HCImage Getting Started Guide

Transcription

1 HCImage Getting Started Guide Release 4.4 October 2016 FCD-0332-HCIAQ41016

2 This guide, as well as the software described in it, is covered under license agreement and may be used or copied only in accordance with the terms of the license agreement. The information in this manual is subject to change without notice and may not be reproduced without Hamamatsu's permission. Hamamatsu has carefully prepared this manual, however, no responsibility is assumed for possible inaccuracies or omissions. Some images are simulated. HCImage is a registered trademark of Hamamatsu Corporation. DCAM-API and ORCA are trademarks of Hamamatsu Photonics K.K. All product and brand names are trademarks or registered trademarks of their respective companies. Hamamatsu Corporation 360 Foothill Road, Box 6910 Bridgewater, NJ USA sales@hamamatsu.com Software Support hcsupport@hamamatsu.com

3 Table of Contents Installing HCImage 3 Adding Devices to a Profile 4 Parallel Port Card Installation and Configuration 6 Image Display 7 Histogram 8 Contrast 9 Line Profile 11 TTL Input & Output 12 Filter Control with the NI USB Filter Control using TTL 14 The Capture Pane 15 Capture Presets 15 Camera Control 16 Binning and SubArray 16 Trigger Modes, Speed and Registration 17 Advanced Camera Properties 18 Processing 18 Capture an Image 20 Capture with the ORCA-Flash4.0 V3 or LT and the W-VIEW 21 Capture a Time Lapse Image Sequence 23 High Speed Streaming 28 Scheduler Example 31 Multiple Site Time Lapse 33 Montage - Stitch 35 DIA Overview 37 DIA Analysis Examples 41 Analyze a Single Image - Advanced Mode 43 Sequence Intensity Analysis - Simple Mode (IPA) 44 Analyze Objects Inside of a Region of Interest 45 Advanced Analysis (IPA & Analysis) 46 Create a Workfile to Track Moving Objects (IPA & Analysis) 47 Object Tracking (IPA & Analysis) 48 How to Merge Several Monochrome Images 49 Batch Export 50 Batch Export to AVI 50 Batch Export W-VIEW Images

4 Installing HCImage 1. Insert the HCImage installation DVD into the DVD-ROM drive. If autoplay is enabled, the HCImage setup will run automatically. If autoplay fails to start, locate your DVD-ROM drive and double-click on setup.exe. 2. Click Yes, if prompted by the User Account Controls. 3. To begin the installation wizard, click Next. 4. Review the Software License information and click Yes. 5. Review the README section for up-to-date information on software compatibility and support. When you are ready, click Yes. 6. On the Personalize screen, enter your registration information and click Next. 7. Choose the Destination Folder and click Next. It is recommended to install the software in the default path. 8. If you are ready to proceed with the installation, click Install. 9. Follow the instructions on each installation page. 10. Securely connect the dongle ( ) to a USB port after the software installation has finished. 11. Install the appropriate DCAM-API drivers, see the instructions below, then turn your camera on prior to launching HCImage. 12. Click the HCImage icon on your Desktop to launch HCImage. 13. Register the software to receive technical support, please go to and click Register. Install DCAM-API Drivers Before installing the camera driver, make sure that the camera is turned off. 1. Open Windows Explorer, go to HCImage installation DVD, expand the Drivers folder, open the Cameras folder and open the DCAM folder. If you downloaded HCImage, please go to and download the DCAM-API drivers for Windows. 2. Double-click Setup.exe to launch the DCAM-API Setup dialog. 3. Click Yes, if prompted by the User Account Controls. 4. Select the appropriate driver for your Hamamatsu camera from the DCAM-API Setup dialog. If you are unsure of which driver to install, please consult the DCAM-API Compatibility Note or contact your local Hamamatsu representative. To view DCAM- API Compatibility Note, select Others and then click Compatibility Note. 5. Click Next to begin the installation. 6. Follow the instructions on each installation page and click Finish when the installation is complete. 3

5 Adding Devices to a Profile Add and setup peripheral hardware devices to the profile. Supported hardware devices include: cameras, stages, filters, shutters, I/O and microscope devices. For a list of supported devices, please visit the our website at Adding a Camera 1. Install DCAM. For information on installing DCAM-API, see "Installing HCImage" on the previous page. 2. Turn camera on and then launch HCImage. Go to File > Current Profile > Device Control. 3. Select Image Capture Devices > Click Add > select Single Camera > OK. 4. Select the available camera, click OK and then click OK to close the Profile window. 5. Go to the Capture pane and select the camera from the Camera drop-down menu. Adding a Filter Wheel and a Shutter 1. Go to File > Current Profile > Device Control > Filter Devices > Add 2. Select the type of Filter Wheel and check Add Shutter. 3. Click the Com-Port tab and select the Com-Port where the filter wheel and shutter are connected. 4. Click the Filter Setup tab and click Apply to test communication and verify filter positions. 5. Customize the filter list. To modify an item in the filter list, highlight it and change its name in the name field and click Modify. 6. Click on the Shutter Setup tab. Click Apply > Click Test to verify communication with the shutter. 7. Click OK once all of the filter names have been added and the devices are communicating properly. If a communication error message appears, ensure that the device is connected to the correct Com-Port. 8. Configure Filter and Shutter settings. Click the Devices pane > Filter Setup > Add > Enter filter name > OK. 9. With the new name highlighted, select the filter wheel installed under the Filter-Shutter tab > double-click Don t Care or the name under Position. Select the filter position or wavelength from the pop-up window. Adding a XYZ Stage 1. Go to File > Current Profile > Device Control > Stage Devices > Add 2. Select the type of stage to be added. 3. Click the Com-Port tab and select the Com-Port. 4. Click the Stage Setup tab, then click Apply to test communication. If you get an error message make sure that the correct Com-Port is selected, and that you have connected everything properly. Click OK. 4

