Extending the temporal and spatial capabilities of single-molecule methods

项目摘要/摘要(最多30行) 本研究响应PAR-19-253《聚焦技术研究与开发》 旨在开创生物光学显微镜的新进展。方法，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)和超分辨显微镜使分子水平的研究成为可能 越来越复杂的活细胞。单分子方法使研究人员能够观察到动力学 用整体方法观察不到的路径和瞬变状态。尽管最近取得了进展，但现有的 光学探测器有其局限性。荧光蛋白在大小上与它们标记的蛋白和 快速漂白。胞浆蛋白的原位标记是可能的，但体外标记方法很多。 首选的，并且没有可靠的方法将这些蛋白质引入细胞的胞浆。 本研究将通过从根本上扩展工具箱来解决这些重大挑战 光学显微镜。Aim 1将开发新的方法来引入体外标记的有机蛋白质 染料直接进入细胞胞浆和染料标记的膜蛋白插入细胞 膜，从而扩大了光学探针在新的生物系统中的应用。这些 将使用方法将上转换纳米颗粒(UCNP)探针插入活细胞中，以允许长时间的- 以纳米空间分辨率对特定的单个蛋白质进行从几分钟到几个月的术语跟踪。 这项技术还将允许对具有多个基因的细胞进行可控的转基因。目标2将 从根本上提高SMFRET到≤100𝜇𝑠的时间分辨率，并发展SMFRET方法 它可以跨越细胞膜。AIM 3将扩展生物光学显微镜以访问 分子运动的时间和空间尺度。在这里，UCNP将被用来衡量连续 背根神经节神经元中动力蛋白对货物的转运 在900𝜇𝑚距离上的毫秒时间分辨率。使用等离子体光学探头，这项工作旨在 在活细胞中达到~100𝑛𝑠的时间分辨率和&lt；1𝑛𝑚的空间分辨率。 到4年资助期结束时，将展示一种能够引入 可控数量的纳米颗粒、蛋白质、多个基因和启动子进入数千个细胞 高存活率。细胞将被转移到适合的显微镜盖玻片或微流控细胞上 用于高分辨率光学显微镜。一台100𝜇𝑠的smFRET仪器将被用来 研究G蛋白偶联受体(GPCRs)的动力学。将建造另一台仪器来改进 亚纳米运动的时间分辨率高达~100𝑛𝑠。有了这个仪器，实时的 分子系统运动的可视化是可能的。

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.