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: 9382019
• 负责人: Matthew D Lew
• 金额: $ 35.06万
• 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/9382019
• 关键词: Affect; Alpha Cell; 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; 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。项目摘要 五维单分子纳米镜检查，用于感测和成像动态功能 生物分子 该项目将实施五维（5D）单分子（SM）纳米镜检查以检测和可视化 活细胞内具有纳米级分辨率的生物结构的动力学。这个项目将是第一个 能够解决3D位置和2D的超分辨率（SR）荧光成像的演示 活生物系统中的单分子的方向（即X，Y，Z，音调和YAW）。五维 需要测量以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；他们的方向和/或构象认真地决定了它们如何相互作用。 超级分辨荧光显微镜，授予2014年诺贝尔化学奖，也称为光学 纳米镜检查，在活细胞内产生结构图像，分辨率超过光学衍射极限 （可见光〜250 nm）。 SR显微镜的一个基本缺点是无法测量活动 分子的功能（例如结合，构象，结构障碍等），因为它的图像仅描绘 荧光标签的2D或3D空间位置。这种限制是传统显微镜设计的结果， 无法测量光的相或极化。在这里，将开发5D SM纳米镜检查以测量 通过创新和结合两种协同方法的生物分子的动态活性：1）使用结合 - 活化的荧光问题用于成像生物结构和2）设计和使用集成的光学 用于可视化3D位置（X，Y，Z）和2D方向的硬件和图像分析软件（θ和φ 荧光问题的球形坐标。光学纳米镜将不再简单地将光线聚焦到相机上 创建2D图像；相反，成像系统中的荧光灯将是“弯曲”的，以便 分子位置和方向可以直接从相机捕获的图像中测量。 人们认为脂质纳米域可以控制生物分子在细胞膜上的运输。批判 理解这些活动的障碍是我们无法直接可视化这些领域。五维SM 纳米镜检查将通过使用荧光分子可视化细胞膜的纳米极性和纳米浮肿 传感器扩散，碰撞和暂时与细胞中感兴趣的生物分子结合，在此过程中照明。 这些问题的旋转迁移率将直接衡量其环境的极性和/或流动性。 Aβ1-42在培养的人神经母细胞瘤细胞（SH-EP细胞）膜上的聚集将研究 具有5D SM纳米镜检查的两种颜色变体以获得纳米级分辨率。同时，脂质筏将是 使用脂质特异性荧光分子传感器可视化。这种方法将揭示动态的纳米级 脂质纳米域与Aβ1-42之间的相互作用，特别是脂质相影响Aβ1-42和 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 仅具有SM敏感性的生物分子，有毒Aβ物种的形成机理和影响 将阐明有关阿尔茨海默氏病进展的膜生理。