High-throughput two-photon volumetric brain imaging in freely moving mice

Imaging neural activities across large volumes at subcellular resolution without impeding animal behaviors remains difficult. Here, we develop a high-throughput miniature Bessel-beam two-photon microscope (miniBB2p) capable of imaging calcium dynamics from neurons and dendrites over a volume of 420 × 420 × 80 μm³ during free behavior. We share a full description of miniBB2p and demonstrate its ability to perform large-scale recordings of the calcium activities of more than 1000 neurons at a time in anterior cingulate cortex and secondary motor cortex of freely moving mice in various behavioral paradigms. Our results position miniBB2p to advance the design of miniature multiphoton microscopes, facilitating precise monitoring of neural activities across large populations within a three-dimensional volume in freely moving animals. Imaging neural activity across large volumes at subcellular resolution in freely behaving animals remains challenging. Here, a miniature Bessel-beam two-photon microscope enables volumetric calcium imaging of 1,000+ neurons in freely moving mice.