Direct optical nanoscopy with axially localized detection

摘要

Evanescent light excitation is widely used in super-resolution fluorescencemicroscopy to confine light and reduce background noise. Herein we propose amethod of exploiting evanescent light in the context of emission. When afluorophore is located in close proximity to a medium with a higher refractiveindex, its near-field component is converted into light that propagates beyondthe critical angle. This so-called Supercritical Angle Fluorescence (SAF) canbe captured using a hig-NA objective and used to determine the axial positionof the fluorophore with nanometer precision. We introduce a new technique for3D nanoscopy that combines direct STochastic Optical Reconstruction Microscopy(dSTORM) imaging with dedicated detection of SAF emission. We demonstrate thatour approach of a Direct Optical Nanoscopy with Axially Localized Detection(DONALD) yields a typical isotropic 3D localization precision of 20 nm.