Longer exposure times will yield higher values because more light is collected by the CCD camera. To test this, get two images of the same field, one with short and one with long exposure time, and measure values with imageJ in the same ROI. After subtracting dark current (image acquired with no light), the values should be proportional to the length of the exposure. 

The answer is yes. Be aware of pixel saturation as well.  If you over saturate your image there no way to properly compare two datasets. The image may look better but quantification cannot be done. 

Thank you all for your input! I compared two images (one that was taken with 1/11sec. exposure and the other 1/5sec exposure and indeed the values were different). In ImageJ, I am running RGB analyses in order to obtain the Green values for analysis. 

In terms of Arbitrary fluorescence units, where can I find a predetermined reference measurement to compare it to? Or can I simply plot the value of the G on the Y axis with my different brain sections on the X?  

Make a test slide with solution of dye used for staining. Image the slide using both exposures in order to find intensity conversion factor.

The camera exposure change made in your case should not do any harm. Only the brightness for the 1/5 exposure thing will be 11/5 times brighter than the other. So depending on what analysis you want to do, you can always start with scaling one (or the other) image using that factor (or its inverse). In any case, in an image, the relative brightness of various pixels would not change if you re-take the image with a different camera exposure. 

And, of course, you will have to subtract your dark noise before you begin your analysis.

If you are using exactly the same optics, imaging settings and data analysis, you can just plot them on the same graph for comparison.

Not sure, how your brain sections look like, but it could be that thicker brain section could give higher fluorescence signal than the thinner one, just because of the thickness (more fluorophores in a thick sample). But if your microscope's focal volume is much thinner than sample thickness - you're protected from this.

Hi Stephen

You should also do some experiments to learn how bleaching the fluorophores influences your results! For example take a series of images with fixed exposure time and analize the change of brightness of your sample over time (some seconds - minutes). You might see that illuminating the sample decreases fluorescence intensity over time. How fast you can find, place and focuse your samples before taking the image might influence your measurement.

Yes it does. The higher the exposure time, photo flux are collected over longer period of time and thus the intensity is also greater. It is recommended to start your experiments with more concentrated sample, and adjust the exposure time in a way it does not oversaturate your picture, use saturation check function in your software and determine exposure time. Use this exposure time for other samples you want to make comparison with.

Always. Light exposure impacts and fades fluorescence over time. Some stains are more resistant, but light exposure always fades the stain and it’s fluorescence properties over time (sometimes in very short time frames, a matter of seconds).This is also true for most standard light microscope stains (most of which carry fluorescence properties). That is why many stains are stored in brown-tented vials.