Losonczy, A.; Mihaila, T. S.; Kong, E.; Negrean, A.; Geiller, T.; Peterka, D. S.

Three-photon microscopy (3PM) has enabled the optical access of neurons~500-1500 micrometer below the brain surface but has been limited to slow imaging frame rates or small imaged area due to the combination of a nonlinear peak power requirement and the need to limit average power below the thermal damage threshold. High sensitivity to laser fluctuation and inherently dim signals introduce additional challenges and add error. Combined with the effects of brain motion in behaving animals, 3P imaging of neuronal activity during animal behavior has remained practically unachievable. Herein, we systematically address these limitations by carefully balancing scanning speed with power requirements, using a deeply cooled silicon photomultiplier detector with Bayesian statistics-based processing to reduce excess noise, and through spatiotemporal shaping of excitation pulses. Our improvements enable rapid (20-30Hz) imaging of calcium activity in the dorsal hippocampal dentate gyrus of behaving mice, allowing the identification of spatially tuned neurons and the recapitulation of established functional properties across different cell types in this brain region. PRED 3-photon imaging provides a new approach for functional characterization of cells deep in the brain that have been inaccessible with two-photon imaging.