tree, select Custom, then Create. This will open a dialog box in which you can name your application and select your settings. Fig. 8-4. Creating a new custom application.
Quantity One User Guide Scan Dimension If an application uses trans illumination, the Scan Dimension buttons become active. The scan dimension is the distance traveled by the transilluminating light source as it scans horizontally across the platen. Fig. 8-5. Scan dimension settings for trans illumination. The full scanning range is 300 mm. Select a smaller range if your sample is small and you do not want to wait while the light source travels over the maximum scan width.
Fluor-S MultiImager Position After you have selected your application, you are ready to center your gel or other object within the camera frame. To do so, click on the Position button. The Fluor-S will begin capturing a “live” image and updating it every second. With the Position button selected, look at the image in the acquisition window while you position your object in the center of the platen. If you have a zoom lens on the camera, you can adjust the magnification while you position.
Quantity One User Guide portion of the sample (this will not affect any zoom lens adjustments you may have made.) When you are finished focusing, click on the Stop button. 8.4 Step III. Set Exposure Time When you are ready to capture an image, you will need to select an exposure time. “Exposure” refers to the integration of image captures on the CCD over a set period of time. The effect is analogous to exposing photographic film to light.
Fluor-S MultiImager Recommended Exposure Times and Lenses Recommended Sample Exposure1 Lens & Filter2 Accessories Used Fluorescent Stain Gel 3–40 sec. Zoom/IR None Fluorescence End-Label Gel 30 sec.–3 min. Zoom/IR None Fluorescent Blot 0.5–5 sec. Zoom/IR None Chemifluorescent Blot 0.5–5 sec. Zoom/IR None Colorimetric Gel 1–10 sec. Zoom/IR White Diffusion Plate Colorimetric Blot 0.5–20 sec. Zoom/IR None X-ray film 1–10 sec.
Quantity One User Guide A preview scan takes only half as long to create as a real scan, because the preview scan does not capture a “dark” image (see the following section on Options). The progress of the exposure will be displayed in the Exposure Status bar at the bottom of the dialog box. You cannot save preview scans. If you want to stop a preview scan that is in progress, click on the Stop button. 8.
Fluor-S MultiImager After an image has been acquired, a separate window will pop up containing the new image. The window will have a default file name that includes the date, time, and user (if known). To save the image, select Save or Save As from the File menu. You can then analyze the image using the analysis functions. Live Acquire Live Acquire allows you to view and preserve intermediate exposures leading up to a full exposure.
Quantity One User Guide Fig. 8-9. Options dialog box. 8.6.a Dark Subtraction Type All CCD cameras accumulate electrons that produce a signal that is indistinguishable from light. This “dark count” adds to the noise in your images. In most cases, you will want to subtract this dark count from your images. Normal The Normal option button selects the default dark subtraction type.
Fluor-S MultiImager Referenced If you do not want to perform a dark exposure with each acquisition, you can take a “reference” dark exposure that will be saved and subtracted from all subsequent acquisitions. Click on the Referenced button to activate this feature. The first time you acquire an image after selecting this option, the Fluor-S will take a 60-second dark exposure that will be saved and used to subtract the dark count from all subsequent acquisitions.
Quantity One User Guide A pop-up box will prompt you to enter a new reference dark exposure time in seconds. Click on OK to implement your change. The new reference dark will be created when you acquire your next image. Note: Because of the high sensitivity of the CCD, fluctuations in background radiation and/or temperature in the room can affect the level of dark count.
Fluor-S MultiImager all your intermediate exposures will be saved as separate files. These files will have the same root name appended by a number indicating the exposure sequence. The final, full exposure will have the root name only, with no exposure number. 8.6.c Save Auto Save After Scan To automatically save any image you create, click on the Auto Save After Scan checkbox.
Quantity One User Guide 8.6.d Imaging Area Size The imaging area is the area of the sample that is captured by the camera and displayed in the scan window. To specify the size of this area, enter a dimension in the appropriate field under Imaging Area. When you change one imaging area dimension, the other will change to maintain the aspect ratio of the camera lens. The imaging area will change depending on your zoom factor. For example, if you have zoomed in on a area that is 4.5 x 3.
Fluor-S MultiImager File Size of Images Image File Size shows the size of the image file you are about to create. This size is determined by whether the image was created in High Resolution or High Sensitivity mode. If you do not have enough computer memory for the specified file size, an error message will appear when you attempt to acquire an image. (Macintosh users can increase the application memory partition. See your Macintosh computer documentation for guidance.
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9. Fluor-S MAX MultiImager Fig. 9-1. Fluor-S MAX MultiImager. Before you can begin acquiring images using the Fluor-S® MAX MultiImager, the imaging system must be properly installed and connected with the host computer. See the Fluor-S MAX hardware manual for installation, startup, and operating instructions. Note: Make sure that the temperature light on the Fluor-S MAX is green before attempting to capture an image.
Quantity One User Guide PC Only: A Note About SCSI Cards The Fluor-S MAX is connected to your computer by a Small Computer System Interface (SCSI) cable. To use the Fluor-S MAX, you must have a SCSI card installed in your PC. If you have an older PC, you may also need to load the SCSI and WinASPI drivers that came with your card. Simulation Mode Any of the imaging device acquisition windows can be opened in “simulation mode.
Fluor-S MAX MultiImager Fig. 9-2. Fluor-S MAX acquisition window. When the Fluor-S MAX window first opens, no image will be displayed. The control panel has been arranged from top to bottom to guide you through the acquisition procedure. There are four basic steps to acquiring an image using the Fluor-S MAX: 1. Select the application. 2. Position and focus the gel or other object to be imaged. 3. Set the exposure time. 4. Acquire the image.
Quantity One User Guide 9.2 Step I. Select Application To set the appropriate filter and other parameters for the type of object you are imaging, click on the Select button under Select Application. The available applications and their associated settings are listed in a tree that expands from left to right. Fig. 9-3. The application tree in the Fluor-S MAX acquisition window. First select your general application, then select the particular stain or medium you are using.
Fluor-S MAX MultiImager Custom Applications If your application is not listed, if you want to use a user-installed filter, or if you want to access Ultra Sensitivity mode (see below), you can create and save your own custom application. From the application tree, select Custom, then Create. This will open a dialog box in which you can name your application and select your settings. Fig. 9-4. Creating a new custom application.
Quantity One User Guide Scan Dimension If an application uses trans illumination, the Scan Dimension buttons become active. The scan dimension is the distance traveled by the transilluminating light source as it scans horizontally across the platen. Fig. 9-5. Scan dimension settings for trans illumination. The full scanning range is 300 mm. Select a smaller range if your sample is small and you do not want to wait while the light source travels over the maximum scan width.
Fluor-S MAX MultiImager 9.3 Step II. Position/Focus Note: When you click on the Position or Focus button, the light inside the Fluor-S MAX box automatically turns off. This is because if you focus with the camera at maximum aperture (see note below), leaving the light on would make it difficult to view the image. To turn the light on while positioning or focusing, hold down the SHIFT key when clicking on the button.
Quantity One User Guide Focus Note: Before focusing, you should adjust the f-stop on the camera to the lowest setting (i.e., the maximum aperture). This reduces the depth of field, allowing you to more accurately focus the camera. Then, after focusing, increase the f-stop to the desired setting. After you have positioned your sample, click on the Focus button and look at the image in the acquisition window while aligning the two focusing arrows on the camera lens.
Fluor-S MAX MultiImager The following table provides recommended exposure times for various applications. Recommended Exposure Times and Lenses Sample Recommended Exposure Lens & Filter1 Accessories Used Fluorescent Stain Gel 1–20 sec. Zoom/IR None Fluorescence End-Label Gel 10 sec.–2 min. Zoom/IR None Fluorescent Blot 0.1–3 sec. Zoom/IR None Chemifluorescent Blot 0.1–3 sec. Zoom/IR None Colorimetric Gel 1–5 sec. Zoom/IR White Diffusion Plate Colorimetric Blot 0.2–10 sec.
Quantity One User Guide Preview For shorter exposures, you can use Preview to test different exposure times. Click on the Preview button create a preview exposure and display it in the acquisition window. A preview scan takes only half as long to create as a real scan, because the preview scan does not capture a “dark” image (see below). The progress of the exposure will be displayed in the Exposure Status bar at the bottom of the dialog box. You cannot save preview scans.
Fluor-S MAX MultiImager Depending on which dark subtraction type you have selected, a dark count may be acquired immediately following image acquisition. See Dark Subtraction Type under Options, below. If you want to stop a scan that is in progress, click on the Stop button. The acquisition will be terminated. After an image has been acquired, a separate window will pop up containing the new image. The window will have a default file name that includes the date, time, and user (if known).
Quantity One User Guide 9.6 Options Click on the Options button to open the Options dialog box. Fig. 9-10. Options dialog box. 9.6.a Dark Subtraction Type All CCD cameras accumulate electrons that produce a “signal” that is indistinguishable from light. This “dark count” adds to the noise in your images. In most cases, you will want to subtract this dark count from your images. Normal The Normal option button selects the default dark subtraction type.
Fluor-S MAX MultiImager The progress of the dark exposure will be displayed in the Exposure Status bar following the regular image exposure. Note: In Normal mode, a dark image is only acquired the first time you perform a scan with particular application and exposure settings. If you perform subsequent scans with the same settings, no dark exposure will be taken.
Quantity One User Guide Fig. 9-11. Reset Reference Dark pop-up box. A pop-up box will prompt you to enter a new reference dark exposure time in seconds. Click on OK to implement your change. The new reference dark will be created when you acquire your next image. Note: Because of the high sensitivity of the CCD, fluctuations in background radiation and/or temperature in the room can affect the level of dark count.
Fluor-S MAX MultiImager The total exposure time will be divided by the number you enter in the Exposure Count field. If you enter an exposure time of 10 minutes and a count of 10, you will create 10 intermediate exposures at 1 minute intervals. Note: Do not enter a count that will result in an intermediate exposure time that is less than the minimum exposure time for the mode you are in.
Quantity One User Guide Make Backup Copy You can automatically create a backup copy of any scan you create. To do so, first select Auto Save After Scan (see above), then select the Make Backup Copy checkbox. With this checkbox selected, when you save a scan, a backup copy will be placed in the same directory as the scanned image. Windows backup files will have an “.sbk” extension. Macintosh backup files will have the word “backup” after the file name.
Fluor-S MAX MultiImager 9.7 Other Features Fig. 9-12. Other Fluor-S MAX acquisition window features. Highlight Saturated Pixels When this box is checked, any saturated pixels in the image will appear highlighted in red in the scan window and in the pop-up image window. To view/hide saturated pixels in the pop-up image window, use the Image > Transform command. File Size of Images Image File Size (below Options) shows the size of the image file you are about to create.
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10.Personal Molecular Imager FX Fig. 10-1. Personal Molecular Imager FX Before you can begin acquiring images using the Personal Molecular Imager® FX, the instrument must be properly installed and connected with the host computer. See the Personal FX hardware manual for installation, startup, and operating instructions. Note: The Personal FX should be turned on and the initialization sequence completed before the host computer is turned on (except in the case of certain Power Macintosh configurations).
