Controlled neuronal firing in vivo using two photon spatially shaped optogenetics

这是一个响应RFA-NS-17-00 4“Brain Initiative：优化变革性的 神经系统的大规模记录和调制技术(U01)“。在概念验证中 我们展示了一种新型的光纤耦合微型显微镜，它实现了三维共聚焦成像 使用电润湿透镜技术进行轴向聚焦的脑组织荧光标记神经元。这么低 重量仪(&lt；2g)适用于外科植入，实现全光清醒行为记录 由于轴向聚焦深入小鼠大脑，与商用设备相比，这是一个重大进步。 在这份提案中，我们展示了一项重大突破，展示了通过光纤耦合的成功成像 显微镜使用双光子显微镜。利用这一结果，我们建议优化我们的证明 使其兼容双光子激发的概念E-FCM (2pE-FCM)，适用于记录和调节清醒行为小鼠的神经活动。我们的2PE-FCM将 将全息双光子光刺激与空间光调制器(SLM)相结合实现快速3D 体积双光子成像和神经元活动的调节。2PE-FCM的优化将是 通过测试版用户与多学科的物理学家、工程师和 神经学家正在同时设计、实施和测试该设备。2PE-FCM的检测 将采用一种循序渐进的方法，旨在有效地产生适合于研究当地的设备 不同物种不同脑区的回路。我们提出了一个坚实的传播计划，与 智能成像创新公司将该设备商业化。2PE-FCM的验证将是一个主要的 神经回路全光询问的进展为理解神经基础创造了一个新的场所 关于行为的。 目的1.技术途径：光遗传激活和同时读出深度 新型变焦电润湿镜光纤耦合显微镜在脑神经元中的应用 不使用活动部件。 目标2.里程碑和时间表：验证和传播2PE-FCM设备 对自由行为动物行为的神经基础的研究。

This is an application in response to RFA-NS-17-00 4 “BRAIN Initiative: Optimization of Transformative Technologies for Large Scale Recording and Modulation in the Nervous System (U01)”. In a proof-of-concept publication we demonstrated a novel fiber-coupled miniature microscope that achieved 3D confocal imaging of fluorescently labeled neurons in brain tissue using electrowetting lens technology for axial focusing. This low weight instrument (<2 g) is suitable for surgical implantation to achieve all optical awake behaving recording with axial focusing deep in the mouse brain, a significant advance compared to commercially available devices. In this proposal we present a major breakthrough demonstrating successful imaging through the fiber coupled microscope using two-photon microscopy. Leveraging on this result we propose to optimize our proof of concept E-FCM (electrowetting-fiber coupled microscope) by making it compatible for two-photon excitation (2PE-FCM) suitable for recording and modulating neural activity in awake behaving mice. Our 2PE-FCM will incorporate holographic two-photon opsin stimulation with a spatial light modulator (SLM) for rapid 3D volumetric two-photon imaging and modulation of neuronal activity. Optimization of the 2PE-FCM will be achieved by regular interactions of beta users with a multidisciplinary group of physicists, engineers and neuroscientists in a parallel process of design implementation and testing the device. Testing of the 2PE-FCM will employ a step-by-step approach designed for effective generation of a device suitable for studying local circuits in different brain areas in different species. We propose a solid dissemination plan partnering with Intelligent Imaging Innovations Inc. to commercialize the device. Validation of the 2PE-FCM will be a major advance in all-optical interrogation of neural circuits creating a new venue for understanding the neural basis of behavior. Aim 1. TECHNICAL APPROACH: Optogenetic activation and simultaneous readout of deep brain neurons using a novel fiber coupled microscope with a variable focus electrowetting lens using no moving parts. Aim 2. MILESTONES AND TIMELINE: Validate and disseminate the 2PE-FCM device for studies of the neural basis of behavior in freely behaving animals.