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

2018; Nature Portfolio; Volume: 9; Issue: 1 Linguagem: Inglês

10.1038/s41467-017-02563-4

2041-1723

Anna‐Karin Gustavsson, Petar N. Petrov, Maurice Y. Lee, Yoav Shechtman, W. E. Moerner,

Cell Image Analysis Techniques

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.