Quantity One User Guide PC Only: A Note About SCSI Cards The Personal FX is connected to your computer by a Small Computer System Interface (SCSI) cable. To use the Personal FX, you must have a SCSI card installed in your PC. If you have an older PC, you may also need to load the SCSI and WinASPI drivers that came with your card. Simulation Mode Any of the imaging device acquisition windows can be opened in “simulation mode.
Personal Molecular Imager FX Fig. 10-2. Personal FX acquisition window The default scanning window is marked by grid lines that divide the area into quadrants. There is also an outer box and inner box marked by thicker lines. This conforms to the sample pad for the standard Bio-Rad Exposure Cassette that is supplied with the Personal FX. The quadrants are numbered 1–16 left to right and lettered A–U top to bottom.
Quantity One User Guide 1. Select the scan area 2. Select the resolution 3. Acquire the image 10.2 Step I. Select Scan Area To select a scan area, drag your mouse within the scanning window. (In the scanning window, your cursor appearance will change to a cross.) The border of the scan area you are selecting is marked by a frame. Drag cursor to define scan area Fig. 10-3. Selecting a scan area. If you are in quadrant mode, note that the frame “locks” onto the next quadrant as you drag.
Personal Molecular Imager FX You can also select the scanning area by entering coordinates in the appropriate fields (Top, Bottom, Left, Right). After you enter a coordinate, the position of the scanning area box will change accordingly. When selecting, be sure to include the entire area of interest, and be generous with borders. You can always crop the image later. 10.3 Step II. Select Resolution The Personal FX acquisition window allows you to scan at 50, 100, 200, or 800 micrometers.
Quantity One User Guide File Size of Images Image File Size (below Select Resolution) shows the size of the scan file you are about to create. If you do not have enough computer memory for the specified file size, an error message will appear when you attempt to scan. If this happens, select a lower resolution or decrease the size of the area to be scanned. (Macintosh users can also increase the application memory partition. See your Macintosh computer documentation for guidance.) 10.
Personal Molecular Imager FX 10.5 Options Auto Save After Scan To automatically save any scan you create, click on the Auto Save After Scan checkbox. With this checkbox selected, when you click on Acquire, a Save As dialog box will open asking you to specify a file name and location for the image you are about to create. The scan will begin when you click on the Save button. Make Backup Copy You can automatically create a backup copy of any scan you create.
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11. Molecular Imager FX Fig. 11-1. Molecular Imager FX Before you can begin acquiring images using the Molecular Imager® FX, the instrument must be properly installed and connected with the host computer. See the FX hardware manual for installation, startup, and operating instructions. Note: The FX should be turned on and the initialization sequence completed before the host computer is turned on (except in the case of certain Power Macintosh configurations). See the hardware manual for more details.
Quantity One User Guide you have an older PC, you may also need to load the SCSI and WinASPI drivers that came with your card. Simulation Mode Any of the imaging device acquisition windows can be opened in “simulation mode.” In this mode, an acquisition window will open and the controls will appear active, but instead of capturing real images, the window will create “dummy” images of manufactured data. You do not need to be connected to an imaging device to open a simulated acquisition window.
Molecular Imager FX Fig. 11-2. FX acquisition window The default scanning window is marked by grid lines that divide the area into quadrants. There is also an outer box and inner box marked by thicker lines. This conforms to the sample pad for the standard Bio-Rad Exposure Cassette that is supplied with the FX. The quadrants are numbered 1–16 left to right and lettered A–U top to bottom.
Quantity One User Guide 2. Select the scan area. 3. Select the resolution. 4. Acquire the image. 11.2 Step I. Select Application To select the appropriate filter and other parameters for the type of object you are imaging, click on the Select button under Select Application. Fig. 11-3. Example of an application tree: Ethidium Bromide gel. Standard Applications The standard applications and associated settings are listed in a tree that expands from left to right.
Molecular Imager FX Standard FX Applications Radioisotopes CS- or BI-Screen (Bio-Rad) K-Screen (Kodak) Fuji-Screen Chemiluminescence Chemi-Screen (Bio-Rad) Chemifluorescence ECL-Plus Attophos DNA Stain Gel Ethidium Bromide Sybr Green I & II Sybr Gold Protein Stain Gel Sypro Orange Sypro Red Nile Red Sypro Ruby Fluorophores Alexa 488 Alexa 532 Alexa 546 FITC FAM CY3 HEX R6G Texas Red Microtiter Plate DNA (Sybr Green I) Protein (Nano Orange) ssDNA (Oligreen) DNA (Picogreen) B-Gal (FDG) GUS (FDG)
Quantity One User Guide Note: Some applications require an external laser. If you choose one of these without having an external laser attached, you will receive a warning. To exit the tree without selecting, press the ESC key. Your selection will be displayed below the Select button. Sample Intensity Many FX applications require that you select a sample intensity (High, Medium, or Low) from the application tree. This is simply a rough estimate of how much sample is visible in your gel or other object.
Molecular Imager FX Fig. 11-4. Creating a custom application. To select a filter (including user-defined) or filter combination, click on the buttons for Filters A, B, and C, and make your choice from each pop-up list. Note: The user-defined filters (User1, User2, etc.) cannot be renamed in the popup list, so be sure to remember which filter you insert into each position in the FX. To use an external laser, click on the Laser button and select it from the popup list.
Quantity One User Guide Fig. 11-5. Selecting a custom PMT voltage. Note: For voltages above 80% of maximum, you will receive a warning message that the high voltage could damage the PMT. If you select Choose Later from the list of PMT voltages, the choices of sample intensity will be displayed when you select your custom application. Finally, enter a name for your application in the Name field and click on OK to implement your changes.
Molecular Imager FX Fig. 11-6. Selecting a custom application. 11.3 Step II. Select Scan Area To select a scan area, drag your mouse within the scanning window. (In the scanning window, your cursor appearance will change to a cross.) The border of the scan area you are selecting is marked by a frame. Drag cursor to define scan area Fig. 11-7. Selecting a scan area.
Quantity One User Guide If you are in quadrant mode, note that the frame “locks” onto the next quadrant as you drag. When you release the mouse button, the border changes to a dashed blue line, indicating a selected area. • To reposition the scanning box you have selected, position your cursor inside the box and drag. The entire box will move. • To resize the box, position your cursor on a box side and drag. The side you have selected will move.
Molecular Imager FX • 50 micrometer resolution should be reserved for images requiring the highest level of detail, e.g., high density in situ samples, 1,536-well microplates, high density arrays, samples with very closely spaced bands. Files scanned at 50 micrometers can be very large. • 100 micrometer resolution is useful for typical gels and arrays. • 200 micrometer resolution is useful for gels with large bands and dot blots.
Quantity One User Guide Saving the Image After the scan is complete, a message will appear asking you if you want to keep the scan. If you select Yes, a separate window will pop up containing the new image. You can then save and analyze the image using the standard menu and toolbar functions. 11.6 Options Auto Save After Scan To automatically save any scan you create, click on the Auto Save After Scan checkbox.
Molecular Imager FX Highlight Saturated Pixels When this box is checked, any saturated pixels in the image will appear highlighted in red in the scan window and in the pop-up image window. To view/hide saturated pixels in the pop-up image window, use the Image > Transform command. Hide Grid To hide the gridlines in the scanning area window, click on the Hide Grid checkbox.
Quantity One User Guide 11-14
12. Viewing and Editing Images This chapter describes the viewing tools for magnifying and optimizing your images. This chapter also describes the tools for cropping, flipping, and rotating images, reducing background intensity and filtering noise, and adding text overlays to images. These tools are located on the View, Image, and Edit menus. Note: 12.1 The following chapters contain instructions for analyzing X-ray films, wet and dry gels, blots, and photographs.
Quantity One User Guide Fig. 12-1. Viewing functions on View menu and main toolbar. Zoom Box Zoom Box allows you to select a small area of the image to magnify so that it fills the entire image window. First, click on the Zoom Box button on your Main toolbar or select View > Zoom Box. Your cursor arrow will change to a cross. Then drag the cursor on the image to enclose the area you want to magnify, and release the mouse button.
Viewing and Editing Images 1. Click on Zoom Box button. 2. Drag box on image. 3. Boxed region is magnified to fill window. Fig. 12-2. Zoom Box tool. Zoom In/Zoom Out These tools work like standard magnifying tools in other applications. Click on the Zoom In or Zoom Out button on your main toolbar (or select from the View menu). Your cursor will change to a magnifying glass.
Quantity One User Guide incrementally within the window. The amount the image shifts is determined by the Pan % setting in the Preferences dialog box. View Entire Image If you have magnified part of an image or moved part of an image out of view, View Entire Image returns to the original, full view of the image. Centering an Image You can center the image window on any point in an image quickly and easily using the F3 key command.
Viewing and Editing Images Tile will resize your windows and arrange them on the screen left to right and top to bottom. Tile Vertical will resize your windows and arrange them side-by-side on the screen. Tile Horizontal will resize your windows and stack them top-to-bottom on the screen 12.2 Density Tools The Density Tools (on the View > Plot Density submenu and the Density Tools toolbar) are designed to provide a quick measure of the signal intensity of the data in your image. Fig. 12-3.
Quantity One User Guide Density in Box Density in Box displays the average and total intensity within a boxed region on the image. Select Density in Box from the Density toolbar or View menu, then define the region you want to measure by dragging your cursor across the image. Plot Density Distribution Plot Density Distribution displays a histogram of the signal intensity distribution for the part of the image displayed in the image window. The average intensity is marked in yellow on the histogram.
Viewing and Editing Images Click on button, then click or drag on the image. Fig. 12-4. Plot Cross-section tool. Plot Vertical Trace Plot Vertical Trace plots an intensity trace of a vertical cross-section of the image centered on the point where you click your mouse. Dragging the mouse with this function selected will continuously update the display. Note: 12.
Quantity One User Guide 12.4 Multi-Channel Viewer You can use the Multi-Channel Viewer to distinguish different types and levels of fluorescence in a gel that has been imaged at different wavelengths. The Multi-Channel Viewer can be used to the merge the information from up to three different images of the same gel. Note: The Multi-Channel Viewer requires that the images being compared are exactly the size. When capturing the images, you should be careful not to move the gel between exposures.
Viewing and Editing Images Note: Note that the color channel used to display an image in the viewer has no relation to the filter used when capturing the image. The red, green, and blue channels are simply designed to distinguish different images. The image name is displayed in the Red name field at the top of the viewer, and the Red channel checkbox will appear selected. To add another image, make sure the image is open and click on the pulldown button next to the Green or Blue name field.
Quantity One User Guide Note: If you deselect this checkbox, any images currently displayed will remain auto-scaled. Click on the Transform button in the viewer and click on the Reset button in the Transform window to undo auto-scaling. Buttons for various viewing tools are included in the Multi-Channel Viewer. Commands such as Zoom Box and Grab will change the display of all the images in the viewer at once.
Viewing and Editing Images Fig. 12-7. Image Stack Tool. In the Image Stack Tool window, all available gels will be listed in the field to the right of the display window. To select an image to display, click on a gel name. The name will appear highlighted with an arrow and the image will appear in the window. Click on another gel name to display that image. Using the controls below the list of names, you can “step” through the images in the stacker.
