The common problem for all fluorescence-based cell detection methods is the cells’ natural fluorescence. Cellular autofluorescence is due to the presence of various biological structures, such as collagen, elastin, NADPH, flavins, mitochondria, and lysosomes, which usually absorb in UV to blue range (355-488 nm) and emit in the blue to green range (350-550 nm). Therefore, autofluorescence interferes with analysis by reducing signal sensitivity and resolution of fluorochromes that operate in that range – FITC, GFP, and Pacific Blue to name a few.
To subtract this background and prevent false positives, it is crucial to include unstained controls and set proper PMTs and gating. However, it is even more important to minimize autofluorescence as much as possible.