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

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

doi:10.1038/s41467-020-20863-0

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

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^*

^*Corresponding author for this work

Randall Centre of Cell & Molecular Biophysics

Research output: Contribution to journal › Article › peer-review

20 Citations (Scopus)

Abstract

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.

Original language	English
Article number	517
Journal	Nature Communications
Volume	12
Issue number	1
DOIs	https://doi.org/10.1038/s41467-020-20863-0
Publication status	Published - Dec 2021

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Szalai, A. M., Siarry, B., Lukin, J., Williamson, D. J., Unsain, N., Cáceres, A., Pilo-Pais, M., Acuna, G., Refojo, D., Owen, D. M., Simoncelli, S., & Stefani, F. D. (2021). Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules. Nature Communications, 12(1), Article 517. https://doi.org/10.1038/s41467-020-20863-0

@article{79bba69ac60f4046a3a1507256a281fa,

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

abstract = "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{\textquoteright}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.",

author = "Szalai, {Alan M.} and Bruno Siarry and Jer{\'o}nimo Lukin and Williamson, {David J.} and Nicol{\'a}s Unsain and Alfredo C{\'a}ceres and Mauricio Pilo-Pais and Guillermo Acuna and Dami{\'a}n Refojo and Owen, {Dylan M.} and Sabrina Simoncelli and Stefani, {Fernando D.}",

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: {\textcopyright} 2021, The Author(s).",

year = "2021",

month = dec,

doi = "10.1038/s41467-020-20863-0",

language = "English",

volume = "12",

journal = "Nature Communications",

issn = "2041-1723",

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Szalai, AM, Siarry, B, Lukin, J, Williamson, DJ, Unsain, N, Cáceres, A, Pilo-Pais, M, Acuna, G, Refojo, D, Owen, DM, Simoncelli, S & Stefani, FD 2021, 'Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules', Nature Communications, vol. 12, no. 1, 517. https://doi.org/10.1038/s41467-020-20863-0

TY - JOUR

T1 - Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

AU - Szalai, Alan M.

AU - Siarry, Bruno

AU - Lukin, Jerónimo

AU - Williamson, David J.

AU - Unsain, Nicolás

AU - Cáceres, Alfredo

AU - Pilo-Pais, Mauricio

AU - Acuna, Guillermo

AU - Refojo, Damián

AU - Owen, Dylan M.

AU - Simoncelli, Sabrina

AU - Stefani, Fernando D.

N1 - 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).

PY - 2021/12

Y1 - 2021/12

N2 - 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.

AB - 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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DO - 10.1038/s41467-020-20863-0

M3 - Article

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AN - SCOPUS:85099830777

SN - 2041-1723

VL - 12

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 517

ER -

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

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