Quantity One User Guide Alternatively, click on the Auto checkbox next to the arrow buttons to begin automatically scrolling through the list. You can adjust the auto-scroll speed using the Slow-Fast slider. Buttons for various viewing tools are included in the Multi-Channel Viewer. These commands will change the display of all the images in the stacker at once (e.g., zooming in on one image will magnify the same relative area in all the images). 12.
Viewing and Editing Images Fig. 12-9. List of Color Groups. Click on a color group in the list to select it. Changing a Color After you have selected the color group to change, click on the specific color button to change. The Color Edit dialog box will open, allowing you to adjust the red-green-blue (RGB) values of the color you selected. Fig. 12-10. Color Edit dialog box. Drag the sliders or enter a value in the fields. The color of the button will change with your adjustments.
Quantity One User Guide Saving/Selecting a Defined Set of Colors After you have changed the colors within color groups, you can save these settings for future use on other images. The Colormap Name field displays the name of a defined set of colors and color groups. There are several predefined colormaps, or you can create your own. To select a predefined colormap, click on the Load button. A user-defined set of colors Fig. 12-11. Selecting a Colormap.
Viewing and Editing Images 12.7 Transform If features in your image are indistinct or fine details appear to be lost in background noise, you can use the Transform functions to optimize your displayed image. To open the Transform dialog box, select Image > Transform, or click on the Transform button on the main toolbar. Fig. 12-12. Transform command.
Quantity One User Guide Preview Window Transform Plot Frequency Distribution histogram Auto-scale Fig. 12-13. Transform dialog box. Note: Any changes made with the Transform controls will only affect how the image is displayed on your screen. They will not affect the underlying data. 12.7.a Transform Subwindows Preview Window The Preview Window in the Transform dialog shows a smaller view of the same image that is displayed in the main image window.
Viewing and Editing Images Transform controls are automatically reflected in the Preview Window. They are only applied to the main image when you click on OK. You can use view tools like Zoom Box and View Entire Image in the Preview Window just as you can in the main image window, to focus on particular regions of interest. You can also use Grab and the ARROW keys to move the image within the Preview Window.
Quantity One User Guide As you drag the sliders, the slider markers on the Frequency Distribution histogram will move. Everything to the left of the Low marker will be remapped to minimum intensity, while everything to the right of the High marker will be remapped to maximum intensity. Using the histogram, you can position the markers at either end of the data range in your image, and use the low slider to cut off the “spike” of background noise.
Viewing and Editing Images Range of actual data in the image is very limited Low slider remaps background noise to white Can better distinguish background noise from real data High slider remaps weaker signals to black Low-High magnifies area between sliders Log scaling enables you to better distinguish peaks Fig. 12-14. Two views of the Frequency Distribution histogram.
Quantity One User Guide 12.7.c Other Features Full Scale/Low-High The Full Scale/Low-High option buttons adjust how the range of data in the image is displayed in the Frequency Distribution histogram and Transform Plot. They do not change how the data is displayed in the image window. Selecting Full Scale adjusts the Frequency Distribution and Transform Plot displays so they show the full intensity range of the image. Selecting From Low to High magnifies the range between the Low and High sliders.
Viewing and Editing Images Highlight Saturated Pixels When the Highlight Saturated Pixels checkbox is selected, areas of the image with saturated signal intensity are highlighted in red. Reset If at any time you want to return to an unmodified view of the scan data, click on Reset. 12.8 Resizing and Reorienting Images Frequently, your images will require some editing prior to analysis. This section describes the features that allow you to change the size and orientation of your images.
Quantity One User Guide 12.8.a Cropping Images To eliminate unwanted parts of an image, you can use the Crop tool. This is also an easy way to reduce the file size of an image. To crop, click on the Crop button on the Image toolbar or select Image > Crop. Your cursor appearance will change to a Crop symbol. Define the region to be cropped by dragging the cursor across the image, creating a box. Everything outside the box will be deleted.
Viewing and Editing Images Fig. 12-16. Crop box and pop-up Crop dialog. If you select the Copy and Crop button, a dialog box will be displayed in which you can enter the name of the cropped image and its version number. Click on the OK button once you have finished. 12.8.b Flipping and Rotating Images If your image is not properly oriented, you can flip and/or rotate the image. Note: These actions will erase any overlays you have created or analysis you have performed on an image.
Quantity One User Guide 90° Rotations Select Rotate 90 Left, Rotate 90 Right, or Rotate 180 from the Image > Rotate menu or Image toolbar to perform the specified rotation. You will be asked to confirm your choice before the command is executed. Custom Rotation If you need to rotate your image in increments other than 90°, you can use the Custom Rotation command. Select Custom Rotation from the Image > Rotate menu or Image toolbar. A green “plus” sign will appear next to your cursor.
Viewing and Editing Images Fig. 12-17. Custom rotation; the arrow points in the direction of the new top of the image. To perform the rotation, position your cursor on the arrowhead and drag. As you drag, the arrow will rotate and the angle in the dialog box will change. Position the arrow so that it points in the direction of the new top of the image. You can “fine-tune” your rotation as much as you like. Note: If you want to center your arrow on a particular point on the image (e.g.
Quantity One User Guide will then have the option of renaming your new image and changing the version number. If you are not satisfied with your rotated image, simply delete it and start over. Note: 12.9 Because an image is composed of square or rectangular pixels, Custom Rotation has to perform some minor smoothing on the image to turn it at a non-90o angle. In addition, any analysis performed on the image cannot be rotated and will be lost.
Viewing and Editing Images Fig. 12-18. Subtract Background dialog box. The Subtract Background dialog box has a Preview Window, which contains a smaller view of the same image that is displayed in the main image window. Changes in the subtract background controls are automatically reflected in the Preview Window. They are only applied to the main image when you click on OK.
Quantity One User Guide You can use view tools like Zoom Box and View Entire Image in the Preview Window just as you can in the main image window, to focus on particular regions of interest. You can also use the ARROW keys to move the image within the Preview Window. A description of each of the available features in the Subtract Background dialog is given below. Auto-scale Clicking on the Auto-scale button will automatically adjust your Dark Contrast and Background settings to optimal levels.
Viewing and Editing Images Example A: without adjustment Example B: with adjustment Fig. 12-19. Images without and with Dark Contrast adjustment. Note that in Example A, with no Dark Contrast adjustment (3.00 O.D.), the surrounding medium appears free of background and image artifacts, but in Example B, with adjustment (0.46 O.D.), artifacts in the image become immediately obvious, indicating that the subtraction levels were not sufficient to remove all background artifacts from the gel image.
Quantity One User Guide Click on the Background Box button, then drag on the image, defining an area that is representative of the background for your entire gel image. The average intensity of the pixels within the box will be used as the background level to be subtracted from your image. Background Stripe This function is useful when the image background is horizontally uniform but changes from the top to the bottom of the image, such as on a gradient gel.
Viewing and Editing Images If you choose to copy and subtract, you will be asked to enter a new name and/or version number for the new copy before the operation is performed. Once this information has been entered, the background subtraction will be performed. 12.10 Filtering Images Filtering is a process that removes small noise features on an image while leaving larger features (like data) relatively unaffected. A wide range of filters are available for removing different types of noise from images.
Quantity One User Guide 12.10.a Filter Wizard The Filter Wizard is designed to guide you through the filter selection process. First, you identify the type of noise in your image. Next, select the size of the filter to use on that noise. Finally, filter the image. To open the Wizard, select Image > Filter Wizard or click on the Filter Wizard button on the Image Tools toolbar. Fig. 12-21. Filter Wizard dialog box. The Wizard contains settings for identifying the different types of noise in the image.
Viewing and Editing Images Step I: Identify Noise Characteristics The first step in the Wizard is to identify the type of noise in your image. Examine both the image and the density distribution histogram, then select one, both, or neither of the following checkboxes: • Salt. This type of noise appears as specks that are lighter than the surrounding background. The density distribution histogram of this type of noise displays noise peaks at the high end of the range (right end of the plot).
Quantity One User Guide The available filter dimensions range from 3 x 3 pixels to 9 x 9 pixels. To select an appropriate size, magnify a background region of your image so that you can see the individual pixels. The filter size you select should be larger than the average noise feature but smaller than your data features. Note: A smaller filter will alter your image less than a larger filter. Large filters can result in better suppression of noise, but can also blur desirable features in the image.
Viewing and Editing Images • Median. Also useful for suppressing salt-and-pepper noise, this filter calculates the median value of the pixels within the filtering window and uses it to replace the value of the pixel being processed. The median filter produces very little blurring if a small-sized window is selected. • Maximum. This filter is useful for eliminating pepper noise in an image (it worsens the effect of salt noise).
Quantity One User Guide Fig. 12-22. Selecting a filter size. The available filter dimensions range from 3 x 3 pixels to 9 x 9 pixels. (See the previous section for guidance on selecting a size.) Because filtering is an irreversible process, a pop-up box will give you the option of filtering the original image, creating a copy of the image to filter, or cancelling out of this operation.
Viewing and Editing Images To invert your image data, select Image > Invert Data or select Invert Data from the Image Tools. You may need to use the Transform function to adjust the appearance of your inverted image. 12.12 Text Overlays If you want to create and display textual notes directly on your image, select Text Overlay Tools from the Edit menu or main toolbar. This will open the Text Overlay Tools toolbar. Select tool Text tool Line tool Copy Paste Alignment tools Fig. 12-23.
Quantity One User Guide To enter your text, simply begin typing in the main field. The buttons in the dialog allow you to select the properties of your text, including format, alignment, and justification. The pull-down boxes (from left to right) allow you to select the font style, font size, color of the text, and color of the background within the text box. Once you have typed your text, click on OK.
Viewing and Editing Images First, you must select the object(s). Click on the Select Tool button on the Text Overlay toolbar. To select a single overlay or line, click on it. To select multiple objects, either drag a box around them or hold down the SHIFT key while you click on them one at a time. When dragging to select a group of objects, make sure that you completely surround all the objects to be selected. Each selected overlay/line will have a green border.
Quantity One User Guide 12.13 Erasing All Analysis from an Image If you want to delete all analysis that has been performed on an image (including lanes, bands, volumes, text overlays, etc.), you can use the Clear Analysis command. Select Clear Analysis from the Edit menu. Since this process is irreversible, you will be prompted to confirm your selection. 12.
13. Lanes Before you can use many of the analysis functions, you must first define lanes and bands on your gel image. This chapter describes the tools for defining lanes. Note: 13.1 If you want to eventually compare bands across lanes (e.g., using standards or band matching), your lane lines should be approximately the same length, with their starting points aligned across the top of the image. This is important for calculating the relative mobility of the bands.
Quantity One User Guide 13.1.a Creating a Lane Frame The fastest way to define all of the lanes in your image is to create a lane frame using the Auto Frame Lanes command. If Auto Frame Lanes does not work well on your images, you can create and place a lane frame manually. Auto Frame Lanes Note: Auto Frame Lanes works best with images with large numbers of clearly defined lanes and bands. Also, the lanes should be reasonably vertical and contain approximately the same amounts of sample.
