Direct optical nanoscopy with axially localized detection

Abstract

Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise. Herein we propose a method of exploiting evanescent light in the context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called Supercritical Angle Fluorescence (SAF) can be captured using a hig-NA objective and used to determine the axial position of the fluorophore with nanometer precision. We introduce a new technique for 3D nanoscopy that combines direct STochastic Optical Reconstruction Microscopy (dSTORM) imaging with dedicated detection of SAF emission. We demonstrate that our approach of a Direct Optical Nanoscopy with Axially Localized Detection (DONALD) yields a typical isotropic 3D localization precision of 20 nm.