Michael Hooker Microscopy Facility (MHMF.ORG)

C-Imaging - SimplePCI - Cell Counting

Index

0. Introduction

An effective way to count cells is to fluorescently label them with the DNA binding dye DAPI.  This will effectively outline the nuclei, which are inherently separated from each other by cytoplasm.  This separation enhances automatic counting.  Below are outlined the typical steps used to count cells in images acquired as tiff images or .cxd images.  DAPI is effective for fixed permeabilized cells.  For live cells several suitable dyes exist. e.g. Hoechst 33342, Draq-5 or Sytox Green.

Care should be exercised to obtain the best quality image possible.  Image exposure should be set to give the best contrast possible. i.e. Brightest nuclei relative to the general back ground.  It may be advantageous to carry out back ground subtraction.  Also staining should be carefully done without over staining with too high a concentration of DAPI, or leaving DAPI in the mounting media, both of which will yield significant background fluorescence, which will reduce contrast.  Overexposure of the nuclei is not necessarily detrimental unless flaring of the image spreads to such an extent that the imaged nuclei touch each other.  Also the fluorescence light path field aperture and aperture should be fully opened in order to give the most even illumination across the field of view.

1. Counting from already acquired image(s)

• Start C-Imaging
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• Open the image
  • File ---> Open ---> Image Document
  • Browse to the image and open it
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  • 
    • Or
  • Click on the image file's name in a "My Computer" window and drag it into SimplePCI window
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• Calibration may not be necessary if a set image field is used.  Otherwise use a pre made C-Imaging.cal file or recalibrate using a standard micrometer image
  • ->
• Click on the "Activate RIO Shapes Layer" button   (click to show complete tool bar )
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• Then clock on "Create Shapes from Overlay" button
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• Since blue objects are going to be selected make the red and green ranges 0 to 255
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• Set the top blue range to 255 and then interactively adjust the minimum blue range to leave the nuclei masked with green.  Be careful leave all the nuclei selected, especially at the corners where Kohler illumination shading will result in those regions being dimmer.
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• Decline "Use Advanced Detection" (this time)
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  • Note: Advanced Detection has many capabilities, e.g. Separation of touching objects
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• Detected nuclei are outline in red
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• Press "Measure Image" button
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• Select "Field" tab and "OBJ_COUNT"
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  • Or select to measure, say, "Object Area" (Although not going to measure area need to select at least one parameter, so that counting is performed)
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• Click "Measure to DataDoc" in order to retain measurements and keep outlines in a file.  (Not really necessary for cell counting)

  • Or press "Measure to SpreadSheet" in order to retain measurements in a table.

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• Scroll down spread sheet to see total object count
• Spread sheet may be saved using "File ---> Save as" (Make sure spread sheet window is highlighted)
• Counts can be noted manually.  Area imaged should be constant. e.g. for a 10x view with the Hamamatsu OrcaER on the side port of the inverted Leica DMIRB the field of view is 1480 um by 1128 um.

		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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