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Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence

The precise spatial localization of single molecules in three dimensions is an important basis for single molecule localization microscopy (SMLM) and tracking. At distances up to a few hundred nanometers from the coverslip, evanescent wave coupling into the glass, also known as supercritical angle f...

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Autores principales: Zelger, Philipp, Bodner, Lisa, Offterdinger, Martin, Velas, Lukas, Schütz, Gerhard J., Jesacher, Alexander
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Optical Society of America 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901312/
https://www.ncbi.nlm.nih.gov/pubmed/33680543
http://dx.doi.org/10.1364/BOE.413018
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author Zelger, Philipp
Bodner, Lisa
Offterdinger, Martin
Velas, Lukas
Schütz, Gerhard J.
Jesacher, Alexander
author_facet Zelger, Philipp
Bodner, Lisa
Offterdinger, Martin
Velas, Lukas
Schütz, Gerhard J.
Jesacher, Alexander
author_sort Zelger, Philipp
collection PubMed
description The precise spatial localization of single molecules in three dimensions is an important basis for single molecule localization microscopy (SMLM) and tracking. At distances up to a few hundred nanometers from the coverslip, evanescent wave coupling into the glass, also known as supercritical angle fluorescence (SAF), can strongly improve the axial precision, thus facilitating almost isotropic localization performance. Specific detection systems, introduced as Supercritical angle localization microscopy (SALM) or Direct optical nanoscopy with axially localized detection (DONALD), have been developed to exploit SAF in modified two-channel imaging schemes. Recently, our group has shown that off-focus microscopy, i.e., imaging at an intentional slight defocus, can perform equally well, but uses only a single detection arm. Here we compare SALM, off-focus imaging and the most commonly used 3D SMLM techniques, namely cylindrical lens and biplane imaging, regarding 3D localization in close proximity to the coverslip. We show that all methods gain from SAF, which leaves a high detection NA as the only major key requirement to unlock the SAF benefit. We find parameter settings for cylindrical lens and biplane imaging for highest z-precision. Further, we compare the methods in view of robustness to aberrations, fixed dipole emission and double-emitter events. We show that biplane imaging provides the best overall performance and support our findings by DNA-PAINT experiments on DNA-nanoruler samples. Our study sheds light on the effects of SAF for SMLM and is helpful for researchers who plan to employ localization-based 3D nanoscopy close to the coverslip.
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spelling pubmed-79013122021-03-04 Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence Zelger, Philipp Bodner, Lisa Offterdinger, Martin Velas, Lukas Schütz, Gerhard J. Jesacher, Alexander Biomed Opt Express Article The precise spatial localization of single molecules in three dimensions is an important basis for single molecule localization microscopy (SMLM) and tracking. At distances up to a few hundred nanometers from the coverslip, evanescent wave coupling into the glass, also known as supercritical angle fluorescence (SAF), can strongly improve the axial precision, thus facilitating almost isotropic localization performance. Specific detection systems, introduced as Supercritical angle localization microscopy (SALM) or Direct optical nanoscopy with axially localized detection (DONALD), have been developed to exploit SAF in modified two-channel imaging schemes. Recently, our group has shown that off-focus microscopy, i.e., imaging at an intentional slight defocus, can perform equally well, but uses only a single detection arm. Here we compare SALM, off-focus imaging and the most commonly used 3D SMLM techniques, namely cylindrical lens and biplane imaging, regarding 3D localization in close proximity to the coverslip. We show that all methods gain from SAF, which leaves a high detection NA as the only major key requirement to unlock the SAF benefit. We find parameter settings for cylindrical lens and biplane imaging for highest z-precision. Further, we compare the methods in view of robustness to aberrations, fixed dipole emission and double-emitter events. We show that biplane imaging provides the best overall performance and support our findings by DNA-PAINT experiments on DNA-nanoruler samples. Our study sheds light on the effects of SAF for SMLM and is helpful for researchers who plan to employ localization-based 3D nanoscopy close to the coverslip. Optical Society of America 2021-01-12 /pmc/articles/PMC7901312/ /pubmed/33680543 http://dx.doi.org/10.1364/BOE.413018 Text en Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. https://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Zelger, Philipp
Bodner, Lisa
Offterdinger, Martin
Velas, Lukas
Schütz, Gerhard J.
Jesacher, Alexander
Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence
title Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence
title_full Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence
title_fullStr Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence
title_full_unstemmed Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence
title_short Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence
title_sort three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901312/
https://www.ncbi.nlm.nih.gov/pubmed/33680543
http://dx.doi.org/10.1364/BOE.413018
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