CAREER: Target-locked single molecule spectroscopy and super-resolution microscopy

在化学系化学测量和成像项目的支持下，以及分子和细胞生物科学系分子生物物理学和细胞动力学集群的共同资助下，杜克大学的Kevin Welsher博士和他的团队正在寻求新的方法，通过高精度测量单分子的功能来增强对化学和生物过程的理解。具体来说，他们正在开发“目标锁定”显微镜工具，以真实的时间跟踪细胞内单个蛋白质和其他细胞物质的快速运动。在这个项目中开发的信息将帮助我们了解生命的规则，帮助我们看到生物细胞内发生了什么，无论是在高速和高分辨率。 Welsher小组还为“家庭光谱学家”开发了一套实验，旨在使用智能手机来说明光谱学和光学的原理。这些实验使用简单的红-绿-蓝值来说明各种样品的颜色分量。正在开发一个在线视频“可教的花絮”，目的是揭开经常被数学掩盖的化学概念的神秘面纱。最后，这项研究正在引导研究生进入显微镜领域，重点是通过学生从头开始建立的相对简单的数值模拟来学习显微镜。传统上，单个蛋白质的内部分子运动是通过单分子福斯特共振能量转移（smFRET）来研究的，这需要蛋白质被束缚在一个表面上，以便它保持在显微镜的焦点体积中。 Welsher小组正在开发目标锁定的smFRET，以实时探测未束缚的单个胞质蛋白，从而能够研究细胞拥挤和非平衡环境对快速蛋白质结构域运动的影响，这些运动逃避了传统的smFRET。 这项工作应该提供一个更完整的图片的分子基础的细胞货物运输提供同时测量的“拔河”运动的个别马达蛋白和本地纳米尺度的细胞骨架结构使用目标锁定超分辨率（TL-SR）显微镜。TL-SR的高速、高精度跟踪和局部亚衍射成像可以区分现有的货物贩运模式。该CAREER提案的更广泛影响集中在使各级学生都能接触到显微镜和光谱学。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, and co-funding from the Molecular Biophysics and Cellular Dynamics Clusters in the Division of Molecular and Cellular Biosciences, Dr. Kevin Welsher and his group at Duke University are seeking new ways to enhance understanding of chemical and biological processes by measuring the function of single molecules with high precision. Specifically, they are developing "target-locked" microscopy tools to follow the rapid motions of single proteins and other cellular matter within cells in real time. The information developed in this project will help us Understand the Rules of Life by helping us see what goes on inside biological cells both at high speed and with high resolution. The Welsher group is also developing a set of experiments for the "home spectroscopists" aimed at using smartphones to illustrate the principles of spectroscopy and optics. These experiments illustrate the color components of various samples using simple red-green-blue values. Online videos "Teachable Tidbits" are being developed with an aim of demystifying chemistry concepts that are frequently obscured by mathematics. Finally, the research is guiding graduate students into the field of microscopy, with an emphasis on learning microscopy through relatively simple numerical simulations that the students build from scratch. Internal molecular motions of single proteins have traditionally been investigated by single molecule Forster Resonant Energy Transfer (smFRET), which requires that the protein be tethered to a surface so that it remains in the focal volume of a microscope. The Welsher group is developing target-locked smFRET to probe untethered single cytosolic proteins in real-time, thereby enabling studies of the effect of cellular crowding and non-equilibrium environments on fast protein domain motions which have eluded traditional smFRET. The work should provide a more complete picture of the molecular underpinnings of cellular cargo transport by providing simultaneous measurement of "tug-of-war" motions of individual motor proteins and the local nanometer-scale cytoskeletal structure using target-locked super-resolution (TL-SR) microscopy. The high-speed, high-precision tracking and local sub-diffraction imaging of TL-SR may enable distinction between existing models of cargo trafficking. The broader impacts of this CAREER proposal focus on making microscopy and spectroscopy accessible to students at all levels.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.