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Planar neural networks and interfaces serve as versatile in vitro models of central nervous system physiology, but adaptations of related methods to three dimensions (3D) have met with limited success. Here, we demonstrate for the first time volumetric functional imaging in a bioengineered neural tissue growing in a transparent hydrogel with cortical cellular and synaptic densities, by introducing complementary new developments in nonlinear microscopy and neural tissue engineering. Our system uses a novel hybrid multiphoton microscope design combining a 3D scanning-line temporal-focusing subsystem and a conventional laser-scanning multiphoton microscope to provide functional and structural volumetric imaging capabilities: dense microscopic 3D sampling at tens of volumes per second of structures with mm-scale dimensions containing a network of over 1,000 developing cells with complex spontaneous activity patterns. These developments open new opportunities for large-scale neuronal interfacing and for applications of 3D engineered networks ranging from basic neuroscience to the screening of neuroactive substances.

Hybrid multiphoton volumetric functional imaging of large-scale bioengineered neuronal networks