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） 这项研究回应了PAR-19-253，“重点技术研发”， 旨在先驱生物光学显微镜的新进展。诸如开发的方法 荧光蛋白，单分子荧光检测，单分子荧光共振 能量转移（SMFRET）和超分辨率显微镜可以实现体外和 复杂性增加的活细胞。单分子方法允许研究人员观察动力学 途径和瞬态状态无法使用大量方法进行观察。尽管最近进步，但现有 光学问题有局限性。荧光蛋白的大小与它们标记的蛋白质相当，并且 迅速光漂白。可以原位的细胞质蛋白标记，但体外标记方法很大 首选，并且没有可靠的方法将这些蛋白质引入细胞细胞溶胶。 这项研究将通过从根本扩展工具箱来解决这些巨大的挑战 光学显微镜。 AIM 1将开发新方法，以引入有机体外标记的蛋白质 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 膜，从而将光学问题的应用扩展到新的生物系统中。这些 方法将用于将纳米颗粒（UCNP）问题插入活细胞中，以允许长期 通过纳米空间分辨率从几分钟到几个月的特定单个蛋白质进行术语跟踪。 该技术还将允许使用多个基因转染细胞。 AIM 2意志 从根本上提高了SMFRET的暂时分辨率为≤100𝜇𝑠并开发SMFRET方法 可以跨越细胞膜。 AIM 3将扩展生物光学显微镜以访问 分子运动的临时和空间尺度。在这里，UCNP将用于测量连续 DRNNEIN在DRG神经元中通过DRNNEIN运输的Cargos，能够用一个分子步骤解决单分子步骤 毫秒的时间分辨率超过900 𝜇𝑚。使用等离子光学问题，此工作旨在 在活细胞中实现〜100𝑛𝑠时间分辨率和<1𝑛𝑚空间分辨率。 到4年筹资期结束时，将证明该设备能够引入 可控制数量的纳米颗粒，蛋白质以及多个基因和启动子到1000 s的细胞中 高存活率。细胞将被转移到显微镜盖玻片或微流体细胞上 用于高分辨率光学显微镜。能够使用100 smfret的仪器 研究G蛋白双门受体（GPCR）的动力学。将建造另一个工具以改进 子纳米计的时间分辨率最高为〜100𝑛𝑠。使用该乐器，实时 可以看到分子系统运动的可视化。