Measuring the Orientation and Re-orientation Dynamics of Single Molecules

Kenneth D. Weston and Lori S. Goldner
Physics Laboratory, NIST

Single molecule fluorescence techniques for the study of macromolecular motion and conformational dynamics are useful for understanding the mechanisms of protein action (i.e., protein catalysts, protein pumps, and motor proteins) and protein folding. Molecular motion is often inferred from fluctuations in the fluorescence intensity, spectrum, or lifetime of a single molecule. Additional information can be acquired by monitoring the orientation of the excitation and/or emission dipole of a fluorophore. Here we report on the direct observation of molecular motion from time-resolved measurements of absorption dipole orientation on single fluorophores embedded in polymeric matrices. Using a polarization modulation technique, we record both the position and dipole orientation of all molecules in an image. For each detected molecule, the orientation as a function of time is also monitored on a millisecond time-scale. These studies reveal large and frequent dipole re-orientations for dye molecules in a variety of polymer matrices. Further applications of this technique to study the rotational dynamics of dye molecules in biomimetic films, in porous media, and linked to proteins will be discussed.