Large-scale two-photon calcium imaging in freely moving mice

We developed a miniaturized two-photon microscope (MINI2P) for fast, high-resolution, multiplane calcium imaging of over 1,000 neurons at a time in freely moving mice. With a microscope weight below 3g and a highly flexible connection cable, MINI2P allowed imaging to proceed with no impediment of behavior in half-hour free-foraging trials compared to untethered, unimplanted animals. The improved cell yield was achieved through a new optical system design featuring an enlarged field of view (FOV) and a new micro-tunable lens with increased z -scanning range and speed that allowed for fast and stable imaging of multiple, interleaved planes as well as 3D functional imaging. A novel technique for successive imaging across multiple, adjacent FOVs enabled recordings from more than 10,000 neurons in the same animal. Large-scale proof-of-principle data were obtained from cell populations in visual cortex, medial entorhinal cortex, and hippocampus, revealing spatial tuning of cells in all areas, including visual cortex. Highlights We developed a lightweight 2-photon miniscope for imaging in freely-foraging mice Activity can be monitored in volumes of over 1,000 visual or entorhinal-cortex cells A new z -scanning module allows fast imaging across multiple interleaved planes Successive imaging from adjacent regions enables imaging from more than 10,000 cells ### Competing Interest Statement The authors have declared no competing interest.