Data repository for "Revisiting NADH Fluorescence Decay and Phasor Analysis under Microenvironmental Changes_Implications for Multiphoton FLIM"

Multiphoton fluorescence lifetime imaging microscopy (MPM-FLIM) of endogenous fluorophores has emerged as a promising strategy for label-free metabolic imaging. NADH is widely explored as a proxy for cellular metabolic state, based on the prevailing interpretation that short and long fluorescence lifetimes correspond to free and enzyme-bound NADH, respectively. However, the molecular mechanisms underlying these lifetime signatures remain insufficiently understood. Here we systematically investigate NADH fluorescence decay under controlled microenvironmental conditions using both one- and two-photon excitation, combining conventional multi-exponential fitting with phasor-FLIM analysis. Our results show that biexponential analysis does not sufficiently capture the complexity of NADH fluorescence decay, whereas triexponential fitting provides a markedly improved description. Moreover, lifetime and phasor shifts induced by enzyme binding overlapped substantially with those caused by increased viscosity. These findings challenge the interpretation of NADH FLIM signals as a simple mixture of “free” and “enzyme-bound” populations. Instead, the observed lifetime trajectories are consistent with broader sensitivity of NADH fluorescence to microenvironmental viscosity, molecular rotational constriction, and confinement. Thus, NADH lifetime and phasor shifts may arise not only from altered enzyme association but also from changes in the local molecular environment, with direct implications for metabolic imaging, while providing fundamental insight into NADH photophysics