Lanes The lane frame contains individual lanes numbered sequentially from left to right. The border and anchor lines of the frame are marked with dashed white lines, the lanes are solid red lines, and each anchor point (interior and corner) is marked with a circle. The top and bottom of the frame are parallel with the top and bottom of the image.
Quantity One User Guide Corner anchor points Frame border Fig. 13-4. Lane frame created using the Frame Lanes command. The lane frame overlay contains individual lanes numbered sequentially from left to right. The borders of the frame are marked with dashed lines, the interior lanes are solid red lines, and each corner anchor point is marked with a circle. 13.1.
Lanes Fig. 13-5. Edit Frame tools. Adjusting the Entire Frame The following commands are located on the Lane > Edit Frame submenu: • To stretch the frame (e.g., if you want to include additional bands at the top or bottom of the image), select Stretch Frame from the submenu and drag an anchor point in or out. The opposite anchor point will remain fixed while the frame expands or contracts.
Quantity One User Guide Still using Add/Adjust Anchors, click anywhere on the dashed lines of the frame border. This creates additional anchor points, both where you clicked and on the other side of the frame. This also creates another dashed line across the frame You can then drag the new anchor points just as you did the corner anchor points, thereby “bending” the frame. Other internal anchor points can be placed along the new dashed line if further adjustments to the lane frame are required.
Lanes 13.1.c Defining a Single Lane You can define individual lanes using the single lane functions. These are located on the Single Lane submenu of the Lane menu or on the Lane Tools toolbar. Fig. 13-7. Single Lane tools. Note: You can use the single lane commands on lanes within a frame; however, the lane will be detached from the frame before the command is executed. To mark an individual lane, select Create Lane, then drag a line from the top to the bottom of the desired lane.
Quantity One User Guide 13.1.e Unadjusting Single Lanes You can undo any of your lane adjustments with the Unadjust Lane command. Select Unadjust Lane from the menu or toolbar, then click on an anchor point you created to remove it. If you remove the anchor points at either end of the lane line, you will delete the line from the image. 13.1.f Deleting Lanes You can delete both single lane lines and lanes from a lane frame.
Lanes When detecting bands, you should select a Lane Width that is slightly wider than the actual lanes in your gel. Note: You must eliminate background intensity from your lanes using the Lane > Lane Background command (section 13.2) before detecting bands. You can adjust the sampling width of all the lanes in your image from within the Detect Bands dialog box (see section 14.2.a). See section 14.1 for a full discussion of the effect of sampling width on band quantitation. 13.1.
Quantity One User Guide The profile is generated by calculating the average intensity of the pixels in every horizontal scan line along the defined lane. The profile shows the data objects in the image, represented by the intensity peaks in the profile, as well as the background noise in the image, represented by the region underneath the intensity peaks. The number of pixels used in this calculation depends on the lane width (see previous section).
Lanes Lanes to Model If you want to apply the background subtraction to all the lanes of your image, click on the All button. Otherwise, select One and enter the number of the lane you want to perform the operation on. Trace Display The profile trace of the lane can be displayed in one of two ways.
Quantity One User Guide If you select Rolling Disk, the dialog box will expand to reveal a field showing the size of the rolling disk. The Disk Size is the disk radius in pixels. You can type in a numerical value for the disk size or you can use the arrows to change the value in 10 percent increments. A disk radius that is too large will result in slow execution and poor removal of background. A disk radius that is too small may subtract nonbackground intensity. Typical values range from 50 to 150.
Lanes 13.3 Compare Lanes The Compare Lanes graph allows you to superimpose the intensity profiles of any number of lanes from any number of open images. Select Compare Lanes from either the Lane menu or toolbar, then click on the first lane you want to display. The Compare Lanes window will open. Note: Your image must have defined lanes for this command to work. Fig. 13-12. The Compare Lanes dialog box.
Quantity One User Guide Adding and Removing Lanes from the Graph To add a lane to the graph, click directly on the lane in the image. The plot of the lane will appear in the graph. Each lane you add will be displayed in one of eight colors, and will be identified by color in a legend underneath the graph. If you add more than eight lanes, the colors will repeat, but each lane will still be identified underneath the trace display. There is no limit to the number of lanes that can be displayed simultaneously.
Lanes Align Band Types The Align Band Types checkbox will become active if any of the lanes being profiled includes defined band types (see section 15.2). If this checkbox is selected, the profiles of all bands that have been identified as the same band type will be stretched and superimposed on one another, so their peaks align.
Quantity One User Guide Printing and Exporting Click on the Print button to print a copy of the Compare Lanes display. Click on the Export button to export the data points in your graph to a spreadsheet. This will open the Compare Lanes Export dialog box. Number of data points Fig. 13-14. Compare Lanes Export dialog box. This Export dialog box includes a field for the number of data points to be taken along the length of each lane.
Lanes 13.4 Lane-based Arrays The lane-based array functions allow you to create a lane frame for the cells in your array. You can then specify the cell dimensions and quantitate them using the Quantity Standards function described in section 15.3. Note: You can quantitate your arrays outside of lanes using volume arrays (see Chapter 16). Open the image of your array. The first step in defining an array is specifying the number of columns and rows in the array and creating an array frame.
Quantity One User Guide Fig. 13-17. Setting the number of array rows. The array matrix will appear on the image. Each column will be marked by a red line, and each cell will be marked by top and bottom brackets. Note: If the cells appear marked by lines instead of brackets, select Band > Band Attributes and select Brackets in the dialog box. When you first create the array matrix, it will probably not be centered on the columns and cells in the actual image.
Lanes Setting Array Cell Height and Width Now you should adjust the cell brackets so that they completely enclose the cells in the array. Select Array Cell Height from the Lane > Lane-based Arrays submenu and enter the height in millimeters of the array cells. Fig. 13-18. Setting the array cell height. When you click on OK, the cell brackets will adjust to the specified height. If you aren’t sure of the exact height, you can experiment with different values.
Quantity One User Guide Analyzing Array Data When the brackets fully enclose each cell in the array, you are ready to analyze the data. You can display various measures of cell quantity on the image using the Band > Band Attributes command. With the Band Attributes dialog open, select from Peak Density, Average Density, Trace Quantity, Relative Quantity, and other measures. You can also report these values by selecting Lane Reports from the Reports menu.
14. Bands Once you have defined the lanes on your gel image, you can automatically identify and quantitate the bands in those lanes using a set of adjustable parameters. Note: You can also quantitate bands outside of lanes using the Volume Tools. See Chapter 16 for details. The tools for band detection are located on the Band menu and on the Band Tools toolbar. Fig. 14-1. Band menu and Band Tools toolbar.
Quantity One User Guide 14.1 How Bands Are Identified and Quantified You can automatically identify all the bands in your image using the Detect Bands command, or you can mark them individually using the Create Band command. Each identified band is defined by brackets above and below the band. You can see these if you select the Brackets option button in the Band Attributes dialog box. (Select Band > Band Attributes.) These brackets mark the boundaries of the band.
Bands Lane Sampling Width Band Height Lane Trace Average pixel intensity across sample width Average intensity Peak Integrate under curve to baseline for band quantitation (intensity x mm). Band Profile Fig. 14-2. Illustration of bracket quantitation. 14.2 Automatically Identifying All Bands The Detect Bands function will find all of the bands in your defined lanes, based on parameters that you select.
Quantity One User Guide Select Band > Detect Bands or click on the button on the Band toolbar to open the Detect Bands dialog box. Band tools buttons are included in the dialog Click here to toggle format Fig. 14-3. Detect Bands dialog box, short and expanded formats, with default values. Note: If you have already manually identified bands (using Create Band, Adjust Band, etc.), the Detect Bands function will overwrite your manual detection.
Bands The Detect Bands dialog box has both a short format and an expanded format, which you can toggle between by clicking the toggle box in the lower right corner of the dialog. To change any of the parameter settings, you can either type in a new value or use the arrows to increase or decrease the setting by 10 percent. You should experiment with various settings to find those best suited to your images. 14.2.
Quantity One User Guide If you select the No button next to the Normalize prompt, the band detection parameters are taken as absolute values and are applied in the same way to every lane on the image. If you select Yes, the parameters are automatically adjusted according to the darkness of the lane. The darkness of the lane is determined by the darkest band in the lane. For example, suppose that all but one of the lanes on an image contain bands with an intensity of up to 50,000 counts.
Bands If the sensitivity is set too high, background noise will be erroneously detected as bands. If the setting is too low, real bands may be missed. The default sensitivity setting is 10.00. If your gel image has faint bands (e.g., O.D. < 0.05, counts < 2,000), you may want to increase this value to 20.00. Lane Width The Lane Width determines the width along the lane lines that will be sampled for band detection and quantitation.
Quantity One User Guide Minimum Density When Normalization is turned off, this is the lowest signal intensity value that will be counted as a band. Before entering a value for Min. Density, use the Lane > Plot Lane command to plot a trace of a lane that includes some faint bands. Then enter a value that is lower than the intensity of the peak of a faint band but is still above the background.
Bands Shoulder Sensitivity Normally, band detection tries to distinguish shoulders as separate bands. When looking at a lane trace, these bands appear as flat or gently sloping abutments to darker, better-defined bands (i.e., there is no dip on the trace between the two bands). Increasing the Shoulder Sensitivity value will result in more shoulders being detected as bands. Changing this setting to zero will result in no shoulders being recognized as separate bands.
Quantity One User Guide Fig. 14-5. Parameter set controls in the Detect Bands dialog box. If you have not already entered a name for your parameter set in the Parameter Set Name field, you will be prompted to do so. Loading Parameters To load a previously saved set of parameters, click on the Load button. Select the parameter set that you wish to load from the list. The values of that parameter set will be displayed in the Detect Bands form.
Bands Fig. 14-6. Create, Adjust, and Remove Bands buttons. Note: When editing individual bands, it is useful to display your bands as brackets using Band > Band Attributes (see above). When you edit bands in brackets mode, a pop-up lane trace will be displayed to help you determine band boundaries. 14.3.
Quantity One User Guide Fig. 14-7. Creating a band in brackets mode. When you release the mouse button, brackets will appear enclosing the region that you have called a band on the image. The corresponding section of the intensity trace will be highlighted. In Lines Mode With your bands displayed as lines, first select Create Band from the menu or toolbar, then click on the center of the band of interest. A line will appear at the center of the band at that point on the lane.
Bands Fig. 14-8. Creating a band in lines mode. In lines mode, no intensity trace will pop up when you click on the image. Note: After identifying several bands, be sure to renumber the bands in your image by selecting Sort and Recalculate from the Edit menu. 14.3.b Adjusting Bands After you identify the bands on your image—either automatically or manually—you may want to go back and change the placement of the band boundaries.
Quantity One User Guide 14.3.c Deleting Bands You can use Remove Band to delete bands from lanes. Select Remove Band from the menu or toolbar, point the cursor at the unwanted band, and click the mouse button. If your bands are displayed as brackets, a trace of the band will be displayed and a pop-up box will ask you to confirm removal of the band. Click on Yes to delete the band or No to cancel the procedure. If your bands are displayed as lines, the band will simply be deleted.
