Ultra-high resolution structural and molecular imaging of cells and tissues

细胞和组织的超高分辨率结构和分子成像

• 批准号: 10205665
• 负责人: Fang Huang
• 金额: $ 43.08万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10205665
• 关键词: 3-Dimensional; Actins; Biological Process; Biomedical Research; Budgets; Cell Lineage; Cell physiology; Cells; Cellular Structures; Chromatin; Development; Dyes; Dynein ATPase; Epigenetic Process; Event; Fission Yeast; Fluorescence; Fluorescence Microscopy; Fostering; Gene Expression Profile; Goals; Growth Cones; Health; Human; Image; Imaging technology; Knowledge; Label; Maps; Methods; Microfilaments; Microscopy; Microtubules; Mission; Modernization; Molecular; Molecular Motors; Myosin ATPase; Phase; Photons; Positioning Attribute; Proteins; Public Health; Research; Resolution; Sampling; Specificity; Specimen; Structural Models; Structure; Surface; Thinness; Three-Dimensional Imaging; Time; Tissues; United States National Institutes of Health; Visualization; analytical method; biological research; cellular imaging; constriction; diffraction of light; imaging system; improved; innovation; insight; instrument; macromolecular assembly; molecular imaging; nanoscale; neuronal growth; novel; optical imaging; public health relevance; reconstruction; single molecule; tool; ultra high resolution

Far-field fluorescence microscopy is a powerful tool in biological research due to its live cell compatibility and molecular specificity. A major hurdle over the last ~100 years has been the limited resolution due to the diffraction of light. Modern super-resolution microscopy methods such as single-molecule localization microscopy (SMLM) overcame this fundamental barrier and improved the resolution of fluorescence microscopy ten-fold by stochastically switching single dyes on and off such that their emission events are separated in time. This allows their center positions to be localized with high precision in space, leading to a reconstructed super-resolved image with a resolution down to ~25 nm. However, current developments and applications of SMLM focus on fixed cells in thin samples and cellular structures that lie close to the coverslip surface. Indeed, the profound impact of SMLM on biomedical studies has yet to fully unfold due to the following limitations: (1) live-cell SMLM is slow and difficult to achieve ultrahigh resolution due to the small photon budget, the insufficient information carried per photon, and the required high excitation power; (2) SMLM through large tissue depths remains difficult, due to the rapidly deteriorating resolution and image fidelity in tissue specimens caused by aberration and fluorescence background; and, (3) molecular resolution (1-5 nm) is yet achievable in whole cells and tissues at low photon flux conditions. Overcoming these hurdles will help reveal the structure, function and dynamics for cellular constituents at the molecular resolution in living specimens, and the reconstruction of nanoscale maps of multiple protein species within a large tissue volume. These capacities will drastically expand the impact of SMLM applications. Our long-term goal is to develop novel optical imaging systems that achieve significant advances in defining the structure and function of cellular constituents in live cells and tissues with molecular resolution. In the next five years, we will focus on two research directions: (1) We will develop novel single molecule super-resolution imaging technologies and a phase-encoded localization method to enable molecular-resolution 3D imaging in live cells under low photon flux conditions. The innovations will enable us to capture 3D dynamics with 1-5 nm resolution and construct time-evolved structural models of macromolecular assemblies in live cells. (2) We will develop novel instruments and analytical methods to allow ultra-high resolution, multiplexed mapping of fluorescently labeled targets in large tissue volumes. We will apply these developments to reveal the molecular organization and functions of networks of actin filaments and myosins during the formation and constriction of the cytokinetic contractile ring in live fission yeast. Also, we will determine the precise subcellular localization of molecular motors like dynein with respect to both microtubule and actin in neuronal growth cones. We will also explore the correlation between nanoscale topology of chromatin loci with defined epigenetic content and cell lineage and changes in gene expression profile.

远场荧光显微镜因其活细胞兼容性和 分子特异性。过去约 100 年来的一个主要障碍是由于衍射而导致的分辨率有限 光。现代超分辨率显微镜方法，例如单分子定位显微镜（SMLM） 克服了这一基本障碍，并将荧光显微镜的分辨率提高了十倍 随机地打开和关闭单一染料，以便它们的发射事件在时间上分开。这允许 它们的中心位置在空间中高精度定位，从而产生重建的超分辨率 图像分辨率低至~25 nm。 然而，SMLM目前的发展和应用主要集中在薄样品和细胞中的固定细胞。 靠近盖玻片表面的结构。事实上，SMLM 对生物医学研究的深远影响 由于以下限制，尚未完全展开：（1）活细胞SMLM速度慢且难以实现超高 由于光子预算小、每个光子携带的信息不足以及所需的高分辨率 励磁功率； (2) 由于组织的快速恶化，SMLM 穿过大的组织深度仍然很困难。 由像差和荧光背景引起的组织样本的分辨率和图像保真度；并且，(3) 在低光子通量条件下，整个细胞和组织仍可实现分子分辨率（1-5 nm）。 克服这些障碍将有助于揭示细胞成分的结构、功能和动态。 活体标本的分子分辨率，以及多种蛋白质物种纳米级图的重建 在大的组织体积内。这些能力将极大地扩大 SMLM 应用的影响。 我们的长期目标是开发新型光学成像系统，在定义光学成像方面取得重大进展 通过分子分辨率了解活细胞和组织中细胞成分的结构和功能。在接下来的 五年内，我们将重点关注两个研究方向：（1）开发新型单分子超分辨率 成像技术和相位编码定位方法可实现分子分辨率 3D 成像 低光子通量条件下的活细胞。这些创新将使我们能够捕捉 1-5 nm 的 3D 动态 分辨率并构建活细胞中大分子组装体的时间演化结构模型。 (2) 我们将 开发新颖的仪器和分析方法，以实现超高分辨率、多重映射 大组织体积中的荧光标记目标。 我们将应用这些进展来揭示肌动蛋白网络的分子组织和功能 活裂殖酵母中细胞因子收缩环的形成和收缩过程中的细丝和肌球蛋白。 此外，我们将确定分子马达（如动力蛋白）的精确亚细胞定位 神经元生长锥中的微管和肌动蛋白。我们还将探讨纳米级拓扑之间的相关性 具有明确表观遗传内容和细胞谱系的染色质位点以及基因表达谱的变化。