6 Adding an Automated Microscope 1. Install the manufacturer s software and configure the microscope accordingly. 2. Go to File > Current Profile > Device Control > Microscope Devices > Add > Select type of microscope > Click on the Com-Port tab and select the Com-Port where the microscope is connected. 3. Click the Controller tab > Click Auto to read the microscope configuration file created in Step 1. The component list will be updated. Click OK to accept the current settings and close the window. 4. Click on the Devices pane > expand the Microscope Setup panel and select the component to be controlled. 5. Microscope components listed are accessible through the Filter Setup panel in the Devices pane to synchronize activity during automated image capture. Calibrate an Image from Pixels to Microns 1. Open or capture an image with some known distance, for example a micrometer. 2. Click on the Calibration Properties icon on the Analysis toolbar. 3. Enter a Title for the calibration file (e.g., 10x). Select Microns from the Units drop-down menu. 4. Click Calibrate. Move the cursor to the start of a known distance; click and drag a line to span the distance to measure. 5. Enter the known distance of the line and click OK. 6. Click Add and repeat the previous steps for adding additional calibrations. 7. [Save Calibration] Click OK > Select file path > File name > Save. The Spatial Calibration is displayed in the lower right-hand corner of the image file. Calibrate an XY Stage 1. Before calibrating the stage, make sure to load the correct scale factor for the selected objective. Click the Devices pane > Click the XYZ Setup panel > Click the Stage Setup tab > Click Step Size Center an object to the green crosshair box and click OK. The green crosshair will move to the bottom-right, center the same object and click OK. 4. If the object moved back to the center of the top crosshair, the calibration for X, Y has been successful, click OK. If it did not move to the same location, check the camera orientation to the microscope and repeat Steps 2 &

7 Parallel Port Card Installation and Configuration We recommend the SIIG CyberParallel PCIe port card ( The installation guide and driver are also available from the SIIG website Install the parallel port card and driver as per the instructions provided with the card. 2. Launch the Device Manager, right-click on Computer, select Manage, click Device Manager and expand Ports (COM & LPT). 3. The SIIG card should be visible, right-click on the card and select Properties. 4. Go to the Port Settings tab, the LPT Port Number should be LPT1 if there are no other parallel ports on the system or LPT2 if there is an on-board parallel port. If there are no other parallel ports on the system and the LPT Port Number is not set to LPT1 or LPT2, select LPT1 from the drop-menu. 5. Go to the Resources tab, in Resource Settings, on the first row, highlight and copy the first four digits under Settings. 6. Launch HCImage, go to File, select Current Profile, click Device Control, select IO/LED Devices and click Add. 7. Select Parallel I/O, go to the LPT-Port tab, and select the LPT Port for the SIIG card. 8. Highlight the last three digits after 0x and paste the content. 9. Click OK, the parallel port has been added as an IO/LED device in the profile. 6

8 Image Display The Image Display provides feedback from live and captured images and is essential for setting up and adjusting devices and lighting conditions. The window can be hidden or shown using the Toggle Image Display button ( ) in the Main Toolbar. Understanding the Image Display The Image Display provides feedback about the acquisition, including: Display Image: The drop-down menu determines the image to be displayed when capturing in monochrome multi-channel mode. The drop-menu is only available when in that capture mode. Frame Rate: The current frame rate that the camera is capturing images. Position: Displays the X and Y coordinates of the cursor. Gray Levels: Displays the pixel intensity value for each channel at the cursor location. Histogram: Click ( ) to toggle the histogram on and off. Contrast Hi/Lo: Click ( ) to apply a contrast function based on the minimum and maximum intensity values of each channel. Image Navigator: Click ( ) to display the entire image in separate window with navigation and zoom controls. Zoom Controls: Use the buttons to change the zoom factor of the active image or select a value from the drop-down menu. Calibration: Displays the calibration factor for the current selected calibration. Note: When using the ORCA-Flash4.0 LT, ORCA-Flash4.0 V2 and the ImagEM X2, the pixel values displayed in the status bar are reported in gray levels and electrons. 7

