Combining 3D single molecule localization strategies for reproducible bioimaging

Nat Commun. 2019 Apr 30;10(1):1980. doi: 10.1038/s41467-019-09901-8.

Abstract

Here, we present a 3D localization-based super-resolution technique providing a slowly varying localization precision over a 1 μm range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently to provide a weakly anisotropic 3D resolution over the imaging range. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several-μm range by direct merging of multiple acquisitions at different depths.

MeSH terms

  • Algorithms
  • Anisotropy
  • Imaging, Three-Dimensional / methods*
  • Microscopy / methods*
  • Nanotechnology / methods*