Advanced Optical Microscopy Core

这个核心的主要目的是为研究人员提供一种新型光学仪器的设施 计划项目。核心的主要职能将是协助需要专业知识和 各个次级项目实验室没有专门的设备。核心成员将 为计划项目的不同成员提供使用光学陷阱的指导和专家帮助， 全内反射显微镜(TIRF)和全内反射偏振荧光显微镜 (PolTIRF)。这种最先进的共享设施提供了测量机械性能的方法 单个运动蛋白及其亚区荧光探针的空间定位 基因工程蛋白质。核心将通过测试几种方法进一步开发这些技术，以获得 来自TIRF图像的三维数据，通过将陷阱与PolTIRPF相结合来测量 单个马达分子在不同的负荷下通过酶循环，改善了长时间 项的空间分辨率和陷阱的刚度。单个野生型和工程化马达的体外分析 分子将毫不含糊地测量马达的基本属性，如刚性、方向变化、 作为应变的函数的一元力、步长和动力学，并将这些特性与结构特征相关联 蛋白质的含量。

The major aim of this Core is to provide a facility for novel optical instrumentation to the investigators of this Program Project. The primary function of the Core will be to assist in experiments that require expertise and specialized equipment not available in the individual sub-project laboratories. The members of the Core will provide guidance and expert help to the different members of the Program Project in using Optical Trapping, Total Internal Reflection Microscopy (TIRF) and Total Internal Reflection Polarized Fluorescence Microscopy (polTIRF). This state of the art shared facility provides methods for measuring the mechanical properties of individual motor proteins and the spatial orientation of fluorescent probes linked to subdomains of individual engineered proteins. The core will further develop these techniques by testing several methods for obtaining three dimensional data from TIRF images, by combining the trap with polTIRPF to measure the orientation of single motor molecules as they proceed through their enzymatic cycles under varying load, improve the long term spatial resolution and stiffness of the trap. In vitro analysis of single wild type and engineered motor molecules will unambiguously measure fundamental motor properties such as stiffness, orientation changes, unitary force, step length and kinetics as a function of strain and relate these properties to structural features of the protein.