Ch. 1 - Installation Guidelines

Ch. 1 - Installation Guidelines Table of Contents Ch. 1 - Installation Guidelines Introduction... 8 The Image-Pro Driver Interface... 8 Installing the SPOT Image-Pro Driver... 8 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 7

Installing the SPOT Image-Pro Driver Ch. 1 - Installation Guidelines Introduction The following section describes only the steps for installing the SPOT Image-Pro driver. However, before you install the driver, you must install the SPOT stand-alone software. Refer to the Installation Guidelines chapter of the User Guide to the SPOT Software (included with your camera) for a list of the software installation steps. Note: In addition to software installation instructions, the Installation Guidelines chapter contains system requirements, the contents of the shipping box, instructions for connecting the SPOT camera to a microscope and computer, and instructions for accessing the PDF version of the User Guide to the SPOT Software. The Image-Pro Driver Interface The SPOT Image-Pro driver interface is completely new beginning in 2005. Instead of being written by Media Cybernetics, the new Spot Image-Pro driver has been written using Media Cybernetic s DDK for Image-Pro 5.X. The SPOT TWAIN interface is similar to the previous appearance of the TWAIN as seen in previous manuals. CAUTION! Do not open the Image-Pro Driver interface and the TWAIN interface at the same time. Both the SPOT Image-Pro driver interface and the SPOT TWAIN interface allow you to define image capture options and capture images with the SPOT camera. However, because the SPOT Image-Pro driver is designed specifically to interface with Image-Pro, it has three important advantages over the TWAIN interface: The SPOT Image-Pro driver allows you to define SPOT image capture options within the new Image Setups interface and then save these options as an Image-Pro Settings (.vpf) file. The SPOT Image Setup window only is used with the TWAIN interface. This allows for greater ease of use in the Image-Pro environment. The SPOT Image-Pro driver allows you to use Image-Pro s IPBasic macro language in the image capture process and with most of the image setups in the Spot driver. With the SPOT Image-Pro driver, you can capture sequential images and perform Workspace Preview functions. Installing the SPOT Image-Pro Driver To install the SPOT Image Pro driver, follow these steps: 1. If you have not already done so, install PC version 4.5.8 of the SPOT stand alone software. 2. Connect the SPOT camera to the microscope and the computer. Refer to the Installation Guidelines chapter of the User Guide to the SPOT Software (included with your camera) for a list of the hardware installation steps. 3. Ensure that the serial number of the SPOT camera/software matches the serial number on the Image- Pro Driver CD. To check the serial number, click on the Help menu in the SPOT software. 4. Download the new SPOT Image-Pro Driver Installer [ ] from the Diagnostic Instruments website into the Image-Pro Plus directory. Once downloaded, double-click on the Installer to install the new driver. The new Installer will look for any valid Image-Pro installations and allow the user to select one of all of these Image-Pro installations using checkboxes for updating to 8 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 1 - Installation Guidelines Installing the SPOT Image-Pro Driver the new driver. Once all locations for driver installation have been selected, the Install button is pressed. The new Driver Installer selection interface is shown below: The default location of the Image-Pro directory is C:\IPWin4 (for version 4.1 or 4.5) or C:\IPWin5 (for version 5.0) or C:\IPWin51 (for version 5.1). You can verify that the latest version of the Spot driver for Image-Pro has been installed by pressing the About button at the bottom, middle of the new SPOT Image-Pro driver stack that is used to access the Spot functions as indicated below. When the About button is pressed, the splash screen for Version 4.5.9 should be indicated as seen below with a Build Date of June 15, 2005. This version of the driver uses the IP Capture Version 5.1.0.8, the latest version of the Image-Pro capture driver. Clicking on the splash screen removes it from the working environment. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 9

Installing the SPOT Image-Pro Driver Ch. 1 - Installation Guidelines NOTE: The user should note that using any previous IP Capture Version (previous to Version 5.1.0.8) with Diagnostic Spot cameras has been shown to cause problems with proper camera function. This new Diagnostic Instruments driver for Image-Pro Plus has been written by Diagnostic Instruments, Inc. using Media Cybernetic s own Device Development Kit (DDK) and the latest version of their Software Development Kit (SDK). Prior versions of the Spot driver were written by Media Cybernetics, Inc. and, if installed, will automatically revert back to older versions of the Spot driver that do not contain the functionality for any of the new Diagnostic Instruments, Inc. cameras. 10 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Table of Contents Ch. 2 - Preparing to Take a Picture Introduction... 11 Working in the SPOT Image-Pro Interface... 11 Setup... 14 Load Settings... 15 Save Settings... 16 Default Settings... 18 About Driver... 18 Video Format Selection...Error! Bookmark not defined. 8-bit Gray Scale Acquire... 18 Configure...Error! Bookmark not defined. Image... 22 New Image... 22 Active Image... 22 Sequence... 22 File/Set Filename... 22 Multiple Images... 22 Preview... 23 Integration... 23 Signal...Error! Bookmark not defined. Macros... 23 Start Preview... 23 Snap... 25 Minimal Dialog...Error! Bookmark not defined. Configuring Settings Files... 27 Name...Error! Bookmark not defined. Pixel Bit Depth... 28 Exposure... 29 Auto-Exposure vs. User-Defined Exposure... 29 Filter Color (8, 12 and 14 bit mono)... 34 Use: Red Green Blue (24, 36 and 42 bit RGB)... 34 Color Order (24, 36 and 42 bit RGB)... 36 Binning... 37 Image Type (Auto-Exposure)... 38 Auto-Gain Limit (Auto-Exposure, 8 bit and 24 bit)...error! Bookmark not defined. Adjustment Factor (Auto-Exposure)... 39 White Balance (Auto-Exposure, 24, 36 and 42 bit RGB)... 39 Exposure Time(s) (User Defined Exposure)... 43 Gain (User-Defined Exposure, 8 bit and 24 bit)... 47 Chip Imaging Area... 47 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 9

Table of Contents Ch. 2 Preparing to Take a Picture Post-Processing... 53 Black Level Subtract (12 and 14 bit mono)... 54 Fluorescence Color Handling...Error! Bookmark not defined. Chip Defect Correction... 55 Noise Filter... 55 Image Size Double... 55 Gamma Adjust... 55 Rotate... 58 Horizontal Flip... 58 Vertical Flip... 58 Background Subtract... 58 Flatfield Correct... 62 Locked... 64 Cameras... 64 10 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Working in the SPOT Image-Pro Interface Introduction This chapter is divided into two sections: Working in the SPOT Image-Pro Interface Configuring Settings Files Working in the SPOT Image-Pro Interface To establish the SPOT Image-Pro Driver interface, follow these steps: 1. Open Image-Pro. 2. From the Acquire menu, select Video/Digital. The Video/Digital interface window appears. The user can also open the Spot Image-Pro Driver using the Video/Digital icon [ ]. The title of this window matches the Current Driver selection on the Setup page, e.g., Diagnostic Instruments SPOT. The Current Driver also includes the version of the new driver in parentheses, in this case being 4.5.8 as shown below. 3. Click on the Setup tab. 4. If it is not already selected, select Diagnostic Instruments SPOT(4.5.8) from the Current Driver dropdown list. It is required by Image-Pro version 5.0 and above to establish at least one named Settings file (.VPF) for camera settings in order for any changes to the Spot Image-Pro driver setups to be Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 11

