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

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 Fluorescence Imaging of Genetic Material: Toward the Molecular Limit

Chromosomes, DNA, and single fluorescent molecules are studied using an aperture-type near-held scanning optical microscope with tuning fork shear force feedback. Fluorescence in situ hybridization labels o­n repetitive and single copy probes o­n human metaphase chromosomes are imaged with a width of 80 nm, allowing their localization with nanometer accuracy, in direct correlation with the simultaneously obtained topography. Single fluorophores, both in polymer and covalently attached to amino- silanized glass, are imaged using two-channel fluorescence polarization detection. The molecules are selectively excited according to their dipole orientation. The orientation of the dipole moment of all molecules in o­ne image could be directly determined. Rotational dynamics o­n a 10-ms to 100-s timescale is observed. Finally, shear force imaging of double-stranded DNA with a vertical sensitivity of 0.2 nm is presented. A DNA height of 1.4 nm is measured, which indicates the nondisturbing character of the shear force mechanism

Multi-detection and polarisation contrast in scannning near-field optical microscopy in reflection

A new type of NSOM probe has been developed, with a design based o­n the probes used in Atomic Force Microscopy. The probe consists of a cantilever with at its end a conical tip. This tip has been metal-coated to provide an aperture. With the cantilevered probe, the problem of breaking of the tip due to high normal forces is solved. In operation, the tip is scanned in contact with the sample while regulating the force between the tip and the sample with a beam deflection technique, which allows to simultaneously make an optical and a topographical image of the sample. The probes are made using micromechanical techniques, which allows batch fabrication of the probes. Testing of the probes is done in a transmission NSOM set-up in which the sample is scanned while the tip and the optical path are kept fixed. Using an opaque sample with submicron holes, the new probes have been tested, resulting an optical image with a simultaneously measured topographical image

Near-field Optical Microscopy

Near-field scanning optical microscopy (NSOM) is one of the most recent scanning probe techniques. In this technique, an optical probe is brought in the vicinity of the sample surface, in the near-field zone. The microscope can either work in illumination mode, in which the probe consists of a sub-wavelength light source, or in collection mode, in which the probe acts as a sub-wavelength detector. By scanning the probe over the sample surface and measuring the optical signal at each position, an optical image can be created. Because the probe has to be kept in the near-field zone at constant distance to the sample, in order to avoid intensity changes, a probe-sample distance regulation scheme is used to maintain a constant distance between probe and sample. The application of a distance regulation scheme results in the capability to measure a topographical image simultaneously with the optical image, an important asset of a near-field scanning optical microscope. This thesis reports the development of two types of illumination mode near-field scanning optical microscopes. The microscopes use aperture type near-field probes, consisting of a sub-wavelength aperture which is illuminated from one side. The light transmitted through the aperture serves as sub-wavelength light source illuminating the sample. Chapter 1 presents a brief introduction into the theory of optical image formation, leading to the resolution limit in conventional far-field optical microscopy. A near-field scanning optical microscope overcomes this limit by probing the near-field over the sample surface. A short overview of the instrumental and experimental accomplishments in nearfield optical microscopy is given in section 1.4. One of the most important parts of the near-field scanning optical microscope is the optical probe. In the experiments metal coated tapered fibers as well as newly designed cantilever probes, with a subwavelength aperture, have been used. Chapter 2 describes the fabrication process and emission characteristics of metal coated tapered optical fibers. Additionally, the micromechanical fabrication of a new type of probe, based on atomic force microscope probes, is described. The probe consists of a silicon nitride cantilever with a solid transparent conical tip. The probes are tested in a newly built near-field scanning optical microscope system. Although the prospects of using cantilever type probes are good, fiber probes are still favored because of the superior optical properties of the aperture. The distance regulation scheme in a fiber based near-field microscope, the shear-force control mechanism, is examined in chapter 3. The dynamics of this shear-force feedback system, based on piezoelectric quartz tuning forks, has been investigated. In this system the fiber is attached to the tuning fork and excited externally at its resonance frequency. Experiments reveal that the resonance frequency of the tuning fork changes upon approaching the sample. Both amplitude and phase of the oscillation 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. The topography of a sample consisting of DNA strands on mica was imaged using phase feedback. A near-field scanning optical microscope with two polarization detection channels, operating with tuning fork shear-force feedback, has been used to observe rotational and translational diffusion of single molecules. The molecules were dispersed on glass or embedded in polymer. In successive images the fluorescence of single molecules was followed over about one hour, with 10 ms integration time, until photodissociation. 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. Different sets of molecules could be selectively excited by rotating the excitation polarization. Monitoring DiI molecules in PMMA over one hour, rotation of less than 10 degrees for the majority of molecules was found, while incidental fast rotation and transition to a dark state occurred. The fluorescence intensity was observed to be molecule dependent, which is an indication for out-of-plane orientation and different local photophysical environment. Interactions between sample and probe, other than due to the light source character of the probe, have been observed in some of the single molecule experiments. Chapter 5 shows some examples of these interactions, such as sample manipulation by the probe and fluorescence quenching. Finally, the results in this thesis are discussed in a broader perspective and an outlook into future developments in near-field optical microscopy is given