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4Pi-RESOLFT nanoscopy

By enlarging the aperture along the optic axis, the coherent utilization of opposing objective lenses (4Pi arrangement) has the potential to offer the sharpest and most light-efficient point-spread-functions in three-dimensional (3D) far-field fluorescence nanoscopy. However, to obtain unambiguous i...

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Autores principales: Böhm, Ulrike, Hell, Stefan W., Schmidt, Roman
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740410/
https://www.ncbi.nlm.nih.gov/pubmed/26833381
http://dx.doi.org/10.1038/ncomms10504
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author Böhm, Ulrike
Hell, Stefan W.
Schmidt, Roman
author_facet Böhm, Ulrike
Hell, Stefan W.
Schmidt, Roman
author_sort Böhm, Ulrike
collection PubMed
description By enlarging the aperture along the optic axis, the coherent utilization of opposing objective lenses (4Pi arrangement) has the potential to offer the sharpest and most light-efficient point-spread-functions in three-dimensional (3D) far-field fluorescence nanoscopy. However, to obtain unambiguous images, the signal has to be discriminated against contributions from lobes above and below the focal plane, which has tentatively limited 4Pi arrangements to imaging samples with controllable optical conditions. Here we apply the 4Pi scheme to RESOLFT nanoscopy using two-photon absorption for the on-switching of fluorescent proteins. We show that in this combination, the lobes are so low that low-light level, 3D nanoscale imaging of living cells becomes possible. Our method thus offers robust access to densely packed, axially extended cellular regions that have been notoriously difficult to super-resolve. Our approach also entails a fluorescence read-out scheme that translates molecular sensitivity to local off-switching rates into improved signal-to-noise ratio and resolution.
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spelling pubmed-47404102016-03-04 4Pi-RESOLFT nanoscopy Böhm, Ulrike Hell, Stefan W. Schmidt, Roman Nat Commun Article By enlarging the aperture along the optic axis, the coherent utilization of opposing objective lenses (4Pi arrangement) has the potential to offer the sharpest and most light-efficient point-spread-functions in three-dimensional (3D) far-field fluorescence nanoscopy. However, to obtain unambiguous images, the signal has to be discriminated against contributions from lobes above and below the focal plane, which has tentatively limited 4Pi arrangements to imaging samples with controllable optical conditions. Here we apply the 4Pi scheme to RESOLFT nanoscopy using two-photon absorption for the on-switching of fluorescent proteins. We show that in this combination, the lobes are so low that low-light level, 3D nanoscale imaging of living cells becomes possible. Our method thus offers robust access to densely packed, axially extended cellular regions that have been notoriously difficult to super-resolve. Our approach also entails a fluorescence read-out scheme that translates molecular sensitivity to local off-switching rates into improved signal-to-noise ratio and resolution. Nature Publishing Group 2016-02-01 /pmc/articles/PMC4740410/ /pubmed/26833381 http://dx.doi.org/10.1038/ncomms10504 Text en Copyright © 2016, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Böhm, Ulrike
Hell, Stefan W.
Schmidt, Roman
4Pi-RESOLFT nanoscopy
title 4Pi-RESOLFT nanoscopy
title_full 4Pi-RESOLFT nanoscopy
title_fullStr 4Pi-RESOLFT nanoscopy
title_full_unstemmed 4Pi-RESOLFT nanoscopy
title_short 4Pi-RESOLFT nanoscopy
title_sort 4pi-resolft nanoscopy
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740410/
https://www.ncbi.nlm.nih.gov/pubmed/26833381
http://dx.doi.org/10.1038/ncomms10504
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