Working in the SPOT Image-Pro Drive Interface Ch. 2 Preparing to Take a Picture saved. A default value of Insight_Firewire01 has been saved in the example above. Any changes to the Image Setups will now be recorded in this.vpf file. 5. The first time the new Spot Image-Pro driver is used, the Spot driver dialog must be activated by selecting the Acquire tab and pressing the Show Camera Dialog button at the bottom left in the Acquire tab dialog box as indicated below. The Show Camera Dialog button will open the new Spot Camera driver dialog box as indicated below. The new Spot Camera driver is a stack of buttons with functions very similar to the Image Setup functions found in the Spot software. 12 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Working in the SPOT Image-Pro Interface Both the Image-Pro Acquire Setup dialog and the Spot driver dialog box can be used together or separately depending on the user preference. The Image-Pro Acquire dialog is not needed, however, and the Basic Dialog can be selected if desired or the Acquire dialog Closed. The user will notice that the new Spot Camera dialog now has a Start Preview button and a Snap button which function the same as those buttons in the Image-Pro Acquire dialog. If the user would like the Spot Image-Pro driver dialog box to open automatically each time the Video/Digital menu or icon is activated, the user just needs to activate the check box at the bottom of the Spot driver dialog box next to the title Automatically show this window. The interface to the SPOT camera is now established. The following sections explain how the buttons work on the new SPOT Image-Pro driver. These buttons are: Image Settings Preferences Compute Exposure Recall Previous Exposure Compute White Balance Set Chip Imaging Area Set Spot Metering Area Get Bias Frame Get Background Image Get Flatfield Start Preview Snap Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 13

Working in the SPOT Image-Pro Drive Interface Ch. 2 Preparing to Take a Picture Setup (Image-Pro Plus Version 4.5 or Advanced Capture Dialog in 5.0, 5.1 or Higher Versions) The Setup tab allows you to create, modify, and recall Settings files. With the SPOT Image-Pro driver, a Settings file serves two crucial purposes: It defines the way an image will be taken. It defines the way an image will be modified after it is taken. Settings files can be created to optimize the image taking process for different types of images or microscopy techniques. Settings files have a.vpf extension. The currently active Settings file is indicated next to the Current File in the Settings dialog area and above the Load, Save, SaveAs and Setting Options buttons. Until you create new Settings files, the factory default default.vpf is the only available option. To create a new Settings file, you must modify an existing Settings file and save the modified file with a different name. New Settings files can be created from only the Video/Digital window (Setup tab) beginning in Image-Pro Version 5.0 and above. To create a new Settings file from the Video/Digital Window (Setup tab) in Image-Pro Plus Version 4.5 or the Advanced Setup in Image-Pro Plus Versions 5.0, 5.1 or higher, follow these steps: 1. Click on Setup tab. The Settings window appears in the center of the dialog box. 14 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Working in the SPOT Image-Pro Interface 2. Modify the image capture options as needed in the Image-Pro dialog or make changes to the settings in the new Spot Camera dialog, and click on OK. 3. Click on the Save or Save As button. The Save As window appears. 4. Rename the file (e.g., default) in the Current File box. If you don t change the file name, the original file is overwritten with the new settings. 5. Click on Save. Note: Settings (.vpf) files are, by default, saved to the C:\IPWin5 or IPWin51 folder when using Image-Pro 5.0 or 5.1, respectfully. They can be saved to other folders, as desired. Note: New Settings (.vpf) files should be created or changed while the Live Preview is not activated. Changing settings files during the middle of a live session may cause errors to be saved to the current settings file or may cause the Live Preview to be reset to full screen. Settings Select Settings from the Active Folder: Pulldown After you have created one or more Settings files (in addition to the default), you can use the Select Settings from the Active Folder: pulldown or spinbox selector to select the one that you want to make active (current). Follow these steps: 1. Click on the spinbox arrow button next to the box under the title Select Settings from the Active Folder:. The Open window appears. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 15

Working in the SPOT Image-Pro Drive Interface Ch. 2 Preparing to Take a Picture 2. Select the Settings (.vpf extension) file that you want to use, and click on Open. The program returns to the Video/Digital interface window. The name of the file that you selected appears as the Current File. 3. Selecting the button next to the spinbox selector allows the user to select settings files from anywhere on the hard drive. Settings Save Use the Settings Save option to save changes made to an existing Settings file. Settings SaveAs Use the Settings SaveAs option to save changes as a new file name. Follow these steps: 1. After you have modified a Settings file, click on Settings SaveAs button. The Save As window appears: 16 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Working in the SPOT Image-Pro Interface Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 17

Working in the SPOT Image-Pro Drive Interface Ch. 2 Preparing to Take a Picture 2. Enter the name/file path for the.vpf file, if necessary. To overwrite the original Settings file with the changes that you made, rename the Settings file. 4. Click on Save. The program returns to the Video/Digital interface window. The name of the new/modified.vpf file appears as the Settings file. Settings Options Use the Settings Options button to reset the Settings File to the default.vpf file by pressing the Settings Options button and pressing the button called Restore factory defaults and then the OK button. About Driver Click on the About Driver button to view information about the SPOT Image-Pro Plus Driver. 8-bit Gray Scale Check Box Checking the 8-bit Gray Scale check box in the Setup tab sets the Pixel Bit Depth in the currently active Settings file to 8 bit (mono). By default, this box is checked in Image-Pro but can be unchecked or checked without any adverse operation. Checking the box will always capture an 8-bit gray scale image (whether the camera is a color mosaic or 3-shot color camera or not). For the best results, leave this box unchecked, and make your Pixel Bit Depth selections on the Image Settings window of the Spot Camera driver dialog. Setup (Basic Dialog of Capture Dialog in Image-Pro Plus 5.0, 5.1 or Higher Versions only) Beginning in Versions 5.0 and 5.1, Settings files (.vpf) can also be created in the Basic Dialog of capture by pressing the Basic Dialog button ( below. ) at the bottom of the Advanced Capture dialog as seen 18 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Working in the SPOT Image-Pro Interface In order to Save settings in the Basic Dialog, the user must first press the More>> button at the bottom of the Basic Dialog to open the right panel for additional options as seen below. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 19

Working in the SPOT Image-Pro Drive Interface Ch. 2 Preparing to Take a Picture Settings created while the Basic Dialog is open can be saved using the Save or Save As buttons. Current Settings may also be selected from this dialog similar to the method in the advanced capture dialog. At any time, the user may close the right panel of the Basic Dialog by pressing the Less>> button. Alternately, the user may also designate their most common settings files in the Basic Dialog by selecting a settings file to be associated with a numbered button at the top of the Basic Dialog. By default, the number 1 button is depressed when only one Settings file has been selected. The other available settings selections will all have an NA or Not Assigned designation on the button until a settings file has been associated with the button. The Settings files in the left panel of the Basic Dialog will be numbered consecutively from the left. Only six numbered Settings are currently available in this dialog. In order for the user to see what settings file has been assigned to the numbered button, the tip of the mouse arrow must be passed over the button and a red arrow with a + sign will appear with the name of the file name and location indicated in a box just under the red arrow. 20 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Working in the SPOT Image-Pro Interface The Camera Options button in the Basic capture dialog functions the same as the Acquire Tab Show Camera Dialog button in the Advanced dialog mode. This opens the Spot Camera dialog if it is not already open. The Configure button has no enabled function when used with the Spot cameras with the Diagnostics driver. At any time, the user can return to the advanced capture dialog (default capture dialog) by pressing the Advanced button at the bottom of the Basic Dialog. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 21

