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: 10445025
• 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/10445025
• 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.

远场荧光显微镜是生物研究中的有力工具，这是由于其活细胞相容性和 分子特异性在过去的100年里，一个主要的障碍是由于衍射而导致的有限分辨率 光明现代超分辨率显微镜方法，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， 随机切换单个染料的开启和关闭，使得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以高精度定位，导致重建的超分辨 图像分辨率低至~25 nm。 然而，目前SMLM的发展和应用集中在薄样品和细胞中的固定细胞上， 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，SMLM尚未完全展开：（1）活细胞SMLM缓慢且难以实现 由于小的光子预算，每个光子携带的信息不足，以及所需的高分辨率， （2）通过大组织深度的SMLM仍然很困难，因为SMLM的快速恶化 由像差和荧光背景引起的组织样本中的分辨率和图像保真度;以及（3） 分子分辨率（1-5 nm）在低光子通量条件下在整个细胞和组织中仍然是可实现的。 克服这些障碍将有助于揭示细胞成分的结构，功能和动力学， 活体标本的分子分辨率，以及多个蛋白质种类的纳米级图谱的重建 在大的组织体积内。这些能力将大大扩大SMLM应用程序的影响。 我们的长期目标是开发新的光学成像系统，在定义 活细胞和组织中细胞成分的结构和功能的分子解析。未来 五年来，我们将重点关注两个研究方向：（1）开发新型单分子超分辨 成像技术和相位编码定位方法，使分子分辨率的三维成像， 低光子通量条件下的活细胞。这些创新将使我们能够在1-5纳米的范围内捕捉3D动态 分辨率和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 开发新的仪器和分析方法，以实现超高分辨率，多路复用映射 在大的组织体积中的荧光标记的目标。 我们将应用这些发展来揭示肌动蛋白网络的分子组织和功能 在活的分裂酵母中，在细胞动力学收缩环的形成和收缩过程中的纤维和肌球蛋白。 此外，我们将确定分子马达如动力蛋白的精确亚细胞定位， 微管和肌动蛋白。我们还将探讨纳米级拓扑结构之间的相关性， 染色质基因座与确定的表观遗传内容和细胞谱系和基因表达谱的变化。