FLIM of metabolic coenzymes, as NAD(P)H and FAD, is now widely accepted to be one of the most important imaging methods for cell metabolism. However, new algorithms are needed to circumvent various problems and to image cell metabolism and redox state from fluorescence lifetimes. The significance of a metabolic index based on NAD(P)H FLIM will be explained and compared with the fluorescence lifetime induced redox ratio (FLIRR). The importance of FMN will be discussed and the FLIRR approach will be extended. Using a three channel TCSPC system simultaneous metabolic NADH/FAD/FMN FLIM and oxygen PLIM/dFLIM (delayed fluorescence) could be realized.
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