Michael Hooker Microscopy Facility (MHMF.ORG)

MBW ImageJ DAB "brown" Analysis

Purpose:

• Measure fraction of DAB brown area in a selected area of brain section
• Note that DAB density is not proportional to epitope concentration.  Also DAB color changes with product density.
• Note experimental section images should be acquired at the same magnification and exposure time and gain as control sections.

Outline:

• Power up microscope - set lamp intensity to 10 1/2 (do not change since this will upset color balance)
• Ensure dichroic filter wheel is set to position 4.  Also check that no filter is istalled in position 4
• Setup color camera the Micropublisher (usually do nothing, make sure USB cable plugged into camera is labeled \\Lister)
• On condenser:
  • Open field aperture
  • Minimize (lower) aperture
  • Switch in NCB filter ("Neutral color balance" the blue one), both neutral density filters
  • Leave in polarizer (unless collagen or other polarizing material is in sample)
• Focus on sample and setup Kohler Illumination
• Run C-Imaging (or Q-Capture)
• Do color balance - then do not change lamp power, move NCB filter or polarizer
• Acquire images & save (or analyze)
• If low magnification objective is used it is best to acquire a background image and do background shading correction
• Transfer files to computer
• Hand outline area/brain region where DAB "brown" is to be quantified
• Threshold & Measure
• Calculate - area of brown and area measured in pixels and then calculate fraction of brown pixels in selected region
• Calibration:
  • Make note of which objective was used.  Suggestion: include obj. mag in file name
• Sample image file: GFAP-9-CMV-5x-CROPPED.tif

Protocol

• Install ImageJ, if necessary.
• Run ImageJ
  • Open "Computer" window
  • Load image (if necessary)
    • either File ---> Open
    • or click on image name in Computer window and drag onto ImageJ
    • Flatten image, if background subtraction has not been done in C-Imaging
      • 
      • In Subtract Background:
        • Check Light background
        • Check Sliding paraboloid
        • Radius should be bigget than largest feature that needs to be measure (best do trial and error to find best radius)
        • 
• Load calibration (do once per session)
  • Image must be open
  • Image ---> Properties (ctrl + shift + P)
  • Enter 1 as Pixel Width & Pixel Height & pixels at Unit of length (Easiest since measuring DAB as a fraction of area of interest.  mm is tedious since switching between 5x & 10x objective views)
  • Check Global
  • 
• Set measurements to make
  • Analyze ---> Set Measurements
  • 
  • Choose Area and perhaps Integrated Density and uncheck the rest
  • 
  • Hit OK
• Measure overall area
  • Select Freehand or Polygon or other desired selection tool
  • Press ^M
• Threshold DAB cells
  • Image ---> Adjust ---> Color Threshold
  • 
  • Set Threshold Color: Red
  • Keep hue at 0 to 255
  • Keep saturation at 0 to 255
  • Adjust upper brightness to include stainnig - move slider back down to see darker areas, then back to desired level. Press Filtered to see results. Red area is selected area.  Note small dots slected can be excluded when measuring particles where small sizes can be rejected.
  • Hit Select
  • Hit Stack
  • 
• Image ---> Type ---> 8 bit (Don;t forget to do this!)
• Manually remove undesired slected blobs with e.g. rectangle tool & Edit ---> Clear
• Analyse ---> Analyze Particles
  • Select Summarize and Record Stats
  • 
  • Hit OK
  • Area of blobs appears in Summary window
  • The summary window will have the area in the Total Area column

• 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!

   

• Notes
  • Don't like a measurement number?  Then highlight the line the number in the Results window and press ctrl + X (cut)
  • Don't forget to save Summary and do calculation brown area / area of highlighted region
• Useful short cut keys
  • ctrl + up/down arrows will zoom in/out

		Copyright 2001-2015  Dr. M. Chua, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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