Multiplexed Volumetric CLEM enabled by antibody derivatives provides new insights into the cytology of the mouse cerebellar cortex.

Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and serial electron microscopy on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (GFP, GFAP, calbindin, parvalbumin, Kv1.2, VGluT1, PSD-95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with linear unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure and superimposition of the different fluorescence channels. We document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of ion channels. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable a wide range of connectomic studies.