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

Andreas Müller,Jean-Marc Verbavatz,Anna Ivanova,Michele Solimena,Thomas Kurth,Anke Sönmez,Susanne Kretschmar,Martin Neukam,Yannis Kalaidzidis,Carla Münster

doi:10.1038/s41598-017-00033-x

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.

Highlights

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments
We show that the number of newly-generated insulin secretory granules (SGs) reduces starting at an age of 2.7 days, with 60% of labeled insulin being degraded in multi-granular bodies (MGBs) after 5 days
We demonstrate that tagging of several cytosolic, membrane-associated, nuclear and cytoskeletal proteins with either SNAP or CLIP followed by labeling with fluorescent substrates enables their detection in Epon epoxy resin sections by Fluorescence light microscopy (FLM) after high pressure freezing (HPF) and freeze-substitution (FS) and even after chemical fixation

Summary

Introduction

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence 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. We preserve the initial fluorescence of SNAP-substrates TMR-Star and 505-Star in ultrathin frozen Tokuyasu sections We examine these specimens with structured illumination microscopy (SIM) and are able to correlate SIM images to their corresponding EM images with high precision. We demonstrate that tagging of several cytosolic, membrane-associated, nuclear and cytoskeletal proteins with either SNAP or CLIP followed by labeling with fluorescent substrates enables their detection in Epon epoxy resin sections by FLM after high pressure freezing (HPF) and freeze-substitution (FS) and even after chemical fixation. We thereby demonstrate the versatility of our approach, which can be applied to a variety of other proteins than insulin

Methods

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Conclusion