Label Free Fluorescence Lifetime FRET Imaging

Abstract

Multiphoton microscopy with endogenous contrasts in biological tissues have primarily focused on detecting signals from the reduced nicotinamide adenine dinucleatide (NADH), its dinucleatide phosphate (NADPH), riboflavins, and tryptophan. All these fluorophores have emission in the wavelength range of 400-600 nm. According to an earlier studies on the autofluorescence spectroscopy of ex vivo leukocyte samples under linear (one photon) excitation conditions, signals from the tryptophan (TRP) moieties in cellular proteins should be much stronger than signals from NAD(P)H on the per cell basis. In recent years, considerable interest has been developed in the effect of various diseases upon the metabolic pathway of L-tryptophan and its derivatives. We describe a new method for imaging tumorigenic and non-tumorigenic cell lines by 3-photon exciting of the endogenous protein fluorescence in the ultraviolet (UV) spectral region, where tryptophan is the major fluorophore. Through systematic analysis of FLIM data from live cells, a statistically significant decrease in the fluorescence lifetime of TRP was observed in response to the increase in protein-bound NADH as cells were treated with glucose. Addition of glycolytic substrates significantly quenches tryptophan lifetime. The results demonstrate the potential use of 3P-FLIM-FRET as a tool for label free screening of the change in metabolic flux occurring in human diseases or other clinical conditions.