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: 10405123
• 负责人: William E Moerner
• 金额: $ 61.96万
• 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 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10405123
• 关键词: 3-Dimensional; Address; Affect; Bacteria; Behavior; Biological Models; Biotechnology; Caulobacter crescentus; Cells; Cellular biology; Chromatin; Collaborations; Complex; Cryo-electron tomography; DNA; Dependence; Development; Disease; Electron Microscopy; Environment; Enzymes; Fluorescence; Fluorescence Microscopy; Image; Label; Laboratories; Light; Mammalian Cell; Measurement; Measures; Methodology; Methods; Microscope; Microscopy; Motion; Motivation; Motor; Oligonucleotides; Optics; Organism; Parasites; Phase; Positioning Attribute; Problem Solving; Proteins; Pupil; RNA; Research; Research Methodology; Resolution; Speed; Structure; Three-Dimensional Imaging; Time; Toxoplasma gondii; Work; base; biomedical imaging; cell behavior; cellular development; cellular imaging; cryogenics; fascinate; fluorescence imaging; high dimensionality; imaging capabilities; imaging modality; invention; mathematical analysis; nanomachine; nanoscale; optical imaging; particle; programs; reconstruction; research and development; single molecule; small molecule; tool

Project Summary 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 nanomachines of the cell, and their work on proteins, oligonucleotides, and small molecules. Visible fluorescence microscopy has been a useful tool capable of non-invasively exploring cellular behavior, but the diffraction-limited resolution of conventional imaging has severely restricted the information obtainable on structures on a scale below ~200 nm. Because the primary biomolecular players in cells are in the size range on the order of 10 nm, comprehensive 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 nanoscale position information down to the 10-40 nm regime and below. In addition, the complementary method of single- molecule tracking provides access to the details of motions of cellular components such as motor-driven transport 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 technical limitations, which will lead to unprecedented spatial and temporal information in fixed and living cells. Research in the Moerner laboratory broadly seeks to address the limitations of super-resolution imaging and single-particle tracking in cells by physical and mathematical analysis as well as by invention of new methods. The deep motivation here is to ask the fundamental question: how can the information available from each single molecule be maximized? Two key new microscopes are under development: 3D imaging over large axial ranges using pupil plane phase modulations and a tilted light sheet, and a correlative method to use cryogenic single-molecule fluorescence localizations to annotate cryo-electron tomography reconstructions. The methodological developments of this research will be applied to a variety of critical problems in cell biology by continuing established collaborations and by developing new collaborations with well-known biologists. The bacterium, Caulobacter crescentus, remains as a useful model system for cellular development needing elucidation of the superstructures and motions of biomolecules to understand the origins of asymmetric division. The Toxoplasma gondii parasite is another fascinating organism which needs exploration with super- resolution methods. The organization of chromatin on all scales remains to be fully understood. These and other cell biology questions with implications for both normal and diseased function will be explored by the application of the advanced imaging methods of this research program.

项目摘要 蜂窝环境既强大又复杂，这取决于结构组织 微米比例尺降低到纳米尺度，以及一大型阵列的动态时间依赖性 酶，细胞的纳米机及其在蛋白质，寡核苷酸和小分子上的工作。 可见的荧光显微镜已成为一种有用的工具，能够非侵入性探索细胞行为，但 常规成像的衍射限制分辨率严格限制了可在 规模低于200 nm的结构。因为细胞中的主要生物分子玩家在大小范围内 按照10 nm的顺序，在这种尺寸规模的生活系统中需要进行全面的测量。极好的- 分辨率显微镜，是基于单分子荧光成像和发射的控制 浓度或刺激的发射耗尽，通过访问纳米级解决了此问题 将信息定位到10-40 nm及以下。另外，单个的互补方法 分子跟踪可访问细胞组件的运动细节，例如电动机驱动 转运或DNA或RNA的运动。结合高级三维（3D）成像，单粒子 跟踪可以在高速上观察到特定的蜂窝玩家的全部运动。它 这是这项工作的主要力量，以开发和增强3D超分辨率成像和3D单粒子 通过突破方法的边界并发明克服新策略来跟踪细胞中的跟踪 技术局限性将导致固定和活细胞中前所未有的空间和时间信息。 Moerner实验室的研究广泛寻求解决超分辨率成像的局限性 通过物理和数学分析以及新的新细胞跟踪单粒子跟踪 方法。这里的深刻动机是提出一个基本问题：如何从 每个分子被最大化？开发了两个主要的新显微镜：大型成像 轴向使用瞳孔平面相调制和倾斜光板，以及使用的相关方法 低温单分子荧光定位，以注释冷冻电子断层扫描重建。 这项研究的方法论发展将应用于细胞中的各种关键问题 通过继续建立合作并通过与知名人士开发新的合作来生物学 生物学家。细菌，花椰菜牙齿牙齿菌仍然是细胞发育的有用模型系统 需要阐明生物分子的上层建筑和运动以了解不对称的起源 分配。 Toxoplasma Gondii寄生虫是另一种引人入胜的生物，需要超级探索 解决方法。在所有尺度上的染色质的组织仍有待完全理解。这些和其他 细胞生物学问题对正常功能和患病功能有影响 该研究计划的先进成像方法。