Five-Dimensional Single-Molecule Nanoscopy for Sensing and Imaging the Dynamic Functions of Biomolecules

五维单分子纳米显微镜用于生物分子动态功能的传感和成像

• 批准号: 9543531
• 负责人: Matthew D Lew
• 金额: $ 32.3万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9543531
• 关键词: Affect; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Award; Binding; Biological; Cell membrane; Cells; Chemistry; Color; Computer software; Development; Diffuse; Dimensions; Disease; Environment; Fluorescence Microscopy; Fluorescent Probes; Human; Image; Image Analysis; Light; Lighting; Lipids; Measurement; Measures; Membrane; Membrane Microdomains; Microscope; Microscopy; Molecular; Molecular Conformation; Morphology; Motion; Nanoscopy; Nobel Prize; Optics; Pathologic; Phase; Physiology; Positioning Attribute; Process; Proteins; Research; Resolution; Rotation; Structure; Technology; Variant; Visible Radiation; biological systems; combat; design; fluidity; fluorescence imaging; imaging probe; imaging system; innovation; interest; nano; nanoscale; nanoscope; neuroblastoma cell; new technology; novel diagnostics; novel therapeutics; optical nanoscopy; polarized light; sensor; single molecule; tool; trafficking

7. PROJECT SUMMARY Five-Dimensional Single-Molecule Nanoscopy for Sensing and Imaging the Dynamic Functions of Biomolecules This project will implement five-dimensional (5D) single-molecule (SM) nanoscopy to detect and visualize the dynamics of biological structures within living cells with nanoscale resolution. This project will be the first demonstration of super-resolution (SR) fluorescence imaging capable of resolving the 3D position and 2D orientation (i.e., x, y, z, pitch, and yaw) of single molecules in living biological systems. Five-dimensional measurements are needed to elucidate biomolecular interactions because molecules are not simple isotropic spheres; their orientation and/or conformation critically determine how they interact with each other. Super-resolved fluorescence microscopy, awarded the Nobel Prize in Chemistry 2014 and also termed optical nanoscopy, produces images of structures within living cells with resolution beyond the optical diffraction limit (~250 nm for visible light). One fundamental drawback of SR microscopy is its inability to measure the activity and function of molecules (e.g., binding, conformation, structural disorder, etc.) since its images only depict the 2D or 3D spatial positions of fluorescent tags. This limitation is a consequence of traditional microscope designs, which cannot measure the phase or polarization of light. Here, 5D SM nanoscopy will be developed to measure the dynamic activities of biomolecules by innovating and combining two synergistic approaches: 1) use binding- activated fluorogenic probes for imaging biological structures and 2) design and utilize integrated optical hardware and image analysis software for visualizing the 3D position (x, y, z) and 2D orientation (θ and φ in spherical coordinates) of fluorescent probes. Optical nanoscopes will no longer simply focus light onto a camera to create 2D images; rather, the fluorescent light within the imaging system will be “bent” specifically so that molecular position and orientation can be directly measured from the images captured by the camera. Lipid nanodomains are thought to control the trafficking of biomolecules across the cell membrane. A critical barrier to understanding these activities is our inability to directly visualize these domains. Five-dimensional SM nanoscopy will visualize nano-polarity and nano-fluidity of cell membranes by using fluorescent molecular sensors to diffuse, collide, and temporarily bind to biomolecules of interest within a cell, lighting up in the process. The rotational mobility of these probes will directly measure the polarity and/or fluidity of their environment. The aggregation of Aβ1-42 on the membranes of cultured human neuroblastoma cells (SH-EP cells) will be studied with a two-color variant of 5D SM nanoscopy to obtain nanoscale resolution. Simultaneously, lipid rafts will be visualized using lipid-specific fluorescent molecular sensors. This approach will reveal the dynamic nanoscale interactions between lipid nanodomains and Aβ1-42, especially how lipid phase affects the binding of Aβ1-42 and how the accumulation of Aβ1-42 remodels membrane morphology. By visualizing the nanoscale dynamics of both biomolecules simultaneously with SM sensitivity, the formation mechanism of toxic Aβ species and the impact of membrane physiology on the progression of Alzheimer’s Disease will be elucidated.

7. 项目概要 五维单分子纳米显微技术用于传感和成像动态功能 生物分子 该项目将实施五维（5D）单分子（SM）纳米显微镜来检测和可视化 该项目将是第一个以纳米级分辨率研究活细胞内生物结构的动力学。 演示能够解析 3D 位置和 2D 的超分辨率 (SR) 荧光成像 五维生物系统中单个分子的方向（即 x、y、z、俯仰和偏航）。 需要测量来阐明生物分子相互作用，因为分子不是简单的各向同性 球体；它们的方向和/或构象决定了它们如何相互作用。 超分辨荧光显微镜，荣获2014年诺贝尔化学奖，也被称为光学显微镜 纳米显微镜，产生活细胞内结构的图像，其分辨率超出光学衍射极限 （可见光约为 250 nm）。SR 显微镜的一个基本缺点是它无法测量活性。 和分子的功能（例如，结合、构象、结构紊乱等），因为它的图像仅描述了 荧光标签的 2D 或 3D 空间位置是传统显微镜设计的结果。 无法测量光的相位或偏振，这里将开发 5D SM 纳米镜来测量。 通过创新和结合两种协同方法来研究生物分子的动态活动：1）使用结合- 用于生物结构成像的激活荧光探针，2) 设计和利用集成光学 用于可视化 3D 位置（x、y、z）和 2D 方向（θ 和 φ）的硬件和图像分析软件 荧光探针的球坐标）将不再简单地将光聚焦到相机上。 为了创建 2D 图像；相反，成像系统内的荧光将被专门“弯曲”，以便 可以从相机捕获的图像直接测量分子位置和方向。 脂质纳米结构域被认为是控制生物分子跨细胞膜运输的关键。 理解这些活动的障碍是我们无法直接可视化这些领域。 纳米显微镜将通过使用荧光分子来可视化细胞膜的纳米极性和纳米流动性 传感器扩散、碰撞并暂时与细胞内感兴趣的生物分子结合，并在此过程中发光。 这些探针的旋转移动性将直接测量其环境的极性和/或流动性。 将研究Aβ1-42在培养的人神经母细胞瘤细胞（SH-EP细胞）膜上的聚集 同时，使用 5D SM 纳米显微镜的双色变体获得纳米级分辨率。 使用脂质特异性荧光分子传感器进行可视化，这种方法将揭示动态纳米级。 脂质纳米结构域和 Aβ1-42 之间的相互作用，特别是脂质相如何影响 Aβ1-42 和 Aβ1-42 的结合 Aβ1-42 的积累如何重塑膜形态通过可视化两者的纳米级动力学。 生物分子同时具有SM敏感性，有毒Aβ物种的形成机制及其影响 将阐明膜生理学对阿尔茨海默病进展的影响。