AlphaImager™ IS-2200 For Windows 2000/XP
PN: 94-11814-00, Rev. D ® AlphaImager 2200 – AlphaEaseFC User’s Manual Ignore all the camera and acquisition references if you are an AlphaEaseFC Stand Alone software user Table of Contents Page Introduction and Setup Introduction to AlphaImager 2200 .............................................................................… AlphaImager Imaging System Setup ............................................................................. Computer, Camera and Peripherals Installation ….....................
Alpha Innotech User’s Manual, version 4 Drop Down Menus File Menu ……................................................................................................................ File Open File Overlay File Close File Save and Save As Print Setup Print Logoff Exit Edit Menu ……............................................................................................................... Edit Activation Copy and Crop Reset and Clear Image Menu ...............................................................
Table of Contents Image Enhancement The Zoom Tool …........................................................................................................... The Rotate / Flip Tool ………………............................................................................ Histogram ....................................................................................................................... Automatic Enhancement ….............................................................................................
Alpha Innotech User’s Manual, version 4 Scanning the Image Adjusting Automatic Peak Detection Parameters Editing Peaks Adjusting the Baseline Interpreting 1D-Multi Data Molecular Weight, Mass and Band Scoring Integrated into 1D-Multi Outputting Quantitative Data Output 1D-Multi Data Exiting 1D-Mult Auto Lane Data Table Editing of Auto Lane Gel Smiling Correction Band Matching, Similarity Matrix, and Dendrograms Spot Densitometry ...............................................................................
Alpha Innotech User’s Manual, version 4 Chapter 1: Introduction and Setup 1.1 AlphaImager 2200 Imaging System The AlphaImager Imaging System is a powerful digital imaging system, ideal for instant photography of a wide variety of samples. The CCD camera allows imaging of low-light samples in UV-illuminated, and fluorescent applications. The instrument is controlled by AlphaEaseFC software, which is designed with ease-ofuse in mind.
Chapter 1: Introduction and Setup 1.1.1 Mouse Functions The mouse supplied with the AlphaImager System has two buttons. The left button is used to activate functions and otherwise make selections when using the software. In some cases, the right mouse button is used to recall or reactivate the function that was most recently assigned to the left button. 1.1.
Alpha Innotech User’s Manual, version 4 1.2 AlphaImager Imaging System Setup 1.2.
Chapter 1: Introduction and Setup 1.3 Computer, Camera and Peripherals Installation All software, peripheral drivers and operating systems have been pre-installed at the factory. During the system installation all components should only need to be plugged into the correct ports 1.3.1 Setting up the Power Strip/Surge Protector Turn the power switch on the power strip/surge protector off. Plug the power strip/surge protector into a wall outlet (preferably a dedicated circuit) and then turn the power on.
Alpha Innotech User’s Manual, version 4 1.3.3 Camera Installation 1) Connect the BNC and serial style connectors to the camera. 2) Complete the camera connection by attaching the other end of the Data Cable to the computer interface card that came preinstalled in the computer system. 3) A small flat head screwdriver should be used to tighten the connection on top of the camera and the back of the computer.
Chapter 1: Introduction and Setup 1.3.4 Connecting the Printer The Mitsubishi P91DW (UB) printer connections are color coded for convenience (see figure on previous page). Plug the USB cable into the back of the printer and into the proper USB connector on the back of the computer. Plug in the standard 3 prong power cable to the back of the printer and to the power strip, then turn the power on.
Alpha Innotech User’s Manual, version 4 1.3.6 Starting AlphaImager System with AlphaEaseFC software To start the AlphaImager 2200 Imaging System software from Windows, double-click the AlphaImager System icon on the windows desktop.
Chapter 1: Introduction and Setup 1.4 AlphaImager System Quick Guide Note: This is intended as a quick reference guide for acquisition. For more detailed information on the individual features reference section 1.6 of this manual. 1. Power on the system: a. Turn on the computer, monitor, and optional printer. b. After the computer has booted up completely to the Windows desktop, turn on the power to the cabinet. c. The AlphaImager 2200 software is activated by double clicking on the AlphaImager icon. 2.
Alpha Innotech User’s Manual, version 4 b. Enter a file name and select the directory to which it should be saved (the directory path should be less than 100 characters). c. Specify the file format (TIF, BMP, PCX, MAC, color TGA) d. Click OK to save the file 5. Enhance the display [optional] a. Adjust the black, white and gamma levels by moving the slider bars at the right of the image in the “Contrast Adjust” window, or select auto contrast. b.
Chapter 1: Introduction and Setup 1.5 System Information To display system information, select the About option in the Help menu. This button accesses a pop-up box. ABOUT Pop-Up Box This box shows the instrument serial number (where appropriate) and the Software version number. Use the information specific to your instrument and software when calling Alpha Innotech for technical support, software upgrades, etc. To close the box, click on the OK button.
Alpha Innotech User’s Manual, version 4 1.6 Acquiring an Image In the TOOL BAR window, click on the Camera Acquire icon: Once the Camera Acquire Icon is pressed, the CAMERA SETUP AND PREVIEW window is activated to provide exposure control of the camera, all lighting and filter wheel position controls, contrast display options and cabinet door open/close indicator using a software ‘virtual control’ menu. Also, Movie Setup is controlled and activated from this window.
Chapter 1: Introduction and Setup 1.6.1 Focusing on an Image Focusing on an image in the AlphaImager 2200 AlphaEaseFC software is easy with the real time display. Simply adjust the parameters on the manual lens, or, if the lens is controlled via the software, click on the appropriate software control button. 1) To open the aperture, turn the lens's aperture ring counterclockwise for more light.
Alpha Innotech User’s Manual, version 4 Turning the Optional Motorized Zoom Lens Software Controls On/Off: The Software controls for the Motorized Zoom Lens can be turned on and off in the Innotech.def file. This file is located in the appropriate program directory. Alpha Innotech’s production facility will turn the controls on or off depending on whether or not the optional Motorized Zoom lens is purchased.
Chapter 1: Introduction and Setup 1.6.3 Auto Exposure and Auto Exposure Setup The acquisition window for the AlphaImager 2200 imaging system includes a check box and setup window for Auto Exposure. Auto Exposure Setup (Auto Exposure Compensation Setting): 4 choices available • Normal Exposure for image saturation: Use this choice for normal Colorimetric and Fluorescent imaging.
Alpha Innotech User’s Manual, version 4 • • • Under Exposure for chemilumenescence Over Exposure for faint band detection. Custom Exposure Compensation: user definable Exposure Value (EV). The up/down arrows allow the user to change the EV value by whole units (left up/down arrows) or by 1/8th (right up/down arrows). Auto Exposure works in both “Expose Preview” and “Acquire Image” modes.
Chapter 1: Introduction and Setup 1.6.4 Cabinet Controls - Activating the Light Source and Selecting a Filter Use the mouse to click on the desired light source in the ‘virtual’ cabinet control interface or you can use the controls on the cabinet itself. Both mechanical and software controls are linked and communicate display setting selections. Note: There is a slight delay when the button is depressed until the light source is fully activated.
Alpha Innotech User’s Manual, version 4 1.6.5 Use Expose Preview to Set Exposure Time Once the sample has been positioned and the camera has been focused, close the door to the MultiImage light cabinet and make sure that the appropriate illumination source is turned on. Also make sure the cabinet door indicator in the Cabinet Control software interface indicates CLOSED.
Chapter 1: Introduction and Setup 1.6.6 Optimizing the Gray Scale for Saturation and Contrast Displays If an image will be analyzed, it is important that it not be over-saturated (too light) or under-exposed (too dark). Using Show Saturation, the user can see the areas of the image that are assigned to each end of the gray scale spectrum, and can adjust the imaging controls accordingly.
Alpha Innotech User’s Manual, version 4 1.6.7 Acquire and Transfer the Image Once the appropriate image is displayed on the screen, click on the red ACQUIRE IMAGE button to capture and transfer the image to the AlphaEaseFC software package for enhancement, archiving, or analysis. The acquired image will be automatically adjusted by a zoom factor based on the resolution setting to display the image optimally. Once a satisfactory image has been captured, we suggest saving it as an original file.
Chapter 1: Introduction and Setup 1.6.8 Capturing Images Using the Movie Mode If kinetic, multiplex, color, or chemiluminescence experiments are desired where you wish to have the system automatically capture several images at preset exposure times, preset time delay between images, preset lighting sources, and preset filter choices, the MOVIE box can be clicked in ToolBox, Enhancement Tools. (Movie Setup is also accessible in camera setup acquisition screen.
Alpha Innotech User’s Manual, version 4 Once MOVIE SETUP is selected, a display box will appear for independent control of all lighting, filters, and exposure delay for each image frame. The TOTAL FRAMES setup provides you with the ability to determine how many individual frames (images) you want for the movie. There is a maximum of 50 frames (images) and a minimum of 1 frame that can be captured with each movie. The FRAME selection is used for setting up the conditions for each frame (image).
