Metabolic Imaging by Simultaneous FLIM of NAD(P)H and FAD

Abstract

We describe a metabolic imaging system based on simultaneous recording of lifetime images of NAD(P)H and FAD. The system uses one-photon excitation by ps diode lasers, scanning by galvanometer mirrors, confocal detection, and two parallel TCSPC FLIM recording channels. Two lasers, with wavelengths of 375nm and 410 nm, are multiplexed to alternatingly excite NAD(P)H and FAD. One FLIM channel detects in the emission band of NAD(P)H, the other in the emission band of FAD. For both channels, the data analysis delivers images of the amplitudes of the decay components, a1 and a2. We show that these are robust parameters to characterize the metabolic state of cells. FLIM results obtained from excised human-bladder tissue were in perfect agreement with histology.