Orientational imaging of single molecules by using azimuthal and radial polarizations

Three-dimensional molecular orientations of single fluorescence molecules in polymeric thin films were measured by focused azimuthally and radially polarized light, in which we found that the fluorescence intensity was dependent on the depth position of the molecule with respect to the film surface. We found that the fluorescence intensity for a molecule which is 80 nm deep in the film excited by radial polarization is appreciably larger when compared with the fluorescence intensity for a molecule which is also excited by radial polarization but which is closer to the polymer/air interface, a feature which leads to different fluorescence intensities, under excitation by radial polarization, for molecules with the same polar orientation but with different depths inside the film. We also found that the variation of fluorescence intensity from a molecule inside an 80 nm film in radial polarization is appreciably larger compared with one in azimuthal polarization. These findings were confirmed by comparing experiments using different thickness films with theoretically calculated electric field distributions.