Ultra-high resolution, multiplexed single molecule nanoscopy and functional characterization of neural circuits

超高分辨率、多重单分子纳米显微镜和神经回路的功能表征

• 批准号: 10025878
• 负责人: Alexander Chubykin
• 金额: $ 242.47万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-20 至 2024-08-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10025878
• 关键词: 3-Dimensional; Antibodies; Atlases; Bar Codes; Behavior; Brain; Calcium Channel; Color; Comb animal structure; Complex; Cytoskeletal Proteins; DNA; Defect; Dendritic Spines; Detection; Deterioration; Development; Image; In Situ; In Vitro; Interneurons; Label; Lateral; Light; Lighting; Link; Maps; Measurement; Methods; Microscopy; Molecular; Molecular Analysis; Molecular Structure; Morphologic artifacts; Mus; Nanoscopy; Neurodevelopmental Disorder; Neurons; Neurotransmitter Receptor; Optics; Phenotype; Presynaptic Terminals; Proteins; Public Health; Radial; Research; Resolution; SNAP receptor; Sampling; Schizophrenia; Slice; Specimen; Structure; Synapses; Synaptic Cleft; Synaptic Vesicles; System; Technology; Thick; Tissue Sample; Tissues; Vertebrates; Visualization; adaptive optics; antibody conjugate; area striata; autism spectrum disorder; brain circuitry; brain tissue; density; design; imaging modality; imaging system; instrument; macromolecular assembly; molecular assembly/self assembly; nanobodies; nanoscale; neural circuit; neuronal circuitry; novel; optogenetics; patch clamp; postsynaptic; protein complex; public health relevance; relating to nervous system; scaffold; single molecule; ultra high resolution

The complex behaviors of all vertebrates are determined by the brain where neurons are connected by synapses. The average volume of synapses corresponds to a sphere of ~400 nm radius—a size scale that can barely be resolved using conventional optical microscopy methods. Synapses are tightly packed with molecular assemblies of synaptic vesicles, synaptic and cytoskeletal proteins and neurotransmitter receptors. These protein complexes are assembled in a much smaller scale—15-60 nm in the three-dimensional tissue space. Current state-of-the-art imaging methods including optical super-resolution microscopy and expansion microscopy remain limited in terms of its achievable resolution in 3D, resolution deterioration in thick tissues, multiplexing capability and the access to the link between functional and structural connectivities. We propose to develop a light-sheet illuminated, adaptive optics assisted, ultra-high resolution (10-15 nm 3D resolution) 4Pi/interferometric single-molecule super-resolution nanoscopy system for thick tissue specimens (Aim 1). We will perform in vitro functional optogenetic neural circuit mapping using automated patch clamp followed by the imaging of the same brain slices using our novel nanoscopy system to autonomously trace and resolve probed circuit (Aim 2). To provide the highly multiplexed in situ protein detection capability (up to 8 targets), we will combine Exchange-PAINT that utilizes DNA-barcode conjugated antibodies with the developed system (Aim 3). Our system will allow comprehensive mapping of ultrastructural features of neuronal circuits (such as pre- and postsynaptic proteins in different types of synapses) in an functionally analyzed brain tissue with 10-15 nm optical resolution in all three dimensions throughout a cortical microcircuit.

所有脊椎动物的复杂行为都是由大脑决定的，大脑中的神经元是通过 突触突触的平均体积相当于一个半径约为400纳米的球体， 用传统的光学显微镜方法几乎无法分辨。突触紧密地包裹着分子 突触囊泡、突触和细胞骨架蛋白以及神经递质受体的组装。这些 蛋白质复合物在三维组织空间中以小得多的尺度（15-60 nm）组装。 当前最先进的成像方法，包括光学超分辨率显微镜和扩展 显微术在其3D中可实现的分辨率方面仍然有限，在厚组织中的分辨率恶化， 多路复用能力和对功能和结构连接性之间的链接的访问。我们提出 开发光片照明、自适应光学辅助、超高分辨率（10-15 nm 3D分辨率） 4Pi/干涉单分子超分辨率纳米显微系统，用于厚组织标本（目标1）。我们 将使用自动膜片钳进行体外功能性光遗传学神经回路映射， 使用我们的新型纳米显微镜系统对相同的大脑切片进行成像， 电路（目标2）。为了提供高度多重的原位蛋白检测能力（多达8个靶标），我们将 联合收割机将利用DNA-条形码缀合抗体的Exchange-PAINT与开发的系统（Aim （3）第三章。我们的系统将允许全面映射神经元回路的超微结构特征（如前 和不同类型突触中的突触后蛋白质）在功能分析的脑组织中用10-15 nm 在整个皮层微电路的所有三个维度上的光学分辨率。

项目成果

Restoration of Visual Function and Cortical Connectivity After Ischemic Injury Through NeuroD1-Mediated Gene Therapy.

• DOI: 10.3389/fcell.2021.720078
• 发表时间: 2021
• 期刊: Frontiers in cell and developmental biology
• 影响因子: 5.5
• 作者: Tang Y;Wu Q;Gao M;Ryu E;Pei Z;Kissinger ST;Chen Y;Rao AK;Xiang Z;Wang T;Li W;Chen G;Chubykin AA
• 通讯作者: Chubykin AA

Deep learning-driven adaptive optics for single-molecule localization microscopy.

• DOI: 10.1038/s41592-023-02029-0
• 发表时间: 2023-11
• 期刊: NATURE METHODS
• 影响因子: 48
• 作者: Zhang, Peiyi;Ma, Donghan;Cheng, Xi;Tsai, Andy P.;Tang, Yu;Gao, Hao-Cheng;Fang, Li;Bi, Cheng;Landreth, Gary E.;Chubykin, Alexander A.;Huang, Fang
• 通讯作者: Huang, Fang