9 Histogram The Histogram Display shows the current intensity distribution of an image and may be toggled on/off by clicking on the Histogram icon ( ) in the Status Bar. For color images, multiple histograms are overlaid, one for each color. Areas where the histograms overlap are represented by the resultant color (i.e., red + green = yellow, red + blue = magenta and green + blue = cyan). The histogram reflects the bit depth of the image, a 16-bit image for example, consists of 65,535 possible intensity values. Statistics including: the range, mean and standard deviation are displayed above the histogram and may be displayed in a separate window by enabling Stats. Histogram Display Features Roi: displays the intensity of the ROI, if a Region of Interest has been defined. Log: displays the Log of the Intensity Histogram is displayed. Sat: the Saturation function will display yellow representing a range of 4% from saturation. Red will be displayed when the image intensity reaches saturation. At the other end of the range when the image intensity is within 4% of zero (black) cyan is displayed. Blue will be displayed when zero is reached. Stats: displays the statistics in a separate window that may be undocked and enlarged. Channel Display: for color images, use the channel display to select which channel or channels to display in the histogram. The selected channel(s) are displayed in color in the histogram, the other channel(s) are displayed as gray. Current Display Depth: displays the bit-depth of the image, changing the display depth of the image does not affect the actual data, but can improve viewing clarity. It also affects the maximum slider value in the contrast dialog, identify dialog, and qualify dialog. The actual bit-depth of captured data can be controlled in Capture tab. Note: When using the ORCA-Flash4.0 LT, ORCA-Flash4.0 V2 or the ImagEM X2, the histogram statistics are reported in gray levels and electrons. This feature is only available when using the ORCA-Flash4.0 LT, ORCA-Flash4.0 V2 and the ImagEM X2. 8

10 Contrast Contrast stretching, sometimes referred to as scaling, redistributes the range of intensity values used to display the image based on a range that you specify. Adjusting the upper and lower limits of the range will result in a decrease or increase in the brightness of the image. For color images this may be done per channel (unlocked) or uniformly (locked). By default, the range is based on the bit depth of the camera and is displayed in the histogram. For example, a 16-bit camera will acquire images with 65,535 possible intensity values (gray-levels). In some instances, all of the pertinent image data is contained within a range in the lower end of the intensity values. By adjusting the upper and lower limits of the range in an image, you can more easily see variations in brightness that might otherwise be indistinct. Another option is to change the display depth using the Current Display Depth drop-menu ( viewing clarity. ). This does not affect the actual data, but can improve the Note: Intensity profile data is not affected by the contrast enhancement. The contrast enhancement function is an image display tool rather than an image processing operation and operates only on the display data. If it is desirable to work on the contrast-enhanced image, copy the Display Image to an image document. Contrast Settings The contrast tools are incorporated into the histogram display and can be enabled by selecting Contrast below the histogram. Adjust the range of intensity values by dragging the highlighted upper or lower limit bars located on either end of the histogram. The selected range is displayed above the histogram just below the standard deviation and is also displayed in the Stats windows when enabled. Select from the various monochrome and pseudocolor enhancements in the Curve drop-menu to draw attention to specific aspects of the image that would otherwise be indistinct. For color images, various contrast stretches or pseudocolor look-up table (LUT) enhancements may be made on each channel separately (Unlocked mode) or simultaneously on all channels (Locked mode). 9

11 Making Adjustments When it comes to making contrast enhancements, some data sets require an added level of control in order to fine tune settings for optimal display. HCImage provides several methods for fine tuning the selected range used for the contrast enhancements, as well as the ability to customize LUTs. The histogram may be undocked and enlarged to provide more detail when making adjustments. Setting the Range For more control, there are several methods for making adjustments to the selected range of the contrast enhancement. The examples below show each method, followed by a description. 1. Manually Enter - highlight one of the selected range values and then enter the range value. 2. Spin Box - hover over the selected range values and then click on the up and down arrows to adjust the values. 3. Scroll - select the range to change and then scroll up or scroll down using the mouse scroll wheel. 4. Click and Drag - hover over the upper or lower range limit bar (upper limit bar shown above), the cursor will change to a left right arrow and then click and drag the cursor to the left to decrease the range limit or to the right to increase it. 5. Ctrl and Drag - similar to the method above, pressing the Ctrl key provides greater precision in that mouse movements result in minimal incremental changes in range. Press and hold CTRL, then click and drag the cursor to the left to decrease the range limit or to the right to increase it. Custom LUT Custom LUTs provide an added level of precision for fine tuning contrast enhancements. Users can tweak one of the predefined LUTs or create their own. To customize a LUT, click on an area of the histogram to define a point in the curve, then click and drag to stretch the curve. 10

12 Line Profile The Line Profile is a useful tool that allows users to draw a line on the image and see the corresponding intensity values plotted on a graph. The line profile may be used on a live or a captured image or image sequence. For two and three channel color images, an intensity profile is plotted for each channel as it's respective color. Click the Line Profile icon ( ) and then draw a line on the image. See the Line Profile Properties below for a list of drawing tools. The Line Profile may be toggled on/off by clicking the Line Profile icon. Line Profile Properties The Line Profile icon is located in the Annotations toolbar, click the Line Profile drop-menu to view the line properties. The Line Profile Properties are defined below. Line Thickness: adjust the line thickness from one to five pixels, the selected thickness is displayed on the toolbar icon Single: left-click, hold and draw a single straight line Segmented: create an open polygon by left-clicking to define a starting point, then left-click to create an end point for each segment, and right-click to complete the line Free-hand: left-click and hold, then using the mouse trace the line on the image Clear: delete the current line profile Viewing the Data The intensity values for the line are plotted in a graph. The graph can be undocked and resized for optimal viewing. It is continually updated, when live or during playback. The intensity data can be exported to a spreadsheet and saved as a.csv (comma separated values) file. If a calibration was used, the scale factor values will be included in the exported data. There are two options for exporting data to a spreadsheet: 1. Export Data to Spreadsheet: export the intensity values for the current image 2. Export Data to Spreadsheet (all fields): export the intensity values for all of the fields in the data set Note: When using the ORCA-Flash4.0 LT, ORCA-Flash4.0 V2 or the ImagEM X2, the pixel values for line profile will be plotted in gray levels and electrons for monochrome images. For color images, the user can toggle between the two by clicking the Electron Count icon ( values will be exported. ). Both pixel 11

