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 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.
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Clément Cabriel, Nicolas Bourg, Pierre Jouchet, Guillaume Dupuis, Christophe Leterrier, et al.. Combining 3D single molecule localization strategies for reproducible bioimaging. Nature Communications, Nature Publishing Group, 2019, 10 (1), pp.1642. ⟨10.1038/s41467-019-09901-8⟩. ⟨hal-02116025⟩

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