Three-dimensional orientation of single molecules observed by far- and near-field fluorescence microscopy

We present a simple and straightforward method for determining absolute spatial orientations of transition dipole moments of single fluorescent molecules. Far-field polarization microscopy provides angles of the dipole moments projected in the plane of the sample. Optical field near total internal reflection surfaces has a strong component perpendicular to the sample and, for a given in-plane angle, provides unambiguous orientation of the molecular dipole moment. Experimentally, both excitation modes are alternated to monitor real-time conformational dynamics of tetramethylrhodamine molecules covalently attached to a quartz substrate.