Image Normalization at Different Time Points

protein-expression
fluorescence
Tags: #<Tag:0x00007fb883cd05f8> #<Tag:0x00007fb883cd01e8>

#1

Hi,

I want to quantify protein fluorescence for confocal microscope images taken at different day time points. The confocal microscopy parameters are consistent (laser intensity, gain etcetera) except for numerical aperture and magnification as well as depth (sample gets larger). The objective is set at only 2x and 5x and the magnification has a range of 1-1.5 and 1.45-1.5 for 5x and 2x, respectively. We are going to run a control experiment on the same specimen on the same day and take images at the different magnifications and objectives to see its effect on intensity. I found online that Image Brightness is proportional to (NA/Magnification)^2. Is there a good way to account for the differences in magnification and objective to standardize intensities??