Cortex-Wide, Cellular-Resolution Volumetric Imaging with a Modular Two-Photon Imaging Platform

Abstract

Mapping cortex-wide neuronal activity at single-cell resolution has been limited by the physical trade-off between numerical aperture and field-of-view (FOV) in two-photon microscopes. We present Meso2P, a modular two-photon platform that decouples excitation and detection by introducing a lateral paraboloid fluorescence collector. The design sustains an effective NA 0.87 over a contiguous 6 x 6 mm2 FOV at high speed (2,048 x 2,048 pixels at 7.67 Hz). The modular platform can be upgraded with optional modules for simultaneous multi-plane imaging (1-4 planes at full resolution and speed), volumetric imaging (6 x 6 x 0.5 mm3, 2,048 x 2,048 x 28 voxels at 1 Hz capturing> 210,000 neurons), and holographic two-photon optogenetic stimulation for targeted perturbations. To handle the resulting large-scale data, we provide an open-source deep-learning pipeline that automates motion correction, segmentation, and spike inference. We demonstrate cortex-wide sensory responses, layer-specific network synchrony during anaesthesia, and in-vivo tracking of micro and nanoplastic distribution. Meso2P therefore provides a reproducible route to high-throughput volumetric imaging across almost the entire cortex with high detection efficiency.