A.G.T. Ruiter

Tuning fork shear-force feedback

Investigations have been performed on the dynamics of a distance regulation system based on an oscillating probe at resonance. This was examined at a tuning fork shear-force feedback system, which is used as a distance control mechanism in near-field scanning optical microscopy. In this form of microscopy, a tapered optical fiber is attached to the tuning fork and scanned over the sample surface to be imaged. Experiments were performed measuring both amplitude and phase of the oscillation of the tuning fork as a function of driving frequency and tip-sample distance. These experiments reveal that the resonance frequency of the tuning fork changes upon approaching the sample. Both the amplitude and the phase of the tuning fork can be used as distance control parameter in the feedback system. Using the amplitude a second-order behavior is observed, while with phase only a first-order behavior is observed. Numerical calculations confirm these observations. This first-order behavior results in an improved stability of the feedback system. As an example, a sample consisting of DNA strands on mica was imaged which showed the height of the DNA as 1.4 +/- 0.2 nm.

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near‐field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1. 77. p1–79 and the plasmid probe α‐spectrin. The hybridization signals are very well resolved in the near‐field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.

Detection of fluorescence in situ hybridization on human metaphase chromosomes by near-field scanning optical microscopy

Fluorescence in situ hybridization signals on human metaphase chromosomes are detected by a near-field scanning optical microscope. This makes it possible to localize and identify several fluorescently labeled genomic DNA fragments on a single chromosome with a resolution superior to traditional fluorescence microscopy. Several nucleic acid probes have been used. The hybridization signals are well resolved in the near-field fluorescence images, and the exact location of the probes can be correlated to the topography as it is afforded by the shear-force feedback.

Near-field optical and shear-force microscopy of single fluorophores and DNA molecules

Photodynamics of individual fluorescence molecules has been studied using an aperture-type near-field scanning optical microscope with two channel fluorescence polarisation detection and tuning fork shear-force feedback. The position of maximum fluorescence from individual molecules could be localised with an accuracy of 1 nm. Dynamic processes such as translational and rotational diffusion were observed for molecules adsorbed to a glass surface or embedded in a polymer host. The in-plane molecular dipole orientation could be determined by monitoring the relative contribution of the fluorescence signal in the two perpendicular polarised directions. Rotational dynamics was investigated on 10 ms-1000 s timescale. Shear-force phase feedback was used to obtain topographic imaging of DNA fragments, with a lateral and vertical resolution comparable to scanning force microscopy. A DNA height of 1.4 nm has been measured, an indication of the non-disturbing character of the shear force mechanism.

Optical contrast in near-field techniques

In this paper results of experiments with a scanning near-field optical microscope with shear-force feedback are presented. The setup will be described and the shear-force signal as function of distance is shown. Images of latex spheres and Langmuir- Blodgett layers of pentacosa-acid with about 100 nm lateral resolution are presented which show a true optical contrast due to fluorescence and polarization.

Fluorescence Lifetime Contrast Combined with Probe Microscopy

Fluorescence lifetime imaging is combined with atomic force microscopy in an integrated scanning microscope using a silicon-nitride probe. The time decay of fluorescence is measured at each image position with a resolution of 50 ps by time correlated single photon counting using a frequency doubled mode-locked Ti-Sapphire laser and fast electronics. Images of a mixture of fluorescence labelled latex spheres are presented where the lifetime contrast of the different spheres can be directly correlated to the topography as detected by force microscopy.