Direct Supercritical Angle Localization Microscopy for Nanometer 3D Superresolution

Abstract

3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. Supercritical angle fluorescence strongly depends on the z-position of the fluorophore and can be used for z localization in a method called supercritical angle localization microscopy (SALM). Here, we realize the full potential of SALM by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying new fitting routines to extract precise intensities of single emitters, resulting in a four-fold improved z-resolution compared to the state of the art. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.