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

 描述（由申请人提供）：细胞环境既强大又复杂，既取决于从微米尺度到纳米尺度的结构组织，也取决于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作用。可见荧光显微镜已经成为一种能够非侵入性地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以下的尺度上可获得的结构信息。由于细胞中的主要生物分子参与者的尺寸范围在10 nm左右，因此在生命系统中需要在此尺寸尺度上进行测量。超分辨率显微镜，无论是基于单分子荧光成像和控制的发射浓度，或受激发射耗尽，已经解决了这个问题，使访问空间分辨率下降到10-40 nm制度和以下。此外，单分子跟踪的补充方法提供了对细胞成分运动细节的访问，例如分子马达或DNA或RNA的运动。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运动员的完整运动。这项工作的主要目的是通过推动这两种方法的界限并发明新的策略来克服关键限制，从而开发和增强细胞中的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时间信息。Moerner实验室的研究广泛地解决了超分辨率成像和细胞中单粒子跟踪的局限性。一个关键的工具涉及使用宽视场显微镜的光瞳平面修改来提供高级功能，例如在前所未有的轴向范围内进行3D成像或在单分子水平上进行分子取向成像。这里的深层动机是问一个基本问题：如何最大限度地利用每个分子的信息，既要测量新的变量，又要检查过程的各个方面，并发明新的方法来消除任何系统误差。本研究的方法学发展将通过继续与知名生物学家建立合作并开发新的合作来应用于细胞生物学中的各种关键问题。细菌，新月柄杆菌，仍然是一个强大的模型系统，需要阐明的超结构和生物分子的运动，以了解不对称分裂的起源。初级纤毛是一个微小但重要的细胞器，它充满了蛋白质的运动和相互作用，需要在纳米尺度上进行探索。染色质在所有尺度上的组织仍有待完全理解。这些和其他细胞生物学问题与正常和病变功能的影响将是本研究计划的先进成像方法的应用的重点。

 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.