Chapter 1: Introduction and Setup Once all images have been captured, the above Movie display box will become displayed. The remaining buttons will perform the following tasks: REC PLAY STOP PAUSE REW (rewind) REV (reverse) FWD (forward) DEL (delete) LOAD SAVE Move to Camera Setup and Preview, Movie Record setup functions to record a movie. Display a continuous loop ‘movie’ of all of the captured images.
Alpha Innotech User’s Manual, version 4 Movie Mode: Save/Load Movie Mode setup routines Two buttons “Save Setup” and “Load Setup” allow you to save and load all Movie Mode setup parameters. Files are saved as *.mvf files. Frame Stacking At the top of the Movie Set-up window is an option for stacking frames. If this selection was chosen during acquisition of the image Stack Frames will use all previous exposure information to sequentially add images to one another.
Chapter 1: Introduction and Setup Capturing a Color Image Using the Movie Function A color image can be generated by acquiring three images each taken with a red, green and blue emission filter. Once saved, these images are then combined in the Overlay pull down menu. Open the image captured with each of the three filters as instructed and a RGB (red, green, blue) true color image will be generated.
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Chapter 2: Getting Started – Basic Imaging Functions Chapter 2: Getting Started - Basic Imaging Functions When the AlphaEaseFC system computer is powered up, you can click on the AlphaEaseFC icon to automatically open the AlphaEaseFC software.
Alpha Innotech User’s Manual, version 4 2.1 Contrast Adjustment The Contrast Adjustment window allows for the best visualization possible of a sample utilizing the black, white, and gamma adjustments, as well as, image reverse and auto contrast. The image on the screen is made up of picture elements (pixels) in an array. Each pixel is assigned a brightness (or a gray scale value) level between black and white.
Chapter 2: Getting Started – Basic Imaging Functions Imaging Display Tools: Black Level, White Level, Gamma Setting with B/W/G, Linear, Log, and Equalize options To adjust any of these settings, place the cursor on the slider. Click and hold down the left mouse button while dragging the slider to a new setting. As the slider is moved along the scale, the image display is updated, along with the change in numeric value.
Alpha Innotech User’s Manual, version 4 Black Level set at 0 Black Level set at 60 White Level Adjustment The number beneath the White Level scale also corresponds to a gray scale value. When the slider is at the very bottom of the scale, this number is 255. As the slider is moved upwards along the scale, the number decreases and the image becomes progressively lighter. This is because all pixels at the specified gray level value and above are shown on the screen as white pixels.
Chapter 2: Getting Started – Basic Imaging Functions Gamma set at 1.0 Gamma set at 0.55 The Auto Contrast Selection The Auto Contrast feature will automatically scale the black and white values of an image to more tightly fit the gray scale intensity profiles (histogram). This selection will use different black and white values for different images depending upon their unique histograms.
Alpha Innotech User’s Manual, version 4 The Reverse Button The Reverse button inverts the gray levels of the displayed image, converting a positive image to negative, or vice versa. For instance, an image with black bands on a white background is converted into an image with white bands on a black background by simply clicking the Reverse button. Clicking the button a second time returns the image to its original form.
Chapter 2: Getting Started – Basic Imaging Functions Making Linear, Log, or Equal Adjustments Original image of film with default BWG settings Image of film with linear Contrast Adjustments selected. Linear provides minimum and maximum adjustment tools from 0 to 100%. Linear stretches the grayscale range of the displayed image to the maximum system dynamic range of 0-65,535 grayscales. Image of film with log Contrast Adjustments selected log provides minimum and maximum adjustment tools from 0 to 100%.
Alpha Innotech User’s Manual, version 4 Using the Contrast Adjustment Tools for Color Images AlphaEaseFC software can also import RGB color images. The AlphaEaseFC Software automatically detects this process and the Contrast Adjustment tools are configured for color image adjustments.
Chapter 2: Getting Started – Basic Imaging Functions 2.2 Tool Bar The Tool Bar window provides intuitive icons for the most common functions in AlphaEaseFC. Tool Bar Display Window The Open icon functions identically to the File Open function in the upper menu bar. This function is used to open previously saved images. Detailed instructions are available in Chapter 3. The Save and Save As icons function identically to the File Save and File Save As functions in the upper menu bar.
Alpha Innotech User’s Manual, version 4 The Saturation icon allows for a quick image display of saturation. Completely saturation black regions (gray scale 0) will turn green and saturated white regions (ie. gray scale 255, 4095, 65,535) will turn red. This is a useful tool to check for linearity of an image before analysis occurs. Saturation is a feature that is most important during the acquisition stages and is thoroughly detailed in the acquisition features of the system manuals.
Chapter 2: Getting Started – Basic Imaging Functions Clear removes any overlays currently displayed on the image. This function can be useful if annotations or other displays obscure parts of the image. Once an image is displayed, it can be printed on the default printer by clicking the Print icon in the Tool Bar window display. Most printers can be configured through the Windows operating system to be the default printer.
Alpha Innotech User’s Manual, version 4 2.3 Tool Box The Tool Box window contains an intuitive interface for performing all image enhancement and analysis functions. Tool Box Display Window The Enhancement Tools option contains the controls for enhancing and adjusting the image. This includes software filtering, false colors, zoom factors and other unique featuers.
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Chapter 3: Drop Down Menus Chapter 3: Drop Down Menus Across the top of the screen is a Windows menu bar containing several system operation functions. These include file saving and loading, edit, image, setup, overlay, file utilities, view and help functions. AlphaEaseFC Menu Bar 3.
Alpha Innotech User’s Manual, version 4 File Open This function opens an image, which has been previously saved as a TIF, GLP, BMP, PCX, TGA, PIC, JPG or Macintosh® TIFF (MAC) file. File Open Dialog Box Using the left mouse button, click on the name of the file to be loaded. That name is then highlighted in the list and appears in the text box below the File Name prompt. Alternate disk drives can be accessed using the “Look In” dialog box.
Chapter 3: Drop Down Menus File Overlay To superimpose images, use the OVERLAY function under the File menu. This function will display separate multiplexed images or a RGB color image as a compiled image with the appropriate color channel images added together. A simple way to acquire multiple images for this function is to use the Movie Mode function in image acquisition and acquire a series of identical images.
Alpha Innotech User’s Manual, version 4 File Save and Save As Save allows original images to be saved in several different formats. Save As allows images that have previously been saved to be saved in a different location or as a different file type without affecting the original image. AlphaEaseFC has the ability to save files in several formats, see the following figure: (i) File Formats: *.tif, *.bmp, *.glp, *.pcx, *.tga, *.pic, *.jpg, *.
Chapter 3: Drop Down Menus File Types: TIFF is the default file format for AlphaEaseFC files. TIFF is an acronym for "tagged image file format" and was developed as a flexible and machine-independent graphic file format. Saving as a TIFF file will allow users to double-click TIFF files from Windows Explorer and automatically launch the application on any machine that has AlphaEaseFC loaded on it. Users may customize this in the preferences section covered in section 3.
Alpha Innotech User’s Manual, version 4 An image that is saved as a Modified file permanently retains the changes to the image's Black level, White level, and Gamma setting. Annotations and any filtering performed are also saved with the image, replacing original image information with the new information. Note: If the image is saved as a Modified file it is converted to an 8-bit image. Print Setup This function displays a dialog box in which the settings for the parallel printer are specified.
Chapter 3: Drop Down Menus Print This function sends the image to the default printer specified in Print Setup. Logoff This function logs the current user out of AlphaEaseFC when security features are in use. For more information on security features, see Section 3.3. The Exit Function The Exit function closes AlphaEaseFC. To restart AlphaEaseFC from Windows, double-click on the AlphaEaseFC icon.
Alpha Innotech User’s Manual, version 4 3.2 The Edit Menu The Edit menu provides the ability to copy, crop and remove any annotations or filters that have been added to the original image. Edit Pull down menu To activate the Copy and Crop functionality, place a check mark next to EDIT ACTIVATION. This will turn the mouse cursor into a + sign that will allow you to highlight the region of interest for the image. After Edit Activation is highlighted, the desired area of interest is drawn using the mouse.
Chapter 3: Drop Down Menus Photoshop, etc.). CROP will display just the region of interest as the active window in the AlphaEaseFC interface. AlphaEaseFC interface after CROP has been selected Reset and Clear The Reset option configures the Black, White, and Gamma settings to default settings. Clear removes any annotations that are present on the image.
Alpha Innotech User’s Manual, version 4 3.3 The Image Menu The Image menu option provides the ability to perform a variety of image processing functions. Image Pull down menu Equalize The equalize option performs a duplicate function to the EQUAL option in the Contrast Adjustment Window. This is a useful function for detecting faint bands on a sample. Arithmetic The Arithmetic function is used to add, subtract, average and divide several images together to generate a compiled image.
