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 () and Fernando D. Stefani ()
Additional contact information
Alan M. Szalai: Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)
Bruno Siarry: Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)
Jerónimo Lukin: Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET-Partner Institute of the Max Planck Society
David J. Williamson: King’s College London
Nicolás Unsain: Instituto Investigación Médica Mercedes y Martín Ferreyra-INIMEC-CONICET-Universidad Nacional Córdoba
Alfredo Cáceres: Instituto Investigación Médica Mercedes y Martín Ferreyra-INIMEC-CONICET-Universidad Nacional Córdoba
Mauricio Pilo-Pais: University of Fribourg
Guillermo Acuna: University of Fribourg
Damián Refojo: Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET-Partner Institute of the Max Planck Society
Dylan M. Owen: King’s College London
Sabrina Simoncelli: King’s College London
Fernando D. Stefani: Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)

Nature Communications, 2021, vol. 12, issue 1, 1-13

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-020-20863-0

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