The number of photons you collect depends on the photon flux and the time you collect the photons. The photon-flux is the parameter you are interested because it should be ideally proportional to then number of fluorescent molecules.
In wide-field fluorescence microscopy the time you collect photons is called the exposure time. Thus in order to compare images with different exposure times you have to normalize regarding the exposure time.
In confocal microscopy the time you collect photons is usually called dwell time. For most (if not all) of the commercial systems the vendors have already normalized the signal (thus you do not see any difference in pixel intensity if you change the scan speed).
In general: if you want to do quantitative fluorescence microscopy the difference in exposure/dwell time is only one parameter to take in to consideration. There are other ones which might be less obvious but to my understanding even more critical. Thus I suggest reading the following articles:
J. Pawley, The 39 Steps: A Cautionary Tale of Quantitative 3-D Fluorescence Microscopy.
http://labs.pbrc.edu/cellbiology/documents/39steps.pdfJ.C. Waters, Accuracy and precision in quantitative fluorescence microscopy.
http://www.ncbi.nlm.nih.gov/pubmed/19564400Regards
Arne
> -----Original Message-----
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> streptopelia
> Sent: dimanche 2 mars 2014 02:47
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> Subject: Exposure time
>
> Hi,
> is there a way to adjust for different exposure times when comparing sets of
> experiments (for quantitative fluorescence analysis)?
> Any help would be very appreciated...thanks!
>
>
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