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Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy
Single molecule localization microscopy (SMLM) is one of the key techniques that break the classical resolution limit in optical imaging. It is based on taking multiple recordings of a sample, each showing only a sparse arrangement of spatially well separated fluorescent molecules which can be local...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Optical Society of America
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041438/ https://www.ncbi.nlm.nih.gov/pubmed/32206395 http://dx.doi.org/10.1364/BOE.375678 |
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author | Zelger, Philipp Bodner, Lisa Velas, Lukas Schütz, Gerhard J. Jesacher, Alexander |
author_facet | Zelger, Philipp Bodner, Lisa Velas, Lukas Schütz, Gerhard J. Jesacher, Alexander |
author_sort | Zelger, Philipp |
collection | PubMed |
description | Single molecule localization microscopy (SMLM) is one of the key techniques that break the classical resolution limit in optical imaging. It is based on taking multiple recordings of a sample, each showing only a sparse arrangement of spatially well separated fluorescent molecules which can be localized at nanometer precision. While localizing along the lateral directions is usually straightforward, estimating axial positions at a comparable precision is known to be much harder, which is due to the relatively large depth of focus provided by the microscope optics. Whenever a molecule is sufficiently close to the coverslip, it becomes feasible to draw additional information from near field coupling effects: super-critical angle fluorescence (SAF) appears and can be exploited to boost the axial localization precision. Here we propose defocused imaging as a SMLM strategy that is capable of leveraging the information contained in SAF. We show that, regarding axial localization precision, our approach is superior to established SAF-based approaches. At the same time it is simple and can be conducted on any research-grade microscope where controlled defocusing on the order of a few hundred nanometers is possible. |
format | Online Article Text |
id | pubmed-7041438 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Optical Society of America |
record_format | MEDLINE/PubMed |
spelling | pubmed-70414382020-03-23 Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy Zelger, Philipp Bodner, Lisa Velas, Lukas Schütz, Gerhard J. Jesacher, Alexander Biomed Opt Express Article Single molecule localization microscopy (SMLM) is one of the key techniques that break the classical resolution limit in optical imaging. It is based on taking multiple recordings of a sample, each showing only a sparse arrangement of spatially well separated fluorescent molecules which can be localized at nanometer precision. While localizing along the lateral directions is usually straightforward, estimating axial positions at a comparable precision is known to be much harder, which is due to the relatively large depth of focus provided by the microscope optics. Whenever a molecule is sufficiently close to the coverslip, it becomes feasible to draw additional information from near field coupling effects: super-critical angle fluorescence (SAF) appears and can be exploited to boost the axial localization precision. Here we propose defocused imaging as a SMLM strategy that is capable of leveraging the information contained in SAF. We show that, regarding axial localization precision, our approach is superior to established SAF-based approaches. At the same time it is simple and can be conducted on any research-grade microscope where controlled defocusing on the order of a few hundred nanometers is possible. Optical Society of America 2020-01-13 /pmc/articles/PMC7041438/ /pubmed/32206395 http://dx.doi.org/10.1364/BOE.375678 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. Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License (http://creativecommons.org/licenses/by/4.0/) . Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. |
spellingShingle | Article Zelger, Philipp Bodner, Lisa Velas, Lukas Schütz, Gerhard J. Jesacher, Alexander Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy |
title | Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy |
title_full | Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy |
title_fullStr | Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy |
title_full_unstemmed | Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy |
title_short | Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy |
title_sort | defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041438/ https://www.ncbi.nlm.nih.gov/pubmed/32206395 http://dx.doi.org/10.1364/BOE.375678 |
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