Extending the temporal and spatial capabilities of single-molecule methods

项目概要/摘要（最多30行） 这项研究是对PAR-19-253“重点技术研究和开发”的回应， 旨在开创生物光学显微镜领域的新进展。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）和超分辨率显微镜使体外和 复杂性不断增加的活细胞。单分子方法允许研究人员观察动力学 路径和瞬态不可观察的散装方法。尽管最近取得了进展， 光学探针具有局限性。荧光蛋白的大小与它们标记的蛋白质相当， 光漂白很快。细胞质蛋白的原位标记是可能的，但体外标记方法很多， 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。 这项研究将通过从根本上扩大工具箱来解决这些重大挑战， 光学显微镜目的1将发展新的方法，在体外引入有机标记的蛋白质 染料直接进入细胞的胞质溶胶和染料标记的膜蛋白插入细胞 膜，从而将光学探针的应用扩展到新的生物系统。这些 方法将用于将上转换纳米颗粒（UCNP）探针插入活细胞中，以允许长时间的 以纳米空间分辨率对特定单个蛋白质进行数分钟至数月的长期跟踪。 该技术还将允许具有多个基因的细胞的可控转染。目标2将 从根本上提高smFRET的时间分辨率至≤ 100μ m，并开发smFRET方法 可以跨越细胞膜。目标3将扩展生物光学显微镜， 分子运动的时空尺度。在这里，UCNP将用于测量连续的 DRG神经元中动力蛋白的货物转运，能够用一个分子步骤分辨单个分子步骤 毫秒时间分辨率超过900毫秒的距离。使用等离子体光学探针，这项工作的目的是 在活细胞中实现约100微秒的时间分辨率和< 1微秒的空间分辨率。 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，多种基因和启动子进入1000个细胞， 较高的存活率。将细胞转移到显微镜盖玻片或合适的微流体细胞上。 用于高分辨率光学显微镜。将使用能够进行100μmFRET的仪器， 研究G蛋白偶联受体（GPCR）的动力学。将建造另一种仪器来改善 亚纳米运动的时间分辨率高达~ 100微秒。有了这个仪器， 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.