This research involves the design and construction of a confocal microscope for single-molecule fluorescence-detected resonance energy transfer (FRET) experiments. Many biological systems exhibit multiple conformations, and those of most interest may be very rare, such that typical spectroscopic techniques that average over many molecules are unable to resolve them. Single-molecule FRET avoids this averaging and allows structural variation as well as millisecond and slower timescale dynamics to be resolved. 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. In this poster the design of a home-built single-molecule fluorescence microscope is presented along with early FRET data on multiple systems.

Single-molecule florescence spectroscopy using a home-built microscope

247th National Meeting (2014)

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