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Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

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

Original languageEnglish
Article number517
JournalNature Communications
Issue number1
PublishedDec 2021

Bibliographical note

Funding Information: This work has been supported by: CONICET, ANPCYT projects PICT2013-0792 and PICT-2014-0739, the Royal Society project IEC\R2\181018, the BBSRC grant BB/ R007365/1, FOCEM (Fondo para la Convergencia Estructural del Mercosur) grant COF 03/11, and Swiss National Science Foundation through the National Center of Competence in Research Bio-Inspired Materials. S.S. acknowledges financial support from the Human Frontier Science Program Organization and the Royal Society through a HFSP (LT001463/2017-C) and Dorothy Hodgkin (DHF\R1\191019) fellowship, respectively. F.D.S. thanks the support of the Max-Planck Society and the Alexander von Humboldt Foundation. D.R. acknowledges the support of the Max-Planck Society and the Volkswagen Stiftung. Publisher Copyright: © 2021, The Author(s). Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

King's Authors


Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

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