Bands Fig. 14-10. Bands in Lane command. 14.5 Band Attributes Once you have identified bands in your image, you can display information about them using the Band Attributes command. Select Band > Band Attributes to open the dialog box.
Quantity One User Guide Fig. 14-11. Band Attributes dialog box. Click on any of the buttons in the Band Labels group to display that particular attribute next to each defined band on your image. 14.5.a Summary of Band Attributes Each band attribute is summarized below. • None—No attribute is displayed. • Band number—The sequential number of a band in lane, as counted from the top of the lane.
Bands • Molecular weight/Isoelectric Point/Base Pairs/other units—This value is determined by the type of standards defined for the gel, the band’s position in the lane, and any modeling performed on the gel (via band matching or multiple lanes of standards) to compensate for gel distortion or smiling. • Peak density—The intensity value of a band’s peak. • Average density—The total intensity of the rows of pixels used to generate the profile of a band, divided by the number of rows.
Quantity One User Guide • Band model—Displays the modeling lines across the gel that are generated by band matching, standards, or both. These lines are used to compensate for gel distortion or smiling. • Tandem repeats—The number of repeated base-pair units in a band that has been analyzed using the VNTR Calculations function.
Bands Fig. 14-12. Band Information dialog box. The Lane and Band number of the band you clicked on are listed at the top of the dialog. Enter new numbers in these fields to display information and a trace overlay for a different band. If the band is part of a band set and is of a known band type, this will be noted in the form. The following information is listed for each band: • Relative front—The distance of a band from the top of a defined lane to the bottom, divided by the total length of the lane.
Quantity One User Guide Note: Note that Normalized Rf is derived from Relative Front; however, Normalized Rf is calculated only for bands that have been modeled using standards or band sets, and can change based on the modeling.
Bands curve you want to use. (See section 15.3 for information on calibration curves.) 14.7 Gauss-Modeling Bands If your bands are closely spaced or overlapping, Gaussian modeling can provide more accurate quantitation than regular band detection. Gaussian modeling will “fit” a Gaussian curve to each band profile, then calculate band quantity from the area under the curve.
Quantity One User Guide Fig.14-13. Profiles of two overlapping bands, without Gaussian modeling (top) and with Gaussian modeling (bottom). Note how modeling better resolves the individual quantities of the bands. To model your bands using Gaussian fitting, first detect your bands as you would normally, then select Gauss-model Bands from the Band menu. Note: Gaussian modeling will not create bands nor will it eliminate detected bands.
Bands A small dialog box will open prompting you to select all your lanes or one of your lanes to model. Fig. 14-14. Gauss-model Bands dialog box. If you select One, type the number of the lane you want to model into the field. Click on OK to begin modeling. A status box will display the progress of the modeling. Reviewing the Results of Gaussian Modeling Bands that have been Gauss-modeled will appear as normal bands in the image.
Quantity One User Guide Adjusting Bands in a Gauss-modeled Lane If you use any of the individual band commands—Create Band, Delete Band, Adjust Band—in a lane that has been Gauss-modeled, the modeling will be automatically removed from that lane. This is because the Gaussian models in a lane are interdependent: changing a single band will invalidate the modeling. After you have made your band changes, you can always remodel the lane you have changed.
Bands 14.8 Irregularly Shaped Bands in Lanes If the bands in your lanes are irregularly shaped, you can use the contour or drawing features to define them. These functions give you more control over defining your bands than either Detect Bands or Create Bands. Note: These tools are similar to the Volume Contour Tool and the Volume Freehand Tool on the Volume menu, except that they are lane-dependent. If you want to quantify objects without defining lanes first, see Chapter 16, Volume Tools.
Quantity One User Guide The functions needed to contour and draw bands are found on the Contour and Draw Band submenus of the Band menu, as well as on the Contour Tools toolbar. Before using any of these functions, we suggest that you magnify the image so that the individual pixels in the band are clearly visible. This allows you to position the cursor more accurately. 14.8.a Contouring Bands Fig. 14-16. Contour tools.
Bands Contour Information To display information about the contour, select Contour Information while the contour is highlighted. A pop-up box will display the area, total intensity, and average intensity of the contour. Converting a Contour into a Band When you are satisfied with the contour, you can redefine the contour as a band with the function Contour to Band. Note: Before you can convert a contour into a band, you must define at least one lane on your gel image.
Quantity One User Guide Density in Region Use Density in Region to get intensity and area information about any region on an image. Select Density in Region from the Band > Draw Band menu or Contour Tools toolbar. The cursor will change to an “information cursor.” Drag the cursor as if it were a pen to enclose a region of interest. Fig. 14-19. Density in Region tool. When you close the border, it will change color and a pop-up box with information about the enclosed area will be displayed.
Bands Drag the cursor around the perimeter of the region you want to define as a band. A line will appear, defining the boundary. Fig. 14-20. Draw Band Boundary tool. If you make a mistake and need to retrace part of the band boundary, simply backtrack with the cursor; the previous path will be erased and you can redraw it. Note: It is important to magnify the image before drawing a boundary.
Quantity One User Guide Editing Band Boundaries If your drawn band needs minor adjustments, you can fix it using Edit Band Boundary. Select Edit Band Boundary from the Band > Draw Band menu or Contour toolbar. Your cursor will change to a pencil symbol. Drag the cursor across the previously defined boundary; a line will appear. When you recross the old boundary, the line will change colors and the new boundary will be created.
15. Standards and Band Matching After you have defined the lanes and bands in your image, you can define the molecular weight, base pair, or other standards and determine the values of the experimental bands using those standards. You can also match your experimental bands across lanes for comparisons of lane similarity. Finally, you can identify bands of known quantity in your lanes and use these to generate a calibration curve for absolute quantitation of your unknown bands.
Quantity One User Guide 15.1 Defining and Applying Standards If you are using molecular weight, isoelectric point, base pair, or other mobility standards on your gels (including predefined Bio-Rad standards), this section will describe how to define them on your images and automatically calculate the values of unknown bands. Note: Multiple standard lanes in your gels will facilitate the band matching process outlined in the following sections. However, they are not required.
Standards and Band Matching Fig. 15-3. Selecting a set of standards. A list will open, prompting you to select new standards, previously created standards (if any), or predefined Bio-Rad standards. Sets of Bio-Rad molecular weight and base pair standards are included in your software installation. Selecting Predefined Standards If you are using Bio-Rad or other predefined standards, select them from the pop-up list. The Standards form will open, displaying the values of the standards.
Quantity One User Guide Fig. 15-4. Specifying units. Click on the Units button to specify your units. This opens a dialog box in which you can select from a list that includes Base Pairs, Isoelectric Point, Molecular Weight, and Normalized Rf. Note: Rf (relative front) expresses the distance a band has traveled down a lane as a fraction of either the total length of the lane or the vertical distance from the top of the lane to the bottom (the calculation method can be specified in Preferences).
Standards and Band Matching 15.1.a Standards Form Fig. 15-5. Standards form. The Standards form contains the values of your standards, and includes tools for applying them to the appropriate lanes in the image and displaying and adjusting the standards regression curve. The form will open with a default name for the standards. For new standards, this will be a generic name (e.g.
Quantity One User Guide be the descriptive name of the standards. You can change the name by typing in the text box next to the Standards prompt. You can enter additional information next to the Comment prompt. The buttons and fields can be used to further define your standards. Entering Standard Values If you are creating new standards, you must enter the values of your standard bands in the table in the middle of the form. Predefined standards will already have these values entered.
Standards and Band Matching To remove a standard value, click on the triangle button at the beginning of the row and select Delete from the pop-up box. The remaining standards will be renumbered. Applying Standard Values to Lanes To apply the values to the standard lanes on your image, click on the Apply to Lane button, then click on a lane. Fig. 15-7. Click on the Apply to Lane button, then click on the lanes containing standards. The values will be applied to the bands in the lane you select.
Quantity One User Guide click on the band to be assigned the standard value. The subsequent bands in the lane will be numbered sequentially based on the initial assignment. Click here to apply this standard value to a particular band on your image Fig. 15-8. Applying standard values. After you have applied the standard values to a lane, the bands in that lane will change color to blue, indicating that they are now standards.
Standards and Band Matching bands. (Note: You must apply the values to a lane before you can view and adjust the regression curve.) Click on the Standard Curve button in the Standards form, then click on a lane. A graph of the standards regression curve is displayed on the image, and the Standard Curve Options dialog is displayed as well. Fig. 15-9. Displaying the standard curve and options dialog box. As you click on different lanes, the curve is displayed for each lane in turn.
Quantity One User Guide Standards Regression Models Select different regression models in the options dialog box while you study the standard curve with the standard points displayed (see display options below). Then choose a curve that best fits the data points. (The correlation coefficient—see below—provides another measure of curve fit.
Standards and Band Matching Display Options The following options will change how the curve graph is displayed: Show Standard Points displays the standard data points on the graph. The standard points in that lane will be marked on the graph as triangles. Note that known band types will appear marked as standards. Show Calculated Points displays the calculated points on the graph. The calculated points in the lane will be marked on the graph as circles.
Quantity One User Guide To save the standard values with the image, click on the Close button to close the Standards form, then save the image. When you open the image again, the standards will be available when you select Standards. If you want to save these standards for use on other images, click on the Archive button. The standards will be saved in an archive file separately from the image.
Standards and Band Matching After you remove this file, a new, empty ONEDPREFS.DBS database will be automatically created the next time you open an image. You can use this to begin a new archive. 15.2 Band Matching To compare the similarity of the lanes in your image (using the phylogenetic tree, similarity matrix, etc.), you must first identify all the bands in the gel and match identical bands in different lanes. The functions for doing this are located on the Match menu and toolbar.
Quantity One User Guide If you have defined standards in the gel, a pop-up box will warn you that the regression model for calculating band values will be restricted to point-topoint semi-log. If you selected a different regression model when defining standards, it will be changed. The first time you click on an experimental lane with the Match command, a pop-up box will prompt you to specify the matching tolerance. Fig. 15-11.
Standards and Band Matching Yellow bands are bands that the software cannot accurately match. The matching algorithm is deliberately conservative to avoid incorrect labeling, so a number of yellow bands may appear on the image. Your next step will be to identify the yellow bands as either new band types, or existing band types that could not be automatched. To summarize: • Green bands are known band types, based on your identification.
Quantity One User Guide Note: Band types should only be added to areas of the gel image that are modeled well. The modeling lines are designed to give you guidance on adding new band types. • To create a new band type from an unknown (yellow) band, select Match and click on the unknown band. • To change a band to a particular band type, select Match and first click on the red or green band with the desired band type. This band type will be assigned to your mouse.
Standards and Band Matching To open the form, select Matched Band Set from the Match menu or toolbar, then click anywhere on the image. Fig. 15-12. Band Set form. The form will open with a default name for the band set (e.g., Band Set 1). You can type a new name in the Band Set field at the top of the form, and add any comments or category/attribute information you want to associate with the band set.
Quantity One User Guide List of Band Types The values of the individual band types are listed within the dialog box in a table format. These values reflect any standards you have defined (e.g., molecular weight, base pairs, etc.). If you have not defined standards, Normalized Rf units are used. Band type value Name of band type Assign band type to the mouse Delete band type Fig. 15-13. Applying and editing band type values.