Working in the SPOT Image-Pro Drive Interface Ch. 2 Preparing to Take a Picture Image There are two groups of options under the Image tab: The first four selections (New Image, Active Image, Sequence, and File) specify how captured images are to be displayed or stored. Select one of these options. The Multiple Images section lets you specify how many images are to be captured each time the Snap button is clicked. New Image Create a new, sequentially numbered, Untitled window for each image. Active Image Create a single Untitled window. Each new image will replace the previous image in that window. Sequence Store a sequence of images in a single sequence file. A sequence file in Image-Pro is a collection of sequentially acquired images stored as a single TIFF file. Sequence files are viewed using the Sequencer Toolbar, which can be accessed by selecting Sequencer Tools > Toolbar from the Acquire menu. Refer to the Image-Pro manual for further details on viewing and editing sequence files. File/Browse Button Save the captured image to file without displaying it on-screen. The default file name and location (C:\IPWIN5\images\series0.tif in the below illustration) appears next to this option. To change the file path options, click on the Browse button. The following dialog box appears: Enter the desired file information. Multiple Images Specify the parameters for capturing one or more images with each click of the Snap button.. Number of Images Specify the number of images to be captured. 22 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Working in the SPOT Image-Pro Interface Time between images Either click on the Set Time button to specify the precise time settings between the start of image captures, or check the Use minimum possible interval box to minimize the time between image captures. If you check this box, the Set Time button becomes unavailable. Preview/WorkSpace Preview The options under the Preview tab are designed to control the size and refresh rates for live images. The Spot camera settings can be changed here to include Binning, a Gray scale preview, the Preview Area defined and Workspace Preview can be activated here. Workspace Preview allows the user to sample digital information from the live image but, therefore, slows the live camera speed considerably. Workspace Preview may also be activated from the Basic Dialog of capture by pressing the Basic Dialog button and checking the Workspace preview checkbox at the bottom of this dialog. Integration The purpose of the integration function is to average multiple frames in order to average out random noise. The Spot camera can be used in this Image-Pro function to perform image integrations if so desired. Checking the Accumulate box and pressing the Accumulate Settings button will set the number of images used in integration. Macros Use the options under the Macros tab to specify the pre and post snap operations that you want to use with your image captures. Note that no macros are available until you define them. Refer to Ch. 4 Macros for a list of the SPOT related macro functions in Image-Pro. Start Preview Click on the Start Preview button to call the SPOT Image-Pro Plus Driver window. The Start preview button can be pressed either in the Image-Pro Setup dialog (bottom left side) or in the new Spot Camera dialog (second large button from bottom). The new Live Preview dialog appears almost as it does in the Spot software, as seen below. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 23

Working in the SPOT Image-Pro Drive Interface Ch. 2 Preparing to Take a Picture The only exception is that the Spot Camera live dialog has a Live Snap (instead of a Snap) button to distinguish it from the Snap button in both the Spot Camera and Image-Pro Acquire dialogs which captures images based on the settings file only. The Live Snap button is controlled independently using the Control button settings in the Live Preview as indicated below with Autoexposure controls or with Manual exposure controls as seen in the diagram below that. 24 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Working in the SPOT Image-Pro Interface From this window, you can: Capture a preview image. Transfer the preview image into Image-Pro Focus the camera. Frame your image (by positioning your specimen). Determine manual exposure times and gain. Acquire a white balance. Set a chip imaging region (for using less than the full CCD area for imaging). Acquire background images and flatfield files. Modify the Settings (.vpf) file that you used to capture the image. Refer to the Configuring Settings Files section of this chapter and to Ch. 3 Taking a Picture for more details on the SPOT Image-Pro Plus Driver window. Snap and Live Snap Click on Snap (in either the Image-Pro Setup dialog or the bottom large button in the new Spot Camera dialog stack) to capture an image using the current settings file settings and the image will be captured directly to Image-Pro workspace for further editing. Click on the Live Snap button in the Spot Live Preview to capture an image directly into the Image-Pro workspace based on settings from the Spot Live Preview dialog. Settings for the Spot Live Preview can be changed by pressing the Controls button in the Spot Live Preview dialog. Refer to Ch. 3 Taking a Picture for more details. Restart Resample the brightness of the image, compute new exposure times, and restart the live image display. This is useful for updating exposure times when specimen brightness has changed. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 25

Working in the SPOT Image-Pro Drive Interface Ch. 2 Preparing to Take a Picture Pause/Resume Freeze the live image window. This is useful when you are changing a specimen and want to preserve the last view. In addition, to use the Transfer option (see below), you must first Pause the live image view. Click on Resume to restart the live image view. Close Close the Spot Live Preview window. Transfer This allows the user an alternate method of capturing an image into the Image-Pro workspace but first pressing the Pause button and then the Transfer button. Under normal operating conditions with the Pause button inactivated, this button is grayed out. Reset Area Return to the non-zoomed view of the live image. Under normal zoom conditions, when a smaller than normal area of interest in the live image has not been selected, this button is grayed out. Faster Readout/Acceleration Faster Readout or Acceleration enables the user to speed up the live image display (higher frame rates) in cases where the user is willing to sacrifice some resolution for a higher frame rate. 26 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Configuring Spot Image Settings in Spot Camera Dialog New Spot Image Settings are created by modifying the currently active Settings and saving the modified file under a different name, as described in the Setup section above. The Image Settings window can be accessed by pressing the top button in the Spot Camera dialog (stack of buttons). When you click on the Image Settings button for the first time, the window appears with the settings of the default.vpf file, with Exposure dialog indicated as shown here: The following sections detail the options on the SPOT Image Settings window, which consists of five areas: Exposure (Auto-Exposure and Manual Exposure) TTL Output Chip Imaging Area Image Orientation Post Processing Exposure The Exposure dialog displays the general exposure settings including the Acquired Image Bits per Pixel (monochrome or RGB bit depths) and the Exposure settings including Auto and Manual Types, the White Balance Red, Green and Blue settings (set manually or through the Auto White Balance command) and a check box for setting the Auto TTL Output Control and Delay. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 27

