Kompetenzzentrum für Mikro- und Nanotechnologien

3D single molecule localization microscopy (SMLM) has emerged as a powerful method for structural cell biology, as it allows probing precise positions of proteins in cellular structures at a resolution of tens of nanometers in both 2D and 3D. Popular approaches to 3D imaging include PSF engineering to encode the position of single-emitters in its shape. However, such methods lead to worse localization precision axially than laterally. Complex localization microscopy techniques, such as iPALM, can provide isotropic resolution at the cost of a complex instrument. Supercritical angle fluorescence strongly depends on the z-position of the fluorophore and can be used for axial 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 several 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.