Standards and Band Matching Band Set Toolbar The toolbar in the Band Set form contains all the commands needed for matching. Apply to Lane Create Band Clear from Lane Match Remove Band Propagate Band Set Show band models Unmatch Outlier Show band types Standard curve Fig. 15-14. Band set toolbar. Apply to Lane applies the band set to any lane in your gel image. Click on the button, then click on the lane. The bands in that lane that can be matched will change to red.
Quantity One User Guide Outlier excludes a known (green) band from the band set model. However, the band will still be marked as known. Show Band Types displays all the red, green, and yellow bands on the image, with the band type numbers next to the matched bands. Show Band Models displays the band set modeling lines across the gel image. Standard Curve displays the Standards Regression Curve (see section 15.1.b). Click on the button, then click on any lane in the image.
Standards and Band Matching Propagate Band Set is a feature that not only simplifies assigning band types, it allows the software to do some optimizations that will significantly speed up modeling. Choose a reference sample lane that you want to apply the band set to. Start by assigning one or two band types in the lane that are easy to identify, then click on the Propagate Band Set button and click on the lane to assign the remaining band types to the lane.
Quantity One User Guide Fig. 15-15. Match Graphs submenu. From the Match Graphs submenu, select the match graph you want to display, then click on a matched band. The bands in the matched group will be displayed, as will the histogram that you chose. Fig. 15-16. Example of a match graph. • Average displays a histogram of the average densities of the bands in a band type.
Standards and Band Matching • Contour displays a histogram of the density of each contoured band in a band type. This function only works with contoured bands. • Normalized Quantity displays a histogram of the normalized quantities of the bands in a band type. See section 15.2.d for instructions on how to normalize for quantity. • Peak displays a histogram of the peak densities of the bands in a band type.
Quantity One User Guide Note: Relative quantity expresses the quantity of each band as a percentage of either all the bands in a lane or the total intensity of a lane (depending on the setting in Preferences). This can tell you that there is twice as much of Band X than Band Y; however, it does not tell you how many micrograms there are of Band X in the gel. Absolute quantity is calculated based on the relative quantity, standards, and a calibration curve that you create.
Standards and Band Matching Fig. 15-18. Quantity Standards dialog box. The dialog box will open with a default name for the quantity standards (e.g., Cal 1). Enter a new name and specify the quantity value units (e.g., µg). You can enter descriptive information next to the Description prompt. The calibration curve is generated by plotting the known quantity values you provide against the intensity-based quantitation. There are several intensityderived values that can be used for the Y axis of the plot.
Quantity One User Guide Selecting Bands of Known Quantity The dialog box includes three buttons that are used to select bands on the gel image with which to generate the calibration curve. These buttons are located next to the Select prompt. Fig. 15-19. Buttons for selecting bands of known quantity. • To select bands one at a time, click on the Band button, then click on each band of known quantity. Each band will be highlighted.
Standards and Band Matching Fig. 15-20. Entering the known quantities. After you have entered a few quantities, the status of the remaining bands in the list may change to O.R., meaning that the remaining bands are out of the current range of values (based on their intensity and what you have already entered).
Quantity One User Guide To remove a band from the quantity standards and omit it from calibration curve calculations, click on the Remove option. All the information regarding that band will be deleted from the quantity standards file. Alternatively, you can select Outlier from the pop-up list. This retains the information about the band in the calibration file but does not include it when calculating the calibration curve. 15.3.
Standards and Band Matching Fig. 15-21. Calibration curve with titles displayed, linear regression, and extrapolation. Points used to calculate the curve are enclosed in circles. Points that you identify as Outliers are enclosed in squares. • To change the status of a point on the graph, click on it. The status will toggle between Known and Outlier. • To see the legend for the different types of points on the graph, click on the Titles button. • To print this graph, click on the Print button.
Quantity One User Guide 15.3.c Applying the Calibration Curve Once you have selected known bands and entered values for them, you are ready to choose the bands whose quantities you want to calculate with the calibration curve. This is done using the buttons found next to the Calibrate prompt. Fig. 15-22. Buttons for calibrating bands of unknown quantity. • To quantitate individual bands, click on Band, then click on the band of interest.
Standards and Band Matching In the Quantity Standards dialog box, next to the band that came from the undiluted stock solution, enter the known quantity. In the Dilution Factor column, type “stock.” Next to each of the other bands in the dialog box, enter the dilution factor in the Dilution Factor column. Fig. 15-23. Entering a dilution series. The quantity of each band will be automatically calculated. 15.3.
Quantity One User Guide When you make your selection, the values associated with it will be displayed in the Quantity Standards dialog box. Each standard value will be labeled “Import” in the Band column. Checking the Imported Curve If the quantity of one or more bands in the image is known, you can use this information to double-check the imported calibration curve to ensure that it accurately quantifies the bands on this image.
16. Volume Tools You can use the Volume tools to quantitate bands, spots, arrays, and other image data. What is a Volume? A volume is the intensity data inside a defined boundary drawn on your image. Volume = Sum of the intensities of the pixels within the volume boundary x pixel area Volume units = intensity units x mm2 Volumes are similar to band contours (see section 14.8.a), except that they are not dependent on lanes and bands.
Quantity One User Guide Fig. 16-1. Volume tools. Note: When using any of the following tools, be careful to completely surround the data you want to quantitate. You should also adjust for background intensity (see section 16.4 below). You may want to experiment with several different volumes drawn around the same object before selecting the one that gives you the best quantitation data.
Volume Tools Fig. 16-2. Volume contour. The contour should completely surround the data you want to quantify. To edit the contour, position your cursor on the border. Your cursor will change to a pencil tool. Drag across the line; a new white line will appear. When you recross the old line, a new contour will be created. Volume Freehand Tool This tool allows you to draw your own volume boundary.
Quantity One User Guide Volume Rect Tool Volume Rectangle Tool will create a volume box around an object. To use this tool, click on the Volume Rect button, then drag a box around the object to be quantified. When you release the mouse button, the volume is created. Fig. 16-4. Volume rectangle. To resize the box, click on it to select it, then position your cursor on one of the corner anchor points and drag.
Volume Tools Fig. 16-5. Volume circle. The volume circle should completely surround the data you want to quantify. To resize the circle, click on it to select it, then position your cursor on the circle border and drag. Volume Labels When you first create your volumes, they will appear unlabeled. You can display the volume labels using the Show/Hide Volume Labels command on the Volume menu or toolbar. Your volumes will have default labels U1, U2, U3, etc.
Quantity One User Guide To reselect the volume, click on it again. If you move your cursor over the volume—selected or unselected—the border will change to gold. You can create as many volumes on an image as you want. After you create a volume, you can view your data (area, density, etc.) by selecting the Volume Analysis Report from the Reports menu. Tips The volume you draw should completely surround the data you want to quantitate.
Volume Tools • To move the selected volume or volumes, position your cursor over the selection and drag. • To copy within an image, hold down the CTRL key while dragging the selected volume or volumes. The copy will be created and dragged to the new position. • To delete the selected volume or volumes, press the DELETE key.
Quantity One User Guide Fig. 16-6. Volume Properties dialog box. Select the Standard option button, then enter the concentration in the Concentration field. (Do not include units.) Click on OK to close the dialog box. Your standard volumes will have default names S1, S2, S3... etc., based on their creation sequence. You can display/hide volume names using the Show/Hide Volume Labels command (see previous section for more about volume labels).
Volume Tools 16.4 Volume Background Subtraction When you draw a volume, you will probably include some non-data background pixels inside the volume. These background pixels will usually have an intensity value that you do not want to include in your volume quantitation. There are two ways of calculating this background intensity for your volumes: local and global. The background subtraction method is selected in the Volume Report Options dialog box (section 19.5.a).
Quantity One User Guide Any pixels inside the volume that have the same intensity as the background pixels will be reduced to zero, thereby eliminating them from the quantitation. Global Background Subtraction Note: If you select Global Background Subtraction in the Volume Report Options dialog, but do not define a background volume as outlined below, you will effectively select no background subtraction. Global background subtraction calculates a single background intensity for the entire gel.
Volume Tools The average intensity of the pixels in that background volume will be calculated and subtracted from each pixel in all standard and unknown volumes. Any pixels inside the volumes that have the same intensity as the average background will be reduced to zero, thereby eliminating them from the quantitation. If you create more than one background volume, all the pixels in those background volumes will be used to calculate the average background.
Quantity One User Guide What Is a Volume Array? A volume array is a matrix of volume circles or rectangles that can be sized/ positioned as a group and overlaid on images of blots, wells, or cells for easy quantitation. The individual cells in the array have the same functionality as standard volumes. You can define cells as background volumes, standards, and/or unknowns, as described in the sections above. You report your array data as you would standard volumes, using the Volume Analysis Report.
Volume Tools Select the shape of the wells/cells (Circular or Rectangular) and click on OK. The array overlay will be created and displayed on the image. Fig. 16-10. Array overlay. Like regular volumes, array volumes are initially displayed without labels. To show the labels of the individual wells/cells, click on the Show/Hide Volume Labels button on the toolbar. Like regular volumes, array volumes are initially labeled U1, U2, U3, etc.
Quantity One User Guide Moving an Array To reposition an array overlay, move your cursor over any individual cell until the cursor changes to a multidirectional arrow and the cell border turns yellow. Then hold down the cursor and drag the entire array to a new position. Fig. 16-11. Moving an array. Resizing an Array To resize an array overlay, make sure it is selected, then position your cursor over the dot at the center of one of the corner cells.
Volume Tools Hold down your mouse button and drag the array frame in or out to compress/expand the array. Resizing the Array Cells To resize the individual cells in the array, zoom in on any individual cell and move your cursor over the cell border (or corner anchor point, in the case of rectangles) until it changes to a cursor with an adjustment symbol. Hold down the mouse button and drag to move the border in or out. All the cells in the array will be resized accordingly. Fig. 16-13.
Quantity One User Guide Ungrouping an Array You can ungroup the individual cells in an array, so they behave like normal, stand-alone volumes. With the array selected, select the Array Ungroup command from the menu or toolbar. This command cannot be undone, and you will be warned before the operation is completed. When your array has been ungrouped, it will appear deselected (i.e., as blue volumes). You can then move the cells individually, and perform all normal volume operations on the individual cells.
17. Colony Counting You can use Quantity One to automatically count the number of white, blue, or plaque colonies in a Petri dish image. Note: For best results, when capturing the image of a Petri dish with an imaging device, the dish should fill the imaging window. Also, images with colonies should not have asymmetric pixels. (Asymmetric pixels can be generated by densitometers and the Reduce File Size command.) The colony counting function will not work properly on images with asymmetric pixels.
Quantity One User Guide Fig. 17-1. Colony Counting control panel. The Colony Counting control panel has been arranged from top to bottom to guide you through the procedure. 17.1 Defining the Counting Region First, you must define the region you want to count in the Petri dish image. Click on the Define Counting Region button in the dialog box and position your cursor at the center of dish image. Drag the cursor outward.
