Multiphoton fluorescence lifetime imaging of NADH reveals spatio-temporal patterns in cell metabolism during collective migration

Fluorescence lifetime imaging (FLIM) is a powerful tool to quantify local changes in cell metabolism without loss of spatial resolution. Here, we adopted FLIM to characterize spatio-temporal metabolic changes occurring during collective cell migration. Using an established in vitro system, we measured biomechanical and metabolic changes during migration of epithelial cell monolayers. We developed a custom image analysis pipeline that combines machine learning segmentation and curve fitting analysis to analyze FLIM data. Our findings – which were validated via separate measurements of cytoplasmic redox ratio, glucose uptake, and mitochondrial membrane potential – are consistent with a glycolytic shift during collective cell migration.