CoVET: Cryo-Electron Tomography guided by optical electrophysiology

Neurons exhibit varying electrophysiological properties due to dynamic changes in spatiotemporal molecular networks. In situ cryo-electron tomography (cryo-ET) provides unique advantages for high-resolution visualization of macromolecular complexes within their cellular context. Although correlation with fluorescent labeling allows cryo-ET to target specific cellular regions, it does not adequately reflect the electrophysiological properties of heterogeneous neurons. To bridge high-resolution molecular imaging with electrophysiological properties of individual neurons, we developed a Correlative-Voltage Imaging and cryo-ET (CoVET) technique. The nondestructive nature of voltage imaging is compatible with cryo-ET, enabling a direct correlation between neuronal electrophysiology and molecular structures. Neurons were clustered based on their electrophysiological properties, allowing for single-cell-guided structural analysis using cryo-ET. We analyzed the translational landscapes of individual neurons and found distinct translational profiles and contextual information among ribosomes from different electrophysiological clusters. Our results highlight the importance of the correlation between the electrophysiological properties and molecular structures.