Integrated Single-Molecule Fluorescence Sensitive Microscope

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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 .