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

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

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

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

Biological Applications of Near-field Optical Microscopy

Presents several biological applications of near field optical microscopy, in combination with force microscopy. Aperture near field scanning optical microscopy (NSOM) with fluorescence detection gives (bio)chemical specificity and orientational information, in addition to the simultaneously acquired force image. This technique has large potential for DNA sequencing, molecular organization in monolayers, and study of the role of the cytoskeleton in cellular mobility in cell growth, cell migration, formation of protrusions, etc. Fluorescence NSOM gives high resolution on flat, not too deep surfaces. Fluorescence NSOM induces virtually no bleaching, as opposed to confocal fluorescence microscopy. Bright field NSOM in transmission generally yields a complicated contrast, caused by a mixture of dielectric and topographic contributions. Shear force feedback is essential in aperture NSOM operation with fibers, and operates on soft surfaces of cells and chromosomes. Ultimately, aperture NSOM is limited by low efficiency with a source brightness of typically 100 pW to 10 nW. Thus, in spectroscopic applications (fluorescence, Raman, etc.) photon noise will be a fundamental limit in the speed of imaging. Photon tunneling in combination with force microscopy allows routine scanning with a high optical lateral resolution. However, interference effects can be dominant on surfaces which display extensive scattering. As such, the application potential of PSTM to biological surfaces is rather limited. Clearly, the virtues of optics, non-invasiveness, high spectral resolution, and high time resolution all apply to the near field optical domain with its high spatial resolution, which adds extensively to the potential of scanning probe microscop

Development of an integrated NSOM probe

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

Microfabrication of near-field optical probes

Near-field optical microscopy generally uses a tapered optical fiber, which is metal coated, to form a sub-wavelength sized light source. Here, a technique for the fabrication of a new type of probe is described. The new design is based o­n atomic force microscope probes and consists of a silicon nitride cantilever with a solid transparent conical tip. The probes are made using micromechanical techniques, which allow batch fabrication of the probes. A near-field scanning optical microscope system was built to test the probes. This system features force detection by a beam deflection technique and subsequent force feedback together with a conventional optical microscope. A major advantage of the apparatus is the ease at which images are obtained. Results o­n a test sample show that an optical resolution of 300 nm can be obtained together with a simultaneous height image. (C) 1996 American Vacuum Society