Abstract text (incl. figure legends and references)
Introduction

In-resin correlative light and electron microscopy (CLEM) facilitates a high-precision registration of the fluorescence signal upon the EM image. Current approaches most often employ acrylic resins, such as Lowicryl HM20, for preserving fluorescence while compromising the ultrastructural preservation (Buerger et al., 2021). Epoxy resins, such as EPON 812, on the other hand are the optimal choice for preserving ultrastructure. However, embedding samples in epoxy resins leads to a reduction in antigenicity and fluorescence. Nevertheless, it was shown that on-section CLEM following a conventional sample preparation including chemical fixation, post-fixation with osmium tetroxide, dehydration at room temperature and embedding in epoxy resins is possible with partly specifically adapted fluorescent proteins or protein tags labelled with certain dyes (Müller et al., 2017; Peng et al., 2022).

We set out to further test the broad applicability of different fluorescent dyes in a conventional in-resin CLEM workflow with embedding in EPON. We plan to demonstrate the robustness of this by application to different cellular organelles, which allows us to achieve a high-precision registration with minimal effort in terms of sample preparation.

Cell lines and cell culture

HeLa cells expressing the HaloTag fused to various markers were labelled with different fluorescent ligands, imaged by confocal laser scanning microscopy (cLSM), if needed, and further processed for EM.

Sample preparation for on-section CLEM

Cells were fixed with 2.5 % glutaraldehyde and post-fixed for 30 min using 1 % OsO4 and 1.5 % K4[Fe(CN6)] on ice. Samples were dehydrated and embedded in EPON. After polymerization, 250 nm thin sections were cut, collected on mesh grids and imaged at a cLSM.

The fluorescence of mitochondria, Golgi apparatus and Salmonella effector proteins labelled with TMR or JFX554 is preserved after conventional sample preparation and embedding in EPON.

The in-resin CLEM workflow devised in this study offers a very promising tool for cases in which Lowicryl embedding cannot or does not need to be used, but a registration of the LM to the EM image with high accuracy is desirable. Live-cell imaging and direct fixation on the LM stage can be easily integrated.

References

Buerger, K., Schmidt, K.N., Fokkema, J., Gerritsen, H.C., Maier, O., de Vries, U., Zaytseva, Y., Rachel, R., and Witzgall, R. (2021). Chapter 8 - On-section correlative light and electron microscopy of large cellular volumes using STEM tomography. In Methods Cell Biol, T. Müller-Reichert, and P. Verkade, eds. (Academic Press), pp. 171-203.

Müller, A., Neukam, M., Ivanova, A., Sönmez, A., Münster, C., Kretschmar, S., Kalaidzidis, Y., Kurth, T., Verbavatz, J.-M., and Solimena, M. (2017). A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags. Scientific Reports 7, 23.

Peng, D., Li, N., He, W., Drasbek, K.R., Xu, T., Zhang, M., and Xu, P. (2022). Improved Fluorescent Proteins for Dual-Colour Post-Embedding CLEM. Cells 11, 1077.

Figure Legends

Fig. 1: Conventional in-resin CLEM with EPON embedding of mitochondria labelled with fluorescent dyes. Fluorescence of Tom20-HaloTag stained with JaneliaFluorX 554 (A-C) or TMR (D-F) is preserved after conventional embedding in EPON. Scale bars: 2 µm.