High-throughput deep tissue two-photon microscopy at kilohertz frame rates

High-speed laser scanning microscopes are essential for monitoring fast biological phenomena. However, existing strategies that achieve millisecond time resolution with two-photon microscopes (2PMs) are generally technically challenging and suffer from compromises among imaging field of view, excitation efficiency, and depth penetration in thick tissue. Here, we present a versatile solution that enables a conventional video-rate 2PM to perform 2D scanning at kilohertz frame rates over large fields of view. Our system is based on implementation of a scan multiplier unit that provides inertia-free multiplication of the scanning speed while preserving all the benefits of standard 2PM. We demonstrate kilohertz subcellular-resolution 2PM imaging with an order of magnitude higher imaging throughput than previously achievable and penetration depths exceeding 500 μm, which we apply to the study of neurovascular coupling dynamics in the mouse brain.