Retroreflective beam multiplexing enables high-throughput two-photon voltage imaging in vivo

Voltage imaging demands technical innovations to realize its full potential in neuroscience. Although two-photon microscopy is widely used in brain research, its imaging speed is insufficient for high-throughput voltage recordings. To overcome this limitation, we developed an all-optical beam multiplexing engine via retroreflection from two near-parallel mirrors. This approach integrates seamlessly with standard two-photon microscopes, enabling kilohertz-frame-rate imaging across a 16-fold expanded field of view while maintaining synaptic resolution. Operating at over 220 megapixels per second, it enables submillisecond imaging of dendritic propagation of action potentials and simultaneous voltage recording from more than 200 hippocampal neurons in head-fixed, behaving mice. This advancement unlocks new possibilities for large-scale, high-speed functional studies in neuroscience.