Cellular Molecular Dynamics Revealed Through Autofluorescence and Fluorescence Lifetime Imaging Microscopy (abstract)

Fluorescence lifetime imaging microscopy is now widely regarded as one of the most effective tools in addressing the challenges to elucidate cellular molecular dynamics in living cells and organisms. It is used to show when and where genetically or biochemically defined molecules, signals, or processes appear, interact, and disappear in time and space. Among all lifetime imaging techniques, time‐correlated single photon counting (TCSPC) has demonstrated the highest recording efficiency and temporal resolution. In addition, the photon counting technique allows detection of very weak fluorescence signal, such as autofluorescence or a single molecule, which is beyond the threshold of conventional confocal or fluorescence microscopy due to the relatively insensitive detectors commonly employed. By integrating two‐photon excitation and TCSPC detection, we show that the lifetime of autofluorescence from cellular nicotinamide adenine dinucleotide (NADH) can be used to reveal the status of cells, particularly during apoptosis. We present the results of system deployment as well as the applications for monitoring cellular apoptosis.