3D Scanning Two-photon Fiberscope Technology for Simultaneous Multi-region Multi-cell-type Imaging in Freely-moving Rodents

项目总结 大脑活动涉及神经元产生快速传播的信号，以编码和传递信息 动态神经网络。神经学家渴望在不受限制的动物身上获得这样的网络 具有高时空分辨率的模型(例如啮齿动物)，这将阐明基本工作 大脑的机制。光学成像，特别是多光子显微镜，在 这一努力。在过去的十年里，已经见证了令人印象深刻的进步，从头戴式台式显微镜 通过虚拟导航到大视野显微镜进行神经元群体成像，三光子显微镜用于深度成像 脑成像和双光子(2P)微型显微镜，用于自由行走(但有限旋转)小鼠的体内成像。 尽管有这些令人兴奋的技术进步，但同时、大规模和高分辨率的工具 自由行为的啮齿动物的多个大脑区域的成像仍然缺乏。成功地开发了 这样的工具可以加速揭示工作大脑中神经网络的一般原理的过程 在近乎自然的条件下。自由移动的成像风格将最大限度地减少 实验控制的行为和自然的自发行为，从而允许精确检查 神经网络功能。两个相互关联的神经群体的同时成像能力 将提供一个全面和精确的时间线的神经回路动力学与各种 在细胞和种群水平上的行为。 我们提出的研究是出于对具有上述特征的此类成像工具的需求。这个 主要目标是开发一种3D扫描、超薄和轻型2P光纤显微镜技术，以使高分辨率 分辨率，同时成像的动态神经活动在两个大脑区域的大视场在自由- 移动中的啮齿动物。为了达到我们的目标，我们提出了以下目标： (1)研制大视场(500um)级联放大率快速扫描2P纤维内窥镜 策略，同时保持紧凑的探头尺寸(直径2.5 mm)。更大的视野将通过使用 创新的微光学设计。此外，将在2P中实施模块化扫描头设计 纤维内窥镜，以提高在高扫描频率(例如，~2.8 kHz)下体内成像的探头稳定性； (2)开发一种可以集成到我们的2D扫描光纤显微镜中的微型(直径2 mm)可调透镜 用于实现150微米以上的深度(聚焦)扫描/选择。聚焦扫描可以方便地选择 适当的一层或一群神经元的。该可调透镜可以在以下情况下产生弯曲的折射率分布 适用于低电压(&lt；10V，安全)电气驱动。与其他可调镜头相比，该可调镜头 将非常紧凑和轻巧，对于成像自由移动的啮齿动物至关重要。一种集成了光纤显微镜和 可调谐透镜将使用幻影、荧光组织切片和活体小鼠模型进行开发和测试。 (3)发展双探头系统，可同时进行两个脑区的2P成像。 行走/旋转的老鼠。光纤内窥镜的超紧凑尺寸和重量允许两个光纤内窥镜 安装了一个小鼠头，允许同时对两个大脑区域(皮质或深部脑)进行成像。一部小说， 将首次开发主动式双探头光电换向器(DpOEC)，以检测和 补偿光纤显微镜中积累的扭矩，允许鼠标在成像过程中自由行走/旋转； (4)评估双探头2P技术用于探索神经网络动力学的可行性。 在社会决策过程中，两个不同的大脑区域同时存在。社交行为涉及到 感觉、认知和运动功能，因此依赖于许多神经元的相互作用，但到目前为止还没有 技术可用于记录大量神经元，其亚细胞分辨率超过多个 在行为自由的小鼠中相互连接的区域。在这里，我们选择研究动态神经连接 在初级运动皮质(M1)和关键感觉信息传递节点--中脑导水管周围灰质之间 (PAG)。这两个领域都与社会行为密切相关，但这些相互关联的地区如何协同 过程信息仍然几乎完全未知。除了测试2P的性能之外 通过纤维镜检技术，这一目的也可以阐明社会偏好是如何编码的。作为控制，我们将 在移动(但非社交)活动(旋转机器人跑步)期间监视这些区域，其信息 在M1上，独立于PAG的版本已经可用。 总而言之，拟议研究的成功完成将建立一种新的双光子光纤成像技术 神经科学界实现神经网络同时高分辨率成像的平台 自由行为啮齿动物不同脑区不同细胞类型的动态变化。此外，聚焦/深度 扫描将成为可能。纤维内窥镜可以容易地安装到鼠标头上和从鼠标头上拆卸， 允许重复使用的。虽然超出了当前提议的范围，但该技术还可以有许多 翻译应用，包括用于诊断或指导干预的内腔器官成像。

PROJECT SUMMARY Brain activities involve neurons generating fast-propagating signals to encode and relay information within dynamic neural networks. Neuroscientists aspire to obtain access to such networks in unconstrained animal models (e.g., rodents) with high spatiotemporal resolution, which will shed light on the fundamental working mechanisms of the brain. Optical imaging, particularly multiphoton microscopy, has played a significant role in this endeavor. The past decade has seen impressive progresses, from head-restrained benchtop microscopy with virtual navigation to large FOV microscopy for neuron population imaging, three-photon microscopy for deep brain imaging, and two-photon (2P) miniscopy for in vivo imaging in freely-walking (but limited rotation) mice. Despite these exciting technological advances, tools for simultaneous, large-scale, and high-resolution imaging over multiple brain regions in freely-behaving rodents are still lacking. Successful development of such tools can accelerate the process of uncovering general principles of neural networks in a working brain under nearly natural conditions. The free-moving style for imaging would minimize the differences between experimentally controlled actions and natural spontaneous behaviors, thus allowing for precise examination of neural network functions. The capability of simultaneous imaging over two interconnected neural populations would provide a comprehensive and precise timeline of the neural circuit dynamics associated with various behaviors at both cellular and population levels. Our proposed research is motivated by the need for such imaging tools with the above-mentioned features. The main objective is to develop a 3D-scanning, ultrathin and light 2P fiberscope technology for enabling high- resolution, simultaneous