We describe a metabolic imaging system based on simultaneous recording of lifetime images of NAD(P)H and FAD. The system is based on the bh DCS-120 confocal scanning FLIM system. It uses one-photon excitation by ps diode lasers, scanning by galvanometer mirrors, confocal detection, and two parallel TCSPC FLIM recording channels. The two lasers, with wavelengths of 375 nm and 405 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. The FLIM data are processed by SPCImage data analysis software. For both channels, the data analysis delivers images of the amplitude-weighted lifetime, tm, the component lifetimes, t1 and t2, the amplitudes of the components, a1 and a2, and the amplitude ratio, a1/a2. Moreover, it delivers the fluorescence-lifetime redox ratio (FLIRR), a2nadh/a1fad. We demonstrate the performance of the system at the example of human bladder cells. Normal cells and tumor cells were discriminated by the tm images, the a1 images, and the FLIRR images.
Keywords: FLIM, Metabolic Imaging, Label-Free, NADH, FAD, Tumor Detection, FLIRR