Immunofluorescence Intensity Measurement in Tissue Sections

Sample image andControl KM001-14.tif (2.3 MB)Treatment KM08-166.tif (2.2 MB) /or code

Hi Everyone,
I know people have posted several topics regarding IF intensity measurements but mostly are related to cell intensity measurements. Attached are two IF-stained tissue sections. My original images are CZI and can be directly opened with fiji, but I had to convert them into Tiff to upload them here.

I want to measure the IF intensity in the red and green channel and I use the following protocol.
• Drag CZI image to ImageJ toolbar to open the image.
• A window titled “Bio-Formats Import Options” will open. Ensure, hyperstack, composite and autoscale is selected. Click OK
• Image-Colour-Split channels
• Close all other images and keep GREEN/RED image only.
• Image-Duplicate-OK
• Image-Adjust-Threshold-Apply
• On the threshold image: Edit-Selection-Create Selection
• On the clean image: Edit-Selection-Restore Selection
• Analyse-Set measurements: Tick AREA, INTEGRATED DENSITY, MEAN GRAY VALUE, ok
• Analyse-Measure and use Raw Integrated Density Value.

However, whilst I can clearly see that the IF intensity is higher in the Treatment image compared with Control image, the integrated density values that I get are actually the opposite.

Could you please advise me on

  1. If my IF intensity measurement protocol is correct.
  2. Why are the IF intensity values obtained not corresponding to the image IF intensity seen.


If you are using integrated intensity, that is based on the area. If you have a smaller area in one image, then it would have to be much brighter to give a higher integrated density.

Comparing integrated density between samples is not really a measure of brightness, but of “total protein content” or some similar idea.

Your mean gray values should be giving you a comparison of brightness within an area.