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.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:29195 |
| Date | 04 April 2017 |
| Creators | Müller, Andreas, Neukam, Martin, Ivanova, Anna, Sönmez, Anke, Münster, Carla, Kretschmar, Susanne, Kalaidzidis, Yannis, Kurth, Thomas, Verbavatz, Jean-Marc, Solimena, Michele |
| Publisher | Nature Publishing Group |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:article, info:eu-repo/semantics/article, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 10.1038/s41598-017-00033-x |
Page generated in 0.002 seconds