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.

项目概要 细胞环境既强大又复杂，取决于结构组织 从微米尺度到纳米尺度，以及大量阵列的动态时间依赖性 酶，细胞的纳米机器，及其对蛋白质、寡核苷酸和小分子的作用。 可见荧光显微镜是一种能够非侵入性探索细胞行为的有用工具，但是 传统成像的衍射极限分辨率严重限制了可获得的信息 尺度低于~200 nm的结构。因为细胞中的主要生物分子参与者都在尺寸范围内 在 10 nm 量级，生命系统中需要对这个尺寸进行全面测量。极好的- 分辨率显微镜，基于单分子荧光成像和发射控制 浓度或受激发射损耗通过实现纳米级解决了这个问题 位置信息低至 10-40 nm 范围及以下。此外，单的补充方法 分子追踪提供了对细胞组件运动细节的访问，例如电机驱动 DNA 或 RNA 的运输或运动。结合先进的三维 (3D) 成像、单粒子 跟踪允许在实际环境中高速观察特定细胞玩家的完整运动。它 这项工作的主要目标是开发和增强 3D 超分辨率成像和 3D 单粒子 通过突破两种方法的界限并发明新的策略来克服细胞跟踪 技术限制，这将导致固定细胞和活细胞中前所未有的空间和时间信息。 莫尔纳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发明的细胞内单粒子跟踪 方法。这里的深层动机是提出一个基本问题：如何从 每个单个分子最大化？两种重要的新型显微镜正在开发中： 大尺寸 3D 成像 使用光瞳平面相位调制和倾斜光片的轴向范围，以及使用的相关方法 低温单分子荧光定位注释低温电子断层扫描重建。 这项研究的方法论进展将应用于细胞中的各种关键问题 通过继续已建立的合作并与知名人士开展新的合作 生物学家。新月柄杆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结构和运动以了解不对称的起源 分配。弓形虫寄生虫是另一种令人着迷的生物体，需要用超级技术来探索。 解决方法。所有尺度上染色质的组织仍有待充分了解。这些和其他 该应用程序将探索对正常和患病功能都有影响的细胞生物学问题 本研究项目的先进成像方法。