Configuring Settings Files Ch. 2 - Preparing to Take a Picture Exposure-Bits per Pixel Box Bits per Pixel is the number of bits per pixel (bpp) that the camera uses to create an image. The SPOT program provides up to five options depending on the camera that is being used: 8 bpp monochrome 12 bpp - monochrome 24 bpp- color 36 bpp color 42 bpp -- color The 24 bpp RGB color setting is the most common selection because it is an industry standard color format that is used by Windows and most printers. Most monitors cannot display more than 24 bpp. Select a pixel bit depth by clicking on the Bits per Pixel drop-down box. Because your selection determines whether your image will be monochrome or color, it also determines the available exposure options. The Exposure section of the Image Settings window changes accordingly. The following table lists the available image depth options and describes some of the potential uses, advantages, and disadvantages associated with each setting. Pixel Bit Depth Settings Characteristics 8 bpp (monochrome) The 8 bit setting is often used in cases where 256 gray levels are adequate. The lower number of bits per pixel results in smaller, more manageable file sizes. A typical 8 bpp image is 1.29 Mb 12 bpp (monochrome) The greater number of bits per pixel in the 12 bit setting provides users with 4096 gray levels. This provides a more accurate means of measuring brightness in a monochrome image, and helps to reduce banding problems that often appear when an image is modified. One disadvantages of the 12 bpp setting is that all pictures are taken at a gain of one. Because higher gain levels cannot be used, exposure times for dim images will be longer and file sizes will be larger. A typical 12 bpp image is 1.94 Mb. 24 bpp (RGB color) The 24 bit setting provides 8 bits per color, per pixel (i.e., 8 x 3 = 24). As noted, 24 bpp is the most common choice because it is an industry standard in terms of printing, the Windows operating system, and monitor display. A typical 24 bpp image is 3.88 Mb. 36 bpp (RGB color) The 36 bit setting provides 12 bits per color, per pixel (i.e., 12 x 3 = 36). 36 bpp is used in cases where images are heavily modified. The 36 bit setting provides users with 4096 brightness levels per color and thereby reduces banding problems when images are stretched. As with the 12 bit setting, all pictures are taken at a gain of one. A typical 36 bpp image is 5.83 Mb. 42 bpp (RGB color) The 42 bit setting provides 14 bits per color, per pixel (i.e., 14 x 3 = 42). 42 bpp is used in cases where images are heavily modified. The 42 bit setting provides users with 16,384 brightness levels per color and thereby reduces banding problems when images are stretched. As with the 12 bit setting, all pictures are taken at a gain of one 28 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files all pictures are taken at a gain of one. A typical 42 bpp image is??? Mb. Exposure-Exposure Box The Exposure box options tell the camera what settings to use when taking a picture. Carefully considered definition of these settings helps to ensure that your images come out as planned. Note: The available exposure options vary according to the selected Bits per Pixel. Auto-Exposure vs. Manual Exposure With the SPOT camera, you can either allow the camera to calculate exposure time (Auto - Exposure), or you can calculate the exposure on your own (Manual Exposure). When you select the Auto Exposure option, the camera samples light levels (as the initial part of the image capture) and uses the values to calculate the final exposure time. When you select the Manual Exposure option, you have three options: You can type in values for the red, green, and blue exposure times. You can use the Compute Exposure toolbar button to calculate and insert the exposure times (and gain) for you. You can use the Recall Previous Exposure toolbar button to recall and insert the exposure times and gain used for the last picture. The following table compares the available exposure options for automated and Manual exposures at all four image depth settings. Pixel Bit Depth = 8, 12 or 14 bpp (mono); Monochrome or Slider Cameras Auto- Exposure Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 29

Configuring Settings Files Ch. 2 - Preparing to Take a Picture Manual Exposure Pixel Bit Depth = 8, 12 and 14 bpp (mono); Flex camera; Binning Required Auto- Exposure Setting Auto- Exposure Changes 30 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Manual Exposure Setting Manual Exposure Changes Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 31

Configuring Settings Files Ch. 2 - Preparing to Take a Picture (cont.) Pixel Bit Depth = 24, 36 and 42 bpp (RGB); Flex Camera; Normal Resolution Auto- Exposure Setting Auto- Exposure Changes Manual Exposure Setting 32 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Manual Exposure Changes Pixel Bit Depth = 24 bpp (or 36 and 42 bpp) (RGB); Flex Camera; 4-Shot and 16- Shot Modes Auto- Exposure Flex 4- Shot Auto Exposure Flex 16- Shot The sections that follow detail all of the Exposure options. If an option is available only with certain Pixel Bit Depth settings, or if an option is associated only with automated exposure or user-defined exposure, this is indicated parenthetically in the heading. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 33

Configuring Settings Files Ch. 2 - Preparing to Take a Picture Filter Color (8, 12 and 14 bit mono) Filter Color specifies the color of the filter that will be used when taking a monochrome picture. Slider Models Users of cameras with an optional Slider color filter installed can slide the camera s color filter out of the light path to produce an entirely unfiltered image. With the filter out of the light path, it does not matter which color is selected. To ensure a high image quality, SPOT users should select the color that is closest to the color of the specimen. For example, if you are looking at a single stained fluorescence, selecting the color filter that most closely matches the color of the stain will shorten the time it takes to acquire the image. To select a filter color for capturing a monochrome image, follow these steps: 1. Ensure that the Pixel Bit Depth is set to either 8 (mono), 12 (mono) or 14 (mono). 2. Click on either the Auto-Exposure or the User-Defined Exposure option. 3. Select one of the following options: Red Green Blue Use: Red Green Blue (24, 36 and 42 bit RGB) Use: Red Green Blue specifies the colors that will be exposed. For most situations, you should select all three colors. However, in situations where you know that your specimen lacks one or two of the colors, you can avoid the additional exposure and download time by turning off (deselecting) the missing colors. For example, if you have a pumpkin colored fluorescence sample with no blue in it, you can deselect the blue value. The camera will only expose the red and green values. To set the color filters for capturing a color (RGB) image, follow these steps: 1. Ensure that the Pixel Bit Depth is set to either 24 (color) or 36 (color). 2. Click on either the Auto-Exposure or the User-Defined Exposure option. 34 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files 3. Deselect one or two of the color option boxes, or leave all three checked. Red Green Blue To save the Settings file, at least one color must be selected. Slider Models If capturing an unfiltered image, it does not matter which color you select. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 35

Configuring Settings Files Ch. 2 - Preparing to Take a Picture Please be aware that when the RT Slider cameras are used for the first time, the Live Image Controls filter setting may be set, by default, to one of the monochrome settings (see below). If this is the case, even though the Image Settings Exposure may be set to RGB (24RGB or higher) and all the R, G and settings are checked, the Live Image Controls may show a gray or monochrome live image. Pressing the Control button in the Live Preview and re-setting the Filter setting to RGB (as seen in the figure below) will restore the Live image to color. 36 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Color Order (24, 36 and 42 bit RGB) In addition to allowing you to specify specific exposure colors, the SPOT camera allows you to specify the order in which the colors are exposed. This feature is particularly useful for triple stained fluorescence samples, where one color dye might fade before the others. For example, if a green dye fades first, you can set green to be the first exposure, thereby catching the green parts of the sample before they fade. To set the Color Order for an image capture, follow these steps: 1. Select the color or colors that you want to expose for the picture. 2. Click on the Color Order scroll bar. 3. Select the Color Order that you want to use. Note: The default Color Order setting for a full color image is RGB. However, since a color LCD filter can control each filter color independently, any color filter order may be selected. All the order selections are available in the pull-down box. Binning Binning is a pre-exposure option that tells the camera to combine adjacent pixels on the CCD chip. The camera treats each set of combined pixels as one large pixel. The large pixels accumulate electrons faster and are therefore more sensitive to light. Binning has three effects on image processing: Light sensitivity is increased. Download time is decreased. Resolution is decreased. Binning is particularly helpful when you want to drastically reduce exposure and download times for dim images and you don t mind a decreased resolution. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 37

