Michael Hooker Microscopy Facility (MHMF.ORG)

Capturing a basic image

Acquiring Images Using SimplePCI with the OrcaER (b/w) or Cool Snap HQ2 (b/w) or MicroPublisher (color) camera

• Log on the computer
  • enter username (and press tab to move to-)
  • enter password (case is important)
  • choose domain MHMICROSCOPY (not local computer e.g. FRESNEL)
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• Start SimplePCI
  • ensure OrcaER camera control box is powered on before starting SimplePCI
  • run SimplePCI (C-Imaging)
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  • During SimplePCI startup a warning box will appear (in versions of SimplePCI before 6.0)
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  • Press OK or ignore messages concerning registry
  • Main SimplePCI window will appear
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• Start Capture mode in SimplePCI
  • Double click on camera icon
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  • Two additional windows will open
  • The  capture box window (for camera control)
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  • The Image Display window
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  • Ensure desired camera is chosen in top right selection box (01)
    • MicroPublisher for transmitted color
    • Hamamatsu ORCA-ER for fluorescence
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  • Choose number of color channels (02)
    • for MicroPublisher choose Three Color Image
    • for Hmamatsu B&W, choose the number of different fluorophores in your sample
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  • For multi-channel fluorescence, select the appropriate Filter for each Color Channel using the drop down menu
  • Ensure fluorescent or transmitted light is on at the microscope and camera port is open
  • Press Focus button, which starts rapid acquisition of an image to the display window (03)
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  • Increase or decrease Exposure Time in order to get an image (03)
    • For Fluorescence:  It may be desirable to increase camera gain to maximum (04) and decrease exposure time in order to make focusing easier.  Note that image may be noisy.  Black level should not be changed
    • For Fluorescence: Repeat for all color channels
  • Focus while viewing image on monitor
  • If desired, return camera Gain to 1 (04) and increase Exposure Time (03) in order to reduce noise
  • Press Stop (05) or Abort
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  • Press capture 1 (06) to acquire - to ensure exposure is correct, check intensity histogram on the right side of the Image window

   

  For Color transmitted light views captured with the MicroPublisher camera

  • The image will have a greenish hue.  This is technical issue due to the MicroPublisher being a Bayer Filter color CCD chip
  • The hue can be corrected by doing a white balance (10)
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  • Uncheck locked (11)
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  • Hit auto (12)
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  • Image will have a better color balance.  Corrected balance will be applied to subsequently captured images until the auto contrast button (10) is clicked off
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  • Save the image
  • To save the color corrected image - right mouse click on image (or hot the floppy icon, 20) , then hit Save Image to file ---> Display Image and choose file name and save as Tiff
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  • To save to uncorrected image - right mouse click on image (or hot the floppy icon, 20) , then  Save Image to file ---> All components and choose file name and save as Tiff

  For images captured with the black and white Hamamatsu camera

  • Save the Image
    • Right click on the image
    • Select "Save Image to File"
      • For Monochrome (single channel) images
        • ... "Original Image" -- saves the image as monochrome
        • ... "Display Image" -- saves the image as index color
          • only available if you have changed the color display in the image window
      • For Multi-channel images
        • ... "All Components" -- saves all components to a single image, each channel is the color in which it was acquired
        • ... "Red/Green/Blue Component" -- saves a monochrome image of the color channel selected
        • ... "Display Image" -- saves a 24bit color image of what is displayed in the Image window
           
• Shut down SimplePCI and log off when you are finished

		Copyright 2001-2014  Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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