A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags

Abstract Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and...

Descripción completa

Guardado en:
Detalles Bibliográficos
Autores principales: Andreas Müller, Martin Neukam, Anna Ivanova, Anke Sönmez, Carla Münster, Susanne Kretschmar, Yannis Kalaidzidis, Thomas Kurth, Jean-Marc Verbavatz, Michele Solimena
Formato: article
Lenguaje:EN
Publicado: Nature Portfolio 2017
Materias:
R
Q
Acceso en línea:https://doaj.org/article/069d03a33d2f4c818999027d415bc62e
Etiquetas: Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
id oai:doaj.org-article:069d03a33d2f4c818999027d415bc62e
record_format dspace
spelling oai:doaj.org-article:069d03a33d2f4c818999027d415bc62e2021-12-02T11:52:21ZA Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags10.1038/s41598-017-00033-x2045-2322https://doaj.org/article/069d03a33d2f4c818999027d415bc62e2017-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-00033-xhttps://doaj.org/toc/2045-2322Abstract Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.Andreas MüllerMartin NeukamAnna IvanovaAnke SönmezCarla MünsterSusanne KretschmarYannis KalaidzidisThomas KurthJean-Marc VerbavatzMichele SolimenaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-13 (2017)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Andreas Müller
Martin Neukam
Anna Ivanova
Anke Sönmez
Carla Münster
Susanne Kretschmar
Yannis Kalaidzidis
Thomas Kurth
Jean-Marc Verbavatz
Michele Solimena
A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags
description Abstract Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.
format article
author Andreas Müller
Martin Neukam
Anna Ivanova
Anke Sönmez
Carla Münster
Susanne Kretschmar
Yannis Kalaidzidis
Thomas Kurth
Jean-Marc Verbavatz
Michele Solimena
author_facet Andreas Müller
Martin Neukam
Anna Ivanova
Anke Sönmez
Carla Münster
Susanne Kretschmar
Yannis Kalaidzidis
Thomas Kurth
Jean-Marc Verbavatz
Michele Solimena
author_sort Andreas Müller
title A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags
title_short A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags
title_full A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags
title_fullStr A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags
title_full_unstemmed A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags
title_sort global approach for quantitative super resolution and electron microscopy on cryo and epoxy sections using self-labeling protein tags
publisher Nature Portfolio
publishDate 2017
url https://doaj.org/article/069d03a33d2f4c818999027d415bc62e
work_keys_str_mv AT andreasmuller aglobalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT martinneukam aglobalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT annaivanova aglobalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT ankesonmez aglobalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT carlamunster aglobalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT susannekretschmar aglobalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT yanniskalaidzidis aglobalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT thomaskurth aglobalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT jeanmarcverbavatz aglobalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT michelesolimena aglobalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT andreasmuller globalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT martinneukam globalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT annaivanova globalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT ankesonmez globalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT carlamunster globalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT susannekretschmar globalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT yanniskalaidzidis globalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT thomaskurth globalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT jeanmarcverbavatz globalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
AT michelesolimena globalapproachforquantitativesuperresolutionandelectronmicroscopyoncryoandepoxysectionsusingselflabelingproteintags
_version_ 1718395026725142528