Configuring Settings Files Ch. 2 - Preparing to Take a Picture The SPOT camera offers four binning options, as illustrated below: Binning Options Combined Pixels on the CCD Chip None 2 x 2 (4 pixels = 1) 3 x 3 (9 pixels = 1) 4 x4 (16 pixels =1) The default Binning option is set to None. Image Type (Auto-Exposure) When using Auto-Exposure, the SPOT software provides 11 image type settings that correspond to various types of microscopy. Image Type Brightfield-transmitted light Darkfield-transmitted light Fluorescence Nomarksi DIC/Hoffman Phase contrast Polarized light Brightfield-reflected light Darkfield-reflected light Stereo/macro no glints Stereo/macro shiny glints Stereo/macro-metallic glints Description Stained specimens on glass slides using transmitted light microscopes. Bright specimens on dark backgrounds using transmitted light darkfield technique. Fluorescence specimens not fluorescing in a single red, green, or blue primary color. The camera expose images using all three colors. Nomarski and Hoffman are polarized light techniques that require proper camera orientation. Refer to the note following this table for details. Specimens being viewed using the phase contrast technique. Polarized light techniques require proper camera orientation. Refer to the note following this table for details. Polished metallurgical specimens and semi-conductors using a microscope with a vertical illuminator. Bright areas of specimens being viewed with darkfield, reflected light techniques. Matte specimens without bright glints. Shiny specimens with bright glints. Metallic specimens with intense glints. 38 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Note: Because the color filter in the SPOT camera uses a polarizer, the camera must have the correct rotational orientation when installed on a microscope that uses polarized light techniques. This is accomplished by rotating the camera while using the SPOT program s focus function. Correct orientation is achieved when the image is brightest. Slider Models When the liquid crystal color filter has been moved out of the light path, users of Slider models may notice a diffuse bright area on non-fluorescence image captures. This is due to infrared light that is transmitted when the filter is out of the light path. Our infrared filter is bonded to the liquid crystal color filter: when the color filter is moved out of the light path, the infrared filter is also moved. To correct this problem, you should use an external infrared filter. Adjustment Factor (Auto-Exposure) Use the Adjustment Factor option to modify the automated exposure settings determined by the SPOT camera. The Adjustment Factor defaults to a setting of 1 (i.e., 1x the exposure times determined by the Auto-Exposure setting). The Adjustment Factor is used to deliberately over-expose or under-expose an image. If an image that you captured (using the default Auto-Exposure settings) comes out too bright or too dark, you can re-capture the image using a different Adjustment Factor to compensate for the excess brightness or darkness. If you use the SPOT camera to take pictures of printed circuit boards or other metallic objects, the Adjustment Factor allows you to over-expose the image in order to burn out the glints that appear when capturing images of metallic objects. Over-exposing the glints is necessary to correctly expose the non-glinted areas. To set the adjustment factor, enter a number between.25 and 32. White Balance (Auto-Exposure, 24, 36 and 42 bit RGB) White balance is the ratio of red, green, and blue exposure times necessary to achieve the proper color rendition for an image. Many factors impact the coloration of an image capture, including lamp voltage, the coloration of the glass used in the slide, and the coloration of the glass used in the lenses of the microscope s objectives. To minimize the impact of these factors, use the Compute White Balance feature. When you perform a white balance calculation, SPOT samples the color of the light from your light source after it has passed through the slide and the objective, and then calculates the exposure values needed to produce images where the white areas are white and the color areas are true. You should perform a new white balance calculation when: Starting a new image capture session. Changing the lamp voltage. Switching to a different objective on the microscope. When you open an Image Settings file, the Image Settings window displays the white balance values that were last saved to that file. If you capture an image without computing a new white balance, SPOT uses these default values. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 39

Configuring Settings Files Ch. 2 - Preparing to Take a Picture To compute new white balance values for the currently active Image Settings file, follow these steps: 1. Set the illumination and the objective that you will use for the image capture. 2. Show the camera a sample of white light. This step varies according to microscopy technique and sample type, as shown in the table below: Image Type Brightfield-transmitted light Darkfield-transmitted light Fluorescence Nomarksi DIC/Hoffman Phase contrast Polarized light White Balance Technique Position the slide so that the specimen is not in the field of view, but light is going through the slide near the specimen. Perform the white balance using the brightfield mode on your microscope. See Brightfield transmitted light above. Start off with the white balance in the default.vpf Settings file, or acquire a white balance with transmitted light brightfield using a daylight filter. This should get you close. Colors can then be tweaked by manually adjusting the red, green and blue white balance values in the Settings file. For Nomarski DIC: Adjust the Nomarski prism until the image is in either the brightfield or the gray scale view. For Hoffman: Perform the white balance directly on the Hoffman image. Because both of these techniques use polarized light, and because the color filter in the SPOT camera uses a polarizer, the camera must have the correct rotational orientation with respect to the polarized light microscope. This is accomplished by rotating the camera while using the SPOT program s focus function. Correct orientation is achieved when the image is brightest. Position the slide so that the specimen is not in the field of view, but light is going through the slide near the specimen. Pull all polarizers and compensators out of the light path and then position the slide so that the specimen is not in the field of view, but light is going through the slide near the specimen. Because the color filter in the SPOT camera uses a polarizer, the camera must have the correct rotational orientation with respect to the polarized light microscope, when acquiring final images. This is accomplished by rotating the camera while using the SPOT program s focus function. Correct orientation is achieved when the image is brightest. 40 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files (cont.) Image Type Brightfield-reflected light Darkfield-reflected light Stereo White Balance Technique Replace your sample with a mirror. Perform the white balance using the brightfield mode on your microscope. See Brightfield reflected light above. Using oblique illumination (shining a light on the top of your specimen from off to one side), replace your sample with a white sheet of paper. 3. Open the SPOT Camera window in Image-Pro. a) From the Acquire menu in (IPP 4.5), select Video acquire or Setup Acquire and Acquire Tab Show Camera Dialog button or just use the Video/digital capture icon in the toolbar and the Spot Camera dialog will open when Automatically show this window has been checked. b) From the Acquire menu in (IPP 5.x), select Capture Options Setup Acquire and Acquire Tab Show Camera Dialog button or just use the Video/digital capture icon in the toolbar and the Spot Camera dialog will open when Automatically show this window has been checked. c) If the Spot camera driver dialog has been previously set up to open automatically, the Spot Camera dialog will appear. d) Click on Start Preview. The SPOT Live Preview window appears: Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 41

Configuring Settings Files Ch. 2 - Preparing to Take a Picture 4. From the Spot Camera dialog button stack, click on the Compute White Balance button: The Compute White Balance window appears: 42 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files 5. Position the slide according to the microscopy technique that you are using. Refer to the table in the beginning of this section for details. 6. Click on Begin to start the calculation. The message Computing white balance values displays. When the camera finishes calculating the white balance values, SPOT displays the computed white balance values, as shown here: Note: Due to the inherently poor blue light sensitivity of CCD chips and the energy distribution of halogen bulbs, the blue value is often much higher than the red and green values. 7. Click on OK. The program returns to the SPOT Live Preview window. 8. To edit the white balance values that you saved, open Image Settings by clicking on the button in the Spot Camera stack. The values should appear under White Balance in the Exposure dialog with the values in the Red, Green, and Blue spin boxes. Edit the values as needed. ------------ Exposure Time(s) (Image Settings-Manual Exposure) There are three options for entering manual exposure times. With the SPOT camera, you can: Manually enter the value(s). Allow the camera to calculate the value(s). Recall the exposure times and the gain used for the previous image capture. When you select the Manual option on the Image Settings window, default values appear for the Exposure Time(s). Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 43