Chapter 3: Drop Down Menus chemiluminescent samples. Subtracting images is often used to remove noise from a sample by running dark images first and subtracting them out of the final image. The most common application for quotient is for those technical users who run their own flat field corrections. This can be done using the Flat Field Calibrate selection under the Image pull down menu which will be described in detail later in this section.
Alpha Innotech User’s Manual, version 4 Flat Field Calibrate Flat Field Calibrate is a function that is used to ‘flatten’ the image so that the pixel data is even across the entire image area. This is a function that is useful for large gels and other applications that use the entire field of view for an image. Creating flats can be art in itself; there are many documents on the internet that can help users interested in this arena to create the ideal flat for the application.
Chapter 3: Drop Down Menus Image Resize The image resize function is to resize an image to a specific dimension for use in graphical presentations. You have the option to ‘Preserve aspect ratio’ to avoid image dimensional distortion, or you can deactivate this function and configure the image resolution to the desired Width and Height dimensions. Image Resize dialog box Note: It is recommended that you DO NOT perform quantitative analysis on resized images.
Alpha Innotech User’s Manual, version 4 3.4 The Setup Menu This menu customizes the system settings by allowing users to save default parameter preferences and customize the software settings. Setup pull down menu Default Parameters Default parameter files eliminate the need to readjust the system settings each time the program is used, and can be especially useful if more than one person works with the program. The default file used by AlphaEaseFC when it initially loads is called INNOTECH.DEF.
Chapter 3: Drop Down Menus Save Defaults Dialog Box Once any of the system settings have been changed, a new default file can be created. To save a default file, enter a file name by typing in the text box below the File Name prompt. If it is necessary to change the directory or drive to which the file will be saved, select a different directory under the Save In pull down menu. AlphaEaseFC will automatically add the appropriate 3-character extension. Click on the SAVE button.
Alpha Innotech User’s Manual, version 4 Security When the system is shared by a number of people or laboratories, a method of keeping track of its use may be helpful. The Security feature allows various levels of security and user log functions. We suggest one user be designated as the supervisor of the system. This individual should refer to Appendix C, which describes the security features in detail.
Chapter 3: Drop Down Menus Print Mode AlphaEaseFC software provides custom printing options. Printing can be achieved in three different methods. Setup Print Image Info Dialog Box Full Image: Prints the original image. Does not print zoomed images or images overlaid with data screens. Screen Dump: Prints the imaging area. Well suited for printing images overlaid with data screens and/or graphs, zoomed images, etc. Image Window: Prints the highlighted window.
Alpha Innotech User’s Manual, version 4 Preferences In order to change the preferences of the system, you will need to find the administrator of the AlphaEaseFC software program to log in. If you do not have an administrator of the AlphaEaseFC software program, see Appendix C in this manual.
Chapter 3: Drop Down Menus 2) Image Acquire – Inverts the image seen by the camera and adjusts the ROI values (For the FluorChem 5500 only). Caution: We do not recommend that you make any changes to these settings without contacting technical support first. Some features on this tab are not currently implemented in the software. 3) Cabinet Settings – Used for adjusting the port settings and customizing filter positions on the cabinet.
Alpha Innotech User’s Manual, version 4 4) Auto Enhancements – Used to customize the Auto Enhance Levels located in the Image Enhancement tool box. To make changes to the preferences, select your change by typing in a new value or select/de-select the appropriate box with a check mark and then select apply. Some settings may require that the software be re-started for the change to take effect.
Chapter 3: Drop Down Menus 3.5 The Overlay Menu The Overlay menu provides a means of saving and retrieving annotation overlays. This is especially useful when a standard gel format is run repeatedly. Lane numbers, molecular weight marker sizes, and other pertinent information can be stored as an Overlay file and retrieved at a later date. This eliminates the need to re-enter the information each time a new image is captured.
Alpha Innotech User’s Manual, version 4 The current directory is the one in which the new overlay file will be saved. necessary, change the directory or drive as described in Section 3.1. If Once a name has been entered and the appropriate directory has been accessed, click the SAVE button to save the overlay file. Loading an Overlay The Load Overlay function allows Overlay files to be retrieved and applied to the image currently displayed.
Chapter 3: Drop Down Menus Loading/Saving Spot Denso Overlays When the Spot Denso function in the Analysis Tools is being used, the overlay menu will contain two additional functions to load and save Spot Denso overlays: Overlay Menu for Spot Denso Analysis Tools Spot Denso Overlays work the same as the annotative overlays described above except that they have a different 3-character extension: Overlays = .ovr Spot denso overlays = .
Alpha Innotech User’s Manual, version 4 3.6 The Utilities Menu A number of functions are now handled by Windows programs. To access many of these programs while in AlphaEaseFC, open the Utilities menu and select the program of choice. Utilities pull down menu Explorer Windows Explorer allows access to files and other information saved on the local machine or the network, if applicable.
Chapter 3: Drop Down Menus Notepad The Notepad is a blank screen that allows the user to make notes about the experiment and save them as an ASCII file. The Notepad is useful for saving any imaging comments or experimental conditions with the saved image for future reference.
Alpha Innotech User’s Manual, version 4 3.7 The View Menu The View function provides the ability to control the display of the on-screen control tools as well as provide image enhancement abilities.
Chapter 3: Drop Down Menus Status Bar ToolBox Window These control windows automatically open when AlphaEaseFC is launched for additional ease of use and to generate a common ‘look and feel’. However, if you would like to remove any of these windows they can be turned off in the View menu by just deactivating the check mark next to the item that you would like to remove from the screen.
Alpha Innotech User’s Manual, version 4 3.8 The Help Menu Help pull down menu On-Line Note On-line help is available in the ON-LINE NOTE section of the help menu. Common tips are included for both Enhancement Tools and Analysis Tools detailed in Chapters 4 and 5 respectively. About To display system information, select the About option in the Help menu. This button accesses a pop-up box. This box shows the system serial number and software version number.
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Chapter 4: Enhancement Tools Chapter 4: The Image Enhancement Tools in the Toolbox Image enhancement tools are contained within the Tool Box as indicated. This tool set allows the user to zoom the image, rotate-flip the image, show the image histogram, perform automatic image enhancement, annotate on the image, display false colors, apply software filters, and activate the Movie function. 4.
Alpha Innotech User’s Manual, version 4 On-screen Zoom tools is also available located on the main ToolBar Window in. This function duplicates the Zoom tool in Tool Box, and also allows for Image Drag to easily pan the image during any analysis functions located in Tool Box Analysis Tools. Zoom icons in ToolBar Image Drag Icon in ToolBar 4.2 The Rotate / Flip Tool The Rotate / Flip Tool The Rotate / Flip tool is found in Tool Box, Enhancement Tools.
Chapter 4: Enhancement Tools Rotated 11 degrees clockwise Rotate / Flip box at 11 degrees Rotated -11 degrees (Counterclockwise) Rotate / Flip box at –11 degrees Image rotated 11 degrees and flipped vertically Rotate / Flip box at –11 degree and vertical Flip button pressed 4.3 Histogram The histogram is a graphical display of the proportion of pixels assigned to each of the 4,095 gray levels. This tool is found in Tool Box, Enhancement Tools.
Alpha Innotech User’s Manual, version 4 A Coomassie blue-stained protein gel visualized with a white light box has a histogram reflecting mostly bright pixels: Histogram of a typical Coomassie gel Most of the pixels are found in the light portion of this histogram. The dark bands represent a small number of pixels and include a variety of gray values, and therefore do not show up as a single peak. The histogram function is particularly useful to verify that an image spans the maximum range of gray levels.
Chapter 4: Enhancement Tools Original Image Auto Enhance Level 2 Image Original Auto Enhance Toolbox Auto Enhance Toolbox with level 2 4-5 Auto Enhance Level 9 Image Auto Enhance Toolbox with level 9
Alpha Innotech User’s Manual, version 4 4.5 Annotations The annotation tools, found in ToolBox, Enhancement Tools, include a number of different options for adding text (including Greek symbols), drawing arrows and otherwise marking an image. Note that these tools are for annotation only. For information on drawing objects for quantitation purposes, see Chapter 5.
Chapter 4: Enhancement Tools Line Thickness The PEN WIDTH menu specifies the thickness of lines when using the freehand, lines, box and circle drawing tools. Click on the appropriate checkbox for the desired width. All annotations subsequently entered will appear at that width. Pen Width Selection Tools Line Types The PEN STYLE menu specifies the style of lines when using the freehand, lines, box and circle drawing tools. Click on the appropriate checkbox for the desired style.
Alpha Innotech User’s Manual, version 4 Text Background and Font The TEXT STYLE menu specifies the style of text. Click on the appropriate checkbox to show text with or without a background. An opaque background is useful if annotations will be made on an image that has wide variations in gray scale. By using an opaque background, text will not be “lost” in the background of the image. Text Style Selection Tools This is also the window in which specific font is chosen.