imaging of dynamic neural activities over a large FOV at two brain regions in freely- moving rodents. To achieve our objective, we propose the following aims: (1) To develop a fast scanning 2P fiberscope of a large FOV (Ø500 um) using a cascaded magnification strategy while maintaining a compact probe size (Ø2.5 mm). The larger FOV will be achieved by using an innovative micro-optics design. In addition, a modular scanner head design will be implemented in the 2P fiberscope to improve the probe robustness for in vivo imaging at a high scanning frequency (e.g., ~2.8 kHz); (2) To develop a miniature (Ø2mm) tunable lens that can be integrated into our 2D scanning fiberscope for enabling depth (focus) scanning/selection over 150 um. Focus scanning allows for convenient selection of a proper layer or population of neurons. The tunable lens can create a curved refractive index profile when applied with a low-voltage (<10 V, safe) electrical drive. Compared with other tunable lenses, the tunable lens will be extremely compact and light, critical for imaging freely-moving rodents. A fiberscope integrated with a tunable lens will be developed and tested using phantoms, fluorescent tissue slides, and a mouse model in vivo. (3) To develop a dual-probe system, enabling simultaneous 2P imaging of two brain regions in freely- walking/rotating mice. The ultracompact size and lightweight of the fiberscope permit two fiberscopes to be mounted a mouse head, allowing for simultaneous imaging of two brain regions (cortex or deep brain). A novel, proactive, dual-probe optoelectrical commutator (dpOEC) will be developed for the first time to sense and compensate the torque built up in the fiberscopes, allowing the mouse to walk/rotate freely during imaging; (4) To assess the feasibility of the dual-probe 2P technology for exploring neural network dynamics in two different brain regions simultaneously during social decision making. Social behavior involves sensory, cognitive, and motor functions and thus depends on the interactions of many neurons, but until now no technology is available to record from a large population of neurons with subcellular resolution over multiple interconnected regions in freely-behaving mice. Here we choose to study the dynamic neural connectivity between the primary motor cortex (M1) and a critical sensory information routing node, periaqueductal gray (PAG). Both areas are critically involved in social behavior, but how these interconnected regions synergize to process information remains almost completely unknown. In addition to testing the performance of the 2P fiberscopy technology, this aim could also shed light on how social preference is encoded. As a control, we will monitor these regions during a locomotion (but nonsocial) activity (Rotarod running), for which the information on M1 that is independent of PAG is already available. In summary, successful completion of the proposed study will establish a new two-photon fiberscope imaging platform for the neuroscience community to enable simultaneous high-resolution imaging of neural network dynamics of different cell types over different brain regions in freely-behaving rodents. In addition, focus/depth scanning will be made possible. The fiberscope can be easily attached to and detached from the mouse head, permitting repeated use. Although beyond the scope of current proposal, the technology can also have many translational applications, including internal luminal organ imaging for diagnosis or guidance of intervention.