Label-free fluorescence lifetime imaging of cells nuclei: A sensitive readout of metabolic response

Fluorescence lifetime imaging microscopy (FLIM) using endogenous fluorescence of NADH (reduced nicotinamide adenine dinucleotide) and its phosphorylated form NADPH represents a powerful tool for monitoring cellular metabolic states. For a more nuanced interpretation of the FLIM data, investigating NAD(P)H in different cell compartments is crucial. In this study, we demonstrate that a weak NAD(P)H fluorescence in cell nuclei, which is often ignored, can be reliably analyzed using a phasor plot approach and provides a sensitive readout of metabolic responses. Using colorectal cancer cells HCT116 treated with the metabolic inhibitors rotenone and 3-bromopyruvate, as well as the chemotherapeutic agent 5-fluorouracil (5-FU), we show that nuclear NAD(P)H fluorescence decay changes in response to treatment. In the case of 5-FU, the phasor analysis of nuclear NAD(P)H reveals heterogeneous cellular responses with two subpopulations differing in NAD(P)H fluorescence decay parameters, fluorescence intensity, and cytoplasm-to-nucleus intensity ratio. Notably, nuclear and cytoplasmic responses are strongly correlated, indicating tight coupling of their NAD(P)H pools. Overall, our results establish nuclear NAD(P)H fluorescence as a robust, label-free indicator of cellular metabolism and highlight its potential for metabolic monitoring in conditions where analysis of NAD(P)H fluorescence is limited by spectral overlap with exogenous fluorescent labels.