Fluorescence intensity and lifetime redox ratios detect metabolic perturbations in T cells

The auto-fluorescent coenzymes reduced nicotinamide dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) allow label-free detection of cellular metabolism. The optical redox ratio, which is traditionally computed as the ratio of NADH and FAD intensities, allows quantification of cell redox state. In addition to multiple formulations of the optical redox ratio from NADH and FAD intensity measurements, a fluorescence lifetime redox ratio (FLIRR) based on the fractions of protein-bound NADH and FAD was developed to overcome the limitations of experimental factors that influence fluorescence intensity measurements. In this paper, we compare fluorescence-intensity computations of the optical redox ratio with the fluorescence lifetime redox ratio for quiescent and activated T cells. Fluorescence lifetime images of NAD(P)H and FAD of T cells were acquired with a two-photon fluorescence lifetime microscope. Metabolic perturbation experiments, including inhibition of glycolysis, oxidative phosphorylation, glutaminolysis, and fatty acid synthesis revealed differences between the intensity and lifetime redox ratios. Statistical analysis reveals that the FLIRR has a lower standard deviation and skewness (two-tail T-test, P value = 0.05) than the intensity redox ratio. Correlation analysis revealed a weak relationship between FLIRR and intensity redox ratio for individual cells, with a stronger correlation identified for activated T cells (Linear regression, R-value = 0.450) than quiescent T cells (R-value = 0.172). Altogether, the results demonstrate that while both the fluorescence lifetime and intensity redox ratios resolve metabolic perturbations in T cells, the endpoints are influenced by different metabolic processes.