Colony Counting Drag cursor out from center to define region Fig. 17-2. Defining a counting region. If you make a mistake in defining your counting region, click on the Reset button at the bottom of the dialog box to start over. Position the blue border until it is just inside the interior edge of the Petri dish. Note: If your border disappears when you release the mouse button after dragging, check to make sure that the Show Data Area checkbox is checked.
Quantity One User Guide 17.2 Counting the Colonies After you have positioned the circle, you are ready to detect colonies. If you are counting plaques, click on the Plaque checkbox. (Because plaques appear as clear circles on a darker background, this checkbox must be selected for proper detection.) Before counting, you may want to adjust the Sensitivity and Averaging parameters described below. When you are ready to count, click on the Count button.
Colony Counting 17.3 Displaying the Results After you click on Count, the number of detected colonies will appear in the Results section of the dialog box in the White column. Fig. 17-3. Example of a dish with white colonies. The colonies will also appear marked as gold triangles on the image itself. Note: If your colonies are not marked on the image, check to make sure that the Mark White Colonies checkbox at the bottom of the dialog is checked.
Quantity One User Guide 17.4 Making and Erasing Individual Colonies If automatic colony detection has missed or erroneously detected some colonies, you can manually mark or unmark them directly on the image using the buttons under Tools/Options. Fig. 17-4. Colony counting tools. To mark a colony, click on the Make Colony button, then click on the spot on the image that you want to identify as a colony.
Colony Counting Colonies Versus Background Noise If there is a clear peak on the left end of the colony counting histogram, it is probably due to background noise in the image. (For information on subtracting background from entire images, see section 12.9; for information on filtering noise from images, see section 12.10.) If this background noise is being detected as colonies, you can use the histogram and Cutoff slider to correct this.
Quantity One User Guide White peak Blue peak White/blue division mark Fig. 17-6. Using the White/Blue slider. Drag the White/Blue slider to the left until it is positioned between the two peaks. The white colony data range is indicated by gold on the bar beneath the histogram, and the blue colony data range is marked with blue. As you drag the slider, the numbers of white and blue colonies will change in the dialog box and in the text box on the image.
Colony Counting Click on the Ignore Region button, then position your cursor on one edge of the region you want to ignore. Drag your cursor on the image, defining the full region you want to ignore. Fig. 17-7. Marking a region to ignore. As you drag, you will create a “pie slice” marked with red cross-hatching. Any colonies in this region will not be considered in the final count. When you have defined the region, release the mouse button.
Quantity One User Guide The adjusted count is an estimate of the total colony count in the Petri dish; it uses the known colonies to extrapolate the number of colonies that might have appeared in the ignored region if it had not been damaged. The adjusted count is calculated based on the area of the ignored region and the density distribution of colonies in the rest of the circle. 17.7 Saving/Resetting Your Count A colony count can be saved to the image and/or a separate spreadsheet file.
Colony Counting Fig. 17-8. Batch Mode controls. Creating/Opening a Batch File To create a new batch file, click on the New Batch button. This will open a dialog box in which you can specify the name and location of the spreadsheet you want to create. Fig. 17-9. Creating a batch file. When you click on Save, the new batch file name will be displayed in the Colony Counting control panel. To open an existing batch file, click on the Open Batch button. This will open a similar dialog box.
Quantity One User Guide Naming/Saving a Count Enter a name for the count you want to save in the Count Name field, or use the default name (the file name plus a time stamp). Enter any comments in the Count Comment field. This data will be included in the spreadsheet. To save the currently displayed count to the batch file, click on the Save Count button. The number of colonies, as well as associated count settings, will be added to the spreadsheet.
18. Differential Display and VNTRs This chapter describes the tools in Quantity One for studying sequence expression in PCR gels and counting VNTRs or other repeated elements in 1-D gels. These analysis functions are located on the Analysis menu. Fig. 18-1. Analysis menu. 18.1 Differential Display Differential Display is a technique using mRNA and PCR amplification to study gene expression. Note: You must match the bands in your gel image before using this function. See Chapter 15 for more information.
Quantity One User Guide Normalization To assign a normalized band type, select Normalize from the Match menu and then click on a matched band that appears in every lane of your gel. The quantity of that band in each lane will be set to 100, and the quantities of the other bands in the lane will be normalized to that band. (If the band is not present in a lane, the normalized quantity for that lane is zero.) See section 15.2.d for more information. 18.1.
Differential Display and VNTRs 1. nq > ratio x mean and nq > mean + quantity 2. nq < (1/ratio) x mean and nq < mean – difference ratio = 1.0 + (percentage/100) Select the Trend button to search for increasing or decreasing gene expression, represented by trends in normalized quantity across a band type. In Trend mode, a linear regression of (nq) versus lane # is computed for each band type. The leftmost and rightmost lanes containing that band type are determined.
Quantity One User Guide 18.2 Variable Number Tandem Repeats If your experiments involve the use of microsatellites, VNTRs, or other repeated elements, you can calculate the number of times a repeated element occurs in a band. Note: You must define the standards in the image before you can use this function (see Chapter 15). To calculate the VNTRs on your gel image, select VNTR Calculations from the Analysis menu.
Differential Display and VNTRs In the text box next to the Flanking Region Size prompt, enter the size (in base pairs or other repeated units) of the part of the fragment that does not include any repeated elements. This would include primer length and the length of any sequences that fall between the end of the primer region and the beginning of the stretch of repeats. PCR Primer Sequence between primer and VNTR VNTR repeat unit Fig. 18-4. Diagram of components of a DNA fragment.
Quantity One User Guide Finally, specify whether the tandem repeat numbers that meet the ambiguity criteria should be marked with an asterisk (*) or left unmarked. When you have entered all the information in the dialog box, click on the Done button. The number of tandem repeats will be displayed next to the bands. If the numbers have been concealed (e.g., by the Hide Overlays command), you can redisplay them by selecting VNTR Display.
19. Reports Quantity One can display and print a variety of different analysis reports. You can format the reports to include different kinds of data. The reporting features are located under the Reports menu. Fig. 19-1. Reports Menu. 19.1 The Report Window All of the reports except Phylogenetic Tree share the same basic report window.
Quantity One User Guide Click here to close the report window Print One Page Print All Pages Controls for scrolling through screen pages Export Reformat Fig. 19-2. Example of a report window. The standard report window has buttons for printing the report, scrolling through the screen pages of the report, and exporting the report to a spreadsheet application. Some report windows also have a Reformat button for displaying different report data.
Reports Scrolling Through the Screen Pages If your report has multiple screen pages, the scroll buttons in the report window will become active. You can use them to scroll to the next page, previous page, first page, or last page of the report. You can also enter a specific page number in the text field. Printing Your Report You can print your report using the Print One Page or Print All Pages commands in the report window.
Quantity One User Guide Note: If you select Print All Pages, the number of pages printed may be less than the number of screen pages listed in the report window. This is because the print command reformats the data to make maximum use of the paper size. Exporting Your Report Data You can export your data to spreadsheet applications for further computation and analysis. Click on the Export button to open the Export Report dialog box. Fig. 19-4. Export Report dialog box.
Reports Reformatting Your Data If you change your mind about the data to display in your report window, click on the Reformat button (not available in all reports). This will reopen the options dialog box for your particular report. 19.2 Lane and Match Reports There are four different lane and match reports: Lane Report, All Lanes Report, Match Report, and All Matches Report. Lane Report generates a report on any you lane you select.
Quantity One User Guide Fig. 19-5. Report Options form. Select the data you want to display in the report by clicking on the appropriate checkboxes. Note: If you change your mind about the data to display, you can change the report options from within the report itself by clicking on the Reformat button. Select the Font Size, Column Spacing, and Line Spacing settings to be used in the report by clicking on the button next to each field and selecting from the list of options.
Reports To load or delete previously saved settings, click on the button next to the Settings to Load or Delete field and select from the list of saved settings. Then click on the Load button to load them, or the Delete button to delete them. To display the selected data, click on the Report button. The report will be displayed in a standard report window (see section 19.1 above for information about standard report window options). 19.
Quantity One User Guide Note: If you change your mind about the data to display, you can change the report options from within the report itself by clicking on the Reformat button. To display the selected data, click on the Report button. The report will be displayed in a standard report window (see section 19.1 above for information about standard report window options). 19.
Reports Band Weighting means that band intensity as well as position will be used when comparing the similarity of your samples. Select Yes to use band weighting when generating the report. Select No to use only band position as the basis for comparing lane similarity. Comparison Method The method for computing similarity in Quantity One is the Dice Coefficient.
Quantity One User Guide si = 0 if the lane does not have a band assigned to the i'th type. Otherwise: si = The quantity of the band assigned to the i'th band type. si = 0 if the lane does not have a band assigned to the i'th type. 19.4.a Compare Lane Images The Compare Lane Images command displays the lanes of your gel image in decreasing order of similarity to a lane that you select. Select Compare Lane Images from the Reports menu, then click on the lane that you want to compare your other lanes to.
Reports Fig. 19-9. Compare Lane Images report window. The Compare Lane Images report will be displayed in a standard report window (see section 19.1 above for information about report window options). You can change the report options from within the report itself by clicking on the Reformat button.
Quantity One User Guide The Print Report dialog for this report contains special fields for entering a title for your report. 19.4.b Phylogenetic Tree Phylogenetic trees are schematic representations of lane similarity. To compare the similarity of your lanes in a phylogenetic tree format, select Phylogenetic Tree from the Reports menu. (You can also use the Phylogenetic Tree Quick Guide under the Help menu to analyze your gel image.
Reports Fig. 19-10. The Phylogenetic Tree report window. The report window for phylogenetic trees is slightly different than the standard report window. The tree appears in one window with scroll bars for moving up and down and left and right. To print the tree, click on the Print button. This will open the standard Print Report dialog box. To close the window, click on the Close box in the upper right corner of the window.
Quantity One User Guide Neighbor Joining This type of phylogenetic tree is computed is based on minimizing the total branch length at each stage of clustering. The method also finds branch lengths between nodes. The approximate distance between any two samples in this tree can be found by adding the branch lengths that connect the samples. Other Methods1,2 The following methods are based on the algorithm below: 1. Begin with n clusters—one cluster for each sample. 2.
Reports np the number of samples in the p'th cluster. nq the number of clusters in the q'th cluster. n the number of clusters in the k'th cluster formed by joining the p'th and q'th cluster. n = np + nq dpq the distance between cluster p and cluster q. Single Linkage This is also called Nearest Neighbor or Minimum Method. d ki = min (d pi ,d qi ) Complete Linkage This is also called the Furthest Neighbor or Maximum Method.
Quantity One User Guide WPGAMA is a special case of UPGAMA that favors the most recent member clusters in forming new clusters. Centroid np nq ( n p ⋅ nq ) d ki = ----- ⋅ d pi + ----- ⋅ d qi – -------------------n n n 2 d pq Median d ki = 0.5 ⋅ d pi + 0.5 ⋅ d qi – 0.25 ⋅ d pq Centroid and Median are similar to UPGAMA and WPGAMA, respectively, but the distance formula contains an additional third term. Centroid and Median methods are not monatomic hierarchical clustering algorithms.
