In vivo imaging of large-scale neuronal activity plays a pivotal role in unraveling the function of the brain’s circuitry.Multiphoton microscopy,a powerful tool for deep-tissue imaging,has received sustained interest...In vivo imaging of large-scale neuronal activity plays a pivotal role in unraveling the function of the brain’s circuitry.Multiphoton microscopy,a powerful tool for deep-tissue imaging,has received sustained interest in advancing its speed,field of view and imaging depth.However,to avoid thermal damage in scattering biological tissue,field of view decreases exponentially as imaging depth increases.We present a suite of innovations to optimize three-photon microscopy for large field-of-view imaging at depths unreachable by two-photon microscopy.These techniques enable us to image neuronal activities of transgenic animals expressing protein calcium sensors in a~3.5-mm diameter field-of-view with single-cell resolution in the deepest cortical layer of mouse brains.We further demonstrate simultaneous large field-of-view two-photon and three-photon imaging,subcortical imaging in the mouse brain,and whole-brain imaging in adult zebrafish.The demonstrated techniques can be integrated into typical multiphoton microscopes to enlarge field of view for system-level neural circuit research.展开更多
基金National Science Foundation NeuroNex(Grant No.DBI-1707312 to C.X.).NIH/NINDS(Grant No.U01NS103516 to C.X.).Cornell Neurotech Mong Fellowship to A.M.
文摘In vivo imaging of large-scale neuronal activity plays a pivotal role in unraveling the function of the brain’s circuitry.Multiphoton microscopy,a powerful tool for deep-tissue imaging,has received sustained interest in advancing its speed,field of view and imaging depth.However,to avoid thermal damage in scattering biological tissue,field of view decreases exponentially as imaging depth increases.We present a suite of innovations to optimize three-photon microscopy for large field-of-view imaging at depths unreachable by two-photon microscopy.These techniques enable us to image neuronal activities of transgenic animals expressing protein calcium sensors in a~3.5-mm diameter field-of-view with single-cell resolution in the deepest cortical layer of mouse brains.We further demonstrate simultaneous large field-of-view two-photon and three-photon imaging,subcortical imaging in the mouse brain,and whole-brain imaging in adult zebrafish.The demonstrated techniques can be integrated into typical multiphoton microscopes to enlarge field of view for system-level neural circuit research.
