Large-Scale, Multiplexed Two-Photon Imaging of Neural Activity In Vivo
Yu CH, Stirman JN, Yu Y, Hira R, Smith SL. Diesel2p mesoscope with dual independent scan engines for flexible capture of dynamics in distributed neural circuitry. Nat Commun 2021;12(1):6639. doi: 10.1038/s41467-021-26736-4.
Background: Imaging neural activity across multiple brain regions with high spatial and temporal resolution is limited by the trade-off between field-of-view and optical performance in conventional two-photon microscopy. Standard systems achieve subcellular resolution only over small areas, preventing simultaneous observation of widely distributed cortical networks. Therefore, a system capable of large-scale, high-speed and flexible imaging of neuronal populations is needed to study distributed neural dynamics.
Hypothesis: This study hypothesized that a two-photon mesoscope incorporating dual, independently controlled scan engines could achieve simultaneous, high-resolution imaging across widely separated brain regions, enabling flexible and efficient capture of neural dynamics in vivo.
Methods: The authors developed and characterized a custom-built two-photon mesoscope equipped with two independent resonant–galvo–galvo scan engines, each coupled to a deformable mirror for adaptive optics and z-focusing. Imaging and data acquisition were controlled using ScanImage. Neuronal calcium activity in transgenic mice expressing GCaMP6s was recorded through a 5-mm cranial window. Neurons were segmented and fluorescence analyzed using Suite2p software, followed by spike inference and correlation analyses.
Results: The system provided subcellular resolution (~1 µm lateral, ~8 µm axial) across a 25 mm2 field with minimal aberration. In vivo imaging resolved dendritic spines and captured calcium dynamics from 5,874 neurons over 15 mm2 with high signal-to-noise ratios. Dual scan engines enabled simultaneous imaging of cortical regions up to 4.4 mm apart with independently set parameters and minimal crosstalk, demonstrating flexible, multiplexed operation.
Conclusions: This study concluded that the Diesel2p mesoscope achieves diffraction-limited, large-scale and independently configurable two-photon imaging across distributed brain regions, enabling new experimental designs for studying neural circuit dynamics in vivo.
