Single Molecule Rotational and Translational Diffusion Observed by Near-Field Scanning Optical Microscopy

Abstract

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.