Abstract
Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of autofluorescent metabolic coenzymes has been widely used to investigate energetic perturbations in living cells and tissues in a label-free manner with subcellular resolution. While the currently used state-of-the-art instruments are highly sensitive to local molecular changes associated with these metabolic processes, they are inherently slow and limit the study of dynamic metabolic environments. Here, a sustained video-rate 2P-FLIM imaging system is demonstrated for time-lapse lifetime imaging of reduced nicotinamide adenine dinucleotide, an autofluorescent metabolic coenzyme involved in both aerobic and anaerobic processes. This system is sufficiently sensitive to differences in metabolic activity between aggressive and nonaggressive cancer cell lines and is demonstrated for both wide field-of-view autofluorescence imaging as well as sustained video-rate image acquisition of metabolic dynamics following induction of apoptosis. The unique capabilities ofthis imaging platform provide a powerful technological advance to further explore rapid metabolic dynamics in living cells.