High-speed two-photon calcium imaging of neuronal population activity using acousto-optic deflectors.

Two-photon calcium imaging of neuronal populations allows optical measurements of spiking activity in living animals. However, laser-scanning microscopes with galvanometric scan mirrors are too slow to capture population activity on a millisecond timescale. This protocol describes a two-photon microscope that is based on two-dimensional laser scanning with acousto-optic deflectors (AODs), enabling high-speed in vivo recording of neuronal population activity at temporal resolutions of several hundred hertz. The detailed construction plan of the AOD-based microscope is accompanied by equally detailed optimization procedures. We also introduce a novel random-access pattern scanning (RAPS) technique for high-speed in vivo measurements of neuronal population activity. AOD-based RAPS can measure calcium transients in neocortical neuronal populations, revealing spike trains with near-millisecond precision. The current limitations of the AOD-based microscope are discussed, and we provide an outlook of its future applications.