Integrated Single-Molecule Fluorescence Sensitive Microscope

集成单分子荧光灵敏显微镜

• 批准号: 431773745
• 金额: --
• 依托单位: Universität Ulm
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/431773745?language=en
• 关键词: Integrated; Single; Molecule; Fluorescence; Sensitive

The requested integrated single-molecule fluorescence sensitive microscope will allow for the observation of the dynamical changes of conformation of macro-molecules and with this will help elucidating the molecular mechanism of important biological machinery. By combining well established single-molecule fluorescence technologies with cutting edge techniques of single spin detection and manipulation, the instrument will serve both, as a work-horse for a larger number of experiments aimed to unravel protein function in genome expression and maintenance, but also as a platform to develop novel ultra-sensitive imaging techniques using micro-wave manipulation of single spin states. Key requirements are:1.) The capability to measure single-molecule fluorescence of immobilised complexes or attached to the membranes of living cells using single-molecule FRET and in particular three colour smFRET in a TIRF, HILO, or Epi fluorescence geometry.2.) The ability to increase the time resolution of fluorescence excitation and detection into the single µs regime by switching in an automated fashion from TIRF/HILO illumination to a confocal excitation scheme, as well as from camera based detection to time-correlated single photon counting (TCSPC), enabling also fluorescence correlation spectroscopy (FCS) and fluorescence cross correlation spectroscopy (FCCS) experiments.3.) The ability to manipulate spins using micro-wave pulses in order to perform DEER experiments with single-molecule sensitivity on biological complexes labelled site specifically .

所要求的集成单分子荧光敏感显微镜将允许观察大分子构象的动态变化，并与这将有助于阐明重要的生物机械的分子机制。通过将成熟的单分子荧光技术与单自旋检测和操作的尖端技术相结合，该仪器将作为大量旨在揭示基因组表达和维护中蛋白质功能的实验的主力，同时也作为一个平台，开发使用微波操作单自旋状态的新型超灵敏成像技术。关键要求是：1.） 使用单分子FRET，特别是TIRF、HILO或Epi荧光几何中的三色smFRET，测量固定化复合物或附着于活细胞膜的单分子荧光的能力。 通过以自动方式从TIRF/HILO照明切换到共焦激发方案，以及从基于相机的检测切换到时间相关单光子计数（TCSPC），将荧光激发和检测的时间分辨率提高到单μs范围的能力，还可以进行荧光相关光谱（FCS）和荧光互相关光谱（FCCS）实验。 使用微波脉冲操纵自旋的能力，以便在生物复合物标记位点上进行具有单分子灵敏度的DEER实验。