Rapid in-EPON CLEM for everyone: Combining fast and efficient labeling of self-labeling enzyme tags with EM resistant Janelia Fluor dyes

Correlative light and electron microscopy (CLEM) allows to link light microscopy (LM) of living cells to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between the LM and EM modalities due to chemical and physical distortions. Post-embedding CLEM enables precise registration of fluorescent structures directly on thin resin sections. However, in-resin CLEM techniques require fluorescent markers withstanding EM sample preparation. Most fluorescent proteins lose their fluorescence during EM sample preparation. Synthetic dyes present an alternative as their photostability and brightness exceed those of fluorescent proteins. Together with self-labeling enzymes (SLE) as protein tags, these fluorophores can be used to precisely label cellular structures of interest. By applying SLE labelling for post-embedding CLEM, we compared Janelia Fluor dyes and TMR to identify most suitable fluorophores. Epithelial cells expressing HaloTag fusion proteins were stained with various ligand-conjugated dyes, and fluorescence preservation was quantified after conventional room temperature sample preparation with embedding in EPON. The results obtained show that only the red dyes TMR, JF549, JFX549 and JFX554 retain their fluorescence in resin, with JFX549 and JFX554 yielding best signal intensity and signal-to-background ratio during in-resin super-resolution microscopy. Since all red dyes possess an oxygen atom within their xanthene structure, our results indicate that this might be a crucial feature making them more tolerant to sample preparation for electron microscopy. Our work reports a rapid in-resin CLEM approach that combines fast and efficient labeling of SLE tags with EM-compatible fluorophores, and serve as benchmarks for experimental planning and future engineering of fluorophores for CLEM.