13 TTL Input & Output The example below will provide a quick overview on how to configure the parallel port to trigger capture events by receiving TTL pulses and how to output TTL pulse based on capture events. Go to the Device panel and click IO/LED Setup. The Capture Events tab is divided into Capture Inputs and Capture Outputs. Capture Events For Capture Inputs, click under Frequency to choose when to trigger. Each click will cycle through the list of choices: For Capture Inputs: Sequence Start - TTL input to trigger the start of a capture event, click under Frequency to choose when to trigger. (Each click will cycle through the list of choices) Each Field - Waits for an input trigger at the beginning of every capture cycle First Field - Waits for an input trigger at the start of the sequence Each Pass - Waits for an input trigger before the first field of each sequence pass Sequence Stop - TTL input to stop a time lapse image sequence. Scan Start - TTL input used by the Scheduler to trigger a scan. (not available in HCImage Live) For Capture Outputs: Sequence On - TTL output for the duration of the sequence. Field On - Will set the chosen state of the pin at the start of a field's exposure. This pin will signal on and off as each field is captured. Scan Pulse On - TTL output used by the Scheduler. Exposure On - TTL output for the duration of each exposure. Configure Capture Events The table below provides the basic steps for setting up capture events. Setup Capture Inputs: Setup Capture Outputs: 1. Select input pin 1. Select the capture output type 2. Set the input pin state 2. Select the output pin 3. Select the capture frequency 3. Set the output pin state 12

14 Filter Control with the NI USB-6501 The National Instruments (NI) USB-6501 is a digital I/O device that is used to control LED light sources using TTL. Adding the NI USB-6501 to a Profile 1. Launch HCImage, go to File and select Current Profile. 2. In the Device Control tab, select IO/LED Devices and click Add. 3. Select National Instruments, click on the Device List tab and USB-6501 should be listed. 4. Go to the IO/LED Setup tab, starting with port0/line0 under Digital I/O, set the status to Active Output. 5. Set the status to Active Output for the rest of the lines. 6. Select the Analog Output tab and set the status to Disabled for each of the lines. 7. Click OK, the NI USB-6501 has been added to the profile and is listed along with the other hardware devices. 8. Click OK to exit. Filter Setup In this example, the LED turns on when the line state is high and off when the state is low. Line 0 controls the Red LED, Line 1 the Green LED and Line 2 the Blue LED. 1. Go to the Devices pane, enable IO/LED Devices under Filter Setup. 2. Next, make sure that Return to Idle on Exit, Return to Idle After Capture and Return to Idle During Delay are enabled. 3. Select Default Idle Positions, then select the first row under Filters in the IO/LED Device tab. 4. Click the ellipses to the right of Don't Care in the first line. 5. Enable pin settings by clicking Don't Care. 6. Click OK. For the default state, all of the lines should be off. 7. Click Add, enter Red as the filter name and click OK. 8. Select the row under Filters in the IO/LED Device tab and click the ellipses. 9. Uncheck Don't Care, enable Line 0 and click OK. 10. Right-click on the filter and select Red from the tint list. 11. Now add the Green filter, enable Line 1 and then tint it Green. 12. Add the Blue filter, enable Line 2 and tint it Blue. 13

15 Filter Control using TTL This example explains how to add the parallel port as an I/O device in HCImage and configure it to control an LED light source. Adding the I/O Device to a Profile 1. Launch HCImage, go to File and select Current Profile. 2. In the Device Control tab, select IO/LED Devices and click Add. 3. Select Parallel-IO, click on the LPT-Port tab and select LPT1. 4. Go to IO/LED Setup, select Output Lines and set the status to Active for all of the pins. 5. Click OK, the parallel port has been added to the profile and is listed along with the other hardware devices. Click OK to exit. Filter Setup In this example, the LED turns on when the pin state is high and off when the state is low. Pin 2 controls the Red LED, Pin 3 the Green LED and Pin 4 the Blue LED. 1. Go to the Devices pane, expand the Filter Setup panel enable IO/LED Devices. 2. Next, make sure that Return to Idle on Exit, Return to Idle After Capture and Return to Idle During Delay are enabled. 3. For the default state, all of the LEDs should be off. Select Default Idle Positions and go to the IO/LED Device tab. 4. Under Shutters, select LPT1 P2, use the drop menu to the right of Don't Care and set the state to Low. 5. Set the pin state to Low for LPT1 P3 and LPT1 P4. 6. Click Add, enter Red as the filter name and click OK. 7. Right-click on the filter and select Red from the tint list. 8. Go to Shutters in the IO/LED Device tab and set the LPT1 P2 state to High and the state to Low for LPT1 P3 and LPT1 P4. 9. Now add the Green filter, tint it green, then set the LPT1 P3 state to High and the state to Low for LPT1 P2 and LPT1 P Add the Blue filter, tint it blue, then set the LPT1 P4 state to High and the state to Low for LPT1 P2 and LPT1 P

