Two photon fluorescence microscopy and the numerous technical advances to it have served as valuable tools in biomedical research. The fluorophores (exogenous or endogenous) absorb light and emit lower energy photons than the absorption energy and the emission (fluorescence) signal is measured using a fluorescence decay graph. Additionally, high spatial resolution images can be acquired in two photon fluorescence lifetime imaging (2P-FLIM) with improved penetration depth which helps in detection of fluorescence signal in vivo. 2P-FLIM is a non-invasive imaging technique in order to visualize cellular metabolic, by tracking intrinsic fluorophores present in it, such as nicotinamide adenine dinucleotide, flavin adenine dinucleotide and tryptophan etc. 2P-FLIM of these molecules enable the visualization of metabolic alterations, non-invasively. This comprehensive review discusses the numerous applications of 2P-FLIM towards cancer, neuro-degenerative, infectious diseases, and wound healing.
Keywords: Flavin adenine dinucleotide (FAD); Fluorescence lifetime imaging microscopy (FLIM); Nicotinamide adenine dinucleotide hydrogen (NADH); Two-photon excitation.