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. In successive images the fluorescence of single molecules was followed over about 1 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 6G molecules on glass during successive images, a diffusion constant of (6.7 ± 4.5) × 10-15 cm2/s was determined. The orientation of the in-plane emission dipole of all molecules in 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 DiI molecules in PMMA over 1 h, we found rotation of less than 10° 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.

Depolarized hyper Rayleigh scattering of /wtf-nitroaniline {Civ symmetry) and nitrocalix[4]arene (CAV symmetry) in solution has been measured. Using linearly and circularly polarized fundamental radiation information about the ratios between the several hyperpolarizability tensor components, including their sign, was obtained. Results are consistent with the theory developed for both symmetry groups. Comparison between experimental depolarization ratios and ratios obtained from ab initio calculated hyperpolarizability tensor components shows good agreement.

The dynamics of a tuning fork shear-force feedback system, used in a near-field scanning optical microscope, have been investigated. Experiments, measuring amplitude and phase of the tuning fork oscillation as a function of driving frequency and tip-sample distance, reveal that the resonance frequency of the tuning fork changes upon approaching the sample. Either amplitude or phase of the tuning fork can be used as distance control parameter in the feedback system. Using amplitude a second-order behavior is observed while with phase only a first-order behavior is observed, and confirmed by numerical calculations. This first-order behavior results in an improved stability of our feedback system. A sample consisting of DNA strands on mica was imaged which showed a height of the DNA of 1.4 nm.

Tuning forks as tip–sample distance detectors are a promising and versatile alternative to conventional cantilevers with optical beam deflection in noncontact atomic force microscopy (AFM). Both theory and experiments are presented to make a comparison between conventional and tuning-fork-based AFM. Measurements made on a Si(111) sample show that both techniques are capable of detecting monatomic steps. The measured step height of 0.33 nm is in agreement with the accepted value of 0.314 nm. According to a simple model, interaction forces of 30 pN are obtained for the tuning-fork-based setup, indicating that, at the proper experimental conditions, the sensitivity of such an instrument is competitive to conventional lever-based AFM.

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