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

摘要

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, andtwo parallel TCSPC FLIM recording channels. The two lasers, with wavelengths of 375nm and 405 nm, are multiplexedto alternatingly excite NAD(P)H and FAD. One FLIM channel detects in the emission band of NAD(P)H, the other inthe emission band of FAD. The FLIM data are processed by SPCImage data analysis software. For both channels, thedata analysis delivers images of the amplitude-weighted lifetime, tm, the component lifetimes, t1 and t2, the amplitudesof the components, a1 and a2, and the amplitude ratio, a1/a2. Moreover, it delivers the fluorescence-lifetime redox ratio(FLIRR), a_(2nadh)/a_(1fad). We demonstrate the performance of the system at the example of human bladder cells. Normal cellsand tumor cells were discriminated by the tm images, the a1 images, and the FLIRR images.