Volumetric Calcium Imaging of 1 Million Neurons Across Cortical Regions at Cellular Resolution Using Light Beads Microscopy

Two-photon microscopy together with genetically encodable calcium indicators has emerged as a standard tool for high-resolution imaging of neuroactivity in scattering brain tissue. However, its various realizations have not overcome the inherent tradeoffs between speed and spatiotemporal sampling in a principled manner which would be necessary to enable, amongst other applications, mesoscale volumetric recording of neuroactivity at cellular resolution and speed compatible with resolving calcium transients. Here, we introduce Light Beads Microscopy (LBM), a scalable and spatiotemporally optimal acquisition approach limited only by fluorescence life-time, where a set of axially-separated and temporally- distinct foci record the entire axial imaging range near-simultaneously, enabling volumetric recording at 1.41 x 10^8 voxels per second. Using LBM, we demonstrate mesoscopic and volumetric imaging at multiple scales in the mouse cortex, including cellular resolution recordings within ~3 x 5 x 0.5 mm^3 volumes containing >200,000 neurons at ~5 Hz, recording of populations of ~1 million neurons within ~5.4 x 6 x 0.5 mm^3 volumes at ~2 Hz as well as higher-speed (9.6 Hz) sub-cellular resolution volumetric recordings. LBM provides an unprecedented opportunity for discovering the neurocomputations underlying cortex-wide encoding and processing of information in the mammalian brain.