1. Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex.
- Author
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Han, Xiaomeng, Lu, Xiaotang, Li, Peter H., Wang, Shuohong, Schalek, Richard, Meirovitch, Yaron, Lin, Zudi, Adhinarta, Jason, Murray, Karl D., MacNiven, Leah M., Berger, Daniel R., Wu, Yuelong, Fang, Tao, Meral, Elif Sevde, Asraf, Shadnan, Ploegh, Hidde, Pfister, Hanspeter, Wei, Donglai, Jain, Viren, and Trimmer, James S.
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NEURAL circuitry ,CEREBELLAR cortex ,ELECTRON microscopy ,MICROSCOPY ,FLUORESCENT probes - 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. Volume EM provides connectomic data but lacks necessary molecular information. Here, authors show fluorescent scFvs enable multiplexed immunolabeling and correlated light and electron microscopy, suggesting potential for molecularly annotated connectomic data. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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