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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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Optical Society of America
2021
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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. |
format | Online Article Text |
id | pubmed-7901312 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Optical Society of America |
record_format | MEDLINE/PubMed |
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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