Correlative STED super-resolution light and electron microscopy on resin sections

Abstract

Correlative light and electron microscopy approaches can reveal the localisation of specific proteins while providing detailed information on the cellular context, thereby combining the strengths of both imaging modalities. The major challenge in combining fluorescence microscopy with electron microscopy is the different sample preparation requirements necessary for obtaining high quality data from both modalities. To overcome this limitation, we combined conventional sample preparation protocols for electron microscopy with post-embedding labelling on ultra-thin sections using antibodies and other specific ligands. We successfully employed STED super-resolution microscopy to image the subcellular distribution of several targets in various specimen including E. coli, T. brucei, S. cerevisiae, human cancer cells and bovine sperm. Thus, we present widely applicable methods facilitating the use of antibodies for correlative super-resolution light and electron microscopy of postembedding labelled targets.

Recent Developments in Correlative Super-Resolution Fluorescence Microscopy and Electron Microscopy

“…They demonstrated this protocol for the imaging of age-defined granule morphology and degradation with high fluorescence contrast. Wurm et al (2019) employed post-embedding immunolabeling rather than pre-embedding immunolabeling to avoid the destructive effects of permeabilization and fluorescence quenching during the embedding process. This method led to a high signal-to-noise ratio of the fluorescence signal; however, the changes in antigenicity resulted in a low labeling density in STED images.…”

Correlative imaging for polymer science

“…7 Since then, superresolution correlative microscopy has been applied extensively to resolve the ultrastructure of cellular samples. Both single-molecule localization microscopies (SMLM) 8 and STED 9 have been correlated with EM for specific protein and cellular content localization.…”

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

“…Conventional chemical fixation, post-contrasting with osmium and dehydration prior to sample embedding in resin for transmission EM often results in partial extraction or denaturation of proteins, loss of fluorescence (in the case of fluorescent proteins) or leads to an inefficient post-embedding immunolabelling due to the destruction or limited accessibility of epitopes on the surface of resin sections. The last decade brought forward advances in sample preparation for CLEM, which demonstrated the feasibility of ultrastructural analysis by EM while maintaining the fluorescence of endogenous proteins with the possibility of super-resolution imaging 1 – 3 or protocols that enable post-embedding on-section fluorescence labelling 4 . There has been considerable progress in endogenous labelling strategies, with different groups successfully developing SMLM-compatible, fixation-resistant fluorescent proteins, such as frSkylan_S 5 or mEos4b 6 .…”