Chapter 4: Enhancement Tools Text Angles In the TEXT ORIENT window, select whether text should be oriented vertically, horizontally, or at an angle (in 15° increments). Text Orient Selection Tools Note: only rotate fonts that are True Type (indicated by TT in front of the name); other fonts (such as Courier and Fixedsys) do not re-scale properly, giving unpredictable results.
Alpha Innotech User’s Manual, version 4 The button labeled with a pencil icon allows the user to draw lines freehand. After clicking on the pencil, move the cursor to the correct position on the image to begin drawing. Press and hold the left mouse button. Using the mouse, move the cursor as if it were a pencil. When finished drawing, release the mouse button. The button labeled with a diagonal line and arrow draws arrows and straight lines.
Chapter 4: Enhancement Tools Annotated image showing: freehand drawings, lines with various characteristics, circles, squares, text with various characteristics The Editing Tools When the cursor is in “edit” mode, it can be clicked on an object to select it. (Note: the cursor can be toggled between “edit” and “drawing” modes by clicking the right mouse button.) When an object is selected, small square boxes appear at the corners.
Alpha Innotech User’s Manual, version 4 Once an object or group of objects has been selected, clicking on the Cut tool deletes it from the image. The Copy tool makes an exact copy of the selected object. The new object becomes the selected object, and can be repositioned by placing the cursor within the object’s boundary and moving it to the desired location. The Horizontal Alignment tool aligns annotations in a straight horizontal line. This is especially useful for labeling lanes, etc.
Chapter 4: Enhancement Tools 4.6 False Color These tools consist of eleven pre-defined color palettes that can be applied to an image. To select a palette, simply click on one of the four buttons labeled GRAY PALETTE, SATURATION, NEXT or PREVIOUS. False Color Selection Box When a palette is selected, its range of colors is displayed to the left of the palette buttons and automatically applied to the image. To apply a different palette to the image, click the NEXT or PREVIOUS buttons.
Alpha Innotech User’s Manual, version 4 Palettes 2 through 11 These are color substitution palettes in which the gray levels are translated into different color ranges. These palettes can be useful to help distinguish features and highlight details on an image. Palette 2 maps the gray scale levels to a red/green/blue palette. Values of 0 are mapped to red; saturated to blue, and values in between to green. Palette 3 maps the gray scale levels to a red/green/blue palette.
Chapter 4: Enhancement Tools 4.7 Image Filters Filters Toolbox with 3-D (contour) selected AlphaEaseFC™ includes a variety of enhancement filters that can improve the appearance of an image. Some filters sharpen detail, others smooth and reduce random noise. Still others help visualize edges and separate closely spaced bands or objects. Depending upon the unique characteristics of an image, the results of each filtering operation vary.
Alpha Innotech User’s Manual, version 4 General Information on How Filters Work To enhance an image, filters change the value assigned to each pixel. The new value assigned to a pixel is determined based on the values of the other pixels in its local vicinity (or “neighborhood”). The neighborhood is a two-dimensional matrix of pixel values, where each dimension has an odd number of elements. The "pixel of interest" is the one at the center of the neighborhood.
Chapter 4: Enhancement Tools Sharpening Filters These filters can increase image sharpness and provide edge enhancement. However image noise may be enhanced as well. These filters accentuate the high-frequency details of an image while leaving the low-frequency content intact. High frequency portions of the image get brighter while low frequency portions become black. Sharpen level 9 (high) has the largest effect on the image. Sharpen level 5 has an intermediate effect.
Alpha Innotech User’s Manual, version 4 Noise Filters These filters are particularly effective in eliminating random noise contained in an image and produce less blurring than the smoothing filter described below. This filtering process uses the values of the pixels contained in the area surrounding a pixel to determine the new value given to the pixel of interest.
Chapter 4: Enhancement Tools Note: After applying the horizontal edge filter the entire image may appear black, and might require reducing the White level in order to better visualize the results. Vertical Edge Filter This filter (Vert. Edge) brightens vertical edges using the approach described for the horizontal edge filter (see above), except that the image is shifted horizontally before the shifted image is subtracted from the original.
Alpha Innotech User’s Manual, version 4 Horizontal Edge Vertical Edge 4.8 Movie Mode If kinetic, multiplex, color, or chemiluminescence experiments are desired where you wish to have the system automatically capture several images at preset exposure times, preset time delay between images, preset lighting sources, and preset filter choices, the MOVIE box can be clicked in ToolBox, Enhancement Tools. (Movie Setup is also accessible in camera setup acquisition screen.
Chapter 4: Enhancement Tools Once MOVIE SETUP is selected, a display box will appear for independent control of all lighting, filters, and exposure delay for each image frame. The TOTAL FRAMES setup provides you with the ability to determine how many individual frames (images) you want for the movie. There is a maximum of 50 frames (images) and a minimum of 1 frame that can be captured with each movie. The FRAME selection is used for setting up the conditions for each frame (image).
Alpha Innotech User’s Manual, version 4 Once all images have been captured, the above Movie display box will become displayed. The remaining buttons will perform the following tasks: REC PLAY STOP PAUSE REW (rewind) REV (reverse) FWD (forward) DEL (delete) LOAD SAVE Move to Camera Setup and Preview, Movie Record setup functions to record a movie. Display a continuous loop ‘movie’ of all of the captured images.
Chapter 4: Enhancement Tools Movie Mode: Save/Load Movie Mode setup routines Two buttons “Save Setup” and “Load Setup” allow you to save and load all Movie Mode setup parameters. Files are saved as *.mvf files. Load Setup Save Setup Frame Stacking At the top of the Movie Set-up window is an option for stacking frames. If this selection was chosen during acquisition of the image Stack Frames will use all previous exposure information to sequentially add images to one another.
Alpha Innotech User’s Manual, version 4 Capturing a Color Image Using the Movie Function A color image can be generated by acquiring three images each taken with a red, green and blue emission filter. Once saved, these images are then combined in the Overlay pull down menu. Open the image captured with each of the three filters as instructed and a RGB (red, green, blue) true color image will be generated.
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Chapter 5: Analysis Tools Chapter 5: The Image Analysis Tools in the Toolbox Analysis Tools in the ToolBox Image analysis tools are contained within the Tool Box as indicated. This tool set allows the user to perform manual and automatic counting of colonies and cells, gel scoring, perform ruler measurements, high density array analysis, molecular weight determinations, 1D-multiple lane densitometry, and spot densitometry. 5.
Alpha Innotech User’s Manual, version 4 ARRAY Toolbox Once these parameters have been set, a template can be saved in a default file and recalled at a later time. (See Section 3.5 for details on saving defaults.) Setting up an ARRAY Template To access the ARRAY tools, open ToolBox, Analysis Tools and click on the ARRAY button. A template is displayed on the image, depicting the layout of the objects to be measured. Up to 10,000 objects in a 100x100 array can be measured.
Chapter 5: Analysis Tools Analyze arrays with Circles or Squares Clicking this button will switch between “Use Square” objects and “Use Circle” objects. The controls labeled Hori. Wells and Ver. Wells let the number of horizontal and vertical objects be set (to a maximum of 100 and 100, respectively). The controls labeled Center and Outer let the size of the center and outer circles be set. Click on an arrow to increase or decrease the number of objects per row or column, or the size of the objects.
Alpha Innotech User’s Manual, version 4 A Skewed Template Specifying the Areas to be Measured For microtiter plate analysis, the toolbox controls labeled Center and Outer specify the size of the inner circle and outer torus scanned to measure each well. The number in the center of each of these controls indicates the diameter of the circles. Click on an arrow to increase or decrease the diameters. As the number is adjusted, the objects on the template grow larger or smaller, reflecting the changes.
Chapter 5: Analysis Tools The values are the average pixel values within the object, adjusted to a scale of 0 to 100. (0 corresponding to black pixel values, and 100 corresponding to white pixel values.) If both a center and an outer circle have been specified, two values are displayed. The upper value corresponds to the inner circle and the lower value corresponds to the torus.
Alpha Innotech User’s Manual, version 4 Output Dialog Box To create a file, click in the circle next to the File prompt and enter a file path and name for it in the text box. To send the data to a printer, click on the Video Printer or Default Printer buttons. To send data to the clipboard for importing to another program, click on the Clipboard button. To Append your data to the same file to allow for easier statistical analysis, click on Append and type in the file name.
Chapter 5: Analysis Tools 5.2 Molecular Weight Determination Introduction The button in ToolBox, Analysis Tools, labeled MOL. WEIGHT, opens a set of tools for entering the values of known molecular weight markers and determining the molecular weights of unknown bands on the image. When the MOL. WEIGHT button is selected, a function box appears in the area to the lower left of the screen, and a data box appears on the image.
