A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.
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.
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.|Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, Dresden, Germany.
Andreas
A
Müller
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.|Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, Dresden, Germany.
Martin
M
Neukam
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.|Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, Dresden, Germany.
Anna
A
Ivanova
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.|Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, Dresden, Germany.
Anke
A
Sönmez
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.|Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, Dresden, Germany.
Carla
C
Münster
Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany.|Biotechnology Center of the TU Dresden (BIOTEC), Dresden, Germany.
Susanne
S
Kretschmar
Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany.|Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow, Russia.
Yannis
Y
Kalaidzidis
Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany.|Biotechnology Center of the TU Dresden (BIOTEC), Dresden, Germany.
Thomas
T
Kurth
Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany.|Institut Jacques Monod, Université Paris Diderot, Paris, France.
Jean-Marc
JM
Verbavatz
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany. Michele.Solimena@tu-dresden.de.|Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, Dresden, Germany. Michele.Solimena@tu-dresden.de.|Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany. Michele.Solimena@tu-dresden.de.
Michele
M
Solimena
D000818:Animals; D002478:Cells, Cultured; D016616:Cryoultramicrotomy; D004853:Epoxy Resins; D005404:Fixatives; D005615:Freezing; D005629:Frozen Sections; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007328:Insulin / Q000235:genetics; D050417:Insulin-Secreting Cells / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000737:chemistry*; D051379:Mice; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods*
D004853:Epoxy Resins; D005404:Fixatives; D007328:Insulin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins