biostudies-literatureRossetta AEuropean Research Council7406https://www.ebi.ac.uk/biostudies/studies/S-EPMC9715684biostudies-literatureUnknown13(1)Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to new single-photon (SP) array detectors, which give access to an entirely new set of single-photon information. Together with the blooming of new SP LSM techniques and the development of tailored SP array detectors, there is a growing need for (i) DAQ systems capable of handling the high-throughput and high-resolution photon information generated by these detectors, and (ii) incorporating these DAQ protocols in existing fluorescence LSMs. We developed an open-source, low-cost, multi-channel time-tagging module (TTM) based on a field-programmable gate array that can tag in parallel multiple single-photon events, with 30 ps precision, and multiple synchronisation events, with 4 ns precision. We use the TTM to demonstrate live-cell super-resolved fluorescence lifetime image scanning microscopy and fluorescence lifetime fluctuation spectroscopy. We expect that our BrightEyes-TTM will support the microscopy community in spreading SP-LSM in many life science laboratories.Nature communicationsThe BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy.PMC9715684818699Fersini FRossetta ADonato MLanzano LDiotalevi FPerego EBarberis ATortarolo GSlenders EVicidomini GKoho SZappone SBruno MCrepaldi MfalseThe BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy.Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to new single-photon (SP) array detectors, which give access to an entirely new set of single-photon information. Together with the blooming of new SP LSM techniques and the development of tailored SP array detectors, there is a growing need for (i) DAQ systems capable of handling the high-throughput and high-resolution photon information generated by these detectors, and (ii) incorporating these DAQ protocols in existing fluorescence LSMs. We developed an open-source, low-cost, multi-channel time-tagging module (TTM) based on a field-programmable gate array that can tag in parallel multiple single-photon events, with 30 ps precision, and multiple synchronisation events, with 4 ns precision. We use the TTM to demonstrate live-cell super-resolved fluorescence lifetime image scanning microscopy and fluorescence lifetime fluctuation spectroscopy. We expect that our BrightEyes-TTM will support the microscopy community in spreading SP-LSM in many life science laboratories.2022-01-01T00:00:00Z2022 Dec2025-04-18T16:56:32.014Z2025-04-07T04:24:59.699ZS-EPMC97156843645657510.1038/s41467-022-35064-0