Configuring Settings Files Ch. 2 - Preparing to Take a Picture To manually enter new exposure value(s) follow these steps 1. On the Image Settings window, ensure that the Manual Exposure box is checked. 2. Either use the selection arrows or type in a value in seconds (sec) or milliseconds (msec) for the exposure time depending on the radio button selected. The exposure times indicated below are camera dependent The minimum exposure time is 80 milliseconds (.080 seconds) per color. The maximum exposure time is 17 minutes (1020 seconds) per color. 3. Click on OK to save the settings. To have the camera calculate the exposure time, follow these steps: 1. From the SPOT Camera dialog stack, click on the Compute Exposure button: The Compute Exposure dialog appears as indicating it is computing exposure: And when completed, the Exposure Settings for the current Settings file appear: If you decide to change the settings, you can do so from the Image Settings dialog by pressing the Image Settings button and then the Manual Exposure setting on the left side of the dialog as indicated below. 44 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Any changes that you make will be saved to the current Settings file. The user may also change the manual settings using the Spot Live Preview dialog and, specifically, the Live Image Controls dialog. This is accomplished by first checking the box next to Use Manual Exposure in the Image Settings dialog under Manual Exposure, as seen above. Then press open the Live Preview dialog and then activate the Control button in the Spot Live Preview dialog. Select the appropriate exposure setting using the spin box controls under Exposure and Time. Wait for the camera to make the adjustment to the live image before changing the settings. The spin box settings are set to millisecond (msec) changes which, in the example below, would only change the 4 th decimal value in the Exposure Time setting indicated. As indicated below, the value of 4060 in msec is actually more than 4 seconds. To change the manual exposure controls in the Live Image Controls box is difficult to use and it is recommended to use Compute Exposure and then change exposure settings by flagging and inserting a whole number (e.g, 4000 msec) close to the original Compute Exposure setting. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 45

Configuring Settings Files Ch. 2 - Preparing to Take a Picture Note: For information on entering Image Type, Auto-Gain Limit, Adjustment Factor, and White Balance, refer to the appropriate section in this chapter. 2. Click on Begin. The Status message changes to Computing exposure When the camera finishes, the Exposure window displays the calculated exposure time and gain: 3. Click on OK. When you re-open the Image Settings window, the new exposure value(s) appear. Note: When you activate the Compute Exposure button or save the calculated exposure values to a Settings file, it automatically changes the Image Settings from Auto Exposure to Manual. For some reason I cannot get the Recall Previous Exposure button to be activated in either IPP 4.5 or 5.x. To recall the exposure times and gain from the previously captured image, follow these steps: 1. From the SPOT Camera, click on the Recall Previous Exposure button. The Exposure window displays the exposure times and gain used for the previously captured image, as shown here: 46 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files 2. Click on OK. The settings are saved to the current Settings file (i.e., default.vpf in the above illustration). Note: When you save the previously used exposure values to a Settings file, it automatically changes the Settings file from Auto Exposure to Manual. Gain (Manual Exposure, 8, 12, 14 bit mono and 24, 36 and 42 bit color) When you define your own Exposure, you can enter the precise gain that the camera will use for an image capture. You can enter a specific gain in three ways: You can manually enter the gain by selecting one of the five gain options (1, 2, 4, 8 and 16) from the select list. You can let the camera compute the best gain (and exposure times) by using the Compute Exposure feature (see the previous section). You can use the Recall Previous Exposure feature to recall the gain from the previous image capture (see the previous section). To manually enter a gain, follow these steps: 1. Ensure that the Manual Exposure option is checked. 2. Ensure that you are using either the appropriate bit image depth setting. 3. Select one of the five gain options from the list. To use the gain that the camera selects, follow the steps (in the previous section) listed for computing exposure times. The Exposure window, in addition to the optimal exposure times, displays an optimal gain. This gain is automatically selected in the current Settings file. TTL Output/Trigger The Spot cameras produce a 5 volt TTL pulse, when an image is captured, to enable an external device to be triggered and timed with image captures. This TTL pulse can be used to open and close an external shutter, for example. The TTL Output section the Image Setup allows the user to specify the active state of the TTL output. There are two TTL level options: Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 47

Configuring Settings Files Ch. 2 - Preparing to Take a Picture High Set the active state of the TTL output to High. Low Set the active state of the TTL output to Low. 48 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Chip Imaging Area SPOT allows you to use any rectangular portion of the CCD chip for image capture. Using a portion of the chip is helpful in cases where you want to: Keep file sizes small. Shorten download times. Avoid areas of the image with optical problems (e.g., vignetting). There are three image area options: Full Chip Capture the entire area of the CCD chip. Center Capture a rectangular part of the CCD chip s area that has the same height - to - width ratio as the full chip, and is centered on the center of the full chip. The percentage that you enter is the percentage of full chip width that the new area s width will be. To capture a centered proportion of an image, follow these steps: a) On the Settings window, check the Center option. b) Enter the percentage of the image that you want to capture. The minimum percentage is 10 and the maximum is 100. c) Click on OK to save the Settings file. Region Capture a specific part of the CCD chip. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 49

Configuring Settings Files Ch. 2 - Preparing to Take a Picture If you have pre-determined the exact pixel values of the area that you want capture, follow these steps: a) On the Image Settings window, check Chip Imaging Area and Region radio button as seen below. b) Enter the pixel values for the Left and Right columns and the Top and Bottom rows that form the boundaries of the region that you want to define. c) Click on OK. The camera only captures the region that you selected if using the Snap button on the Spot Camera dialog or Image-Pro Diagnostic Instruments Spot dialog is used. This Snap button uses the Image Settings Chip Imaging Area settings to capture the image. If you do not know the exact pixel values of the area that you want to capture, do the following: a) On the Image Settings window, click on the Full Chip option. b) Click on OK. c) Click on the Live Preview button to open the Live Preview dialog. d) Position the cursor at the upper left edge of the image region that you want to define then click and drag the mouse over the region of the Live Preview image you want to define. The Live Preview image will take a little time to update to a smaller region. To reset the Live Preview window to the full size of the CCD, use the Reset Area button ( )on the bottom, lower left portion of the Live Preview dialog. Once this button is pressed, give the Live Preview time to reset to the full chip area. Once reset, the Reset Area button will be grayed out as it is in the normal, full chip Live Preview. The Set Chip Imaging Area button ( )on the Spot Camera dialog stack allows the user to observe the chip imaging area once set using the Live Preview. Pressing the button opens the Chip Imaging Area dialog box to view the current Live Preview chip imaging area as seen below. 50 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Note that once the Image Settings dialog is open (so that the Chip Imaging Area is able to be set here), the Set Imaging Area button is disabled. If the Chip Imaging Area dialog box is open, the Image Settings dialog, conversely, cannot be opened. Image Orientation The Image Orientation option allows the user to define an image capture s on-screen orientation based on checking the appropriate checkbox. There are three options: Rotate the Left option rotates the image counterclockwise 90 degrees and the Right option rotates the image clockwise 90 degrees. Horizontal Flip This option flips the image display from left to right. Vertical Flip This option flips the image display from top to bottom. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 51

