Home-Built Single-Molecule Florescence Spectroscopy
Dr. Brent Krueger
This research involves the design and construction of a confocal microscope for single-molecule fluorescence-detected resonance energy transfer (FRET) experiments. Avoiding the averaging inherent to bulk techniques, single-molecule FRET spectroscopy allows us to resolve structural characteristics and dynamics of biological systems that often exist in multiple conformations. Donor and acceptor dyes will be attached to small biologically important molecules such as RNA, DNA, or protein. These samples will be used at low (femtomolar) concentrations, such that either zero or one molecule will be in the focal volume of the microscope at any given time. As the system passes through the focal volume it will be excited by the laser light. Detection of the corresponding fluorescence, originating from either the donor or acceptor, will allow calculation of the FRET efficiency. A function of the distance between the dye molecules, the experimentally-determined FRET efficiencies will be compared to simulated FRET data calculated using molecular dynamics simulations. This comparison will lead to a better understanding of FRET and will improve the process of extracting structural information from raw FRET data.
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