16 The Capture Pane The Capture Pane provides a flexible and comprehensive method to access the camera's features and functionality. The Capture Pane is organized by functionality into panels that can be expanded when in use or collapsed when space is needed. The capture controls at the top of the pane (shown below) are always visible and used for controlling how images are acquired and displayed. Capture Presets Capture settings can be saved as presets and then loaded when needed. Create multiple capture presets to easy change between frequently used capture settings. Capture presets may be selected from a list of saved presets available in the Capture Presets panel, located at the top of the Capture pane. To add, remove or view the settings of a preset, click the ellipses to the right of the list, to open the Capture Presets dialog. Capture presets save basic settings such as the capture mode, channels, filters, exposure times, as well as output trigger settings and advanced camera properties. For a list of the camera settings that are saved, select a capture preset from the Capture Presets dialog and click View. HCImage will load the capture settings from the previous session when launched. Note: Capture presets are not automatically saved before changing presets or exiting the software. To make changes to a saved capture preset, select the capture preset from the list, adjust the capture settings and click Save. 15

17 Camera Control Manage capture settings using the individual channel and exposure controls. Hint: In order to achieve the best possible acquisition speed when acquiring color images, set the same exposure for each channel. Once the exposures have been entered, click the Exposure Lock icon ( ) to lock the exposure settings. Now any exposure adjustments will be made to all of the channels. Binning and SubArray With a CCD camera, 2x2 binning increases the signal to noise ratio by a factor of four and increases the speed of image acquisition by a factor of about two. With an scmos camera binning is purely digital, 2x2 binning increases the signal to noise ratio by a factor of two. Digital binning does not increase the speed of image acquisition. Adjust the spatial resolution using a subarray preset for increased speed and less data throughput. For scmos cameras a subarray must be centered on the camera sensor in order to achieve maximum speed. The subarray preset sizes for in the list are automatically centered (for scmos) but custom arrays are not. To center a custom array, see the example below. 16

18 Define a Custom SubArray for Maximum Speed (ORCA-Flash) Click Live, focus on the sample and move the area of interest into the center of the image. Follow the steps below to define a custom subarray. Trigger Modes, Speed and Registration By default the camera is controlled through software but some cameras offer advanced triggering features allow the camera to control external devices or be controlled by them. The speed, capture mode and output trigger settings can be adjusted based on the needs of the application. The example below describes options for the ORCA-Flash4.0 V

19 Output Trigger Options The camera provides a range of trigger output signals to synchronize with an external instrument where the camera becomes the master and the external instrument becomes the slave. There are three different trigger output functions, as well as a continuous High output (High output fixed) or continuous Low output (Low output fixed). For a detailed description of each of the output trigger options, please see "Camera Trigger Output" on page 1. Advanced Camera Properties DCAM Properties provide a list of camera parameters reported by DCAM. The camera properties and reported values are specific to the connected camera and in some cases provide access to additional functionality based on the capture mode. These properties are referenced in text and screenshots as needed for setting specific camera modes. Most of the camera properties in the list display values that cannot be changed and appear grayed out. Processing The Processing Panel provides the opportunity to enhance images during focus and acquisition by incorporating image-processing operations during or immediately after image Capture. To select an Image Processing operation, first expand the Process Pane and then select the Operation Type. Rolling Average and Frame Integration are used for noise reduction. Use the image arithmetic functions like Shade Correction, Background Subtraction or Image Subtraction to remove artifacts from the incoming image. Clicking Capture1 will initiate image capture with the selected image processing operations applied. Note: For Image Correction or Arithmetic, the user must first choose a source or background image. The image may be the current image saved in a buffer or one previously saved to disk. To use the current image, make sure Processing is OFF, select Buffer, click Capture and then select Shade Correction, Background Subtraction or Image Subtraction. Use the same method when using an image from Disk. 18

20 Hint: Enable Processing ON for correction image when you would like to capture a correction image using Rolling Average or Frame Integration. When you are ready to capture the correction image, select Rolling Average and enter the number of frames, enable Processing ON for correction image and then click the Capture button to the right of Buffer. The captured averaged image is stored in the buffer and ready to use a correction image. How to Setup a Background Subtraction Typically used in fluorescence microscopy, a background subtraction can be used when the image presents a dark non-uniform background. To perform a background subtraction click Live, bring the sample into focus and then move the stage off of the sample so that only the background is visible. Next, follow the steps below, when finished move the stage to bring the sample into view and the background subtraction is applied. Hint: HCImage remembers the capture settings from the previous session, if background subtraction was left enabled, the following message will appear the next time HCImage is launched. 19

21 Capture an Image 1. Go to the Capture pane, select RGB Color: 3-Band from the drop menu. 2. Select Red from the filter drop menu for channel 1, Green for channel 2 and Blue for channel Click Live for a live image. Adjust exposure manually or use Auto Expose. 4. Select which channel to display clicking on 1, 2, or Enable Live Color mode to sequence through the channels displaying a color image while live. 6. Click Capture1 to acquire a 3 channel image. Hint: In order to achieve the best possible speed when acquiring color images, set the same exposure for each channel. Once the exposures have been entered, click the Exposure Lock icon ( ) to lock the exposure settings. Now any exposure adjustments will be made to all of the channels. Using AutoSave Enabling AutoSave will automatically save the current image every time Capture1 is selected. The captured image is saved as a TIFF (or a MPTIFF for mono multi-channel image), based on the file name and destination directory defined in the AutoSave Properties dialog. 1. Enable AutoSave and then click on the ellipses to open the AutoSave Properties dialog. 2. Enter or navigate to the destination directory. 3. Enter the file name and the starting image number, subsequent captures will be number sequentially. 4. Enable Open Document after saving, to open image documents immediately after capture. 5. Click OK. 20