Alpha Innotech User’s Manual, version 4 At the top of the data box is a bar with two menus: Marker and Query. The area below the menu bar is divided into two sections. Molecular weight marker data is displayed in the upper section and the calculated or query data is displayed in the lower section. Entering Known Molecular Weights for Markers An unlimited number of molecular weight standards can be defined, either all in one lane, or in multiple lanes.
Chapter 5: Analysis Tools • • • The position corresponds to the band's location along the y-axis, ranging from 0 to 1030. The value displayed under the Mol. Wt. heading is the molecular weight value entered for a known band or the value calculated for an unknown band. The Rf value for each band is also displayed. Unless otherwise specified, AlphaEaseFC assumes the origin (Rf value = 0.00) is located at the top of the screen, and dye front (Rf value = 1.00) is at the bottom of the screen.
Alpha Innotech User’s Manual, version 4 To delete all the markers and start over, select the Clear Markers function in the Marker menu. Determining Molecular Weights of Unknown Bands After marker values are entered, the molecular weight of any unknown band can be determined. Manually Selecting Bands To indicate unknown bands manually, select the Add Band function from the Query menu. Just as in Add Marker above, a line will appear attached to the cursor.
Chapter 5: Analysis Tools To delete an unknown band indicator, point the cursor at the band and click the left mouse button. The band in question is highlighted. (Alternatively, clicking in the data table highlights the band’s information and selects it on the image.) Click on the Delete Band function in the Query menu. The band will be deleted, as will the band's data in the query data table. Bands entered after the one deleted are renumbered on the image and the data table.
Alpha Innotech User’s Manual, version 4 Once the file has been selected, click on the OK button to load the file. (Alternatively, double-click on the file name.) The dialog box disappears and a new dialog box appears. Auto Load Dialog Box This box gives the file name selected, as well as the number of markers contained within the file. It also give the user the option of auto-loading the file. If Yes is selected, a vertical line will appear attached to the cursor.
Chapter 5: Analysis Tools Adding a Set of Molecular Weight Markers to the Library If the same set of molecular weight markers will be run repeatedly, there is no need to enter values on each new image. Instead, save the molecular weight values in a file and apply them to the marker lane of any subsequent images. To save a set of molecular weight standards, click on Write Markers to File in the Marker menu after the marker values have been entered.
Alpha Innotech User’s Manual, version 4 Output Dialog Box To create a file, click in the box next to the File prompt and enter a path and file name for it in the text box. To send the data to a printer, click on the Video Printer or Default Printer buttons. To send data to the clipboard for importing to another program, click on the Clipboard button. When the appropriate output source has been selected, click on the OK button to send the data.
Chapter 5: Analysis Tools REVERSE button described in Chapter 3, this function does not alter the appearance of the image. This function is especially important when using Snap to Peak. If INVERT is incorrectly set, the cursor will snap to areas between peaks rather than finding peaks. Calculating Rf Values To obtain accurate Rf values, specify the location of the wells (i.e., origin) and the dye front using the functions Set Well Pos./Start and Set Dye Front/End both found in the Marker menu.
Alpha Innotech User’s Manual, version 4 The Graph Tool The Graph function is found in the lower left corner of the Molecular Weight tools box. Molecular Weight Tools Clicking in the Graph box displays the semi-log graph of the molecular weight data that is used to calculate the molecular weights of unknown bands. (To remove the graph window from the display, click on the Graph checkbox a second time.
Chapter 5: Analysis Tools 5.3 1D-Multi (Lane Densitometry) The 1D-Multi button in ToolBox, Analysis Tools accesses a set of densitometry tools with which bands on a gel can be scanned and quantitated in a lane format. There are two different ways in which this can be done, Auto Lane and Auto Grid. Auto Grid allows the user to manually define the lane number, lane shape, and scan width of the Grid.
Alpha Innotech User’s Manual, version 4 Setting up the Lane Template When the 1D-Multi button is clicked, a lane template appears on the image. The red lines indicate the borders of adjacent lanes. The green lines define the Scan Width (described below). Under the heading Grid Controls, the number of lanes can be specified. The number under the title Lanes indicates the number of lanes and can be changed by clicking the right (increase) or left (decrease) arrows.
Chapter 5: Analysis Tools The Invert check box should be selected if the image being analyzed is a dark sample on a white background. These are usually colorimetric gels such as Coomassie Blue. Reversing the image on the Contrast Adjustment window would NOT require the user to select invert. Specifying the Scan Width The Scan Width (the green line within each lane) is the area from which the pixel density is measured. The control labeled WIDTH sets the Scan Width.
Alpha Innotech User’s Manual, version 4 Recommended Scan Width (middle of bands scanned; “smiling” edges excluded) Scanning the Image When the AUTOGRID button is clicked, the densities of each lane are measured, and information is displayed in several quadrants of the screen: • The graphs for each lane scanned are displayed in the lower left. • The active graph is displayed in the upper quadrant of the screen. • Peak information for the active graph is displayed in a data table in the lower right.
Chapter 5: Analysis Tools Graph Display Graphs representing all the lanes on the template are shown in the lower left-hand quadrant. When AUTOGRID is first selected, the active graph is the one corresponding to Lane 1. To select a different lane, simply click on its graph, and it will fill the Active Graph quadrant.
Alpha Innotech User’s Manual, version 4 menu. To change the background color of the graph, choose Background Color from the Options pull down menu. AlphaEaseFC automatically detects the peaks and integrates the area under each peak. Adjustments that can be made manually are described below. The Data Window Once a peak is defined, its integration data and associated information are displayed in a table located in the lower right quadrant of the screen.
Chapter 5: Analysis Tools Adjusting Peak Detection Parameters: Minimum Area, Height and Width Once the graph to be analyzed has been selected and is displayed in the upper right of the screen, the tools in the lower left of the screen change to allow adjustment of the automatic peak finder parameters.
Alpha Innotech User’s Manual, version 4 Minimum Width is 18 Peak Detection Algorithms If these adjustments do not allow AlphaEaseFC to adequately find the peaks on a sample, the peak detection algorithms can be manually adjusted. Select Peak Detection Controls from the Options menu. The following window will appear: Peak Detection Controls Dialog Box Adjust these controls but clicking on the increase and decrease arrows.
Chapter 5: Analysis Tools Use points for angle measure refers to the number of data points that will be scanned at once to determine an angle. The larger the number of points, the less sensitive the algorithm (ie. the fewer peaks that will be defined). Editing Peaks: Editing Peak Boundaries The boundaries of existing peaks can be readjusted to make the peaks wider or narrower. To adjust either peak boundary, click on the peak to select it.
Alpha Innotech User’s Manual, version 4 Visualizing Peak Placements To determine exactly where a peak is located relative to the original image, it is often helpful to see the graph and the gel image in the same orientation. The Show Strip function (found in the Options menu) displays the area of the gel that has been defined as a lane across the length of the x-axis.
Chapter 5: Analysis Tools Adjusting the Baseline: As a default, the baseline value is set to Auto Base. As the baseline is adjusted, the values for all peaks are updated to reflect the new baseline. All of the following functions are found in the Background Subtraction menu: Auto Base This method breaks the red line defining the left boundary of the lane into sixteen regions, and outputs sixteen points which reflect the average backgrounds across those sixteen regions.
Alpha Innotech User’s Manual, version 4 This method requires that you have performed band detection first. The background is taken as the line between the edges of the bands in the lane. You enter a value (see below in setting parameters) of maximum slope in the accompanying entry box to avoid situations where the edge between overlapping bands, which is not at the background intensity, causes the background to climb too high.
Chapter 5: Analysis Tools Reset Base Activating the Reset Base function will move the baseline to the 0 position (no background). Unlike Horizontal Base the baseline cannot be moved. Base Subtract This selection will subtract the current baseline from the intensity value line. Un-check Base Subtract to deactivate this feature. Interpreting 1D-Multi Data Alpha Ease™ provides a variety of tools to help interpret 1D scan data.
Alpha Innotech User’s Manual, version 4 Using the V.LINE The line also contains hash marks, which represent pixel intensities. Use this information for a quick comparison of peak heights. Note: The hash marks are most useful when only four graphs are shown so that enough detail is displayed. To accomplish this, click on the selection button in the upper left corner of the window and deselect Show All Lanes. Alternatively, the intensity value at the point where the V.
Chapter 5: Analysis Tools The Smooth Data function (found in the Data menu) minimizes single pixel background noise in a lane. original graph smoothed graph When this function is selected, the menu option changes to “Unsmooth Data.” Selecting this undoes the smoothing operation. The Invert Button The INVERT function reverses the gray scale assignments, so that zero corresponds to white and 4,096 corresponds to black.
Alpha Innotech User’s Manual, version 4 When the Invert Data function is activated from the pull down menu, the menu option changes to “Uninvert Data.” Selecting this undoes the inverting operation. Unlike the REVERSE button described in Chapter 2, this function does not alter the appearance of the image, but does change the data. Overlaying Graphs When comparing the bands in one lane with those in another it is often convenient to display the graphs so that one is superimposed over another.
