Sample image andControl KM001-14.tif (2.3 MB)Treatment KM08-166.tif (2.2 MB) /or code
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.
• 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
- If my IF intensity measurement protocol is correct.
- Why are the IF intensity values obtained not corresponding to the image IF intensity seen.