Polarization spectroscopy of single fluorescent molecules

Polarization spectroscopy of single fluorescent molecules is used to probe their rotational dynamics. When a molecule is immobilized on a dry surface, its in-plane dipole orientation is precisely determined by utilizing its transition dipole moment. An angular offset between the absorption and the emission dipoles was detected from a single fluorophore revealing its binding geometry to the surface. In an aqueous environment, DNA-tethered fluorophores display dynamics that are well-described by a hindered rotational diffusion model. A detailed description of the model is given, including calculations to estimate depolarization effects resulting from the high numerical aperture objective used to collect fluorescence photons. Protein-conjugated fluorophores display very distinct dynamics with continuous evolution of the rotational profile, possibly reflecting fluctuations in the polypeptide chain, When protein-conjugated fluorophores are allowed to freely diffuse in solution, it is possible to determine the fluorescence polarization anisotropy of each molecule that traverses the laser beam. The anisotropy values could, in principle, be used to identify conformational states of single molecules without the potential artifacts associated with surface immobilization.