22 Capture with the ORCA-Flash4.0 V3 or LT and the W-VIEW The W-VIEW GEMINI uses image splitting optics to output a pair of images, side-by-side onto the camera sensor. HCImage incorporates the W-VIEW mode, a multi-view functionality, allowing the ORCA-Flash4.0 V3 and LT to function in a similar manor as using dual cameras. In W-VIEW mode, the ORCA-Flash4.0 V3 and LT support independent exposure time settings and independent readout directions for the upper and lower halves of the sensor, as well as separate position offsets for subarray. Note: Independent exposure time settings, independent readout directions and separate position offsets during subarray are specific to the ORCA-Flash4.0 V3 and LT and not available when working with other cameras, such the ORCA-Flash4.0 V2 and ImagEM X2 in W-VIEW mode. Add W-VIEW Camera to Profile 1. Go to File and select Current Profile. 2. Go to the Device Control Tab, select Image Capture Devices and click Add. 3. Select W-VIEW Camera and click OK. 4. Select C U S/N: ###### and click OK. (Note: Only properly installed DCAM supported cameras will appear in the Select Camera dialog.) 5. The camera is now listed as a capture device, click OK to add it to the profile. 6. Go to the Capture Pane and select the C U S/N: ###### W-VIEW from the Camera drop-menu. Capture Modes W-VIEW capture modes include: Mono 1 Channel, RGB Color 2-Band and Mono 2 Channel. To select a capture mode go to the Capture panel and click on the drop-menu above the Live button. The Camera Control pane is modified based on the capture mode selected. The capture modes are explained below. Mono 1 Channel In the single channel monochrome mode, the user can select which image to display, only one image will be displayed at a time. Click on the 1 or 2 button to select which image will be displayed. RGB Color 2-Band The RGB Color 2-Band mode displays a merged red-green image from image 1 and

23 Mono 2 Channel In the two channel monochrome mode, both images 1 and 2 are displayed (i.e., the whole camera sensor is displayed). Image Alignment The Camera Registration feature allows the users while Live to flip and rotate the image. Click on the Registration button in the Trigger Modes, Speed and Registration pane to open the Camera Registration dialog. 22

24 Capture a Time Lapse Image Sequence The Time Lapse scan provides flexibility and a variety of options for defining a time lapse to fit the needs of your application. Scan Settings The Scan Settings panel provides multiple options for defining speed, storage, duration and output settings. Scan settings can be saved for future use. Note: Select Enable Maximum to acquire at maximum speed. During maximum speed, items which slow down acquisition will be ignored. 23

25 Auto Save In the AutoSave Properties dialog, the user can determine how and where to store the acquired data. Image data can be saved as a CXD, TIFF or MPTIFF. The example below provides a description of the Auto Save Properties dialog. Note: MPTIFF files have a 65,000 image limit or a 4 GB size limit. For image sequences having more than 65,000 images or larger than 4 GB, multiple MPTIFF files will be saved and numbered sequentially. Storage Options The three options for storing acquired data during a time lapse include saving to Disk, Memory or Temporary Buffer. Save to Disk Acquired data is written directly to the hard drive. Frame rates vary based on the PC configuration, including the type and speed of the hard drive(s) being used. Save to Memory Acquired data is stored in memory and then written to disk when the time lapse is complete or stopped. When the system runs out of memory during a time lapse, acquired data is written to disk for the remainder of the sequence. Saving to memory typically provides a higher frame rate with less timing variation then saving to disk. The maximum number of images that can be acquired depends upon the amount the RAM in the system and the RAM limit set in HCImage. This number is displayed to the right of the memory storage option. When Memory is selected, End Frame automatically displays the maximum number of frames that can be streamed to memory, although any number less than the max can be entered. The Status Bar, located in the bottom left corner of the application window, displays the maximum number of frames that can be streamed to memory. Save to Temporary Buffer Acquired data is stored in memory with the option to review the image sequence before saving or deleting it. Storage is limited to the amount of system memory without the option to write to disk when the memory is full. The maximum number of images that can be acquired depends upon the 24

26 amount the RAM in the system and the RAM limit set in HCImage. When Temporary Buffer is selected, End Frame is automatically enabled and display the maximum number of frames that can be streamed to memory, although any number less than the max can be entered. Note: Streaming to the Temporary Buffer is very useful because it provides the option to review the image sequence when trying to capture specific event and for demonstrating camera speeds. Setting up a Time Lapse This section provides three examples of typical time lapse settings, using each of the storage options. Setup a Time Lapse - Save to Disk The time lapse in this example will acquire an image every 30 seconds for 3 hours and the data will be saved as a cxd. Once your are satisfied with capture settings and the sample is in focus, go to the Sequence pane and follow the steps below. 25

27 Setup a Time Lapse - Save to Memory The time lapse in this example will store images in memory until the acquisition is stopped or runs out of memory at which point the acquired images are saved to disk for the remainder of the time lapse. Once your are satisfied with capture settings and the sample is in focus, go to the Sequence pane and follow the steps below. 26

