Optimization of FLIM imaging, fitting and analysis for auto-fluorescent NAD(P)H and FAD in cells and tissues

Increasingly, the auto-fluorescent coenzymes NAD(P)H and FAD are being tracked by multi-photon fluorescence lifetime microscopy (FLIM) and used as versatile markers for changes in mammalian metabolism. The cellular redox state of different cell model systems, organoids and tissue sections is investigated in a range of pathologies where the metabolism is disrupted or reprogrammed; the latter is particularly relevant in cancer biology. Yet, the actual optimized process of acquiring images by FLIM, execute a correct lifetime fitting procedure and subsequent processing and analysis can be challenging for new users. Questions remain of how to optimize FLIM experiments, whether any potential photo-bleaching affects FLIM results and whether fixed specimens can be used in experiments. We have broken down the multi-step sequence into best-practice application of FLIM for NAD(P)H and FAD imaging, with images generated by a time-correlated-single-photon-counting (TCSPC) system, fitted with Becker & Hickl software and further processed with open-source ImageJ/Fiji and Python software.