Reports All Other Methods Neighbor Joining Only Fig. 19-11. Two forms of the Options dialog box. The box on the left has controls that are specific for the Neighbor Joining method. The box on the right is applicable to all other tree methods. Both boxes share certain features. Each allows you to choose between aligning the tree to the right or proportionally aligning it.
Quantity One User Guide The Min. and Max. sliders are active only for proportionally aligned Neighbor Joined trees. They allow you to adjust the Minimum and Maximum distance values between nodes on branches, so that you can adjust the display of your tree to highlight specific regions of data. Distances below the Min. or above the Max. values will be collapsed to fit on the form.
Reports Fig. 19-12. Similarity Matrix report. The Similarity Matrix report will be displayed in a standard report window (see section 19.1 for information about standard report window options). 19.5 Volume Analysis Report The Volume Analysis Report displays your volume data. To open this report, select Volume Analysis Report from the Reports menu or Volume toolbar. The Volume Report Options dialog box will pop up, allowing you to specify the information that will appear in your report.
Quantity One User Guide When you have selected your report options, click on the OK button. The report will be displayed in a standard report window (see section 19.1) above for information about standard report window options). 19.5.a Volume Report Options These settings appear when you first open the Volume Analysis Report. They can also be accessed from within the report itself by clicking on the Reformat button. Fig. 19-13. Volume Report Options dialog box.
Reports • Type—Unknown, standard, or background. • Volume—Sum of the intensities of the pixels inside the volume boundary x area of a single pixel (in mm^2). • Adj. Vol.—Volume minus the background volume; if there is no background volume, this is simply the volume. • % Volume—The volume expressed as a percentage of all the volumes in the image. • Concentration—The quantity as calculated from the standards and the regression method. If you have not defined standards, this is not calculated.
Quantity One User Guide Other Options The Image Display Options affect how the image is displayed and/or printed on the report. You can choose whether to report on all your volume objects (All objects) or only those objects you have selected (Selected objects) Select the regression method for calculating the Volume Regression Curve (section 19.6). To display the curve, click on the Show Curve button.
Reports Fig. 19-14. Volume Regression Curve window. Each standard volume is marked by a red X and each unknown is marked by a blue triangle. The X axis is the adjusted volume and the Y axis is the concentration, based on the standards you have marked on the image. To display the numbers and names (if any) of your volumes, click on the Show Name checkbox. Select your preferred regression method from the list of option buttons.
Quantity One User Guide To print your curve, click on the Print button. This will open the standard Print Report dialog box. To close the Volume Regression Curve window, click on the OK button. 19.7 VNTR Report The VNTR Report displays your VNTR data. Select VNTR Report from the Reports menu. A pop-up box will ask if you want to display the gel image in the report. Fig. 19-15. Example of a VNTR Report.
Reports The report is displayed in a standard report window (see section 19.1 above for information about standard report window options). The report displays the molecular weight, the raw repeat number, and the rounded repeat number for each band in the gel image. The raw repeat number is the number of repeats calculated using the information that you provided in the Tandem Repeat Calculation dialog box, and is likely to include fractional values.
Quantity One User Guide 19-26
20. Printing and Exporting The commands for printing and exporting images are located on the File menu. Reports (Volumes, VNTR, Phylogenetic Tree, etc.) are printed from within the individual report windows. 20.1 Printing There are four different printing options under File > Print: 1. Print Image prints a copy of the image and any overlays that are currently displayed. 2. Print Actual Size prints an actual-size copy of the image. 3.
Quantity One User Guide File > Print > Print Image opens the Print Image dialog box. Windows version: Macintosh version: Fig. 20-1. Print Image dialog box. At the top of the dialog box, enter the title that you want to appear above the image when you print. Choose whether a margin border should appear around the edge of the image by clicking either the On or Off button next to the Margin Box prompt.
Printing and Exporting Print Settings: Windows Only Click on Printer to display a list of your available printers. After you have selected a printer, you can click on Properties to select the standard Windows print settings for that printer. Note: If you are using Windows 95 with certain printer drivers, you may experience difficulty printing some overlays and overlay symbols on images.
Quantity One User Guide Select Print Actual Size from the File > Print menu. The standard Print Image dialog box will open (see previous section). 20.1.c Printing the Scan Report Scan Report allows you to print out a single-page report of an image and its associated information. The format of the report is designed to provide a concise yet thorough summary of the most relevant features of an image for documentation purposes. The report includes the following information: • The image.
Printing and Exporting 20.1.d Video Printing The Video Print command allows you to print images and reports on a video printer. To create a video printout of the active window, select Video Print from the File > Print submenu. Note: Video printing requires installation of the video board and cable that come with the Gel Doc 1000/2000 and Chemi Doc gel documentation systems. The video board and cable can also be ordered separately.
Quantity One User Guide Fig. 20-2. Export to TIFF Image dialog box. Analysis Export Mode Analysis mode exports the scan data, unmodified by any viewing adjustments you may have made (such as Transform or Zoom). If you select Analysis mode, the other controls in this dialog box will become inactive. Publishing Export Mode Publishing mode exports a TIFF image that looks like the image as it is currently displayed on the screen.
Printing and Exporting Note: This is the only mode available if you are exporting from the Multichannel Viewer. In Publishing mode, you can specify a resolution for the TIFF image by selecting 72 dpi (typical computer screen resolution), 150 or 300 dpi (standard printing resolutions), Same As Scan, or any resolution you Specify (up to the resolution of the scan). If you have log transformed the image, you can specify a linear transform, or preserve the current view.
Quantity One User Guide 20-8
Appendix A Cross-Platform File Exchange It is possible to move image data between Discovery Series software applications on different platforms. There are different protocols depending on the platform (PC or Macintosh) you are transferring from and to. A.1 Macintosh to PC To transfer a file from a Discovery Series application running on a Macintosh to a Discovery Series application running on a PC, you need to tag the file name with the suffix appropriate to the image file type (e.g., 1-D scan).
Quantity One User Guide A-2
Appendix B Other Features The following features are available in Quantity One, but have more utility in its more powerful companion application, Diversity Database. You can examine your gel images in Quantity One and then database them using Diversity Database. B.1 Categories and Attributes User-defined buttons are available in the Standards dialog box, Matched Band Set dialog box, and Gel Layout dialog box.
Quantity One User Guide Fig. B-2. Edit Category dialog box. Type the name of the new category (e.g., “Color”) next to the Category prompt and list attributes of that category in the Attribute fields (e.g., “Red,” “Green,” “Blue,” etc.). The form will automatically sort your attributes alphabetically within the Attribute fields. Categories and attributes can be defined for any characteristics of your gel that would be useful to sort by.
Appendix B. Other Features 1. Select category 2. Click to select attribute 3. Select attribute from list and click on OK to apply 4. Click OK Fig. B-3. Selecting a category and attribute. Select the category to be applied from the available categories on the list (or select ). Click on the Attribute button to specify an attribute. Click OK to apply your selection to the Standards box. B.
Quantity One User Guide Fig. B-4. Gel Layout dialog box. The following information about your gel can be specified at the top of the Gel Layout form: • Category and attribute information (see above) for the whole gel. • A brief description of the gel. • Gel run date. • Number of lanes. • Lane width (see section 13.1.g). • Lane-based background subtraction and disk size (see section 13.2).
Appendix B. Other Features Each lane in your gel has its own line on the Gel Layout form. The following information can be specified for a lane. (See section 15.2 for more information about band sets.) • Band set type (standard or experimental). • Lane number. • Band set name. • Sample name. • Category/attributes information (see above) for individual lanes. The pop-up buttons on the left side of the form offer a number of choices pertaining to the individual lanes on your gel.
Quantity One User Guide B-6
Index A Annotations. See Text Overlays. Application icon ................................................................................................... 1-9 Arrays Lane-based ................................................................................................. 13-17 Volume ....................................................................................................... 16-11 Arrow keys .....................................................................................................
Quantity One User Guide Brackets or lines preference ....................................................................... 2-23 Contouring ................................................................................................. 14-26 Deleting ...................................................................................................... 14-14 Detecting automatically ............................................................................. 14-3 Detection parameters .....................
Index Exposure time ....................................................................................... 3-7, 4-7 Freeze .............................................................................................................. 4-7 Image mode ................................................................................................. 4-11 Imaging area ................................................................................................ 4-15 Invert display .............................
Quantity One User Guide Colors, setting ................................................................................................... 12-12 Compare Lane Images ..................................................................................... 19-10 Compare Lanes ................................................................................................. 13-13 Comparison reports ...........................................................................................
Index F File locations in Windows .................................................................................. 1-8 Filter Wizard ..................................................................................................... 12-32 Filtering image noise ....................................................................................... 12-31 Flipping images ................................................................................................
Quantity One User Guide Imaging area size ........................................................................................ 8-16 Live acquire .................................................................................................. 8-11 Live acquire options ................................................................................... 8-14 Options ......................................................................................................... 8-11 Positioning ..................
Index Saving images ............................................................................................... 3-9 Simulation mode ........................................................................................... 3-2 UV mode ........................................................................................................ 3-9 Video card ...................................................................................................... 3-1 Video display window ........................
Quantity One User Guide Scanning window ......................................................................................... 6-3 SCSI card ........................................................................................................ 6-1 Simulation mode ........................................................................................... 6-1 Step tablet, editing ......................................................................................
Index Colors ......................................................................................................... 12-12 Contrast ...................................................................................................... 12-15 Cropping .................................................................................................... 12-22 Filtering noise from .................................................................................. 12-31 Inverting data ............................
Quantity One User Guide Lanes Adjusting ...................................................................................................... 13-7 Comparing ................................................................................................. 13-13 Defining ........................................................................................................ 13-7 Deleting ........................................................................................................
Index Saturated pixels, highlighting ................................................................. 11-13 Saving the image ....................................................................................... 11-12 Scanning window ....................................................................................... 11-3 Selecting an application ............................................................................. 11-4 Selecting resolution ......................................................
Quantity One User Guide Saving the image ......................................................................................... 10-6 Scanning window ....................................................................................... 10-3 Selecting resolution ..................................................................................... 10-5 Selecting scan area ...................................................................................... 10-4 Simulation mode ...........................
Index Quantity Standards Applying to bands .................................................................................... 15-30 Calibration curve ...................................................................................... 15-28 Checking imported values ...................................................................... 15-32 Creating ...................................................................................................... 15-24 Definition ....................................
Quantity One User Guide Revert to Saved ................................................................................................... 2-12 Rotating images ................................................................................................ 12-23 S Sample images .................................................................................................... 2-10 Saturated pixels, highlighting ........................................................................
Index Starting the program ........................................................................................... 1-9 Status boxes .......................................................................................................... 2-2 Subtract Background. See Background subtraction. T Tandem repeats ................................................................................................ 14-18 Tandem repeats, calculating ...................................................................
Quantity One User Guide U Unable to Obtain Authorization message ...................................................... 1-10 V Video printing ..................................................................................................... 20-5 View Entire Image .............................................................................................. 12-4 Viewing images in multiple channels ............................................................. 12-8 VNTR Report ............................
Index Z Zoom Box ............................................................................................................ 12-2 Zoom In/Zoom Out .......................................................................................... 12-3 Zoom tools Changing behavior .....................................................................................
