Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex
Mapping neuronal networks is a central focus in neuroscience. While volume electron microscopy (vEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide molecular information to identify cell types or functions. We developed an approach that uses fluorescent single-chain variable fragments (scFvs) to perform multiplexed detergent-free immunolabeling and volumetric-correlated-light-and-electron-microscopy on the same sample. We generated eight fluorescent scFvs targeting brain markers. Six fluorescent probes were imaged in the cerebellum of a female mouse, using confocal microscopy with spectral unmixing, followed by vEM of the same sample. The results provide excellent ultrastructure superimposed with multiple fluorescence channels. Using this approach, we documented a poorly described cell type, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.
Authors
- Xiaotang Lu (ORCID: https://orcid.org/0000-0002-8575-5394)
- Karl D. Murray (ORCID: https://orcid.org/0000-0001-8159-6843)
- Shuohong Wang (ORCID: https://orcid.org/0000-0001-5186-4157)
- Daniel R. Berger (ORCID: https://orcid.org/0000-0002-9677-6932)
- Zudi Lin (ORCID: https://orcid.org/0000-0003-4176-8035)
- Yaron Meirovitch (ORCID: https://orcid.org/0000-0002-1946-8012)
- Viren Jain (ORCID: https://orcid.org/0000-0003-1488-3505)
- Peter H. Li (ORCID: https://orcid.org/0000-0001-6193-4454)
- Richard Schalek (ORCID: https://orcid.org/0000-0003-2450-1718)
- Jeff William Lichtman (ORCID: https://orcid.org/0000-0002-0208-3212)
- Hanspeter Pfister (ORCID: https://orcid.org/0000-0002-3620-2582)
- James S. Trimmer
- Yuelong Wu (ORCID: https://orcid.org/0000-0003-0075-1237)
- Jason Ken Adhinarta (ORCID: https://orcid.org/0000-0002-6247-7475)
- Leah M. MacNiven
- Hidde L. Ploegh (ORCID: https://orcid.org/0000-0002-1090-6071)
- Donglai Wei (ORCID: https://orcid.org/0000-0002-2329-5484)
- Xiaomeng Han (ORCID: https://orcid.org/0000-0002-1409-7419)
- Elif Sevde Meral (ORCID: https://orcid.org/0009-0008-3336-3293)
- Shadnan Asraf
- Tao Fang (ORCID: https://orcid.org/0000-0001-8129-8635)
Institutions
- Boston College (US)
- Boston Children's Hospital (US)
- Google (United States) (US)
- Harvard University (US)
- University of Massachusetts Amherst (US)
- Harvard University Press (US)
- Bezmiâlem Vakıf Üniversitesi (TR)
- University of California, Davis (US)
Publication Details
- Journal
- Nature Communications
- Published
- 2024-08-05
- DOI
- https://doi.org/10.1038/s41467-024-50411-z
- Citations
- 8
- Primary Topic
- Advanced Electron Microscopy Techniques and Applications
- Type
- article
- Field-Weighted Citation Impact
- 3.65
Funders
- National Science Foundation
- Harvard University
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology
- National Institutes of Health
- Office of Naval Research