A novel rhodopsin-based voltage indicator for simultaneous two-photon optical recording with GCaMP in vivo.

Genetically encoded voltage indicators (GEVIs) allow optical recording of membrane potential from targeted cells . However, red GEVIs that are compatible with two-photon microscopy and that can be multiplexed with green reporters like GCaMP, are currently lacking. To address this gap, we explored diverse rhodopsin proteins as GEVIs and engineered a novel GEVI, 2Photron, based on a rhodopsin from the green algae . 2Photron, combined with two photon ultrafast local volume excitation (ULoVE), enabled multiplexed readout of spiking and subthreshold voltage simultaneously with GCaMP calcium signals in visual cortical neurons of awake, behaving mice. These recordings revealed the cell-specific relationship of spiking and subthreshold voltage dynamics with GCaMP responses, highlighting the challenges of extracting underlying spike trains from calcium imaging.