Open-source modular field-programmable gate array system for two-photon mesoscope enabling multiarea, multidepth neural activity recording and lifetime imaging

SignificanceLarge field-of-view (FOV) two-photon microscopy enables simultaneous recording across multiple brain regions, but larger FOVs lengthen raster scans and limit temporal resolution. A modular, open-source solution that increases imaging speed and adds fluorescence-lifetime capability on standard systems would broaden access to mesoscale neural measurements.AimWe aimed to develop and validate an open-source, modular field-programmable gate array (FPGA)-based acquisition platform and a circular delay-path (CDP) module that together enable multiarea, multidepth mesoscale two-photon imaging and large-FOV two-photon fluorescence lifetime imaging (2p-FLIM) using an 80 MHz laser.ApproachWe built an FPGA system that digitizes photomultiplier signals at 3.2 GS/s and integrated it with a CDP module for a Diesel2p mesoscope. The CDP temporally multiplexes excitation for four focal planes; the FPGA demultiplexes and reconstructs images. Lifetime imaging was implemented on the same platform.ResultsThe system enabled simultaneous recording of >10,000 neurons across the bilateral dorsal cortex at up to four depths and demonstrated large-FOV 2p-FLIM. All hardware and software are open-source and compatible with existing two-photon microscopes.ConclusionsThis modular, open-source FPGA + CDP system increases throughput of large-FOV two-photon imaging and adds lifetime contrast without specialized lasers, facilitating multiscale in vivo studies and broad biomedical applications.