The BrightEyes-TTM: an Open-Source Time-Tagging Module for Single-Photon Microscopy

Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to the introduction of new asynchronous read-out single-photon (SP) array detectors. These detectors give access to an entirely new set of single-photon information typically lost in conventional fluorescence LSM, thus triggering a new imaging/spectroscopy paradigm - the so-called singlephoton LSM (SP-LSM). The revolutions outcomes are, from one side, the blooming of new SP-LSM techniques and tailored SP array detectors; from the other side, the need for data-acquisition (DAQ) systems effectively supporting such innovations. In particular, there is a growing need for DAQ systems capable of handling the high throughput and high temporal resolution information generated by the single-photon detectors. To fill this gap, we developed an open-source multi-channel timetagging module (TTM) based on a field-programmable-gatearray (FPGA), that can temporally tag single-photon events - with 30 ps precision - and synchronisation events - with 4 ns precision. Furthermore, being an open-access project, the TTM can be upgraded, modified, and customized by the microscopy-makers. We connected the TTM to a fluorescence LSM equipped with a single-photon avalanche diode (SPAD) bi-dimensional array detector, and we implemented fluorescence lifetime image scanning microscopy (FLISM) and, for the first time, fluorescence lifetime fluctuation spectroscopy (FLFS). We expect that our BrigthEyes-TTM will support the microscopy community to spread SP-LSM in many life science labs.