Hello, I have a question regarding fluorescence intensity measurement in imageJ software. The tutorials provide different methods, the simplest one is to use a fixed ROI and measure the mean gray value. But

1. Should the image be split into R,G,B channels? or RGB stacked images are to be used?

2. If a single fluorescent dye is used with a particular fluorescent colour, what is the need for using LUT in grays?

3. Is the mean gray value similar to the mean intensity that is provided in the histogram option?

4. for measuring fluorescence intensity, is there a need to measure the mean intensities of R, G and B values separately?

It would be really helpful if esteemed researchers could provide me with any module or material that can help me resolve all these doubts.