Ronceray, NathanBennani, SalimMitsioni, Marianna FanouriaSiegel, NicoleMarcaida, Maria J.Bruschini, ClaudioCharbon, EdoardoRoy, RahulDal Peraro, MatteoAcuna, Guillermo P.Radenovic, Aleksandra2025-08-202025-08-202025-08-192025-12-0110.1038/s41377-025-01901-22-s2.0-105012545603https://infoscience.epfl.ch/handle/20.500.14299/25294940754532Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment. Traditionally, FLIM uses time-correlated single-photon counting (TCSPC), which is precise but intrinsically low-throughput due to its dependence on point detectors. Although time-gated cameras have demonstrated the potential for high-throughput FLIM in bright samples with dense labeling, their use in single-molecule microscopy has not been explored extensively. Here, we report fast and accurate single-molecule FLIM with a commercial time-gated single-photon camera. Our optimized acquisition scheme achieves single-molecule lifetime measurements with a precision only about three times less than TCSPC, while imaging with a large number of pixels (512 × 512) allowing for the spatial multiplexing of over 3000 molecules. With this approach, we demonstrate parallelized lifetime measurements of large numbers of labeled pore-forming proteins on supported lipid bilayers, and temporal single-molecule Förster resonance energy transfer measurements at 5-25 Hz. This method holds considerable promise for the advancement of multi-target single-molecule localization microscopy and biopolymer sequencing.enfalse1234567890():,1234567890():,1234567890():,1234567890():,text::journal::journal article::research article