Chapter 5: Analysis Tools Molecular Weight (MW), Mass and Band Scoring integrated into 1D-Multi A new “Analysis” menu item has been added to 1D-Multi. Under this menu the user now has the option of calculating Molecular Weight and/or Mass. The user can also access the Band Scoring feature from this menu. This unifies four important analysis tools into one area of the software and reports Mass, Rf, and MW on one report. Molecular Weight and Mass integration: FC v3.
Alpha Innotech User’s Manual, version 4 Checking the checkbox activates the “Enter total mass per lane” field and reveals the choices for calculating the Mass values. Procedure: If you would like to calculate Mass based on your MW Standard Lane follow these steps. 1) Check the “Calibrate Mass using MW std lane” checkbox. 2) Enter the total mass that was loaded into the MW standard lane into the ‘total mass per lane’ field. 3) Select the Calculation method to be used for Mass calculation.
Chapter 5: Analysis Tools The Mass for all bands is now calculated based on the total mass entered for the MW standard lane and reported in one report. Note Legend: Describes which lanes/bands make up the MW and Mass standard curve values. Also notes which regression method has been used to calculate the MW and Mass curves.
Alpha Innotech User’s Manual, version 4 Saving and Loading Mass Standards Mass standard files can be loaded onto any image and saved for future reference. To load a marker, select Mass Standard under the Analysis pull down menu. A Mass Standard dialog box will show on the screen: Select Add Mass Std to add the first marker. Pull the cursor onto the image to select the band of interest and click once on the left mouse button to add the marker.
Chapter 5: Analysis Tools the file to be saved in the directory of interest. Select apply to calculate the unknown values of the markers in the rest of the lanes. Saving a Mass Standard file in Autogrid… And in Autolane Mass Standard files can be loaded by selecting Add Mass Std in either autolane or autogrid. Browse through the directories to find the marker file of interest, then select the file name and click on open.
Alpha Innotech User’s Manual, version 4 Band Scoring Lane #’s to score: Choose the lanes to score by highlighting the lanes to be included. Score based on: Select the parameter to base the scoring on. The choices are Area, Mass, and Height. The Area and Height are calculated automatically in 1D multi. In order to use the Mass as the scoring parameter the user must calculate the Mass of every band by applying a Mass Standard. This can be done separately or in conjunction with MW calculations.
Chapter 5: Analysis Tools • High/Medium/Low/Absent: This method is used to classify bands into groups based on Area, Mass, or Height. The researcher will determine break points for each classification (high, medium, low, and absent). The software will place each band into one of these classifications depending on the user entered criteria. • Additional Criteria: When choosing the High/Medium/Low/Absent method the user must enter in values to use as break points for each of these classifications.
Alpha Innotech User’s Manual, version 4 Outputting Quantitative Data The OUTPUT button (located in the 1D-Multi tools section at the lower left of the screen) transfers the peak integration data table to a printer, to the computer’s clipboard, or to an ASCII file. Click on the appropriate box to specify where the data should be sent. (If creating a file, enter a name for it in the text box.
Chapter 5: Analysis Tools The file can also be exported to other software programs, such as spreadsheets, for further peak analysis. To access the file, enter the desired program (such as Microsoft® Word or Microsoft® Excel). From the program’s “Edit” menu, select “Paste”. The information will be pasted into the open document. Exiting 1D-Multi Activating the EXIT function dismisses the graphs, and exits Scan mode, leaving only the image with the lane template on the screen.
Alpha Innotech User’s Manual, version 4 Vertical Lanes or Horizontal Lanes If the gel image has vertical lanes the Vertical Lanes option should be checked. If the image’s lanes run horizontally then the Horizontal Lanes option should be chosen. The software will not automatically detect the lane orientation so the user should aware of this parameter. Sensitivity Adjustment Bar This feature changes the number of bands the software will find using a sensitivity scale of 0-9.
Chapter 5: Analysis Tools Auto Lane Profile and Data Table Data Table and Editing of Auto Lane When the AUTO LANE button is clicked, the lanes are designated, bands found and the information is displayed in three windows: the upper window is the Active Graph, the lower left is the Graph Display and the lower right is the Data Window. The 1D-Multi tab will also change to display the Auto Lane Editing functions.
Alpha Innotech User’s Manual, version 4 AlphaEaseFC uses the following color-coding: • the active graph is shown in white • graphs that have been viewed already are shown in yellow • graphs that have not yet been viewed are shown in gray All of the lanes can be displayed in the graph display quadrant by selecting “Show All Lanes” under the Options drop down menu in the Profile and Data window. By deselecting “Show All Lanes” only the first several lanes are shown providing more detail.
Chapter 5: Analysis Tools Data The data drop down menu allows for the user to superimpose different lanes onto the Active Window using the Overlay Control selection. (See Auto Grid Overlay Control above for detailed instructions.) Output Data This data can be outputted in several ways. Select Output from the Data menu. This tool allows you to output the 1D-Multi data to a printer, the clipboard, or to a file.
Alpha Innotech User’s Manual, version 4 Baseline Background subtraction can be preformed using this menu. (See Auto Grid Baseline Control described previously in this section for detailed instructions). Please note that background subtraction in AutoLane can have a dramatic effect on how many peaks are recognized as bands. Try changing the background subtraction to optimize band recognition. Options Background Color allows the user to select a variety of different background colors for the Active Graph.
Chapter 5: Analysis Tools The Data Window Once a peak is defined, its integration data and associated information are displayed in a table located in the lower right quadrant of the screen. The data table is updated any time a peak is deleted or added, peak boundaries are redefined, or the background value is reset. Example of an Auto Lane Data Table PEAK is the number assigned to each peak on the graph beginning on the left and moving right.
Alpha Innotech User’s Manual, version 4 Add Lane This feature allows an additional lane to be placed on the image. Once the button is selected the additional lane can be viewed and positioned on the image. A left mouse click will place the lane on the image. The data tables will readjust accordingly, and the bands within the lane calculated. As with most AlphaEaseFC features a right mouse click will reactivate the function and another lane will be ready to be added.
Chapter 5: Analysis Tools Display Options This feature allows the user to change the visual display of the lanes and bands on the analyzed image. • Under Band Representation, the Cross selection designates bands with a cross symbol. The Box selection will display a 2 dimensional box around each of the bands. • Lane Property gives the option of either showing the lane name by number (Numerical) or by letter (Character).
Alpha Innotech User’s Manual, version 4 Gel Smiling Correction with Rf Curve Tool The AutoLane portion of the software contains the Rf Curve tool which allows the user to adjust for gel smiling or curvature. Using this tool will increase the accuracy of Molecular Weight calculations and Automatic Band Matching. After the Rf Curves have been applied a new column of data will be generated, Adjusted Rf. The Rf.
Chapter 5: Analysis Tools Adjust the curve by dragging the yellow anchor points. Adding/Removing Anchor Points Anchor points can be added by clicking on or near any Rf Curve. When the cursor changes to a wand it will add an anchor point to the nearest curve by clicking the left mouse button once. Additionally, when the cursor is a wand, clicking once on the right mouse button will remove the curve.
Alpha Innotech User’s Manual, version 4 enough to an Rf Curve and will apply a new curve. If the cursor is a plus sign, then the cursor is over an anchor point, and clicking once on the right mouse button will remove the point. To move a curve far from its origin click and drag on the curve. Adjusting Anchor Points Anchor points can be adjusted or moved by clicking and dragging the anchor point from one location to the next. Additional Rf.
Chapter 5: Analysis Tools Prerequisites for Band Matching Prior to performing band matching, a number of steps must be followed. 1. Lanes and Bands must be detected and edited through the AutoLane feature. 2. If Molecular Weights are to be calculated, molecular weight markers must be loaded prior to band matching. Refer to the section within 1D-Multi on loading molecular weight markers. 3. (Optional) A new Rf curve tool has been added to AlphaEaseFC.
Alpha Innotech User’s Manual, version 4 Profile and Data window. These values result from the gel smile/curvature rather than the absolute vertical Rf value. Adjusted Rf is calculated based on the addition of M.W. markers and/or the Rf Curve lines which serve to correct the Generic Rf values for gel smiling. Generic Rf Matching: When Generic Rf Matching is selected the software will match bands based on the data values in the Rf column of the Profile and Data window.
Chapter 5: Analysis Tools Manual Band Matching Band Matching Dialog window Manual band matching tools are useful for quickly matching lanes with only a few bands or for manual edits and corrections to Automatic Band Matching results. New Band Type Select the New Band Type tool by clicking the New Band Type button. A Band Type is a band with a unique Rf. or Adj. Rf. Value. Each band within a lane will be part of a band type and no two bands within a lane can be of the same band type.
