Three-dimensional orientation measurements of symmetric single chromophores using polarization microscopy

a complete understanding of any complex molecular system generally requires a knowledge of the three-dimensional (3D) orientation of its components re lative both to each other, and to directional perturbations such as interfa ces and electromagnetic fields, Far-field polarization microscopy is a conv enient and widespread technique for detecting and measuring the orientation of single chromophores. But because the polarized electromagnetic field th at is used to probe the system lacks a significant longitudinal component, it was thought that, in general, only 2D orientation information could be o btained(1-3). Here we demonstrate that far-field polarization microscopy ca n yield the 3D orientation of certain highly symmetric single chromophores (CdSe nanocrystal quantum dots in the present case). The key requirement is that the chromophores must have a degenerate transition dipole oriented is otropically in two dimensions, which gives rise to a perpendicular 'dark ax is' that does not couple to the light field. By measuring the fluorescence intensity from the dipole as a function of polarization angle, it is possib le to calculate both the tilt angle between the dark axis and the sample pl ane, as well as the in-plane orientation, and hence obtain the 3D orientati on of the chromophore.