Configuring Settings Files Ch. 2 - Preparing to Take a Picture Sensor Temperature This window (see below) allows the user to regulate the temperature (Celcius or C) settings of the CCD in cameras that currently support this feature in our software. The Sensor Temperature setting will only be displayed as a setting selection between the Image Orientation and Image Readout functions when the attached camera has this capability. Regulated temperature is selected by activating the checkbox in the interface. Regulated temperatures are maintained + or 0.1 degrees C down to the lowest setting of -42.0 C. Regulated temperatures set to below -42.0 C will not conform to the + or 0.1 degrees C standard. Non-regulated temperatures may be obtained below -42.0 C depending on the experimental room conditions. Live updates of the camera temperature is observed in a box at the bottom of the Spot Camera dialog button stack when cameras supporting this feature are attached as indicated in the picture below. The temperature setting is determined using the up and down arrows in the spin box selection or typing in the necessary temperature in the spin box. Please note that temperature settings are both positive and negative. Since the dark current noise increases as the temperature of the CCD rises, keeping the temperature of the CCD constant will ensure that each image taken has similar dark current characteristics. Temperature regulation also increases the accuracy of the background subtraction. The temperature should be the same when creating background subtraction files as when applying the background correction. Please refer to the section Get Background Subtraction in this chapter for more details. 52 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Note: The Sensor Temperature dialog and display will only be available if the camera model being used is the Pursuit or Xplorer. Currently, these are the only Diagnostic Instruments camera models that have regulated temperature that can be controlled by software. Image Readout The Image Readout window allows the user to specify the rate at which data is read off from the CCD. The faster the sensor is read out, the nosier the image will be. The available sensor readout options will vary depending on the camera model. To select the speed to read out the CCD sensor, select a horizontal readout frequency from the dropdown list. Note: The Image Readout dialog and display will only be available if the camera model being used is the Pursuit or Xplorer. Currently, these are the only Diagnostic Instruments camera models that have variable readout frequencies. Post-Processing The final aspect of defining a Settings file is to determine the post-processing adjustments (corrections) that you want the SPOT camera/software to make. The options are as follows: Black Level Subtract (12 bit mono) Fluorescence Color Handling Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 53

Configuring Settings Files Ch. 2 - Preparing to Take a Picture Chip Defect Correction Noise Filter Image Size Double Gamma Adjust Rotate Horizontal Flip Vertical Flip Background Subtract Flatfield Correct Black Level Subtract (12 bit mono) The amplifier in the Spot camera has been adjusted so that the average black (no light) pixel value is between 20 and 25, rather than zero for 12 bit (4,095 full scale) downloads. This offset is called the black level (sometimes referred to as the pedestal ). Before each picture, the camera samples the black level by taking a picture with the shutter closed. The software then downloads the pixel values in the first row of the CCD chip, averages them, and stores this value as the black level. When capturing 8-bit monochrome, 24-bit RGB and 36-bit RGB images, the software automatically subtracts the black level from all pixel values so that black pixels are 0 (black) rather than 20-25 (dark gray), and colors are represented accurately. When capturing 12-bit monochrome images, you can turn off the automatic Black Level Subtract. This is a useful option in cases where you need high accuracy brightness measurements. For example, you may want to adjust the black level of an image on a pixel-by-pixel basis by subtracting a second picture, taken with no light (a dark frame ), from the original image. When high accuracy measurements are needed, the automatic Black Level Subtract is a problematic because the Black Level is an average of a sampling of black pixel values. Some values are higher, and some values are lower than the average. Subtracting this average value from a black pixel value that is lower than average would normally result in a negative value. But, since there are no negative values, the result is arbitrarily set to zero and the actual value is lost. This diminishes the accuracy of measurements. There are two situations in the SPOT software where the automatic Black Level Subtract is always turned off: When you are acquiring background images (the Get Background Image option) and 12 bit mono is selected as the Pixel Bit Depth on the Settings window. When you are capturing 12 bit mono images and the Background Subtract option is selected on the Settings window. Correct Color Technology Check the Correct Color Technology box to optimize the capture and display of color images. Checking this box enables the software to make the appropriate processing adjustments for color images based on International Color Committee (ICC) settings contained within the camera firmware. 54 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Chip Defect Correction When you capture an image, the SPOT program automatically compensates for CCD chip defects, so that they do not appear on your images. The Chip Defect Correction option allows you to turn off this automatic correction. This can be useful in cases when you are performing quantitative measurements of pixel brightness values and need to see precisely where chip defects are. The Chip Defect Correction box is, as a default, checked in the default.vpf Settings file. To turn off the program's automatic chip correction, deselect the Chip Defect Correction box. Noise Filter The Noise Filter corrects for the effects of electrical or thermal noise. The Filter Noise option eliminates single, isolated pixel values that appear out of place due to electrical or thermal noise by checking each pixel on the CCD chip as described below: 1. The program compares the red value of each pixel with the red values of the eight neighboring pixels. 2. If none of the eight neighboring pixels has a red value within ± x% (x is the Threshold value that you enter) of the red value for the pixel being checked, the red value for the pixel being checked is replaced with the average red value of the eight neighboring pixels. 3. The program repeats this procedure for the green and blue values of the pixel being checked. To activate the Noise Filter, check the Noise Filter box, and enter a value between 10 and 100. The default value is 50%. The program runs the noise filter each time an image is captured with that Settings file. Image Size Double When you select the Image Size Double feature, the camera interpolates between pixels to create an image that is twice as large. The program reads the red, green, and blue brightness values for the original (e.g., 1315 x 1035 pixel) image and interpolates those values to create a new image that has double the number of columns and rows (e.g., 2630 x 2070 pixels). The red, green, and blue values for the additional pixels are calculated based on the RGB values of the original pixels. The Image Size Double feature helps to reduce the pixellation of fine detail that often results when you use lower magnification objectives. When used with low magnification images, the additional pixels created via the Image Size Double feature help to smooth out details; this results in a real perception of higher resolution. Gamma Adjust Gamma Adjust smoothly lightens darker areas of an image without burning out bright areas or lightening black areas. This is especially useful for capturing/editing images with a wide range of brightness values (dynamic range) such as: Fluorescence specimens Macroscopic objects with glints, such as printed circuitry When the bright areas of these types of images are correctly exposed, the darker areas can be so dark that they are in effect invisible. Gamma Adjust can remedy this problem. The below figures illustrate the effect of a gamma adjust on a fluorescent image. The image on the left is the original image, prior to the gamma adjust. The image on the right has been gamma adjusted to a value of 1.75 (a value of 1 indicates Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 55

Configuring Settings Files Ch. 2 - Preparing to Take a Picture that the image has not been gamma adjusted). In this instance, the gamma adjustment results in a better display of detail by lightening the darker areas without burning out bright areas or lightening black areas: Fluorescence image, no gamma adjustment Fluorescence image, gamma adjusted to 1.75 Gamma adjustment corrects an image by creating a new version of the original. To create the new image, the Gamma Adjust function reassigns the RGB values of each pixel in the image according to the curve in the following graph: full scale Pixel Value - New Image gamma > 1 gamma = 1 gamma < 1 0 0 Pixel Value - Original Image full scale The above graph demonstrates the basic principles of gamma adjustment: 56 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Black (pixel value = 0) remains black at all gamma values. White (pixel value = full scale) remains white at all gamma values. Gamma values greater than one lift the darker areas of the original image into the brighter areas of the new image. A gamma curve is smooth: there are no unexpected jumps or cutoffs. This means that when viewing a gamma adjusted image, you will be able to see the details (intensity differences) in both the black and white areas of the image. On the Settings window, you can specify whether or not to automatically apply gamma adjustment to an image capture. To automatically gamma adjust an image after it is captured, follow these steps: 1. Check the Gamma Adjust box on the Settings window. 2. Enter a gamma value between.1 and 4.0. Although there is no formula for determining a gamma value, gamma values are typically between 1.3 and 1.7 Note: Although the gamma adjust option is typically used to lighten the mid-tones of an image, entering a value between.1 and 1 will darken the mid-tones. 3. Select one of the following options: HSL HSV RGB HSL (Hue Saturation Luminance), HSV (Hue Saturation Value), and RGB (Red Green Blue) are three different color spaces. A color space is any coordinate system that is used to mathematically represent color. The HSL color space is an intuitive model based on the three attributes that are common to all colors: hue, saturation, and luminance. The HSL color space assigns a value for each color attribute (e.g., hue, saturation, and luminance). In this color space the brightest saturated color is considered to be 50 % luminance. Above 50 % luminance, saturated colors transition through pastel shades before reaching pure white at 100 % luminance. This process mimics the natural perception of objects illuminated by broadband light sources. The HSV color space is similar to the HSL color space except that it uses value instead of luminance. In this color space, the brightest saturated color is considered to be 100 % value. This allows you to modify images that contain only saturated colors (e.g., fluorescence) without changing the bright, saturated areas to pastel or white. The RGB color space assigns a red, green, and blue value to each pixel in an image. This is the method that color computer monitors use to display images. The process by which the program applies gamma to an image capture differs according to the color space that you select. The following table briefly describes how gamma is applied in each of the three color spaces and lists situations where the use of one color space would be preferable to another. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 57

