FLIM Characterization of Staurosporine-Induced Changes in Endogenous Cellular Autofluorescence

Autofluorescence arising from endogenous fluorophores like Flavins and NADH offers a label-free approach to monitoring cellular metabolism. In this study, we investigated the changes in intrinsic fluorescence under chemically induced mitochondrial stress in live cells. Using confocal imaging, Raman spectroscopy, fluorescence lifetime imaging, and high-performance liquid chromatography, we characterize a reproducible increase in green autofluorescence. Lifetime signatures suggest involvement of flavin adenine dinucleotide (FAD), a key mitochondrial factor. This enhanced autofluorescence response appears both intracellularly and extracellularly and is consistent across three cancer cell lines and HEK293 cells. Our findings highlight autofluorescence as a sensitive, label-free reporter of cellular metabolic state, with potential applications in real-time monitoring of cell stress and death.