Michael Hooker Microscopy Facility (MHMF.ORG)

Alessandra's ImageJ Mucus Analysis

Outline:

• Basal lamina length - hand outline
• Plug areas - hand outline or use fill function, then delete plug
• Mucus staining Area of purple, blue, pink, fuchsia
• Calculate = area (mm^2) * 1000 / 4/pi*length (mm)
• Calibration:
  • Assume at10x image width is 690 um 1280 pixels = 0.539 um / pixel
  • Assume at 4x image width is  ........... 0.00275 mm / pixel
• Sample image file: H6365-7_4x_AB-PAS_2.tif

Protocol

• Install ImageJ, if necessary.
• Run ImageJ
• Acquire image
• Load image (if necessary)
  • either File ---> Open
  • or click on image name in MyComputer and drag onto ImageJ
• Load calibration (do once per session)
  • Image must be open
  • Image ---> Properties (ctrl + shift + P)
  • Enter 0.00054 as Pixel Width & Pixel Height & mm at Unit of length
  • Check Global
  • 
• Set measurements to make
  • Analyze ---> Set Measurements
  • 
  • Choose area and uncheck the rest
  • 
  • Hit OK
• Convert to gray
  • Image ---> Type ---> 8-bit
• Outline length of mucus
  • Select line.  If 1st time right click on line icon and choose
  • 
  • Left click at bends/corners on image, right click at last position.  Click points are movable/correctable with the left mouse button
  • ctrl + M (ignore area &
  • If multiple segments are to be measure then repeat line outline for each additional segment
  • clear any highlighted region by a right mouse click
• Measure any plugs - for each
  • Select Freehand or Polygon selection tool
  • Outline each plug and press ^M for each (length is recorded as 0 mm)
  • Edit ---> Clear for each plug (stops them being thresholded below)
• Threshold mucus label (what was pink, purple, fuscia, blue on basal lamina)
  • Image ---> Adjust ---> Threshold (ctrl + shift + T)
  • 
  • Keep minimum threshold at 0
  • Adjust upper threshold to include stained mucus - move slider back down to see darker areas, then back to desired level. Red area is selected area.
  • (note the auto button may work appropriately)
  • 
  • Hit Apply
  • Select Poly selections tool
  • Outline outside stained mucus (click inside initial point box to complete polygon)
  • 
  • Edit --> clear Outside
  • 
  • 
• Count & measure thresholded mucus regions
  • Analyze ---> Analyze Particles
  • Check Summarize Uncheck Display Results, Clear Results, show Nothing and keep other defaults
  • 
  • 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?  Then highlight the line in the results window and press ctrl + X (cut)
  • Don't forget to save summary and do calculation area (mm^2) * 1000 / 4/pi*length (mm) in Excel
• Useful short cut keys
  • ctrl + up/down arrows - zoom in/out

----

Updated Ale Protocol

• run("Subtract Background...", "rolling=40 light");
• run("Enhance Contrast", "saturated=0.4");
• run("8-bit");
• run("ROI Manager...");
• //run("Threshold...");
• //setTool("polygon");
• \\fresnel\MH1_0\Documents and Settings\livraghi\My Documents\Morphometry Ale\mucus.ijm

		Copyright 2001-2018 Dr. M. Chua, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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