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Here, we propose large-FOV, dual-region, two-photon fluorescence lifetime imaging microscopy (LD-2P-FLIM) for multiparameter and quantitative brain function imaging, with a 3 \u00d7 3 mm\n                    <jats:sup>2<\/jats:sup>\n                    FOV, a uniform lateral resolution of 0.7 \u03bcm, and a FLIM throughput of up to 15.73 megapixels\/s (512 \u00d7 512 pixels, 30 Hz, two regions). We extend the FOV by breaking the limit of commercial objectives with an effective adaptive optics strategy. To alleviate the trade-off between the imaging area and temporal resolution, we use a temporal multiplexing system that enables simultaneous and flexible two-region imaging across the large FOV. Furthermore, we develop a field programmable gate array module to demultiplex fluorescence signals from different regions and perform high-throughput, two-region FLIM. We demonstrate the superior performance of LD-2P-FLIM by simultaneous monitoring of neural activities across multiple cortical areas, synchronous recording of neurovascular coupling under both physiological and pathological conditions, long-term observation of the microglial response to local neuron injury, and quantitative imaging of calcium concentrations across a large neuronal population in vivo.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2536213123","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T17:46:06Z","timestamp":1786124766000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":0,"title":["Large-FOV, dual-region, two-photon fluorescence lifetime imaging microscopy (LD-2P-FLIM) for multiparameter and quantitative brain function 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