Fig. 2.

Higher-speed two-photon microscopy. Optical schematic (a) and scanning patterns (b) for 3D random-access scanning. The system allows conventional raster scanning for structural imaging, discrete point sampling for multiunit recording, and pattern mapping for functional imaging. In the high-speed 3D random-access mode, 2000 points can be scanned in 40 ms, enabling volumetric calcium imaging in hundreds of neurons in vivo. Compensating elements must be used because acousto-optical devices generate high spatial and temporal dispersion. Optical schematic (c) and focusing configurations (d) possible in a spatially- and temporally-multiplexed two-photon system. Ultrafast laser pulses are emitted every 12 ns and divided into four beams with 3 ns relative delay that are simultaneously focused at different positions within a sample. Different imaging configurations include a scan of four imaging planes, a single plane scan with four beams, or a scan of two imaging planes with two beams each. When used with a resonant scanning mirror, fast (250 Hz/plane at 500×500 pixels) calcium imaging in four axial planes is possible.