A New Method for Determining Dipole Moment Orientation of Single Molecules

A new imaging method is proposed to determine the three-dimensional dipole moment orientation of single fluorophore. Far-field microscopy can provide orientational information projected in the sample plane, while total internal reflection fluorescence microscopy (TIRFM) can offer the knowledge perpendicular to the surface because longitudinal electric-field components can be generated in total internal reflection geometry. By comparing fluorescence intensities measured with far-field epi-fluorescence microscopy and TIRFM, the exact information of single-fluorescent-molecule orientation is extracted. Detailed analysis of the method is given with a numerical example.