Re: measuring fluorescent intensity

Posted by alemsqdt on Sep 09, 2015; 6:16pm
URL: http://imagej.273.s1.nabble.com/measuring-fluorescent-intensity-tp5000912p5014294.html

Hello all, I hope you have solved your problem (for the post is quite old) but in case you haven't, I tell you that I had the same problem (including the saturation fact) and I found the answer here: https://digital.bsd.uchicago.edu/resources_files/basic%20image%20quantification.pdf
(if you can't get the archive you can write to me and I will send it to you by e-mail)

About this
"An important point is that there is no such thing as a standard exposure time  that can be applied for all specimens or experiments.    You must empirically determine an exposure that will not saturate your brightest specimen then apply that to all samples being compared."

I agree It would be perfect to have "unsaturated" images, but I think that having saturated images is quite common in the real (not ideal) world. That's because you never know exactly which is the maximum expected intensity before doing the experiment (that's why youŕe doing the experiment!) so you have to guess it (using a few of your samples, and some theoretical background) and based on that information you set the "standard" conditions of the microscope which you must maintain constant in order to be able to compare the values between images thereafter. In other words, it's imposible to "determine an exposure that will not saturate your brightest specimen" because you don't know already which is your brightest specimen.

So, if you have a sample with more intensity than you have expected at the begining of the experiment you can not modify again the setup. But I would never say that if "you do not know the actual intensity for each saturated sample point, which makes quantitation irrelevant". In my case (and maybe in yours too) it's enough to say that there is more intensity in one tissue than in another and you can consider the saturated value as a maximum and compare it with the unsaturated area without problems (in the way described in the pdf)

Hope it helps

Regards!

Alejandro Serrano