Single molecule polarization microscopy for investigating real time structural dynamics of biological macromolecules

To quantitatively monitor structural dynamics of individual biomolecules in real time, a single-molecule polarization microscopy technique has been developed. This novel approach combines the single molecule sensitivity of total internal reflection fluorescence microscopy with orientation measurements by time-resolved fluorescence polarization. With this technique, functionally relevant conformational changes of individual biomolecules can be continuously detected at 10 ms time resolution or better. Initial measurements, using site specific fluorescently labeled skeletal muscle myosin showed changes in the orientation of the light chain domain, which has been postulated to act as a lever arm during chemo-mechanical energy transduction.