Revisiting the origin of intrinsic cellular autofluorescence in metabolic imaging

Metabolic dysregulation is a hallmark of numerous diseases. Multiphoton microscopy (MPM) combined with fluorescence lifetime imaging (FLIM) of the metabolic coenzymes NADH and FAD has emerged as a key method for monitoring metabolic processes in live tissue with sub-cellular resolution. However, recent data demonstrates that spectral crosstalk from other cellular components, such as keratin, is substantial. Yet the contributions from endogenous proteins are often neglected, bringing into question the validity of earlier studies. To address this issue, we propose to systematically scrutinize the fluorescence properties of NADH, FAD and keratin in different states and combinations. This will be done by meticulous investigation of the fluorescence lifetime, spectra and related photophysical properties using MPM and time-resolved spectrophotometry under a range of conditions (pH, temperature, viscosity, protein binding), and in combination with computational chemistry. Based on this knowledge, we will develop approaches that can accurately determine the concentration, diffusion and binding state of NADH and FAD even in the presence of spectral crosstalk from keratin. The strategy is to implement phasor-FLIM analysis, fluorescence lifetime correlation spectroscopy (FLCS) and raster lifetime image correlation spectroscopy (RLICS) to separate the fluorescence contributions of bound and unbound NADH and FAD from the protein background and quantify the spatial distribution of each species with unprecedented spatiotemporal resolution. The developed methodologies will be validated by monitoring cellular metabolism in human primary cells and tumour cell lines. This study will solidify our understanding of NADH and FAD autofluorescence in complex cellular environments and substantially expand the analytical toolset by which quantitative information about metabolic dysregulation can be obtained, thereby advancing MPM-FLIM as a research and diagnostic tool for metabolic diseases.