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

7.项目总结 用于传感和成像的五维单分子纳米显微镜的动态功能 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 以纳米级分辨率研究活细胞内生物结构的动力学。这个项目将是第一个 演示了能够分辨3D位置和2D的超分辨率(SR)荧光成像 生物系统中单分子的取向(即x、y、z、螺距和偏航)。五维 由于分子不是简单的各向同性的，因此需要测量来阐明生物分子间的相互作用 球体；它们的取向和/或构象关键地决定它们如何相互作用。 超分辨荧光显微镜，获得2014年诺贝尔化学奖，也被称为光学 纳米显微镜，在活细胞内产生分辨率超过光学衍射极限的结构图像 (可见光为~250 nm)。SR显微镜的一个根本缺点是它不能测量活动 和分子的功能(如结合、构象、结构无序等)因为它的图像只描绘了 荧光标记的2D或3D空间位置。这种限制是传统显微镜设计的结果， 它不能测量光的相位或偏振。在这里，5D SM纳米显微镜将被开发用于测量 通过创新和结合两种协同方法来研究生物分子的动态活性：1)使用结合- 用于生物结构成像的激活荧光探针和2)设计和利用集成光学 硬件和图像分析软件，用于可视化3D位置(x，y，z)和2D方向(θ和φin 球面坐标)的荧光探针。光学纳米显微镜将不再简单地将光聚焦到相机上 来创建2D图像；相反，成像系统内的荧光将被特别地“弯曲”，从而 分子的位置和取向可以从相机捕获的图像中直接测量。 脂质纳米结构域被认为可以控制生物分子在细胞膜上的运输。一位批评者 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 纳米显微镜将利用荧光分子显示细胞膜的纳米极性和纳米流动性 传感器扩散、碰撞并暂时结合到细胞内感兴趣的生物分子上，在此过程中发光。 这些探头的旋转迁移率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 使用5D SM纳米显微镜的双色变体，以获得纳米级分辨率。同时，脂筏将被 使用脂类特异的荧光分子传感器进行可视化。这种方法将揭示动态纳米尺度 脂类纳米结构域与Aβ1-42的相互作用，特别是脂相对Aβ1-42与A DNA结合的影响 Aβ1-42的积累如何重塑膜的形态。通过将两者的纳米级动力学可视化 生物分子同时对SM的敏感性、A类毒力β物种的形成机制及其影响 膜生理学在阿尔茨海默病进展中的作用将被阐明。

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.