Extending the temporal and spatial capabilities of single-molecule methods

扩展单分子方法的时间和空间能力

• 批准号: 10281044
• 负责人: Steven Chu
• 金额: $ 57.86万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10281044
• 关键词: 3-Dimensional; Address; Animals; Axon; Biological; Cell membrane; Cells; Cellular Structures; Cryoelectron Microscopy; Cytosol; Data; Detection; Development; Devices; Dyes; Dynein ATPase; Endocytosis; Enzymes; Feedback; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; GTP-Binding Proteins; Genes; Hour; Image; In Situ; In Vitro; Individual; Kinetics; Label; Ligand Binding; Light; Measurement; Measures; Membrane Proteins; Methods; Microfluidics; Microscope; Microscopy; Modeling; Molecular; Molecular Motors; Molecular Structure; Motion; Movement; Neurons; Optics; Organism; Pathway interactions; Photobleaching; Photons; Physics; Power stroke; Process; Proteins; Research; Research Personnel; Resolution; Roentgen Rays; Side; Signaling Protein; Spatial Distribution; Surface; Survival Rate; Synapses; System; Technology; Testing; Time; Tissues; Transfection; Vesicle; Virus; Visualization; Work; base; biological research; biological systems; biophysical techniques; cell injury; extracellular; improved; in vivo; infrared microscopy; insight; instrument; instrumentation; laser tweezer; millimeter; millisecond; molecular imaging; molecular scale; nanometer; nanoparticle; nanoscale; new technology; novel; particle; plasmonics; prevent; professor; promoter; prototype; receptor; response; single molecule; single-molecule FRET; technique development; temporal measurement; tool

Project Summary / Abstract (30 line maximum) This research, in response to the PAR-19-253, “Focused Technology Research andDevelopment,” aims to pioneer new advances in biological optical microscopy. Methods such as the development of fluorescent proteins, single molecule fluorescence detection, single molecule fluorescence resonance energy transfer (smFRET) and super-resolution microscopy enabled molecular level study of in vitro and live cells of increasing complexity. The single molecule methods allowed researchers to observe kinetic pathways and transient states unobservable with bulk methods. Despite recent advances, the existing optical probes have limitations. Fluorescent proteins are comparable in size to the proteins they label and photobleach quickly. In situ labeling of cytosol proteins is possible, but in vitro labeling methods are much preferred and there are no reliable methods to introduce these proteins into cytosol of cells. This research will address these grand challenges by fundamentally expanding the toolbox of optical microscopy. Aim 1 will develop new methods to introduce proteins labeled in vitro with organic dyes directly into the cytosol of cells and the insertion of dye-labeled membrane proteins into cell membranes, thereby expanding the application of optical probes to new biological systems. These methods will be used to insert up-converting nanoparticle (UCNP) probes into live cells to allow the long- term tracking of specific individual proteins from minutes to months with nanometer spatial resolution. This technology will also allow the controllable transfection of cells with multiple genes. Aim 2 will fundamentally improve the temporal resolution of smFRET to ≤ 100𝜇𝑠 and develop smFRET methods that can span across cell membranes. Aim 3 will extend biological optical microscopy to access the temporal and spatial scales of molecular motion. Here, UCNPs will be used to measure the continuous transport of cargos by dynein in DRG neurons capable of resolving single molecular steps with one millisecond time resolution over a distance of 900 𝜇𝑚. Using plasmonic optical probes, this work aims to achieve ~ 100 𝑛𝑠 time resolution and < 1 𝑛𝑚 spatial resolution in live cells. By the end of the 4-year funding period, a device will be demonstrated that is able to introduce controllable numbers of nanoparticles, proteins, and multiple genes and promoters into 1000s of cells with high survival rates. The cells will be transferred onto microscope coverslips or microfluidic cells suitable for high-resolution optical microscopy. An instrument capable of 100𝜇𝑠 smFRET will have been used to study the dynamics of G-protein couped receptors (GPCRs). Another instrument will be built to improve the time resolution of sub-nanometer movement to by up to ~ 100 𝑛𝑠. With this instrument, the real-time visualization of the motion of molecular systems may be possible.

项目摘要/摘要（最多 30 行） 这项研究响应 PAR-19-253“重点技术研究与开发” 旨在开拓生物光学显微镜方法的新进展，例如开发 荧光蛋白、单分子荧光检测、单分子荧光共振 能量转移（smFRET）和超分辨率显微镜使体外和体外的分子水平研究成为可能 日益复杂的活细胞使研究人员能够观察动力学。 尽管最近取得了进展，但现有的方法仍然无法观察到路径和瞬态。 光学探针具有其局限性，其大小与它们标记的蛋白质相当。 细胞质蛋白的原位标记是可能的，但体外标记方法有很多。 优选的，并且没有可靠的方法将这些蛋白质引入细胞的胞质溶胶中。 这项研究将从根本上扩展工具箱来应对这些重大挑战 目标 1 将开发新方法来引入体外有机标记的蛋白质。 染料直接进入细胞的细胞质，并将染料标记的膜蛋白插入细胞中 膜，从而将光学探针的应用扩展到新的生物系统。 方法将用于将上转换纳米颗粒（UCNP）探针插入活细胞中，以允许长期 以纳米空间分辨率对特定个体蛋白质进行从几分钟到几个月的长期跟踪。 这项技术还可以实现 Aim 2 细胞的可控转染。 从根本上将smFRET的时间分辨率提高到≤100𝜇𝑠并开发smFRET方法 可以跨越细胞膜的 Aim 3 将扩展生物光学显微镜的范围。 在这里，UCNP 将用于测量连续的分子运动。 DRG 神经元中的动力蛋白运输货物，能够用一个解决单分子步骤 这项工作的目的是使用等离子体光学探针在 900 𝜇𝑚 的距离上实现毫秒时间分辨率。 在活细胞中实现约 100 𝑛𝑠 时间分辨率和 < 1 𝑛𝑚 空间分辨率。 到 4 年资助期结束时，将展示一种能够引入 将可控数量的纳米颗粒、蛋白质以及多个基因和启动子注入数千个细胞中 高存活率。细胞将被转移到合适的显微镜盖玻片或微流体细胞上。 用于高分辨率光学显微镜的仪器将被用于 100𝜇𝑠 smFRET。 研究 G 蛋白偶联受体 (GPCR) 的动力学将建立另一个仪器来改进。 使用该仪器，亚纳米运动的时间分辨率高达 ~ 100 𝑛𝑠。 分子系统运动的可视化是可能的。