Quantifying relative fluorescence between samples not compared to baseline

Looking for a way to quantify anti H3K27me3 antibody staining in zebrafish tail samples on 8-bit confocal images. What are the best parameters to select for quantifying fluorescence? Additionally samples are drug treated at varying concentrations to inhibit methylation and therefore I want data that shows the relative fluorescence i.e. difference between intensity of staining between samples.

Good day,

I’m not a specialist in this field and perhaps some experts will chime in.

However, the first thing you need to do is, find out what mathematical relationship exists between the fluorescence intensity (gray value in an image, provided the camera uses a gamma of one) and (1) the applied quantity of stain and (2) the effect you want to measure (treatment).

In general you can’t assume that the relationships are linear in the mathematical sense.