Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex

Xiaomeng Han (), Xiaotang Lu (), Peter H. Li, Shuohong Wang, Richard Schalek, Yaron Meirovitch, Zudi Lin, Jason Adhinarta, Karl D. Murray, Leah M. MacNiven, Daniel R. Berger, Yuelong Wu, Tao Fang, Elif Sevde Meral, Shadnan Asraf, Hidde Ploegh, Hanspeter Pfister, Donglai Wei, Viren Jain, James S. Trimmer and Jeff W. Lichtman ()
Additional contact information
Xiaomeng Han: Harvard University
Xiaotang Lu: Harvard University
Peter H. Li: Google Research
Shuohong Wang: Harvard University
Richard Schalek: Harvard University
Yaron Meirovitch: Harvard University
Zudi Lin: Harvard University
Jason Adhinarta: Boston College
Karl D. Murray: University of California Davis School of Medicine
Leah M. MacNiven: University of California Davis School of Medicine
Daniel R. Berger: Harvard University
Yuelong Wu: Harvard University
Tao Fang: Boston Children’s Hospital
Elif Sevde Meral: Bezmialem Vakif University School of Medicine
Shadnan Asraf: University of Massachusetts Amherst
Hidde Ploegh: Boston Children’s Hospital
Hanspeter Pfister: University of California Davis School of Medicine
Donglai Wei: Boston College
Viren Jain: Google Research
James S. Trimmer: University of California Davis School of Medicine
Jeff W. Lichtman: Harvard University

Nature Communications, 2024, vol. 15, issue 1, 1-18

Abstract: Abstract 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.

Date: 2024
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DOI: 10.1038/s41467-024-50411-z

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