Controlled neuronal firing in vivo using two photon spatially shaped optogenetics

这是响应RFA-NS-17-00 4“BRAIN倡议： 神经系统大规模记录和调制技术（U 01）。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，实现了3D共焦成像， 使用用于轴向聚焦的电润湿透镜技术在脑组织中荧光标记的神经元。这种低 重量仪器（<2g）适合于外科植入以实现全光学清醒行为记录 轴向聚焦在小鼠大脑深处，与商业上可用的设备相比，这是一个重大的进步。 在这项提案中，我们提出了一个重大突破，证明通过光纤耦合成功成像 双光子显微镜（Two Photon Microscopy）利用这一结果，我们建议优化我们的证明， 概念E-FCM（电润湿-光纤耦合显微镜），使其兼容双光子激发 （2 PE-FCM）适合于记录和调节清醒行为小鼠中的神经活动。我们的2 PE-FCM将 结合全息双光子视蛋白刺激与空间光调制器（SLM）用于快速3D 体积双光子成像和神经元活动的调制。2 PE-FCM的优化将是 通过beta用户与物理学家，工程师和 神经科学家在设计实施和测试设备的并行过程中。2 PE-FCM的测试 将采用一步一步的方法，旨在有效地产生一个适合研究当地的设备， 不同物种大脑不同区域的神经回路。我们提出了一个坚实的传播计划， 智能成像创新公司将该设备商业化。2 PE-FCM的验证将是 神经回路的全光学询问的进展为理解神经基础创造了新的场所 的行为。 目标1。技术方法：光遗传激活和同时读出深度 使用具有可变焦点电润湿透镜的新型纤维耦合显微镜的脑神经元 不使用移动部件。 目标二。里程碑和时间轴：宣传和推广2 PE-FCM设备， 对行为自由的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.