Is it possible that the illumination across your field of view is not even? This may explain it, and it can be fixed, to a degree, by proper alingment of the system. Or, you could just make sure you always measure in the same part of the field.

Hi Namrata,

Besides the non-uniform illumination across the field (due to the curvature of the holder, lens, alignment, flat field correction, etc.) other factors affect the illumination. Some examples include:

- shading correction, is it a cold or a hot start of the microscope, photo-bleaching, alignment of the optical components, focal depth - focusing on the top of the sample or inside the Z volume, etc.

Check this article for guidance and look at Figure 9 b for example (one of the authors in the article, Graham Wright is from 3A*STAR Microscopy Platform (AMP), Skin Research Institute of Singapore - if it helps):

- Tutorial: guidance for quantitative confocal microscopy. Jonkman et al., Article Tutorial: guidance for quantitative confocal microscopy

When it comes to quantitative analysis of intensity, the variability is more than what we can imagine.

Good luck.

@Sathya Srinivasan

Sathya Srinivasan Alexandros A Lavdas Thank you for your suggestion. Appreciate it.

You have hit the nail on the head with your observations as has Sathya: intensity readings through the fluorescence microscope are not reproducible and not quantitative. You might want to read "The 39 Steps: A Cautionary Tale of Quantitative 3D Microscopy" by James Pawley (Article The 39 Steps: A Cautionary Tale of Quantitative 3-D Fluoresc...