A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags
© 2017 The Author(s). 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...
| Main Authors: | , , , , , , , , , |
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| Format: | Journal Article |
| Published: |
Nature Publishing Group
2017
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| Online Access: | http://hdl.handle.net/20.500.11937/72565 |
| _version_ | 1848762784156745728 |
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| author | Müller, A. Neukam, M. Ivanova, A. Sönmez, A. Münster, C. Kretschmar, S. Kalaidzidis, Y. Kurth, T. Verbavatz, J. Solimena, Michele |
| author_facet | Müller, A. Neukam, M. Ivanova, A. Sönmez, A. Münster, C. Kretschmar, S. Kalaidzidis, Y. Kurth, T. Verbavatz, J. Solimena, Michele |
| author_sort | Müller, A. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | © 2017 The Author(s). 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. |
| first_indexed | 2025-11-14T10:53:04Z |
| format | Journal Article |
| id | curtin-20.500.11937-72565 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T10:53:04Z |
| publishDate | 2017 |
| publisher | Nature Publishing Group |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-725652019-03-21T01:14:57Z A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags Müller, A. Neukam, M. Ivanova, A. Sönmez, A. Münster, C. Kretschmar, S. Kalaidzidis, Y. Kurth, T. Verbavatz, J. Solimena, Michele © 2017 The Author(s). 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. 2017 Journal Article http://hdl.handle.net/20.500.11937/72565 10.1038/s41598-017-00033-x Nature Publishing Group unknown |
| spellingShingle | Müller, A. Neukam, M. Ivanova, A. Sönmez, A. Münster, C. Kretschmar, S. Kalaidzidis, Y. Kurth, T. Verbavatz, J. Solimena, Michele A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags |
| title | 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_short | 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 |
| url | http://hdl.handle.net/20.500.11937/72565 |