Single molecule rotational and translational diffusion observed by near-field scanning optical microscopy

We have observed rotational and translational diffusion of single molecules using a near-field scanning optical microscope with two polarization detection channels. The measurements were performed under ambient conditions with the molecules dispersed on glass or embedded in polymer. Tn successive images the fluorescence of single molecules was followed over about I h, with 10 ms integration time, until photodissociation, The position of single molecular fluorescence could be located with an accuracy of 1 nm. From the lateral diffusion of Rhodamine SG molecules on glass during successive images, a diffusion constant of (6.7 +/- 4.5) x 10(-15) cm(2)/s was determined, The orientation of the in-plane emission dipole of all molecules irt one image could be directly determined with an accuracy of a few degrees by simultaneous detection in two perpendicular polarization directions. By rotating the excitation polarization we could selectively excite different sets of molecules and compare their in-plane absorption and emission dipole orientation. Monitoring Dil molecules in PMMA over 1 h, we found rotation of less than 10 degrees for the majority of molecules, while incidental fast rotation and transition to a dark state occurs. The fluorescence intensity was observed to be molecule dependent, which is an indication for out-of-plane orientation and different local photophysical environment.