Alpha Innotech User’s Manual, version 4 Band Matching Results Display Results After bands have been matched a table is generated with the matching results. Click the Display Results button to view the matching results. Band Matching Results window displaying the similarity matrix Viewing Result Tables The match results can be viewed in one of four configurations. Select the configuration desired under the View menu. In each view Band Type is identified in the left most column.
Chapter 5: Analysis Tools Exporting and Printing Band Matching Results Print and Export are selectable under the File menu. Select Print to send the report to a local or network printer. Select Export to export the results to the clipboard or to file. When exporting to a file there are two file types that can be saved, standard text file (*.txt) or a comma separated value file (*.csv). When exporting to Excel, use the *.
Alpha Innotech User’s Manual, version 4 Similarity Matrix The similarity matrix is a graphical display of the similarity between lanes. Exporting and Printing the Similarity Matrix Print and Export are selectable under the File menu. Select Print to send the report to a local or network printer. Select Export to export the results to the clipboard or to file. When exporting to a file there are two file types that can be saved, standard text file (*.txt) or a comma separated value file (*.csv).
Chapter 5: Analysis Tools Distance Matrix Select a method from the Distance Matrix pull down for calculating similarity. The choices are Dice Coefficient, Jaccard Coefficient, Pearson Coefficient, and Frequency Similarity. These are standard statistical algorithms, references can be found in most statistical text books. Cluster Method Select a method for calculating the clustering from the Cluster Method pull down.
Alpha Innotech User’s Manual, version 4 Grab Tool The grab tool allows the user to navigate the entire dendrogram in a zoomed display by clicking and dragging to window. Zoom In/Out Tools The zoom in and out tools allow zoom control over the dendrogram window. Fit to Window Tool Selecting this button will fit the dendrogram to the current window size. 5.
Chapter 5: Analysis Tools in the image area, indicating that AlphaEaseFC is in “drawing” mode. Move the "+" to the corner of the object to be measured, click the left mouse button, move the cursor, and release the mouse button when the box (or circle) surrounds the object. Hint: to draw a perfect circle around a portion of an image, first visualize a square surrounding the area of interest. Position the mouse in the upper left hand corner of the square.
Alpha Innotech User’s Manual, version 4 Magic Wand and AutoSpot These two selections are automated detection features designed to recognize sample in an image and draw Object boxes around it. They are found under the Auto Detect heading in the main Spot Denso. Tab. Once an object is created the dimensions can be altered and the background subtracted similar to manually drawn objects.
Chapter 5: Analysis Tools the spot the software will define. The greater the sensitivity number the more of the object the software will find. **HINT: Selecting a brighter pixel within the band (or spot) will have magic wand draw a tighter area of interest. Correspondingly, a less intense pixel will draw a larger perimeter around the selected spot** Spot Type Under the Spot Type heading the Bright Spots selection should be checked if the image contains bright spots on a dark background (e.g.
Alpha Innotech User’s Manual, version 4 Auto Spot Area of Interest After selecting AutoSpot an area should be drawn on the image by clicking and draging the mouse. The smaller and more defined the Area of Interest is, the better the resulting data. The area can be manipulated using the manipulation tabs at the edge of the red box. Options The Options selection allows either a box outline to be drawn around the spots or a border outline.
Chapter 5: Analysis Tools Auto Spot Options Bright Spot vs Dark Spot The Bright Spots selection should be checked if the image contains bright spots on a dark background (eg ethidium bromide stained fluorescent gels and chemiluminescent blots). The Dark Spots selection should be made when the sample contains dark sample on a light background (film, commassie blue protein gels etc..).
Alpha Innotech User’s Manual, version 4 Target Size The user can input the minimum and maximum area in pixels of the spots or bands of the desired targets. The default values are 1 pixel minimum and no limit for maximum pixels. Get Data Get Data is selected once a satisfactory spot outline is achieved. This will convert all drawn objects into standard Spot Densitometry objects with associated density numbers. Manipulating Objects: Selecting Objects To select an object, click on it with the mouse.
Chapter 5: Analysis Tools To make multiple copies of an object, continue to click on the COPY button until the desired number of copies is displayed. Moving Objects An object can be moved to fine-tune its position simply by clicking on it, holding down the left mouse button and dragging it to the desired location. Deleting Objects To remove an object or set of objects, select the object(s). Then, click on the DELETE button in the toolbox.
Alpha Innotech User’s Manual, version 4 To retrieve the size and position of objects, select Load Spot Denso Overlay from the Overlay pulldown menu. From the dialog box, select the file of interest. The objects will be displayed on the current image. Object Libraries AlphaEaseFC Software contains a library of Spot Denso objects that can be accessed through the Load Spot Denso Overlay function described above: 32SLOT.SPO 48CIRCLE.SPO 6CIRCLE.SPO 96CIRCLE.
Chapter 5: Analysis Tools Example Of A Spot Density Data Window If this window obscures a region of interest, resize and/or move it following Windows® conventions. Alternatively, close the data window by clicking in the checkbox next to the HIDE DATA heading in the toolbox. An “X” appears in the box, indicating that this option is selected. To re-display the data window, click in the checkbox again.
Alpha Innotech User’s Manual, version 4 The INVERT function reverses the gray scale assignments so that 0 corresponds to white and 4,095 corresponds to black. If the image has dark bands and light background, then INVERT should be selected by placing an “X” in its box. If the image has light bands and dark background, the INVERT option should not be activated. Unlike the REVERSE button described in Chapter 2, this function does not alter the appearance of the image.
Chapter 5: Analysis Tools Manual Background Using this option, different region(s) of the image representative of different background densities can be specified. Further, the user can specify exactly which objects of interest should correspond to which background objects. To manually specify the background, use the buttons beneath the BACKGROUND heading in the Spot Denso toolbox to draw a box (or ellipse or freehand drawing) around an area representative of background density.
Alpha Innotech User’s Manual, version 4 Calibration Curves for Quantitative PCR: The button in the Spot Denso toolbox labeled STD CURVE opens a set of tools that create a calibration curve for applications such as quantitative PCR. The calibration curve functions allow quantitation of the bands on a gel based on a set of standards. A minimum of two standard bands must be input, but the accuracy of the calibration curve increases as the number of standard bands and their range of values increase.
Chapter 5: Analysis Tools Click on one of the bands whose value is known. In the dialog box displayed, enter the value for the band, using either the keyboard or the numeric keypad in the dialog box. When the entry is correct click on the OK button. The band number changes from white to green, indicating that it is now a standard. To enter the next value, click on another band.
Alpha Innotech User’s Manual, version 4 To remove a band from the set of standards, click on it again. The band number changes from green back to white, indicating that it is no longer a standard. To de-select all the standard points at once, click on the CLEAR STD PTS button in the toolbox. To change the value of a standard band, click on it twice. In the dialog box, the estimated value for that band is displayed. Accept this value by clicking OK, or enter a new value.
Chapter 5: Analysis Tools of the curve, and its value has been approximated through extrapolation. Standards have an “s” next to the object number, and are highlighted in green to differentiate them from unknown bands. Outputting Quantitation Data The OUTPUT tool provides a way to save the data to a printer, to the clipboard, or to an ASCII file. This ASCII file can then be imported into a spreadsheet for further analysis and/or graphing.
Alpha Innotech User’s Manual, version 4 5.5 Object Counting: Auto Count and Manual Count The Object Count tools in the Tool Box, Analysis Tools make it easy to count the number of objects on an image, such as colonies on a Petri dish or viral plaques. Click on the button under the Tool Box, Analysis Tools heading, then, click either the MANUAL COUNT or the AUTO COUNT button. The following sets of tools will be displayed in the lower left of the screen.
Chapter 5: Analysis Tools Both MANUAL COUNT and AUTO COUNT allow two different types of objects (for example, blue and white colonies on a Petri dish) to be counted. Both counts are initially set to zero (0). Sample with Two Types of Objects When two types of objects are counted, the objects are highlighted in different colors. They can also be marked on the image using a "+" or an “X”.
Alpha Innotech User’s Manual, version 4 Manual Count Some samples are extremely difficult to count because of their shape, size, or lack of intensity variation from the background. To accurately count these samples, we have developed the manual counting feature within our software. To manually count the objects in an image, click on MANUAL COUNT in Tool Box, Analysis Tools. You may use either a green x or a red +.
Chapter 5: Analysis Tools The Sensitivity indicator determines how close the cursor must be to a marker in order to delete it. Higher numbers allow the user to click further away from the marker to delete it; however, if Sensitivity is set too high, it will be impossible to place markers on spots that are close together. Hiding Your Count Markers from the Screen The software will record all counts as you continue clicking on your objects.
Alpha Innotech User’s Manual, version 4 open the circle. When the circle corresponds to the perimeter of the Petri dish, release the mouse. The circle has “handles” around it, and can be moved or re-sized at this point. Hint: to draw a perfect circle around a portion of an image, first visualize a square surrounding the area of interest. Position the mouse in the upper left hand corner of the square.
