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信号以编码和中继信息。 动态神经网络。神经科学家渴望获得无约束动物中这种网络的访问 具有高时空分辨率的模型（例如啮齿动物），它将阐明基本工作 大脑的机制。光学成像，尤其是多光子显微镜，在 这项工作。过去十年来，从头部弯曲的台式显微镜进行了令人印象深刻的进步 通过虚拟导航到大型FOV显微镜进行神经元种群成像，三光子显微镜用于深 脑成像和两光片（2p）Miniscopy用于自由行动（但有限的旋转）小鼠中体内成像。 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 仍然缺乏自由行啮齿动物的多个大脑区域的成像。成功发展 这样的工具可以加速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。成像的自由移动风格将最大程度地减少 实验控制的动作和自然的赞助行为，因此可以精确检查 神经网络功能。在两个相互联系的神经种群上同时成像的能力 将提供与各种神经元电路动力学的全面和精确的时间表 细胞和人口水平的行为。 我们提出的研究是由具有上述功能的此类成像工具的必要性所激发的。这 主要目的是开发一种3D扫描，超薄和光2P 2P纤维镜技术，以实现高 分辨率，在自由区的两个大脑区域的大型FOV上对动态神经元活动的简单成像 移动啮齿动物。为了实现我们的目标，我们提出以下目标： （1）使用级联放大倍率开发大型FOV（Ø500UM）的快速扫描2P纤维尺寸 策略，同时保持紧凑的探针大小（Ø2.5毫米）。通过使用 创新的微观设计。此外，将在2P中实现模块化扫描仪设计 在高扫描频率下（例如，〜2.8 kHz），以提高体内成像的探针鲁棒性； （2）开发一个可以集成到我们的2D扫描纤维镜中的微型（Ø2mm）可调透镜 为了使深度（焦点）扫描/选择超过150 um。聚焦扫描可以方便地选择 适当的神经元层或人群。可调透镜可以在 使用低压（<10 V，安全）电气驱动器应用。与其他可调透镜相比，可调镜头 将非常紧凑和轻巧，对于成像自由移动啮齿动物至关重要。与A集成的纤维镜 可调透镜将使用幻像，荧光组织载玻片和体内小鼠模型进行开发和测试。 （3）开发一个双探针系统，可以在自由的两个大脑区域同时进行2P成像 步行/旋转小鼠。纤维镜的超型尺寸和轻量级允许两个纤维尺寸为 安装了一个小鼠头，可以同时对两个大脑区域（皮层或深脑）进行成像。小说， 将首次开发主动，双探针光电通勤者（DPOEC） 补偿纤维镜中构建的扭矩，使鼠标在成像过程中自由行走/旋转； （4）评估双探针2P技术在探索神经网络动态的可行性 在社会决策过程中，同时同时进行了两个不同的大脑区域。社会行为涉及 感觉，认知和运动功能，因此取决于许多神经元的相互作用，但直到现在还没有 技术可从大量的神经元中记录，这些神经元具有亚细胞分辨率超过多个 自由式小鼠中的相互联系。在这里，我们选择研究动态神经连通性 在主要运动皮层（M1）和关键的感觉信息路由节点之间 （PAG）。这两个领域都涉及社会行为，但是这些相互联系的区域如何协同与 流程信息几乎完全未知。除了测试2p的性能 纤维份技术，此目标还可以阐明社会偏好的编码方式。作为控制，我们将 在运动（但非社交）活动（Rotarod运行）期间监视这些区域，信息 在M1上，与PAG无关。 总而言之，拟议的研究成功完成将建立一个新的两光子纤维镜成像 神经科学社区的平台，使神经科学网络同时进行高分辨率成像 自由式啮齿动物中不同大脑区域的不同细胞类型的动力学。另外，聚焦/深度 扫描将成为可能。纤维镜可以很容易地连接到鼠标头部并分离 允许重复使用。尽管超出了当前建议的范围，但该技术也可以拥有许多 翻译应用，包括用于诊断或干预指导的内部腔内器官成像。