Single-Molecule Florescence Spectroscopy at Hope College
Dr. Brent Krueger
This research involves the design and construction of a confocal microscope for single-molecule fluorescence-detected resonance energy transfer experiments. Single-molecule FRET is useful for determining structural characteristics and structural dynamics of systems under study. Donor and acceptor dyes will be attached to small biologically important molecules such as RNA, DNA, or protein. These samples will be used at very small (picomolar) concentrations, such that either zero or one 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, and the detection of the corresponding fluorescence originating from either the donor or acceptor will allow calculation of FRET efficiency. Experimentally determined FRET efficiencies will be compared to simulated FRET data calculated using MD simulation experiments. This comparison will lead to a better understanding of FRET and will eventually refine the process of extracting structural information from the raw FRET data.
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