Combining 3D single molecule localization strategies for reproducible bioimaging.

ABSTRACT: Here, we present a 3D localization-based super-resolution technique providing a slowly varying localization precision over a 1??m 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-?m range by direct merging of multiple acquisitions at different depths.

SUBMITTER: Cabriel C 

PROVIDER: S-EPMC6491430 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature