Remote focusing system for simultaneous dual-plane mesoscopic multiphoton imaging

We present a dual-plane mesoscopic imaging system capable of simultaneous image acquisition from two independent focal planes. The system was designed as an add-on to a recently introduced large field-of-view two-photon microscopy system, developed by Sofroniew, et al., eLife, 5, e14472, 2016. In this work, we merge two advanced multiphoton imaging technologies, i.e., temporal-division multiplexing and remote focusing, to maintain diffraction-limited resolution at both imaging planes, and achieve a more than 2-fold increase in the system9s overall imaging throughput. We introduce a novel solution to decode temporally interleaved analog signals at nanosecond timescales to achieve high-speed time-multiplexed imaging. Detailed characterization and comparison of the modified and the original two-photon microscopy system was performed.