Photon-Resolved Excitation-Denoised (PRED) Three-Photon Imaging Improves Detection of Neuronal Activity in Awake and Behaving Mice

Three-photon microscopy (3PM) has enabled the optical access of neurons ∼500-1500 µm below the brain surface but has been limited to slow imaging frame rates or small total imaged areas by competing constraints: a high peak power requirement for nonlinear excitation, and the need to limit total average power. Additionally, the high sensitivity to laser power fluctuations and the inherently dim signals introduce additional challenges and add error. When combined with other effects, like brain motion in behaving animals, 3P imaging of neuronal activity during animal behavior has remained inadequate. Herein, we systematically address these limitations by 1) carefully balancing scanning speed with power requirements, 2) using a deeply cooled silicon photomultiplier detector and Bayesian statistics-based processing (PRED-correction) to reduce excess noise, and 3) by 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-3P imaging provides a new approach to functional characterization of cells deep in the brain that were previously inaccessible to two-photon imaging.