Two-photon autofluorescence microscopy of multicolor excitation.

We developed a two-photon autofluorescence lifetime imaging system with excitations selected from the supercontinuum generated from a photonic crystal fiber. The system excites multiple endogenous fluorophores, such as nicotinamide adenine dinucleotide (NADH) and tryptophan, simultaneously and produces coregistered two-photon autofluorescence images of a biological sample. The technology provides a unique approach to investigate the cellular metabolic activity and protein expression in cells that are potentially important for noninvasive precancer diagnostics. We demonstrated that by taking the tryptophan fluorescence as a reference the ratio of NADH to the tryptophan signal serves as a sensitive indicator of cellular metabolism. The ratio can also clearly differentiate normal cells from cancer cells. The tryptophan fluorescence lifetime images of cells shows that the lifetime of tryptophan fluorescence, varying over a wide range, may be highly dependent on the expression and structure of the protein that tryptophan is packed in.