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Tilted light sheet microscopy with 3D point spread functions for single-molecule super-resolution imaging in mammalian cells
Author(s): Anna-Karin Gustavsson; Petar N. Petrov; Maurice Y. Lee; Yoav Shechtman; W. E. Moerner
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Paper Abstract

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered 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. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D superresolution 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 PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Paper Details

Date Published: 20 February 2018
PDF: 10 pages
Proc. SPIE 10500, Single Molecule Spectroscopy and Superresolution Imaging XI, 105000M (20 February 2018); doi: 10.1117/12.2288443
Show Author Affiliations
Anna-Karin Gustavsson, Stanford Univ. (United States)
Karolinska Institutet (Sweden)
Petar N. Petrov, Stanford Univ. (United States)
Maurice Y. Lee, Stanford Univ. (United States)
Yoav Shechtman, Stanford Univ. (United States)
Israel Institute of Technology (Israel)
W. E. Moerner, Stanford Univ. (United States)


Published in SPIE Proceedings Vol. 10500:
Single Molecule Spectroscopy and Superresolution Imaging XI
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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