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

    Access Status
    Open access via publisher
    Authors
    Müller, A.
    Neukam, M.
    Ivanova, A.
    Sönmez, A.
    Münster, C.
    Kretschmar, S.
    Kalaidzidis, Y.
    Kurth, T.
    Verbavatz, J.
    Solimena, Michele
    Date
    2017
    Type
    Journal Article
    
    Metadata
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    Citation
    Müller, A. and Neukam, M. and Ivanova, A. and Sönmez, A. and Münster, C. and Kretschmar, S. and Kalaidzidis, Y. et al. 2017. A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags. Scientific Reports. 7 (1).
    Source Title
    Scientific Reports
    DOI
    10.1038/s41598-017-00033-x
    ISSN
    2045-2322
    School
    School of Pharmacy and Biomedical Sciences
    URI
    http://hdl.handle.net/20.500.11937/72565
    Collection
    • Curtin Research Publications
    Abstract

    © 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.

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