Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits its speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and computationally recovers high-resolution images, attaining voxel rates of over 1 billion Hz in structured samples. Using a static image as a prior for recording neural activity, we imaged visually evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 µm and frame rates over 1 kHz. Dendritic glutamate transients in anesthetized mice are synchronized within spatially contiguous domains spanning tens of micrometers at frequencies ranging from 1-100 Hz. We demonstrate millisecond-resolved recordings of acetylcholine and voltage indicators, three-dimensional single-particle tracking and imaging in densely labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.

2nd Medical Faculty Charles University Prague Czech Republic

Department of Chemistry University of California Berkeley Berkeley CA USA

Department of Molecular and Cell Biology University of California Berkeley Berkeley CA USA

Department of Neurobiology Stanford University Stanford CA USA

Helen Wills Neuroscience Institute University of California Berkeley Berkeley CA USA

Janelia Research Campus Howard Hughes Medical Institute Ashburn VA USA

Vidrio Technologies Ashburn VA USA

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Nat Methods. 2019 Sep;16(9):932. doi: 10.1038/s41592-019-0545-1. PubMed

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