Combining 3D single molecule localization strategies for reproducible bioimaging


  • Cabriel Clément
  • Bourg Nicolas
  • Jouchet Pierre
  • Dupuis Guillaume
  • Leterrier Christophe
  • Baron Aurélie
  • Badet-Denisot Marie-Ange
  • Vauzeilles Boris
  • Fort Emmanuel
  • Leveque-Fort Sandrine


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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