Normalization of fluorescence intensity

Hi, I want to measure the fluorescence intensity profile across a line in one image. I found in image J< analyze<plot profile, and RGB profiler plug-in, however, the intensity values were not correct in the composite. The value in channel 2 were increased during merging the two channels. How can I solve this problem? Should I normalize the fluorescence intensity since I want to have RGB profile of the ROI? Thanks!Image1%20(RGB)

Why don’t you just use the original/raw images (the one of the green channel and one of the red channel) to plot the profile?
Are they grayscale images right?
If you convert them to RGB you may change the original values of the fluorescence…

Thanks, Ematini! The grayscales in separate channel are correct, and I can extract the values and make plot on excel. Do I need to do any normalization of intensity?

this is a good approach, you are not converting and changing in any manner the data coming from the microscope.

this is a really hard question to answer without knowing the goal of your experiment and how you’ve acquired the images.


Thanks, Emartini! We want to use the intensity plot to show 1) the size of the signal, 2) to confirm that the red signal attached on the fiber in the green signal. I read in publication that people present intensity plot by “normalized signal intensity” vs “distance (micron)”. I don’t understand what does “normalized” mean here. How can I normalize the gray value to make our intensity plot? Thanks!

It could be interesting if you can link us those papers, so we can check on material and methods section which kind of normalization they used.

Thanks for your prompt reply! Here is the link of paper: