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

Head-mounted miniscopes have allowed for functional fluorescence imaging in freely moving animals. However, current capabilities of state-of-the-art technology can record only up to two, spectrally distinct fluorophores. This severely limits the number of cell types identifiable in a functional imaging experiment. Here we present a pipeline that enables the distinction of nine neuronal subtypes from regions defined by behaviorally relevant cells during in vivo GCaMP imaging. These subtypes are identified utilizing unique fluorophores that are co-expressed with GCaMP, unmixed by spectral imaging on a confocal microscope and co-registering these spectral fingerprints with functional data obtained on miniaturized microscopes. This method facilitates detailed analyses of circuit-level encoding of behavior.