Simultaneous two- and three-photon multiplane imaging across cortical layers in freely moving mice.

Head-mounted multiphoton microscopes enable imaging of activity from neuronal populations spread throughout the cortical layers in freely moving mice but so far have been restricted to recording from one cortical layer at a time. Here, combining two- and three-photon-based excitation delivered through multiple fibers, we built a head-mounted multiplane microscope enabling near-simultaneous imaging of neuronal activity from five vertically separated planes, spread across multiple cortical layers. Both excitation pathways had remote focusing mechanisms for fine axial adjustments, enabling activity recordings from the same neuronal populations over weeks in freely behaving mice. The lightweight microscope utilized an onboard, two-channel detection system designed to enable activity recordings from neuronal populations spread across visual-cortex layers in both lit and dark conditions as well as imaging activity across posterior parietal cortex layers during complex gap-crossing behaviors. We show that during gap-crossing tasks, layer 5 and 2/3 neuronal subpopulations in the posterior parietal cortex have differential pattern sequences during free decision-making.