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

项目摘要 蜂窝环境既强大又复杂，这取决于来自 微米级到纳米级，以及大量的 酶，细胞的纳米机器，以及它们对蛋白质、寡核苷酸和小分子的作用。 可见荧光显微镜一直是一种有用的工具，能够非侵入性地探索细胞行为，但 传统成像受衍射限制的分辨率严重限制了在计算机上获取的信息 尺度在~200纳米以下的结构。因为细胞中的主要生物分子在大小范围内 在生命系统中，这种大小的尺度需要进行10纳米量级的全面测量。超级- 分辨率显微镜，基于单分子荧光成像和发射控制 浓度，或受激发辐射耗尽，通过使人们能够接触到纳米级的物质来解决这个问题 位置信息向下延伸到10-40 nm区域及以下。此外，补充的方法是单一的-- 分子跟踪提供了对细胞组件运动细节的访问，例如马达驱动 DNA或RNA的运输或运动。结合先进的三维(3D)成像，单粒子 跟踪允许在他们的实际环境中以高速观察特定蜂窝播放器的全部运动。它 这项工作的主要目的是开发和增强3D超分辨率成像和3D单粒子 通过突破这两种方法的边界并发明新的策略来克服 技术限制，这将导致固定细胞和活细胞中前所未有的时空信息。 Moerner实验室的研究广泛地寻求解决超分辨率成像的局限性 以及通过物理和数学分析以及通过发明新的 方法：研究方法。这里的深层动机是问一个根本的问题：如何从 每一个分子都是最大化的？两种关键的新显微镜正在开发中：超大型3D成像 利用光瞳平面位相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用于低温电子断层扫描重建的注解。 这项研究的方法论发展将应用于细胞中的各种关键问题。 通过继续已建立的合作和通过与著名的 生物学家。这种细菌，新月杆菌，仍然是细胞发育的有用模型系统。 需要阐明生物分子的超结构和运动以了解不对称的起源 组织。弓形虫寄生虫是另一种令人着迷的生物，需要用超级... 解决方法。染色质在所有尺度上的组织仍有待完全了解。这些和其他 该应用程序将探索与正常和疾病功能有关的细胞生物学问题 这项研究计划的先进成像方法。

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.