Interpreting NADH fluorescence lifetime across timescales

NADH fluorescence lifetime imaging is a widely used, label-free way to visualize cell metabolism, typically read as a balance between glycolysis and oxidative respiration. This reading assumes that the effective NADH-binding landscape remains sufficiently stable during the experiment. This assumption is generally best protected during brief perturbations, but can fail in two ways: slowly, as prolonged treatments or differentiation reshape the identity and abundance of NADH-binding enzymes over hours to days; and rapidly, as metabolites can transiently alter their engagement with NADH even without changes in protein abundance. In this Perspective, we outline when each concern applies and offer practical guidance for experimental design and interpretation.