Kilohertz frame-rate two-photon tomography

Nat Methods. 2019 Aug;16(8):778-786. doi: 10.1038/s41592-019-0493-9. Epub 2019 Jul 29.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Calcium / metabolism
  • Cerebral Cortex / cytology
  • Cerebral Cortex / physiology*
  • Female
  • Glutamic Acid / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Neurons / cytology
  • Neurons / physiology*
  • Photons
  • Rats
  • Tomography / methods*

Substances

  • Glutamic Acid
  • Calcium