biostudies-literatureDemas JU.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and StrokeNINDS NIH HHSKavli FoundationBristol-Myers Squibb Postdoctoral Fellowship1103-1111https://www.ebi.ac.uk/biostudies/studies/S-EPMC8958902biostudies-literatureUnknown18(9)Two-photon microscopy has enabled high-resolution imaging of neuroactivity at depth within scattering brain tissue. However, its various realizations have not overcome the tradeoffs between speed and spatiotemporal sampling that would be necessary to enable 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 lifetime, where a set of axially separated and temporally distinct foci record the entire axial imaging range near-simultaneously, enabling volumetric recording at 1.41 × 10⁸ voxels per second. Using LBM, we demonstrate mesoscopic and volumetric imaging at multipleNature methodsHigh-speed, cortex-wide volumetric recording of neuroactivity at cellular resolution using light beads microscopy.PMC89589021RF1NS1105011RF1NS113251RF1 NS1132515U01NS103488U01 NS103488RF1 NS110501Barber KKim HDemas JTraub FMManley JChen BTejera FVaziri AfalseHigh-speed, cortex-wide volumetric recording of neuroactivity at cellular resolution using light beads microscopy.Two-photon microscopy has enabled high-resolution imaging of neuroactivity at depth within scattering brain tissue. However, its various realizations have not overcome the tradeoffs between speed and spatiotemporal sampling that would be necessary to enable 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 lifetime, where a set of axially separated and temporally distinct foci record the entire axial imaging range near-simultaneously, enabling volumetric recording at 1.41 × 10⁸ voxels per second. Using LBM, we demonstrate mesoscopic and volumetric imaging at multiple2021-01-01T00:00:00Z2021 Sep2025-04-21T23:29:58.247Z2025-02-18T23:37:13.243ZS-EPMC89589023446259210.1038/s41592-021-01239-8