Abstract
In vivo calcium imaging at multiple depths simultaneously is shown using multifocal two-photon microscopy and spatiotemporal multiplexing. This technique involves scanning the sample with multiple beams in parallel at different axial planes and is applied to monitor neuronal network activity in multiple cortical layers of an anesthetized mouse. In vivo two-photon calcium imaging would benefit from the use of multiple excitation beams to increase scanning speed, signal-to-noise ratio and field of view or to image different axial planes simultaneously. Using spatiotemporal multiplexing we circumvented light-scattering ambiguity inherent to deep-tissue multifocal two-photon microscopy. We demonstrate calcium imaging at multiple axial planes in the intact mouse brain to monitor network activity of ensembles of cortical neurons in three spatial dimensions.

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