Correlative light and electron microscopy for quantitative studies
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. DZD-Reseachers 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.
F.l.: (a) SIM image of Epon section of SOFIA mouse beta cells labeled with 505-Star (green) and TMR-Star (magenta) fixed by HPF followed by FS. As fiducial markers fluorescent beads (blue) were added to the section prior to imaging. Punctate SGs can be unequivocally distinguished by SIM. (b) CLEM image of boxed area in (a). (c) CLEM detail of boxed area in (c) showing perfect overlay of 505+ and TMR+ SGs. Scale bars: (a) 10 µm. (b+c) 1 µm.
Andreas Müller, Martin Neukam, Anna Ivanova, Anke Sönmez, Carla Münster, Susanne Kretschmar, Yannis Kalaidzidis, Thomas Kurth, Jean-Marc Verbavatz & Michele Solimena. A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags. Scientific Reports 7, Article number: 23 (2017). doi:10.1038/s41598-017-00033-x