Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

Head-mounted miniscopes have enabled functional fluorescence imaging in freely moving animals. However, current technology is limited to recording at most two spectrally distinct fluorophores, severely restricting the number of identifiable cell types. Here, we introduce multiplexed neuronal imaging (Neuroplex), a pipeline combining miniscope Ca²⁺ recordings with in vivo multiplexed confocal spectral imaging to distinguish nine projection-defined neuronal subtypes through the same GRIN lens. By co-registering defined neurons with fluorophore-specific spectral fingerprints via linear unmixing, we link projection-defined identities to behaviorally relevant neuronal activity. This approach overcomes spectral constraints of miniscopes, enabling circuit-level dissection of behavior in single animals.