How to measure fluorescent Intensity over multiple tissue spots


I’m having a problem measuring fluorescent intensity on tissue arrays. What I have been doing is subtracting the background and selecting 3 random ROI’s within the tissue and measuring the mean pixel intensity. However, I’m not sure this is the best route. Autofluorescence of the tissue seems to be a big issue here and I’m not sure if I’m measuring autofluorescence or the actual fluorescent marker. I have tried comparing the control (with no fluorescent marker) to the tissue with the marker but in all of these cases the autofluorescence seems to be brighter than the tissue with the fluorescent marker. The mean intensity and the max is always higher in the control. I’m not sure if there is a way to discriminate the two? Does anyone know of a way I can separate the two or measure the marker only?

Thank you,

These are the stitched images with background subtracted that I have been using

Hey Kate -

So… your images worry me a bit. I am not sure image analysis can help you at this point - it seems to me based on your statements and images - that the problem is on the sample-prep side of things. From your images - i’m not sure you are really getting the specific staining that you want… so either - your marker isn’t working - in this case, is there alternative antibody or so you could try? Or even just trying a different fluorophore (might help with autofluor interference)? Or boosting the signal with a tertiary fluorescent marker?? Also - just to be sure sure … during your acquisitions - are you using the same exact settings when acquiring your controls (no marker) and samples - yes ?? Just to be sure…

But once you get these things in order … I am also not sure if the 3 random ROI’s is how you want to analyze these images. It depends on your marker - your biological question really - to determine what is the best way of measuring the signal in this tissue. But no matter what you do - just make sure it is reproducible.



I don’t know this may help you but, we use spectral unmixing on our confocal microscopes when autofluorescence is an issue. If you have a microscope with spectral detectors (and lambda scan or spectral scan function) you can differentiate autofluorescence from your actual signal by using spectral unmixing. I haven’t use Image J to do spectral unmixing but it seems that it is possible to do it with Image J, as well (certainly you need new set of images to do this)

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