Multispectral Fluorescence Lifetime Imaging Microscopy of Endogenous Fluorophores at a Single Excitation Wavelength

We present the experiments on spectral Fluorescence Lifetime Imaging (FLIM) microscopy to interrogate metabolic cofactors such as reduced nicotinamide adenine dinucleotide (NAD(P) H) and flavins with a single wavelength of 750 nm. Global analysis of spectral and fluorescence lifetime data allows to follow the behavior of free and bound NAD(P)H and flavins upon perturbation of HeLa cells and tumor spheroids with metabolic inhibitors. We show that the simplified protocol for data collection allows to get more insights compared to conventional single frequency FLIM.