The application of localization-based super-resolution imaging methods (SMLM) is hindered by the complexity of their implementation. However, putting super-resolution within reach of non-specialists is an indispensable step to fully realize the potential of localization microscopy methods.
A major hurdle is the inherent bi-dimensionality of SMLM. The strategies developed so far to extend the resolution gain to the axial dimension and achieve 3D-SMLM (bi-plane imaging, psf engineering, interferometry) have a major limitation in that their axial information is contingent on the position of the emitter relative to the focal plane. The consequence is that credible experimental repeatability, comparability, and reproducibility of results can be obtained only with complex and time-consuming calibrations.
Scientists at Abbelight, a spin-out from the Institut de Sciences Moleculaires in Orsay (ISMO) have developed a complete solution for 3D-SMLM, which improves performances, experimental repeatability, and reliability of the data, and simplifies access to SMLM for non-specialists
The core of Abbelight’s solution is a dual-view optical setup, which exploits the Supercritical Angle Fluorescence (SAF) emission of fluorophores thanks to the patented DONALD technology (Direct Optical Nanoscopy with Axially Localized Detection, Bourg et al., Nature Photonics, 2015).
Fluorophores within a biological sample emit both a near field and far field component (Figure 1 and view the video here). The evanescent near field waves quickly decay in intensity, while the intensity of the propagative far field remains approximately constant. When the emitting fluorophore is located near the sample/glass coverslip the evanescent emission reaching the interface beyond the critical angle is coupled into propagative waves, which can be collected by high numerical aperture objectives. This component is the SAF emission, which can be distinguished from the far field emission in the back focal plane (BFP) of the objective. Using masks placed in the BFP it is possible to discriminate between the two components. The comparison between the two components permits to retrieve the axial information of the fluorophore position with respect to the glass/water interface, independently of the focus position (absolute axial positioning (FIG). Thus, DONALD technology enables the detection of single fluorophores with a lateral localization precision of typically 5 to 10 nm and an absolute axial precision of 15 nm, yielding quasi-isotropic precision of localization in 3D-SMLM.
Researchers at the IPBS of Toulouse have used DONALD 3D nanoscopy on human macrophages to localize with nanometric precision and obtain a quantitative map of the spatial organization of the podosome components (Bouissou et al., ACS Nano 2017). Podosomes are short-lived adhesive structures, no greater than 0.5 μm in width and 1 μm in depth, found at the surface of myeloid cells where they protrude into, probe and remodel the extracellular matrix by exerting force and releasing proteases.
The position of each molecule super-localized within a given podosome was plotted on a map with coordinates indicating its height and radial distance from the corresponding actin core, and with an intensity inversely proportional to this distance (Figure 2).
Thanks to the absolute nature of DONALD measurements Bouissou et al. could easily combine the density maps of thousands of organelles, yielding an average podosome characterized by the probability density of the radial and vertical position of each component analyzed.
These results represent an excellent example of how isotropic resolution and quantitative treatment of data permits the nano-topography of complex molecular structures, yielding precious structural and functional insights.
http://www.ismo.u-psud.fr/spip.php?article1538&lang=en
https://pubs.acs.org/doi/abs/10.1021/acsnano.7b00622
http://www.ipbs.fr/may-force-be-podosome
https://www.nature.com/articles/nphoton.2015.132