28 Setup a Time Lapse - Save to the Temporary Buffer Once your are satisfied with capture setting and the sample is in focus, go to the Sequence pane and follow the steps below. 27

29 High Speed Streaming High Speed Streaming is used to obtain the fastest acquisition speed from the camera. This scan is optimized for single channel streaming to RAM or directly to the computer's solid state drives (SSD) configured in a RAID 0. Note: Acquisition rates will vary based on the PC configuration, for information about the computer requirements, please see the PC Recommendations for ORCA-Flash4.0 V2. Note: High Speed Streaming does not support multi-channel acquisition, camera registration features (i.e., flip, rotation and pixel shift) or software processing operations (e.g., shade correction and rolling average). Stream to RAM When streaming to RAM, the image data is stored in memory and then the user has the option to save as either CXD, TIFF, MPTIFF or to delete the data. Up to 80% of the systems available memory will be used for storing streamed data. The Status Bar, located in the bottom left corner of the application window, displays the maximum number of frames that can be streamed to memory. In the AutoSave Properties dialog, the user can determine how and where to store the acquired data. Once the acquisition is complete, the data stored in memory can automatically be saved as a CXD, TIFF or MPTIFF. Note: MPTIFF files have a 65,000 image limit or 4 GB size limit. For image sequences having more than 65,000 images or larger than 4 GB, multiple MPTIFF files will be saved and numbered sequentially. Circular Buffer The Circular Buffer stores streamed data in memory, once the frame count has been reached, the previous acquired data is replaced sequentially. The cyclic process repeats until the acquisition is stopped, leaving the most recent images stored in RAM. 28

30 Steps for Streaming to RAM Once your are satisfied with capture settings and the sample is in focus, go to the Sequence pane and follow the steps below. Stream to Disk When streaming to disk, a temporary file (.dcimg) is created to store the data while it is being acquired, the temporary file location needs to be located on the RAID array, SSD drive, or the fastest drive available. Steps for Streaming to Disk Configure the capture settings, go to the Sequence pane and follow the steps below. 29

31 Steps for Streaming to Disk and Leave as DCIMG Once your are satisfied with capture settings and the sample is in focus, go to the Sequence pane and follow the steps below. Steps for Batch Export DCIMG to MPTIFF Go to the File menu, select Batch Export and follow the instructions below. 30

32 Scheduler Example The diagram below provides a partial timeline of the experiment. Each component is broken down and plotted as interval time (red line), with the individual intervals labeled by action. Components are grouped into scans (purple line) and labeled by type. The schedule for the experiment consists of seven scans, the scans are defined below: 1. Scan 0 (Acquire) 340 nm and 380 nm with 50 ms exposures and a field delay of 900 ms for 15 minutes 2. Scan 1 (Pulse) excite at 500 nm for 350 ms single pulse 3. Scan 2 (Wait) delay of 650 ms 4. Scan 3 (Acquire) 340 nm and 380 nm with 50 ms exposures and a field delay of 900 ms for 10 minutes 5. Scan 4 (Pulse) excite at 380 nm for 350 ms single pulse 6. Scan 5 (Wait) delay of 650 ms 7. Scan 6 (Acquire) 340 nm and 380 nm with 50 ms exposures and a field delay of 900 ms for 10 minutes Create the Schedule Before setting up the scans we need to enter the capture settings and configure the TTL settings for triggering pulses. For this example, the light source is controlled using TTL, were pin 1 (340 nm), pin 2 (380 nm), and pin 3 (500 nm). Capture Settings 1. Go to the Capture tab and select RGB Color: 2-Band from the drop-menu. 2. Select 340 nm and 380 nm from the filter drop-menu and enter a 50 ms exposure for each channel. For exposure enter the numerical value followed by units, that is, u = microseconds, m = milliseconds, s = seconds, and t = minutes. I/O Setup 1. Click the I/O Setup button in the Devices tab, the TTL trigger outputs are identified as Scan Pulse On, For 0: Scan Pulse On, click None under Output Pins and select LPT1 P3. 3. For 1: Scan Pulse On, select LPT1 P4, the pin state will automatically be set to high. 31

33 Create Scans 1. Go to the Sequence pane and select Use Scheduler. 2. (Scan 0) Enter new name, double-click the Scan 0 and enter Acquire. To enter the acquisition settings, double-click Time, enter Field Delay 900 ms, End Time 15 min and click OK. Right-click and select Add Subscan. 3. (Scan 1) Change the name Scan 1 to Pulse. Select TTL Out from the Scan Type drop-menu. Double-click TTL Out, and select 1 from the TTL Out Pulse drop-menu for 500 nm excitation. Enter a Pulse Duration of 350 ms and click OK. Right-click and select Add Subscan. 4. (Scan 2) Change the name towait and select Delay from the Scan Type drop-menu. Double-click Delay, enter a Scan Delay of 650 ms and click OK. Right-click and select Add Subscan. 5. (Scan 3) Change the name to Acquire, enter the acquisition settings, Field Delay 900 ms, End Time 10 min and click OK. Right-click and select Add Subscan. 6. (Scan 4) Change the name to Pulse, select TTL Out, select0 from the TTL Out drop-menu for 380 nm excitation and enter a Pulse Duration of 350 ms. Click OK, then right-click and select Add Subscan. 7. (Scan 5) Change the name towait, select Delay, enter a Scan Delay of 650 ms and click OK. Right-click and select Add Subscan. 8. (Scan 6) Change the name to Acquire, enter the acquisition settings, Field Delay 900 ms, End Time 10 min and click OK. 9. Click Start. 32

