Trouble trying to calculate fluorescence of a ROI full of vesicles

Hi all, first post here in this forum, and hope it’ll not be the last :smiley:

So, as suggested by @haesleinhuepf, I’ll share with you a problem that’s been bugging me for quite a lot of time now.

Just as a bit of backstory here: I’m trying to determine if there are differences in fluorescent intensity inside a cell when comparing different mutant strains of C. elegans.
The cell in question is labelled by CFP and contains fairly small vesicles marked with a red fluorescent protein (Pseudo-colored magenta).
The tricky part is that I’m only interested in measuring the fluorescence intensity of vesicles inside a region of interest (the soma of the CFP cell), this soma varies in size and shape from animal to animal (see image below).

Screen Shot 2020-04-03 at 16.02.54.tiff (3.6 MB)

Some comments:

  • The image is a confocal Z-stack acquired with LSM780. Zeiss file format, 8-bit image and 2 channels (CFP and RFP). I am only interested on the red fluorescent quantification.
    -The image I shared is the result of a Z-stack projection. Actually, would like to measure the fluorescence in 2D, as 3D rendering/analysis could make it complicated and tedious, but I’m open to anything.
  • When trying to measure the ROI intensity you’ll see that the value of the Standard deviation is quite high, probably due to big differences between the RFP signal from the vesicles and the dark background. I would need a way to normalize by the background.
  • All images were acquired the same day with same acquisition settings

So knowing all that, which would be the best way to analyze such fluorescent images? A method that could give me Relative Fluorescent values to compare among all my mutant backgrounds that I’m dealing with.

Is anything that I could follow as a step-by-step guide (I could not find that), and also how to calculate and normalize the data that I get from that?

I also share with you the example I’ve shown above in Zeiss format:

Thanks a lot for your help, any advice is highly appreciated.
Adrià R.

PS: Sorry I had to edit, the post was sent sooner than expected.