Laser-induced photobleaching of NAD(P)H fluorescence components in cardiac cells resolved by linear unmixing of TCSPC signals

NAD(P)H fluorescence was investigated by spectrally-resolved lifetime detection, while individual NAD(P)H fluorescence components were resolved by spectral linear unmixing approach. Photobleaching was induced by excitation of a defocused elliptical spot with a 375nm picosecond laser for 30s repeated every 60s for 7min. Our data indicate presence of three individual components in cardiac cell autofluorescence (AF), and we recorded comparable photobleaching of the two resolved NAD(P)H components ("bound" and "free"). Decrease in photon counts during photobleaching was induced by lowering of the component amplitudes, without modification in the fluorescence lifetimes, while the ratio of the two amplitudes remained unchanged. Gathered results are crucial for choosing appropriate light excitation and fluorescence acquisition for prolonged studies of endogenous fluorescence aiming to investigate changes in metabolic oxidative state in living cardiac cells during their contraction.