Axial measurements can also be performed thanks to intensity-based techniques like Supercritical Angle Fluorescence (SAF) 9, 10, 11, 12, 13, 14, which relies on the detection of the near-field emission of fluorophores coupled into propagative waves at the sample/glass coverslip interface due to the index mismatch. These sources of biases often degrade the resolution or hinder colocalization and experiment reproducibility. Still, all PSF shaping methods including astigmatic imaging suffer from several bias sources such as axial drifts, chromatic aberrations, field-varying geometrical aberrations, and sample tilts. Other Point Spread Function (PSF) shaping methods are also available 6, 7, 8, but their implementations are not as inexpensive and straightforward. The most widely used 3D Single Molecule Localization Microscopy (SMLM) technique is astigmatic imaging, which relies on the use of a cylindrical lens to apply an astigmatic aberration in the detection path to encode the axial information in the shape of the spots, achieving an axial localization precision (standard deviation) down to 20–25 nm 5-though the precision sharply varies with the axial position: 300 nm away from the focus, the precision is typically around 60 nm (see Supplementary Fig.
While photoactivated localization microscopy (PALM) and (direct) stochastic optical reconstruction microscopy ((d)STORM) can easily provide a lateral localization precision (i.e., the standard deviation of the position estimates) down to 5–10 nm 1, 2, 3, 4, a great deal of effort is being made to develop quantitative and reproducible 3D super-localization methods. 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.ĭespite recent advances in localization-based super-resolution techniques, nanoscale 3D fluorescence imaging of biological samples remains a major challenge, mostly because of its lack of versatility. 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. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. 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. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. 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.
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