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

By Alan M. Szalai, Bruno Siarry, Jerónimo Lukin, David J Williamson, Nicolas Unsain, Raquel Becerra, Damian Refojo, Alfredo Cáceres, Mauricio Pilo-Pais, Guillermo Acuna, Dylan M Owen, Sabrina Simoncelli, Fernando D. Stefani

Posted 05 Jul 2019
bioRxiv DOI: 10.1101/693994

Here, we present a single-molecule localization microscopy (SMLM) analysis method that delivers sub-10 nm z-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.

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