Research highlight: new and powerful approach to wide-field fluorescence lifetime imaging with single-molecule sensitivity

Abstract Historically constrained by the low throughput of traditional Time-Correlated Single-Photon Counting (TCSPC), a new method utilizes an optimized, time-gated single-photon camera acquisition scheme for achieving single-molecule lifetime precision comparable to TCSPC, but across a wide field of view simultaneously. This high-throughput capability transforms single-molecule FLIM from a specialized, time-consuming effort into a scalable, quantitative assay. The resulting multimodal imaging platform provides unprecedented simultaneous insight into the nanoscale location (SMLM) and local environment (FLIM) of biological molecules, advancing our ability to map complex biophysical parameters like FRET efficiency in living systems.