Michael Hooker Microscopy Facility (MHMF.ORG)

Making Measurements with ImageJ

Outline: 

This measurement protocol provides an example of how to measure the percent of black areas in a field of cells stained with phalloidin.  Images need not be calibrated since a percentage is being estimated.  Black areas are estimated using a intensity threshold. Another possible way to estimate black areas is to manually outline areas by hand.  However this method is not presented here (yet?).

Summary:

• Load image
• Setup x-y calibration (optionally)
• Setup measurements to be made
• Make a duplicate for comparison
• Threshold black areas
• Make area measurement of thresholded regions
• Save measurements as a spreadsheet
• Repeat for next image

Protocol:

• Install ImageJ, if necessary
• Run ImageJ
• Acquire image(s) and save as tiff format (Do not use jpg or any other format which uses lossy image compression) 
• Load image (if necessary)
  • either File ---> Open
  • or click on image name in MyComputer and drag onto ImageJ
• Load calibration if desired (do once when ImageJ is started)
  • Image must be open
  • Image ---> Properties (ctrl + shift + P)
  • Enter e.g. 1.0 as Pixel Width & Pixel Height & pixels as Unit of length or e.g. calculate um per pixel by measuring the pixel length of a calibration graticule
  • Check Global
  • 
  • Hit OK
• Set measurements to make
  • Analyze ---> Set Measurements
  • 
  • Choose area and uncheck the rest (although selecting centroid can be helpful later to find the location of an area which may need to be rejected.)
  • 
  • Hit OK
• Convert to gray if desired/required
  • Image ---> Type ---> 8-bit
• Make a copy of the image for reference (ctrl + shift + D) or Image --->  Duplicate 
• Threshold black area
  • Image ---> Adjust ---> Threshold (ctrl + shift + T)
  • 
  • Keep minimum threshold at 0
  • Adjust upper threshold to include required black areas - move slider back down to see darker areas, then back to desired level. Red area is selected area. 
  •  
  • Hit Apply
  • 
  • Unwanted areas can be erased with the Poly selections tool or Rectangular tool, etc.
    • Outline outside unwanted area
    • 
    • Note: Click on first control point box to finish outlining if using the Poly selections Tool
    • Note: Control points can be moved by clicking on them with and holding down the left mouse button, and then moving them, one at a time.
    • Note: Multiple selection area can be selected by holding down the Shift key.  Shape type can be changed in the process.
    • Edit --> clear (or press the Backspace key)
    • 
    • 
• Count & measure thresholded regions
  • Set size range e.g. "100 - infinity" sq. pixels.  Can select units as pixels if that is more intuitive.
  • Analyze ---> Analyze Particles
  • Check Display Results & Summary, uncheck Clear Results, Show Outlines and keep other defaults
  • 
  • The summary window will have the area in the Total Area column
  • The Results window will have the separate areas for each region.  Note line number

• Repeat with next image
  • Click and drag next image into ImageJ window
  • Box below should pop up   
            Uncheck Disable Global Calibration
            Check Disable these messages
  • 
• Save summary file with results, including fraction of area which is black.

   

• Notes
  • Don't like a measurement number?  Then highlight the line in the Results window and press ctrl + X (cut)
  • Don't forget to save summary.  Calculations can be done in Excel
• Useful short cut keys
  • ctrl + up/down arrows - zoom in/out

		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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