Fluorescence Spectroscopy Core (David Millar, Director)
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The Fluorescence Spectroscopy Core provides state-of-the-art instrumentation and expertise to support a wide range of fluorescence spectroscopic measurement techniques, including steady-state, time-resolved and single-molecule methods. These resources support the development of new imaging technologies to visualize the assembly and disassembly of virus-host complexes and to identify homogeneous complexes suitable for structure determination. The Core is equipped with a steady-state fluorimeter and attached rapid mixing cell for stopped-flow kinetic measurements, a picosecond laser and time-correlated single photon counting system for fluorescence lifetime measurements, a TIRF-based single molecule microscope for FRET and dual color imaging of immobilized molecules, a confocal microscope for FRET measurements of freely diffusing single molecules, and a two-photon excitation microscope for FCS and coincidence measurements of diffusing molecules.
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RNP Assembly at the Single-molecule Level. An RRE fragment capable of binding 4 Rev monomers was immobilized on a quartz surface, fluorescently labeled Rev (grey oval and star) was introduced, and individual complexes monitored over time in a single-molecule TIRF microscope (left). Numbers above each segment of the trajectory indicate the number of bound Rev molecules. Analysis of multiple trajectories yields rate constants for each step of assembly (right). This work was performed in the Millar lab, in a collaboration with the Williamson lab.
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