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

Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between LM and EM modalities. Post-embedding CLEM enables precise registration of structures directly on EM sections, but requires fluorescent markers withstanding EM sample preparation, especially osmium tetroxide fixation, dehydration and EPON embedding. Most fluorescent proteins (FPs) lose their fluorescence during such conventional embedding (CE), but synthetic dyes represent promising alternatives as their stability exceeds those of FP. We analyzed various Janelia Fluor dyes and TMR conjugated to ligands for self-labeling enzymes, such as HaloTag, for fluorescence preservation after CE. We show that TMR, JF525, JF549, JFX549 and JFX554 retain fluorescence, with JFX549 and JFX554 yielding best results overall, also allowing integration of high-pressure freezing and freeze substitution. Furthermore, we found the recently published FP StayGold to resist CE, facilitating dual-fluorescence in-resin CLEM.
Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The declaration of competing interests that you upload should be in a standard and editable format. Please select the suitable option and upload it with this submission. You can download the standard declaration of competing interests form from the following link. https://declarations.elsevier.com/ Please note that all manuscripts submitted to Heliyon are checked for originality using the Crosscheck database (For more information on Crosscheck visit their website at http://www.crossref.org/crosscheck.html). We have studied your work carefully and have come to the conclusion that the textual overlap between your manuscript and previously published articles goes beyond the normal occurrence of standard phrases in your field. The largest overlap is with the following articles: < https://doi.org/10.1101/2023.11.04.565612> You can also find the largest sources of overlap in the attached iThenticate report. Please rewrite your manuscript to eliminate textual overlap with previously published literature before resubmitting. Please be aware that you will have one opportunity to address and reduce the overlap in your manuscript to an acceptable level. If you do not do this we may be forced to reconsider our decision for your submission and issue a reject decision. Please reference all numbered tables in text. Currently, numbered table [4] in the manuscript have not been cited in text. Notes to each check: Plagiarism: Not Okay. 71% Overall; 65% < https://doi.org/10.1101/2023.11.04.565612>
(© 2024 The Authors.)