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Super-Resolution Microscopy Using Standard Fluorescent Proteins in Intact Cells under Cryo-Conditions
[Image: see text] We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, curr...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092024/ https://www.ncbi.nlm.nih.gov/pubmed/24884378 http://dx.doi.org/10.1021/nl501870p |
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author | Kaufmann, Rainer Schellenberger, Pascale Seiradake, Elena Dobbie, Ian M. Jones, E. Yvonne Davis, Ilan Hagen, Christoph Grünewald, Kay |
author_facet | Kaufmann, Rainer Schellenberger, Pascale Seiradake, Elena Dobbie, Ian M. Jones, E. Yvonne Davis, Ilan Hagen, Christoph Grünewald, Kay |
author_sort | Kaufmann, Rainer |
collection | PubMed |
description | [Image: see text] We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400–500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. This enabled us to perform super-resolution imaging of vitrified biological samples and to visualize structures in unperturbed fast frozen cells for the first time with a structural resolution of ∼125 nm (average single molecule localization accuracy ∼40 nm), corresponding to a 3–5 fold resolution improvement. |
format | Online Article Text |
id | pubmed-4092024 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-40920242014-07-21 Super-Resolution Microscopy Using Standard Fluorescent Proteins in Intact Cells under Cryo-Conditions Kaufmann, Rainer Schellenberger, Pascale Seiradake, Elena Dobbie, Ian M. Jones, E. Yvonne Davis, Ilan Hagen, Christoph Grünewald, Kay Nano Lett [Image: see text] We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400–500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. This enabled us to perform super-resolution imaging of vitrified biological samples and to visualize structures in unperturbed fast frozen cells for the first time with a structural resolution of ∼125 nm (average single molecule localization accuracy ∼40 nm), corresponding to a 3–5 fold resolution improvement. American Chemical Society 2014-06-02 2014-07-09 /pmc/articles/PMC4092024/ /pubmed/24884378 http://dx.doi.org/10.1021/nl501870p Text en Copyright © 2014 American Chemical Society Terms of Use CC-BY (http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html) |
spellingShingle | Kaufmann, Rainer Schellenberger, Pascale Seiradake, Elena Dobbie, Ian M. Jones, E. Yvonne Davis, Ilan Hagen, Christoph Grünewald, Kay Super-Resolution Microscopy Using Standard Fluorescent Proteins in Intact Cells under Cryo-Conditions |
title | Super-Resolution Microscopy Using Standard Fluorescent
Proteins in Intact Cells under Cryo-Conditions |
title_full | Super-Resolution Microscopy Using Standard Fluorescent
Proteins in Intact Cells under Cryo-Conditions |
title_fullStr | Super-Resolution Microscopy Using Standard Fluorescent
Proteins in Intact Cells under Cryo-Conditions |
title_full_unstemmed | Super-Resolution Microscopy Using Standard Fluorescent
Proteins in Intact Cells under Cryo-Conditions |
title_short | Super-Resolution Microscopy Using Standard Fluorescent
Proteins in Intact Cells under Cryo-Conditions |
title_sort | super-resolution microscopy using standard fluorescent
proteins in intact cells under cryo-conditions |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092024/ https://www.ncbi.nlm.nih.gov/pubmed/24884378 http://dx.doi.org/10.1021/nl501870p |
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