Intensity within a region of interest (tissue, cells, organelles) can be measured using various tools. Fiji offers a basic tool set for 2D-image processing while Imaris can be used to extract advanced 3D statistics. Should you have vast amounts of data, Cell Profiler can be trained to analyze your data for you. Great care should be taken when recording the data used to quantify intensities within the sample. 


When measuring intensities on a widefield microscope you should make sure that the following parameters are same for each and every sample. It should be noted that the non-LED lamps lose brightness over time which means that two samples measured months apart might not yield same results. 

-Sample thickness
-Exposure time
-Light intensity
-Magnification changer
-Objective magnification and NA
-Field diaphragm


Quantifying intensities with confocal microscopes is not recommended. However if you wish to do it you have to take several things into account. It is important to acknowledge that hybrid detectors are not perfectly linear. A sample with double the fluorophores will not yield double the signal.  Additionally the laser power fluctuates up to 10% during an hour so small differences between samples should be properly scrutinized.

The following parameters should be kept for each and every sample.

-Sample thickness
-Laser power
-Detection window
-Pixels size
-Scanning speed
-Objective magnification and NA