A three-photon head-mounted microscope for imaging all layers of visual cortex in freely moving mice

Recent advances in head-mounted microscopes have enabled imaging of neuronal activity using genetic tools in freely moving mice but these microscopes are restricted to recording in minimally lit arenas and imaging upper cortical layers. Here we built a 2-gram, three-photon excitation-based microscope, containing a z-drive that enabled access to all cortical layers while mice freely behaved in a fully lit environment. The microscope had onboard photon detectors, robust to environmental light, and the arena lighting was timed to the end of each line scan, enabling functional imaging of activity from cortical layer 4 and layer 6 neurons expressing jGCaMP7f in mice roaming a fully lit or dark arena. By comparing the neuronal activity measured from populations in these layers we show that activity in cortical layer-4 and layer-6 is differentially modulated by lit and dark conditions during free exploration.