Three-dimensional total internal reflection fluorescence nanoscopy with sub-10 nm resolution

Here, we present a single-molecule localization microscopy (SMLM) analysis method that delivers sub-10 nm -resolution when combined with 2D total internal reflection (TIR) fluorescence imaging via DNA point accumulation for imaging nanoscale topography (DNA-PAINT). Axial resolution is obtained from a precise measurement of the emission intensity of single molecules under evanescent field excitation. This method can be implemented on any conventional TIR wide-field microscope without modifications. We validate this approach by resolving the periodicity of alpha-tubulin assembly in microtubules, demonstrating isotropic resolution below 8 nm.