Chapter 5: Analysis Tools To set the density threshold, click and drag the sliders. As the sliders are moved, the regions of the image that fall within the gray scale range are highlighted in the image area. Adjust the slider positions until the objects to be counted are highlighted. The numbers next to the Min and Max settings change to reflect the positions of the sliders. These numbers represent the minimum and maximum gray scale values that are currently being detected.
Alpha Innotech User’s Manual, version 4 Displaying Spots Spots are displayed as red (or green) numbers on the image. These can be changed to symbols (“+” or “x”) by holding the key and clicking the right mouse button. Repeat to return to numbers. Spots can be hidden from view by checking the Hide Spots checkbox. Specify the Size Range of the Objects If the image has objects of different sizes but only those in a certain size range are of interest, use the Area Controls.
Chapter 5: Analysis Tools To add a second object, click on the right mouse button to re-enter “add” mode, then click on the desired object with the left mouse button. Using the Erase Spot Tool This function removes any extra spots that were counted either automatically or manually. For example, if a portion of an AOI has high background or other noise, a number of objects may erroneously be included in the count. Draw a box around the object or set of objects.
Alpha Innotech User’s Manual, version 4 To see specific details on each spot, select Spot Details from the View menu. The following information will appear: Selecting the Auto Count Data Window to Show Individual Spot Details The data table gives the individual spot number, the AOI in which it was counted, its coordinates, the integrated density value (IDV) for each spot, the area (number of pixels) for each spot, and the % contribution to the total. Note: manually-added spots show areas of 1 pixel.
Chapter 5: Analysis Tools When the appropriate output source has been selected, click on the OK button to send the data. Saving and Loading AutoCount Parameters To save the size and position of objects for future analyses, select Save Auto Count Overlay from the Overlay pulldown menu. A dialog box will open. Enter a file name and path. AphaEaseFC™ will automatically add the extension .CNT to the file.
Alpha Innotech User’s Manual, version 4 5.6 The Scoring Function The Scoring function is designed for gene expression applications to check for presence or absence of specific samples. The scoring function can be used for a quick manual identification of several different sample types. The software allows for up to three different scores on a gel, blot, or microtiter plate. To score an image, click on the SCORING button in Toolbox 4.
Chapter 5: Analysis Tools Scoring the Sample Three different scoring options are available: score with a positive, negative, or positive / negative. The software will display an “X” if you have a positive sample or three “–“ signs arranged in a vertical fashion if you score with a negative. Nothing will appear if you score with a positive/negative. You may use the right and left mouse buttons to determine different scores. The right mouse button will score the sample with an “X”.
Alpha Innotech User’s Manual, version 4 Outputting Scoring Results Once you have completed scoring your sample, you may output the results. The results can either be printed directly to a printer, or you may export the results as ASCII data for direct importation into Excel or other spreadsheet programs Sending a Screen Image to a Printer Click on the PRINT button on the software interface to printout your image with the scoring marks.
Chapter 5: Analysis Tools 5.7 The Ruler Function Introduction The Ruler function allows the user to create a scale based on a standard and to measure distances from a set origin to a chosen location. The Ruler Tools and Ruler Toolbox Using the Ruler Function To access the Ruler function, click on ToolBox, Analysis Tools and then on RULER. A line with a box at each end and a circle with a cross in it will appear on the screen. Before any measurements can be obtained, a standard scale must be established.
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Appendix A Appendix A: Opening AlphaEaseFC™ Files in Other Software Programs Alpha Innotech-generated files have been tested in the software packages below. For successful imports, the command line is given. Programs for the Macintosh® operating system were tested on a PowerMac® 8100/100AV. Results may vary for different software versions and/or hardware configurations. Adobe Photoshop 2.51 LE (Mac) Adobe Photoshop 3.0 (Mac) Canvas 3.5 (Mac) Microsoft Word 6.0 (Mac) Microsoft Word 6.
Alpha Innotech User Manual, version 4 Using ResEdit™ 1) Save image in IS-1000/500 as a TIFF or MACTIFF 2) Obtain a copy of the freeware ResEdit™ by downloading from Apple Computers through the Internet. 3) Open ResEdit™. An animated startup display will show up and continue until you click on the mouse or any key. 4) A dialog box will appear. Open the TIFF image. Another dialog box will appear asking if you want to add a resource fork, click on ‘OK.
Appendix B Appendix B: AlphaEaseFC Molecular Weight Library Files A library including the following DNA, RNA and protein molecular weight standards has been incorporated into AlphaEaseFC. For information about using these standard files, see Section 5.2.
Alpha Innotech User Manual, version 4 RNA Size Standards Protein Markers (in kD) BRLRNA1 BRLRNA2 BRLPROT1 BRLPROT2 BRLPROT3 BRL10KD NOVEXM12 SIGMAHMW SIGMAPMW 1770 9490 87.0 200.0 43.0 200 200.0 205 190 1520 7460 60.0 97.4 29.0 120 116.3 116 108 1280 4400 53.0 68.0 18.4 110 97.4 97 89 780 2370 46.0 43.0 14.3 100 66.3 84 77 530 1350 40.0 29.0 6.2 90 55.4 66 61 400 240 34.0 18.4 3.0 80 36.5 55 41 280 29.0 14.3 70 31.0 45 36 155 27.0 60 21.5 36 21.0 50 14.4 15.0 40 6.0 6.5 30 3.5 20 2.5 10 B.
Appendix C Appendix C: Security Features This feature should only be used by the purchaser or supervisor of the system. Remove this page from the manual and store it in a safe place. When the system is shared by a number of users, security features may be useful for regulating or maintaining a log of instrument use. To Change the Security Setup When you initially receive the system, all security features are inactive and the password is set to "master." We suggest that the master password not be changed.
Alpha Innotech User Manual, version 4 Security Features Dialog Box If you decide not to edit the security settings at this time, simply click on the EXIT button to dismiss the dialog box. C.
Appendix C Setting Various Security Levels A variety of security levels are available. Each is described below, in increasing order of security. Also shown are the proper settings in the dialog box to choose each security level. Option 1: No security functions No log in or log out is required and anyone can use the system. Option 2: Open system with user name log in and out This security level keeps the system open but requires users to log into the system by typing their name.
Alpha Innotech User Manual, version 4 Option 3: Open system with user name and password log in and out This security level keeps the system open but requires users to log into the system by typing their names and passwords. When someone enters a name and assigns a password to that name, the same password is needed whenever that name is used to log in. Only one password can be assigned to a name. A list of users with their log in and log out times is generated. Note: passwords are case sensitive.
Appendix C Option 5: Closed system with user name and password log in and out The system only accepts authorized names and their passwords at log in. The system supervisor can add or remove names from a list of authorized users as described above. Only one password can be assigned to each name. A user log is generated. NOTE: Passwords are case sensitive. C.
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Appendix D Appendix D: Cabinet Installation Instructions MultiImage™ II Light Cabinet (DE-500) Pictured with the cabinet top (DE-500FC). D.
Alpha Innotech User Manual, version 4 Cabinet Setup When you remove the light cabinet from its shipping carton, it is already partially assembled. The camera mounting assembly is packed separately in the same container. The UV transilluminator and cabinet top are both packed in separate boxes. Make sure you have received all the hardware before discarding the shipping carton. 1. The largest box will be the DE500 cabinet.
Appendix D (DE-500 Power Connect, Power Switch, RS-232 connect) Then connect the other end of the RS-232 cable to COM 1 on the rear of the computer (see below picture). (CPU mouse, keyboard, printer port, RS-232 connectors) WARNING: IF EQUIPMENT IS USED IN A MANNER NOT SPECIFIED BY THE MANUFACTURER, THE PROTECTION PROVIDED BY THE EQUIPMENT MAY BE IMPAIRED. “CAUTION: POWER SUPPLY CORD IS USED AS THE MAIN DISCONNECT DEVICE.
Alpha Innotech User Manual, version 4 Symbols: Ultra Violet Light WARNING: Please use protective equipment when using UV light. UV protective goggles, face shields, long sleeve lab coats can be obtained from major scientific distribution catalogs. (e.g. Fisher Scientific) CAUTION: Risk of electric shock HAZARD, please take appropriate precautions Earth (ground) Terminal ( 250 V 3.15 A ) Fuse symbol Power Ratings: DE 500 MultiImage™ II cabinet is rated at: 250V 50/60 Hz 3A Fuse: 250V 3.15A D.
Appendix D Camera Installation 1. Write down the position of each filter on the magnetic notepad, using the dry erase marker provided. Standard with the MultiImage™ II cabinet is position one: Chemiluminescent Imaging, position two: Ethidium Bromide filter. Please write down appropriate filters for positions three, four and five. Note: if a DE-500 cabinet was purchased, filter positions also need to be customized in the software (see manual for detailed instructions).
