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: 10627987
• 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/10627987
• 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; Disease Progression; Electron Microscopy; Environment; Enzymes; Fluorescence; Fluorescence Microscopy; Image; Label; Laboratories; Light; Mammalian Cell; Measurement; Measures; Methodology; Methods; Microscope; Motion; Motivation; Motor; Oligonucleotides; Optics; Organism; Parasites; Phase; Positioning Attribute; Proteins; Pupil; RNA; Research; Research Methodology; Resolution; Speed; Structure; Three-Dimensional Imaging; Time; Toxoplasma gondii; Work; 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; superresolution imaging; superresolution microscopy; tool; ultra high resolution

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 细胞环境既强大又复杂，既依赖于结构组织， 从微米尺度到纳米尺度，以及一个巨大的阵列的动态时间依赖性， 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。 可见荧光显微镜是一种能够非侵入性地探索细胞行为的有用工具，但 传统成像的衍射极限分辨率严重限制了可获得的信息， 结构在~200 nm以下的尺度上。因为细胞中的主要生物分子 在10纳米的量级上，在生命系统中需要对该尺寸尺度进行全面测量。超- 分辨率显微镜，或者基于单分子荧光成像和发射的控制， 浓度，或受激发射耗尽，已经解决了这个问题，使访问纳米 位置信息低至10-40 nm范围和更低。此外，单一的补充方法- 分子跟踪提供了对细胞成分运动的细节的访问 DNA或RNA的运输或运动。结合先进的三维（3D）成像，单颗粒 跟踪允许在特定蜂窝播放器的实际环境中以高速观察到它们的全部运动。它 是这项工作的主要推动力，以开发和增强三维超分辨率成像和三维单粒子 通过推动两种方法的界限并发明新的策略来克服 技术限制，这将导致固定和活细胞中前所未有的时空信息。 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发明新的 方法.这里的深层动机是问一个基本的问题：如何从 每一个分子都能被最大化？两种关键的新型显微镜正在开发中： 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，以注释低温电子断层扫描重建。 本研究的方法学发展将应用于细胞中的各种关键问题 生物学通过继续建立合作和发展新的合作与著名的 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超结构和运动，以了解不对称的起源， 师.弓形虫寄生虫是另一种迷人的生物体，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探讨的应用程序 这个研究项目的先进成像方法。

项目成果

Fast and parallel nanoscale three-dimensional tracking of heterogeneous mammalian chromatin dynamics.

快速和平行的纳米级三维跟踪异质性哺乳动物染色质动力学。

• DOI: 10.1091/mbc.e21-10-0514
• 发表时间: 2022-05-15
• 期刊: MOLECULAR BIOLOGY OF THE CELL
• 影响因子: 3.3
• 作者: Gustavsson, Anna-Karin;Ghosh, Rajarshi P.;Petrov, Petar N.;Liphardt, Jan T.;Moerner, W. E.
• 通讯作者: Moerner, W. E.

Exploring Cell Surface-Nanopillar Interactions with 3D Super-Resolution Microscopy.

• DOI: 10.1021/acsnano.1c05313
• 发表时间: 2022-01-25
• 期刊: ACS nano
• 影响因子: 17.1
• 作者: Roy AR;Zhang W;Jahed Z;Tsai CT;Cui B;Moerner WE
• 通讯作者: Moerner WE

Identification and demonstration of roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy.

• DOI: 10.1016/j.jsb.2022.107881
• 发表时间: 2022-09
• 期刊: JOURNAL OF STRUCTURAL BIOLOGY
• 影响因子: 3
• 作者: Perez, Davis;Dahlberg, Peter D.;Wang, Jiarui;Sartor, Annina M.;Borden, Julia S.;Shapiro, Lucy;Moerner, W. E.
• 通讯作者: Moerner, W. E.

Super-resolution Microscopy with Single Molecules in Biology and Beyond-Essentials, Current Trends, and Future Challenges.

• DOI: 10.1021/jacs.0c08178
• 发表时间: 2020-10-21
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Möckl L;Moerner WE
• 通讯作者: Moerner WE

Stimulated emission does not radiate in a pure dipole pattern.

受激发射不会以纯偶极模式辐射。

• 发表时间: 2024
• 期刊: ArXiv
• 作者: Barentine,AndrewES;Moerner,WE
• 通讯作者: Moerner,WE