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

3D 扫描双光子纤维镜技术，可对自由移动的啮齿动物进行同步多区域多细胞型成像

• 批准号: 10660682
• 负责人: Xingde Li
• 金额: $ 63.98万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10660682
• 关键词: 3-Dimensional; Acceleration; Animal Model; Area; Behavior; Biomedical Engineering; Brain; Brain imaging; Brain region; Cognitive; Collaborations; Columbidae; Communities; Compensation; Decision Making; Development; Diagnosis; Fiber Optics; Frequencies; Head; Image; Imaging Device; Imaging technology; Intervention; Light; Locomotion; Microscopy; Monitor; Motor; Motor Cortex; Mus; Neural Interconnection; Neurons; Neurosciences; Optics; Organ; Performance; Play; Population; Process; Productivity; Publications; Recording of previous events; Refractive Indices; Research; Resolution; Rodent; Role; Rotation; Route; Running; Scanning; Sensory; Signal Transduction; Slice; Slide; Social Behavior; System; Technology; Testing; Thick; Time; Tissues; Torque; Universities; Walking; Washington; calcium indicator; cantilever; cell type; design; high resolution imaging; histological image; imaging platform; improved; in vivo; in vivo Model; in vivo imaging; innovation; interest; lens; light weight; liquid crystal; microscopic imaging; midbrain central gray substance; mouse model; multiphoton microscopy; neural; neural circuit; neural network; neuroimaging; neuronal circuitry; novel; optical fiber; optical imaging; performance tests; phantom model; preference; programs; restraint; social; spatiotemporal; synergism; three photon microscopy; timeline; tool; translational applications; two-photon; virtual; voltage

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.

项目摘要 大脑活动涉及神经元产生快速传播的信号，以编码和中继内部的信息。 动态神经网络神经科学家渴望在不受约束的动物中获得这种网络 模型（例如，啮齿动物）具有高时空分辨率，这将揭示基本的工作 大脑的机制。光学成像，特别是多光子显微镜，在 这种奋进。在过去的十年里，从限制头部的台式显微镜， 通过虚拟导航到大FOV显微镜进行神经元群体成像，三光子显微镜进行深层成像， 脑成像，以及用于自由行走（但旋转受限）小鼠体内成像的双光子（2 P）微型扫描。 尽管有这些令人兴奋的技术进步，但用于同步，大规模和高分辨率的工具 在行为自由的啮齿类动物的多个大脑区域的成像仍然缺乏。成功发展 这些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 在几乎自然的条件下。自由移动的成像方式将最大限度地减少 实验控制的行动和自然自发行为，从而允许精确检查 神经网络函数在两个相互连接的神经群体上同时成像的能力 将提供与各种神经回路相关的神经回路动力学的全面和精确的时间轴 在细胞和群体水平上的行为。 我们提出的研究的动机是需要这样的成像工具与上述功能。的 主要目标是开发一种3D扫描、扫描和光2 P纤维镜技术， 分辨率，同时成像的动态神经活动在一个大的FOV在两个大脑区域在自由- 移动的啮齿动物为了实现我们的目标，我们提出以下目标： (1)研制一种级联放大的大视场（~ 500 μ m）快速扫描2 P纤维内窥镜 这是一个非常简单的策略，同时保持紧凑的探头尺寸（约2.5 mm）。通过使用 创新的微光学设计。此外，模块化扫描头设计将在2 P中实施 纤维镜以提高探针在高扫描频率下的体内成像的鲁棒性（例如，~2.8 kHz）; (2)开发可集成到我们的2D扫描纤维内窥镜中的微型（约2 mm）可调透镜 用于实现超过150 μ m的深度（聚焦）扫描/选择。焦点扫描允许方便的选择 一个适当的层或群体的神经元。可调谐透镜可以在以下情况下产生弯曲的折射率分布： 采用低压（<10 V，安全）电气驱动。与其它可调谐透镜相比，本发明的可调谐透镜 将是非常紧凑和轻的，对自由移动的啮齿动物成像至关重要。一种集成有 将开发可调透镜，并在体内使用体模、荧光组织载玻片和小鼠模型进行测试。 (3)为了开发一种双探头系统，使两个大脑区域的同时2 P成像自由- 行走/旋转小鼠。纤维内窥镜的超紧凑尺寸和重量轻允许两个纤维内窥镜 安装在老鼠头上，允许同时对两个大脑区域（皮层或深部脑）进行成像。一本小说， 将首次开发主动式双探头光电换向器（dpOEC）， 补偿纤维内窥镜中产生的扭矩，允许鼠标在成像期间自由行走/旋转; (4)为了评估双探针2 P技术用于探索神经网络动力学的可行性， 两个不同的大脑区域同时进行社交决策社会行为包括 感觉，认知和运动功能，因此取决于许多神经元的相互作用，但到目前为止，没有 现有技术可用于从大量神经元中记录多个亚细胞分辨率 相互连接的区域。在这里，我们选择研究动态神经连接 在初级运动皮层（M1）和一个关键的感觉信息路由节点（导水管周围灰质）之间 （PAG）。这两个区域都与社会行为密切相关，但这些相互关联的区域如何协同作用， 过程信息仍然几乎完全未知。除了测试2 P的性能外， 通过纤维镜技术，这一目标也可以揭示社会偏好是如何编码的。作为对照，我们将 在运动（但非社交）活动（旋转棒跑步）期间监测这些区域，其中的信息 独立于PAG的M1上已经可用。 综上所述，成功完成拟议的研究将建立一个新的双光子纤维镜成像 神经科学社区的平台，使神经网络的同步高分辨率成像成为可能 在自由行为的啮齿动物中，不同脑区的不同细胞类型的动态。此外，重点/深度 扫描将成为可能。纤维镜可以容易地附接到鼠标头和从鼠标头拆卸， 允许重复使用。虽然超出了当前提案的范围，但该技术也可以具有许多优点。 翻译应用，包括用于诊断或指导干预的内腔器官成像。