3D single-molecule super-resolution microscopy with a tilted light sheet

Nat Commun. 2018 Jan 9;9(1):123. doi: 10.1038/s41467-017-02563-4.

Abstract

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.

Publication types

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

MeSH terms

  • Cell Line, Tumor
  • HeLa Cells
  • Humans
  • Imaging, Three-Dimensional / methods*
  • Lighting / methods
  • Mitochondria / ultrastructure*
  • Single Molecule Imaging / instrumentation*
  • Single Molecule Imaging / methods*