Quantification of fluorescence in images

I am a beginner in image processing analysis and I have too many free software but I want an advice on the scientific way to quantify the fluorescence in different images (like the attached)
Do I have to split channels?
what is the best image channel ? why?
can I perform the analysis on RGB images of I have to convert it to a grayscale?

too many questions ?
but to give a small hint, I want to make a relation between different concentrations with different fluorescence so I want to determine the fluorescence intensity for each concentration.


Sorry but your question is not really to the point, i.e. we need more specific descriptions of what is what in your sample and what should be compared in the image or images.

Regarding your sample image, there is not only a different intensity for the left and right objects but both also differ significantly in color. The left shows more cyan and the right more magenta.

I have no idea if this is important for your investigations but it shows at least that you must be careful when using color channels.