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.

About samples, giving examples: Optimized Single Molecule Localization Microscopy

“…Drift correction is usually performed in 2D as a focus stabilization is used to avoid Z drift. However, Z drift can nevertheless occur (Cabriel et al, 2019) and can be further corrected by post-processing correction (3D-DAOSTORM, ZOLA-3D). Finally, sequential application of several drift correction steps can lead to progressively better results.…”

Nanometric axial localization of single fluorescent molecules with modulated excitation

“…We first characterize ModLoc axial and lateral localization precisions at various depths. To evaluate the intrinsic precision, we perform repeated localizations over small fluorescent nanospheres with a controlled emission level as previously reported 21,22,26 (see Supplementary Note 5). Figure 2a shows We now focus on ModLoc performances on biological structures ( Supplementary Note 6 and 7).…”

“…The symmetry of the microscope and the optical optimization of the objectives in the lateral direction deteriorate the axial localization precision compared to the lateral one. A large variety of strategies have been developed based on point spread function (PSF) manipulations like multi-focused techniques [11][12][13][14] , PSF engineering approaches [15][16][17][18] or supercritical angle fluorescence detection [19][20][21] . Alternative accurate techniques are based on the coherence of the fluorescence when emitted by a single molecule to retrieve axial localization, like interferometric PALM (iPALM) 22,23 , 4Pi single marker switching nanoscopy (4Pi-SMSN) 24,25 or Self-Interference (SelFi) 26 .…”

Direct Supercritical Angle Localization Microscopy for Nanometer 3D Superresolution

“…1e), experiments on biological samples failed to demonstrate this 14,15,17 . To extend the axial range of vSALM, it has been combined with astigmatism by introducing a cylindrical lens in the UAF channel 18 . Interestingly, SAF has a strong impact on the shape of a defocused PSF.…”

“…1f). In the undercritical path, we insert a cylindrical lens to introduce weak astigmatism 18 . By fitting single molecules in the UAF channel with an experimentally derived PSF model 17 , we obtain their precise x and y coordinates and intensity, as well as less precise z coordinates (referred to as as ).…”