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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...

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Detalles Bibliográficos
Autores principales: Kaufmann, Rainer, Schellenberger, Pascale, Seiradake, Elena, Dobbie, Ian M., Jones, E. Yvonne, Davis, Ilan, Hagen, Christoph, Grünewald, Kay
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
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.
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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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