Label-free metabolic imaging of microbial infections by two-photon fluorescence lifetime imaging microscopy

Accurate, non-invasive visualization of metabolic alterations during infection is critical to understanding host-pathogen interactions and disease progression. We present a comprehensive application of two-photon fluorescence lifetime imaging microscopy (2p-FLIM) for label-free, quantitative imaging of metabolic changes in cells and tissues infected with bacterial and viral pathogens. By detecting the intrinsic autofluorescence of NAD(P)H and resolving differences between its free and protein-bound forms, 2p-FLIM provides real-time insights into cellular metabolic states, particularly shifts between glycolysis and oxidative phosphorylation. In both viral (HIV-1) and bacterial (Pseudomonas aeruginosa) infection models, we observed increased fluorescence lifetimes and protein-bound NAD(P)H fractions, indicating enhanced oxidative metabolism in infected cells and tissues. For bacterial infections, 2p-FLIM coupled with phasor plot analysis enabled spatial and temporal mapping of metabolic responses across microbial strains, infected host cells, and animal tissues. Similarly, in viral infection models, the method allowed clear differentiation between infected and uninfected cells based on NAD(P)H lifetime signatures, capturing metabolic heterogeneity at the single-cell level. This work demonstrates the broad applicability of 2p-FLIM as a minimally invasive tool for studying infection-induced metabolic reprogramming at molecular, cellular, and tissue scales. The ability to perform high-resolution, real-time imaging without exogenous labels makes 2p-FLIM a promising approach for diagnostics, therapeutic monitoring, and basic research across a range of infectious diseases.