Single-Molecule Imaging for Cell Biology and Super-Resolution Microscopy

细胞生物学和超分辨率显微镜的单分子成像

• 批准号: 9920156
• 负责人: William E Moerner
• 金额: $ 63.17万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920156
• 关键词: 3-Dimensional; Address; Affect; Award; Bacteria; Behavior; Biological Models; Biotechnology; Caulobacter crescentus; Cells; Cellular biology; Chromatin; Cilia; Collaborations; Complex; DNA; Dependence; Disease; Environment; Enzymes; Fluorescence Microscopy; Funding Opportunities; Image; Label; Laboratories; Light; Mammalian Cell; Measurement; Measures; Methodology; Methods; Microscope; Microscopy; Modification; Molecular Motors; Motion; Motivation; Oligonucleotides; Optics; Organelles; Organism; Positioning Attribute; Problem Solving; Process; Proteins; Pupil; RNA; Research; Research Methodology; Research Personnel; Resolution; Speed; Structure; Three-Dimensional Imaging; Time; Visible Radiation; Work; base; bioimaging; cell behavior; cellular imaging; fluorescence imaging; high dimensionality; imaging capabilities; imaging modality; light microscopy; molecular imaging; nanomachine; nanoscale; optical imaging; organizational structure; particle; programs; public health relevance; research and development; single molecule; tool

 DESCRIPTION (provided by applicant): The cellular environment is both powerful and complex, depending both on structural organization from the micron scale down to the nanometer scale, as well as on the dynamic time-dependence of a huge array of enzymes, the Nano machines of the cell, and their work on proteins and oligonucleotides. Visible fluorescence microscopy has been a useful tool capable of non-invasively exploring cellular behavior, but the limited resolution of visible light microscopy has severely restricted the information obtainable on structures on a scale below 250 nm. Because the primary bio-molecular players in cells are in the size range on the order of 10 nm, measurements are needed on this size scale in living systems. Super-resolution microscopy, either based on single-molecule fluorescence imaging and control of the emitting concentration, or on stimulated emission depletion, has solved this problem by enabling access to spatial resolutions down to the 10-40 nm regimes and below. In addition, the complementary method of single-molecule tracking provides access to the details of motions of cellular components such as the molecular motors or the motion of DNA or RNA. Combined with advanced three-dimensional (3D) imaging, single-particle tracking allows the full motion of specific cellular players to be observed in their actual context at high speed. It is a primary thrust of this work to develop and enhance both 3D super-resolution imaging and 3D single-particle tracking in cells by pushing the boundaries of both approaches and inventing new strategies to overcome critical limitations, which will lead to unprecedented spatial and temporal information in fixed and living cells. Research in the Moerner laboratory broadly addresses the limitations of super-resolution imaging and single-particle tracking in cells. A key tool involves using pupil plane modification of wide-field microscopes to provide advanced function, such as 3D imaging over unprecedented axial range or imaging of molecular orientations at the single-molecule level. The deep motivation here is to ask the fundamental question: how can the information available from each single molecule be maximized, both by measuring new variables, but also by examining every aspect of the process and inventing new methods to remove any systematic errors. The methodological developments of this research will be applied to a variety of critical problems in cell biology by continuing established collaborations and developing new collaborations with well-known biologists. The bacterium, Caulobacter crescentus, remains as a powerful model system needing elucidation of the superstructure and motions of biomolecules to understand the origins of asymmetric division. The primary cilium, a tiny but important cellular organelle, is filled with protein motions and interactions which need exploration on the nanometer scale. The organization of chromatin on all scales remains to be fully understood. These and other cell biology problems with implications for both normal and diseased function will be the focus of the application of the advanced imaging methods of this research program.

 描述（由应用程序提供）：蜂窝环境既有功能又复杂，这既取决于从微米尺度到纳米尺度的结构组织，以及大量酶的动态时间依赖性，细胞的纳米机器及其对蛋白质和寡核苷酸的工作。可见的荧光显微镜已成为能够非侵入性探索细胞行为的有用工具，但是可见光显微镜的有限分辨率严重限制了在250 nm以下的规模上获得的对结构获得的信息。由于细胞中的主要生物分子参与者的尺寸范围在10 nm的范围内，因此需要在这种尺寸的生命系统上进行测量。超分辨率显微镜基于单分子荧光成像和对发射浓度的控制或刺激的发射耗尽的控制，通过使空间分辨率访问至10-40 nm左右的空间分辨率，从而解决了这一问题。此外，单分子跟踪的完整方法可访问细胞成分的细节，例如分子电机或DNA或RNA的运动。结合高级三维（3D）成像，单粒子跟踪可以在高速下在其实际上下文中观察到特定的蜂窝玩家的全部运动。这是这项工作的主要目的，是通过突破两种方法的边界并发明新策略来克服临界局限性，从而开发和增强细胞中3D超分辨率成像和3D单粒子跟踪，这将导致固定和活细胞中前所未有的空间和临时信息。 Moerner实验室的研究广泛解决了细胞中超分辨率成像和单粒子跟踪的局限性。一个关键工具涉及使用宽视野显微镜的瞳孔平面修饰来提供高级功能，例如在前所未有的轴向范围上进行3D成像或单分子水平上分子取向的成像。这里的深刻动机是提出一个基本问题：如何通过测量新变量，以及检查过程的各个方面并发明新方法来删除任何系统错误，从而最大程度地提高每个分子中可用的信息。这项研究的方法论发展将通过继续建立的合作并与知名生物学家开发新的合作，将其应用于细胞生物学的各种关键问题。染色质的组织Caulobacter Crescentus仍然是一个强大的模型系统，需要阐明生物分子的上层建筑和动作，以了解不对称分裂的起源。原发性纤毛是一种微小但重要的细胞细胞器，充满了蛋白质运动和相互作用，需要在纳米尺度上进行探索。在所有尺度上的染色质的组织尚待充分理解。这些和其他细胞生物学问题对正常功能和否定功能的影响将是该研究计划的先进成像方法的应用。