Article,
Combining 3D single molecule localization strategies for reproducible bioimaging
Nature Communications, 10 (1): 1980 (December 2019)
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 $\mu$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-$\mu$m range by direct merging of multiple acquisitions at different depths.
