Single-molecule orientations determined by direct emission pattern imaging

We demonstrate a new method of determining the three-dimensional dipole orientations of single molecules by direct imaging of the emission patterns in the back focal plane of a high-numerical-aperture objective lens. We compare the reconstructed emission-dipole orientations with a previously established method of absorption-dipole mapping. We find that, for a given number of emitted photons, emission pattern imaging provides better accuracy (1°–2°) than absorption-dipole mapping of single molecules. Compared with some other methods for emission-dipole mapping, the presented method is (1) less sensitive to optical aberrations and adjustment and (2) data analysis is simplified because radiation patterns can be expressed in a simple analytical form.