One for All, All for One: A Close Look at In-Resin Fluorescence Protocols for CLEM

Abstract

Sample preparation is the novel bottleneck for high throughput correlative light and electron microscopy (CLEM). Protocols suitable for both imaging methods must therefore balance the requirements of each technique. For fluorescence light microscopy, a structure of interest can be targeted using: 1) staining, which is often structure or tissue specific rather than protein specific, 2) dye-coupled proteins or antibodies, or 3) genetically encoded fluorescent proteins. Each of these three methods has its own advantages. For ultrastructural investigation by electron microscopy (EM) resin embedding remains a significant sample preparation approach, as it stabilizes the sample such that it withstands the vacuum conditions of the EM, and enables long-term storage. Traditionally, samples are treated with heavy metal salts prior to resin embedding, in order to increase imaging contrast for EM. This is particularly important for volume EM (vEM) techniques. Yet, commonly used contrasting agents (e.g., osmium tetroxide, uranyl acetate) tend to impair fluorescence. The discovery that fluorescence can be preserved in resin-embedded specimens after mild heavy metal staining was a game changer for CLEM. These so-called in-resin fluorescence protocols present a significant leap forward for CLEM approaches towards high precision localization of a fluorescent signal in (volume) EM data. Integrated microscopy approaches, combining LM and EM detection into a single instrument certainly require such an “all in one” sample preparation. Preserving, or adding, dedicated fluorescence prior to resin embedding requires a compromise, which often comes at the expense of EM imaging contrast and membrane visibility. Especially vEM can be strongly hampered by a lack of heavy metal contrasting. This review critically reflects upon the fundamental aspects of resin embedding with regard to 1) specimen fixation and the physics and chemistry underlying the preservation of protein structure with respect to fluorescence and antigenicity, 2) optimization of EM contrast for transmission or scanning EM, and 3) the choice of embedding resin. On this basis, various existing workflows employing in-resin fluorescence are described, highlighting their common features, discussing advantages and disadvantages of the respective approach, and finally concluding with promising future developments for in-resin CLEM.

The synaptic basis of activity-dependent eye-specific competition

“… 36 However, because the sample preparation method is similar to serial-section EM, there is future potential to integrate super-resolution light and EM to achieve dense connectomic reconstructions together with molecular-specific information at individual synaptic connections and cellular contacts. 96 , 97 Similar to EM, STORM images reveal static synaptic features. By comparing eye-specific synapses at different ages, we showed the association of specific subsynaptic properties (particularly vesicle organization) with the future outcome of eye-specific competition ( Figures 2 and 3 ).…”

Diversity of cells and signals in the cardiovascular system

“…Also, of note here are expansion microscopy methods (Wassie et al., 2019; Gallagher & Zhao, 2021) enabling subdiffraction resolutions to be attained using simple hardware and emerging techniques such as minFlux (Gwosch et al., 2020) and minSTED (Weber et al., 2021), which are achieving near‐atomic scale resolutions comparable to electron microscopy. The impressive power of these light microscopy techniques to reveal the molecular workings of cells is further enhanced by the addition of ultrastructural context through correlative light and electron microscopy (CLEM) techniques (Dillard et al., 2018; Mezache et al., 2019; Heiligenstein & Lucas, 2022; Jeong & Kim, 2022; van den Dries et al., 2022). A more detailed review of current super‐resolution methods is provided in the excellent recent review by Jacquemet et al.…”

Application of immuno- and affinity labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells

“…In addition, nonspecific labeling protocols for endogenous targets (nuclei, cell membrane, cytoskeleton, lipophilic domains, entire cells and tissues) have been reported for in-resin CLEM. However, these techniques are only suitable for a very limited number of targets and cannot be used to analyze specific endogenous proteins using in-resin CLEM [ 16 ]. Regarding in-resin CLEM for endogenous protein targets, in-resin correlative super-resolution fluorescent and electron microscopy of immunolabeled mitochondria and cytoskeleton in the epoxy resin-embedded samples has been challenged [ 17 ].…”