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

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • Andreas Müller - , Molecular Diabetology, University Hospital Carl Gustav Carus Dresden, TUD Dresden University of Technology (Author)
  • Martin Neukam - , Molecular Diabetology, University Hospital Carl Gustav Carus Dresden, TUD Dresden University of Technology (Author)
  • Anna Ivanova - , University Hospital Carl Gustav Carus Dresden (Author)
  • Anke Sönmez - , University Hospital Carl Gustav Carus Dresden (Author)
  • Carla Münster - , University Hospital Carl Gustav Carus Dresden (Author)
  • Susanne Kretschmar - , Center for Regenerative Therapies Dresden (CRTD) (Author)
  • Yannis Kalaidzidis - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • Thomas Kurth - , Core Facility Electron Microscopy & Histology, Center for Regenerative Therapies Dresden (Author)
  • Jean-Marc Verbavatz - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • Michele Solimena - , Molecular Diabetology, University Hospital Carl Gustav Carus Dresden, TUD Dresden University of Technology, Max Planck Institute of Molecular Cell Biology and Genetics (Author)

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.

Details

Original languageEnglish
Article number23
JournalScientific reports
Volume7
Issue number1
Publication statusPublished - 2 Feb 2017
Peer-reviewedYes

External IDs

PubMedCentral PMC5428382
Scopus 85013277042
ORCID /0000-0001-5624-1717/work/142239015

Keywords

Keywords

  • Animals, Cells, Cultured, Cryoultramicrotomy, Epoxy Resins, Fixatives, Freezing, Frozen Sections, Green Fluorescent Proteins/chemistry, Insulin/genetics, Insulin-Secreting Cells/ultrastructure, Luminescent Proteins/chemistry, Mice, Microscopy, Electron, Transmission/methods, Microscopy, Fluorescence/methods, Recombinant Fusion Proteins/chemistry, Staining and Labeling/methods

Library keywords