A robotic multi-armed two-photon microscope for imaging neural interactions across multiple brain areas

摘要 BRAIN Initiative最重要的成就之一是对许多新的神经元类型进行了遗传鉴定，并创建了获得这些细胞类型的遗传工具。然而，揭示这些神经元类型的功能作用以及它们如何在大脑区域之间合作以产生哺乳动物行为仍然是一个突出的挑战。因此，如果我们要理解全球大脑动力学，发明方法来监测大量基因识别的神经元如何在大脑的多个区域相互作用至关重要。今天，电记录方法可以跟踪多个区域的神经活动，但不能轻易针对特定类型的神经元。宽视野和双光子介观镜可以在皮层组织的毫米级区域上对识别出的神经元类型的动态进行成像，但无法访问包括大脑感觉、认知或运动回路的主要节点的皮层和皮层下区域的分布式集合。 为了打破这一僵局，我们发明了“Octopus”，这是一种带有多个铰接光学臂的机器人成像系统，每个光学臂都是一个双光子显微镜，可以灵活地定位在大脑周围，以同时记录头部受限行为啮齿动物或灵长类动物的多个浅表或深部区域的神经活动。我们设计，建造和测试了Octopus的初始版本，它有4个手臂，每个手臂都有5个机械自由度，尖端有一个用于双光子成像的微型光学探针。手臂的设计是基于外科机器人技术的想法，并使用远程运动中心运动学来提供一个多功能的机器人手臂运动。使用这个系统，视觉神经学家可以同时对外侧膝状体核、视觉皮层、上级丘和枕中的神经活动进行成像，运动神经生理学家可以对运动皮层、基底神经节、小脑和运动丘脑中的活动进行成像。 在这项计划中，我们会改善每个八达通臂的光学和机械设计，并准备透过开放源码和商业途径广泛披索该系统。每个手臂将获得最先进的双光子显微镜的光学功能，用于成像大规模神经系综活动。具体来说，每个臂将结合光遗传学，并允许在800微米宽的视场内进行双色双光子成像。这些能力将允许神经科学家监测4个大脑区域中每个区域的两种遗传识别的神经元类型，用光遗传学扰乱这些细胞的动力学，并观察这些操作对其他3个区域中动物行为和活动的影响。我们亦会精简机械设计，以简化八达通的初步组装程序，让新用户使用八达通，并使机械手更灵活。新的设计也将是电动的，并将为用户提供高度直观的精确操纵机器人手臂的手段。最后，为了迭代地提高Octopus的性能和可用性，并验证其作为突破性新技术的传播准备，我们将与7个beta测试实验室密切合作，在清醒行为小鼠和绒猴中实施多区域神经成像研究。

Abstract Among the BRAIN Initiative’s most important achievements are the genetic identification of many new neurons- types and the creation of genetic tools to access these cell types. However, uncovering the functional roles of these neuron types and how they cooperate across brain areas to generate mammalian behavior remains an outstanding challenge. Thus, inventing ways to monitor how large populations of genetically identified neurons interact across multiple regions of the brain is crucial if we are to comprehend global brain dynamics. Today, electrical recording methods can track neural activity across multiple areas but cannot easily target neurons of specific types. Widefield and two-photon mesoscopes can image the dynamics of identified neuron-types across millimeter-scale regions of cortical tissue but cannot access the distributed sets of cortical and subcortical regions that comprise the major nodes of the brain’s sensory, cognitive, or motor circuits. To clear this impasse, we invented the ‘Octopus’, a robotic imaging system with multiple articulated optical arms, each a two-photon microscope, that can be flexibly positioned around the brain to record neural activity concurrently in multiple superficial or deep areas of a head-restrained behaving rodent or primate. We designed, built, and tested an initial version of the Octopus with 4 arms, each of which has 5 mechanical degrees of freedom and a micro-optic probe at its tip for two-photon imaging. The design of the arms is based on ideas from surgical robotics and uses remote center-of-motion kinematics to provide a versatile repertoire of robotic arm movements. Using this system, a visual neuroscientist can concurrently image neural activity in the lateral geniculate nucleus, visual cortex, superior colliculus, and pulvinar, and a motor neurophysiologist can image activity in the motor cortex, basal ganglia, cerebellum, and motor thalamus. In this project, we will enhance the optical and mechanical design of each Octopus arm and prepare the system for wide dissemination through open-source and commercial routes. Each arm will gain the optical functionality of a state-of-the-art, two-photon microscope for imaging large-scale neural ensemble activity. Specifically, each arm will incorporate optogenetics and allow dual-color two-photon imaging over an 800-µm- wide field of view. These capabilities will allow neuroscientists to monitor two genetically identified neuron- types in each of 4 brain areas, to perturb the dynamics of these cells with optogenetics, and to observe the effects of these manipulations on animal behavior and activity in the other 3 areas. We will also streamline the mechanical design to simplify the initial assembly of the Octopus for new users and to endow the robot arms with additional dexterity. The new design will also be motorized and will provide users with highly intuitive means of precisely steering the robot arms. Finally, to iteratively improve the performance and usability of the Octopus and to validate its readiness for dissemination as a groundbreaking new technology, we will work closely with 7 beta-tester labs to implement multi-area neural imaging studies in awake behaving mice and marmosets.