Configuring Settings Files Ch. 2 - Preparing to Take a Picture Colorspace Gamma Adjustment Process Recommended Usage HSL 1. RGB pixel values are translated to HSL (hue, saturation, and luminance) values. 2. Gamma is applied to the luminance. The HSL option is ideal for all color images except fluorescence images. 3. The HSL values are translated back to the RGB pixels. HSV 1. RGB pixel values are translated to HSV (hue, saturation, and value) values. The HSV option is ideal for fluorescence images. RGB 2. Gamma is applied to the value. 3. The HSV values are translated back to the RGB pixels. Gamma is applied separately to the red, green, and blue pixel values. The RGB option is ideal for monochrome images. 4. Click on OK to use the Gamma Adjust settings. 5. Click on Save Settings to save the changes to the Settings file. When you capture an image using this Settings file, the program automatically adjusts the image according to the gamma settings that you defined. Rotate The Rotate option allows you to define an image capture s on-screen orientation. There are two options: Right The image is rotated clockwise 90 degrees. Left The image is rotated counterclockwise 90 degrees. Horizontal Flip To flip an image display from left to right, check the Horizontal Flip box.. Vertical Flip To flip an image from top to bottom, check the Vertical Flip box. Background Subtract Background Subtract allows you to subtract a previously acquired Background Image from an image capture. The Background Subtract option is useful when: You want to eliminate the irritating background glow seen in some fluorescence images. Sometimes, you will notice this glow while looking at the specimen under the microscope; other times, you will only notice it after you have already captured an image. You are doing high accuracy measurements in 12 bit monochrome and want to subtract a dark frame from each image to adjust the black level of each pixel. Refer to the Black Level Subtract section of this chapter for more details. 58 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files Note: If 12 bit mono is selected as the Pixel Bit Depth on the Settings window, the automatic Black Level Subtract process is always turned off when acquiring background images. The Background Subtract option works by taking two pictures of a slide, using the same exposure and gain settings: a foreground image that contains the specimen and the background glow and a background image that contains only the background glow. When you select the Background Subtract option, the program subtracts the background image from the image of the specimen, thereby removing the background glow. The figures below illustrate a fluorescence image before and after background subtraction: Fluorescence sample prior to background subtraction Fluorescence sample after background subtraction To perform a background subtraction, you must capture the background image that the camera will subtract to create the final image. Follow these steps: 1. Ensure that the specimen is under the desired objective and in focus. 2. From the SPOT Camera dialog, click on the Background Image button: The Get Background Image dialog box appears: 3. A Status box indicates what the user should do next -- Move to sample for exposure computation of the background image. The default number of image frames to average in determining the background image is three (3) but the user can change the number of images to use in the calculation at any time prior to clicking on the Begin button by using the spin box arrows. The user must capture a minimum of one (1) image and a maximum of 50 images for the background calculation. Please type in the Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 59

Configuring Settings Files Ch. 2 - Preparing to Take a Picture Background File Name prior to clicking on the Begin button and indicate the location of the saved file using the file button (button with 3 dots) to the right of the Background Image File Name field. 4. If a Background Image file has already been captured, the user can select the file using the file button with the three dots to the right of the File Name field. 5. Click on Begin. The Get Background Image dialog box displays the Status Computing Exposure and opens a SPOT Camera dialog box indicating that the user should Move to background or block light as indicated below and press the OK button. 6. Occasionally, the user will encounter background issues (e.g., too bright) and the software will prompt the user to correct the error before continuing in the background exposure calculation. If an error like the one indicted below occurs, please correct the error and press the OK button to continue. If the error is not corrected, the background exposure determination will stop and the user must start the process over again. 7. If the error was corrected successfully, the Status box now indicates Exposing and then Processing as seen below. 60 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files 8. When the computations for the bright light images have been determined, the user will be prompted by another SPOT Camera dialog that indicates Block all light to the camera for use in computing the remaining factor. The user should press the OK button to complete the process and then return the light settings to normal before capturing images. 9. When the program finishes capturing the background image (e.g., the image that will be subtracted from the final image), an image capture should be performed to test for an adequate background image. If the resulting background corrected image is satisfactory, then the user can resume live imaging and capture. The user should note that going back to the Image Settings Post-Processing area in the SPOT Camera button stack will now show the Background Subtract box checked and the name of the background image indicated in the file name box. The file name for background images uses the special format designation as a.tfr file name. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 61

Configuring Settings Files Ch. 2 - Preparing to Take a Picture 10. If the background image is adequate, the user should now use the Save button on the Image-pro Diagnostics Instruments Spot interface to save the final selections to the current or a new Settings file name (.VPF). Because background image files are different for each objective, you might want to name the background file according to the objective's magnification (e.g., 10x). Note: The default file location varies, depending on where you Installed the SPOT program. With typical installations, by default, the background and flatfield correction images will be saved to the C:SpotCam/BkgImgs folder on the computer hard drive. 11. The user may now move the specimen back into the field of view, and perform the image capture with the background subtracted from every image captured. Refer to Ch. 3 Taking a Picture, for more details on the image capture process. Flatfield Correct Flatfield Correct is used to correct for display problems associated with uneven intensity or coloration in your illumination, or artifacts (e.g., dust) in your optical system. It is used mainly with lower magnification brightfield images. Flatfield correction works by taking a picture of the brightfield background without the specimen in the field if view. This image lets the SPOT software know, on a pixel-by pixel basis, which areas in the field of view are receiving less illumination than the brightest areas. When taking a picture that contains a specimen, the software uses this information to accurately increase the brightness of each pixel in the dimly illuminated areas. The resulting image gives the appearance of an even illumination. Flatfield Correct is similar to Background Subtract in that it is a process that involves two images. In the case of flat field correction, the flatfield image and the final image are the two images. Note: Flatfield correction with the SPOT camera refers only to the correction of uneven lighting. It should not be confused with the correction of optical field flatness. 62 Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005

Ch. 2 Preparing to Take a Picture Configuring Settings Files It should not be confused with the correction of optical field flatness. To use Flatfield Correct, follow these steps: 1. Ensure that the specimen is under the desired objective and in focus. 2. From the SPOT Camera dialog button stack, click on the Get Flatfield button. The Get Flatfield dialog box appears and the user types in the Flatfield File Name, determines the final location of the flatfield file on the hard drive and designates the number of image frames to be used in the flatfield correction. 3. Remove the slide from the field of view on the microscope. 4. Click on Begin. The program displays the Status as it computes the exposure times and captures the flatfield image. If the flatfield image is being determined for color images, the Status will indicate Exposing in each of the Red, Blue and Green color planes. Image-Pro Driver Supplement to the SPOT User Guide, 6/1/2005 63