34 Multiple Site Time Lapse The Multi-site XYZ Scan allows images to be captured for display, archiving or processing, while scanning multiple areas of a sample. This gives the ability to collect spatial information on static objects outside of a single field of view. To setup a multi-site scan go to the Sequence pane and select XYZ Scan from the Select Scan Type drop-menu. Once the scan has been setup, use the Save Scan icon for repeated use. The XYZ Scan Setup pane provides users with a variety of ways to define their scan, whether it s selecting individual sites within a region or defining a 10x20 grid to scan a slide, the controls are straight forward and easy to use. Note: Prior to setting up an XYZ scan, it is very important that the stage has been calibrated and that the appropriate scale factor has been selected for the objective. Failure to do so could cause damage to the hardware and/or sample. Setting up a Multiple Site Scan 1. Go to the Capture pane and select number of channels to capture. 2. Select filter(s) from the filter drop-down list in the Camera Control panel. 3. In the Camera Control panel adjust exposure manually or automatically by clicking Auto Expose. 4. Select the scale factor for the current objective from the Calibration Properties drop-down list. Calibrate the XY stage if it s not calibrated. 5. Click the Sequence pane and select XYZ Scan from the Scan Type drop-down list. 33

35 6. [Add Locations] Click on the XYZ Scan Setup > Click on the Live icon ( ) to get a live image > Navigate to a location of interest and click on the Add Position icon ( ) to add the current location, a green square appears on the XYZ Scan Map. Add additional locations. 7. [Review and Modify Locations] Select a location > Right-click and select Feedback to get a live image of the selected location. Modify the current location if necessary. Click the Live feedback button off when done. 8. [Create a Scan Path] Press the Scan Region icon > Create a box around the locations > Right-click inside the box > Select Path > Create. The path is shown as red connecting arrows. 9. [Set Time Delay and Number of Passes] Click on the Scan Settings panel, enter a time delay and the number of passes. 10. Click Start > Select file directory and assign file name > Save > Time-lapse starts. Setting up a Z Scan 1. Click on the Capture pane and select number of channels. 2. Select filter(s) from the filter drop-down list in the Camera Control panel. 3. In the Camera Control panel adjust exposure manually or automatically by clicking Auto Expose. 4. Click Live and move stage to an area of interest. 5. Click on the Sequence pane > Select Z Scan from the Scan Type drop-down list. 6. [Add Locations] Click on the Z Scan Setup panel. Set the current position to zero by clicking the 0 button and clicking Set to 0. Focus to the bottom of the sample and click the Bottom check box. Focus to the top and click the Top check box. Enter Z increment in the increment field and hit Enter. Stop Live image by clicking Stop Live. 7. [Set Time Delay and Number of Passes] Click the Scan Settings panel and enter a time delay and duration. 8. Click Start > Select file directory and assign file name > Save > Start Z Scan. 34

36 Montage - Stitch HCImage Montage provides users with an automated and manual solution for stitching large image data sets. Instructions for using the automated and manual stitching are covered in this topic, as well as how to setup an XYZ scan for optimal stitching. First, lets start with the montage. Once the data set has been acquired, select Field Image Montage in the data tree of the data document to create the montage. The montage is created automatically based on the XY acquisition coordinates. At this point, the montage is tiled images that have not been stitched, if the data set was acquired with the appropriate offset, you will see the corresponding overlap in the X and Y directions on the individual images as shown in the image on the right. Now the user has the option to perform an auto-stitch or to manually stitch the images. Before we look at each stitching method, let's take a look at how to setup an XYZ scan to get the best end result. Image Acquisition For the best stitching results, there should be a 10-25% overlap between images in both the X and Y directions. An easy way to ensure that there is image overlap, is by setting an X and Y offset using Define Scan located in the XYZ Scan Setup panel of the Sequence pane. Note: Prior to setting up an XYZ scan, it is very important that the stage has been calibrated and that the appropriate scale factor has been selected for the objective. Failure to do so could cause damage to the hardware and/or sample. For more information, see "Calibrate an XY Stage" on page Click the Stage Marker icon and move the stage to starting location. 2. Next, click Define Scan, select Get Current to set the origin to the current stage location. 3. Under Field Count, enter the number of fields to acquire in both the X and Y direction. 4. Enter the X and Y offset under Spacing and click OK. 5. Click Stop Live and click Start. How to determine the offset Keep in mind that when using an offset of 10-25%, the total length and width of scan may need to be adjusted by adding additional fields in both the X and Y. The units for the offset are based on the units of the current scale factor. The image dimensions are listed as Width and Height, in the Binning and SubArray panel of the Capture pane. X Offset = (image width x percent of overlap)(current scale factor) Y Offset = (image height x percent of overlap)(current scale factor) Hint: The ORCA-Flash4.0 chip size is 2048 x 2048 pixels, with a pixel size 6.5 microns. At 10x, the scale factor is microns per pixel, for more information, see "Calibrate an Image from Pixels to Microns" on page 5. To calculate a 15% overlap use the following equations: X Offset (2048 x 0.15)(0.571) = ; Y Offset (2048 x 0.15)(0.571) =