Hello Sweta, I think the best option is to first calibrate your measurements by using a reference standart, the one i know is like a fluorescence ruler, like this you guarantee that the fluorescence is equal in the whole optical field, then the best approach is to use an image analyzer program, there're several, some are freeware while others come with the software for the microscope, I have worked with imageJ which is a freeware and is quite good and also with imagepro, this programs will let you quantify the fluorescence of your samples by detecting differences on the intensity of the tones.

Hope it helps.

Good luck!

Background subtraction is a big part of quantifying your fluorescence. That is usually the first step before trying to take any measurements. ImageJ can do background subtraction and the quantification.

Some problems you can run into is autofluorescence of your sample or bleedthrough if you are using other light channels as well.

Usually this is not as big of a problem with confocal microscopes, but out of focus fluorescence can cause some background problems. Deconvolution reduces the background caused by out of focus fluorescence. You can deconvolute your images using a free plugin for ImageJ or commercial software.

Using a secondary staining for another protein, same concept as a housekeeping gene used in other assays. You can eliminate the possibility of uneven staining in different regions of your slide or sample.

A good article on quantitative microscopy:

http://jcb.rupress.org/content/185/7/1135.full

Thanks for your replies.