We apply the concept of tomography to polarization-sensitive optical microscopy of single fluorophores to determine the three-dimensional orientation of molecular absorption dipoles with isotropic sensitivity. Wide-field microscopy provides the opportunity to monitor simultaneously three-dimensional rotation and two-dimensional translation of many molecules in parallel. For orientation determination the molecules are illuminated from different directions on incidence with linearly polarized light. In each exposure the excitation along a particular projection of the absorption dipole on the electric field leads to a distinct fluorescnece intensity. Five exposures are sufficient to determine the full orientation of the fluoreophores. To demonstrate the potential of the method we determine the orientation and position and position of individual immobilized lipid membrane markers. The shot-noise-limited isotropic angular resolution is 2 deg. For time-resolved studies the bandwidth can be expanded up to 200 Hz.
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