Fluorescence 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 i...Fluorescence 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.展开更多
基金support from the EPFL Center for Imaging(A.R.,N.R.,E.C.and C.B.)European Research Council(grant 101020445 to A.R.)+2 种基金the Swiss National Science Foundation(grant 200021-184687 to G.P.A.,grant 200021L-212128 to M.D.P.and grant IZSEZ0-224299 to R.R.)the National Center of Competence in Research Bio-Inspired Materials(NCCR 51NF40-182881 to G.P.A.and A.R.)the European Union Program HORIZON-Pathfinder-Open(grant 101099125 to G.P.A.).
文摘Fluorescence 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.
