Rotational motions of macromolecules by single-molecule fluorescence microscopy

Several complementary techniques have been developed to determine average orientation, dynamics on multiple time scales, and concerted rotational motions of individual fluorescent probes bound to biological macromolecules. In both protein domains and nucleic acids, tilting and wobble are relevant to their functional mechanisms. Here we briefly review methods to detect angles and rotational motions of single fluorophores and give an example of three-dimensional, total internal reflection, single-molecule fluorescence polarization applied to actin as it is translocated by conventional muscle myosin.