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J Vesenka

Publications and source records attributed to J Vesenka.

12 recordsLinked to original sources
PubMed1995

A new DNA nanostructure, the G-wire, imaged by scanning probe microscopy.

G-DNA is a polymorphic family of quadruple helical nucleic acid structures containing guanine tetrad motifs [G-quartets; Williamson, J.R., Raghuraman, M.K. and Cech, T.R. (1989) Cell 59, 871-880; Williamson, J.R. (1993) Proc. Natl. Acad. Sci. USA 90, 3124-3124]. Guanine rich oligonucleotides that are self-complimentary, as found in many telomeric G-strand repeat sequences, form G-DNA in the presence of monovalent and/or divalent metal cations. In this report we use the atomic force microscope (AFM) to explore the structural characteristics of long, linear polymers formed by the telomeric oligonucleotide d(GGGGTTGGGG) in the presence of specific metal cations. In the AFM these polymers, termed G-wires, appear as filaments whose height and length are determined by the metal ions present during the self-assembly process. The highly ordered, controllable self-assembly of G-wires could provide a basis for developing advanced biomaterials.

Base Sequence↗
PubMed1995

Imaging and manipulating chromosomes with the atomic force microscope.

Polytene chromosomes from the salivary gland cells of Drosophila melanogaster were examined by atomic force microscopy. The atomic force microscope (AFM) was capable of resolving chromosomal features down to the limits of the tip sharpness, about 500 A for pyramidal-shaped tips. Resolution was increased to 300 A by using electron beam deposited (EBD) tips with high aspect ratios. This significantly exceeds the resolution obtainable with conventional optical microscopes, but at the cost of compromising the structural integrity of the sample. A reasonable compromise was achieved by using oxide-sharpened tips. In this case high resolution was obtained without sample degradation, but when desired these tips were also capable of sample disintegration with increased scanning force and rate. Thus, oxide-sharpened tips were used to precisely dissect defined chromosomal regions to illustrate their potential use in genetic mapping efforts. This study illustrates the utility of the AFM in the characterization and manipulation of chromosomes and chromosomal DNA.

Animals↗
PubMed1995

Scanning force microscopy of chromatin.

Scanning force microscopy (SFM) is a new method to obtain the topography of surfaces with nanometer-resolution. The ability to image under liquids makes the technique attractive for biological applications, especially for the determination of the ultrastructure of biomolecules under native conditions. One growing field of interest is the investigation of chromatin and chromatin-related structures. Different levels of chromatin condensation were the subject of several previous SFM investigations, from the nucleosomal chain, to the 30-nm fiber, ending with the metaphase chromosome. The SFM yielded new information on such fundamental problems as the core spacing of the nucleosomal chain, the internal structure of the 30-nm fiber and the banding mechanism of metaphase chromosomes. Other investigations dealt with the SFM characterization of polytene chromosomes. This paper reviews the state-of-the-art in SFM chromatin research and discusses future developments in this field.

Animals↗
PubMed1995

Combining optical and atomic force microscopy for life sciences research.

The atomic force microscope (AFM), a three-dimensional imaging tool that can measure structures from the atomic level to micron scale, has been combined with an inverted optical microscope capable of confocal imaging. The robust design of this microscope, termed the BioScope, enables the operator to use fluorescent markers on a wide variety of biological specimens to determine internal structure to 200 nm resolution and determine surface morphology of the same sample to 20 nm resolution while imaging under physiological conditions. In this report we demonstrate the capabilities of the BioScope by examining living Xenopus retinal glial (XR1) cells, Drosophila polytene chromosomes and colloidal gold-labeled plasmid DNA.

Animals↗
PubMed1993

Chirality of DNA supercoiling assigned by scanning force microscopy.

Reproducible images of pBR322 plasmid molecules have been recorded by scanning force microscopy under 1-propanol. Most of the plasmids were found in a coiled state. The supercoiled molecules of our samples look like branched or unbranched interwound superhelixes. This is consistent with available electron microscopy data on circular DNA molecules. By applying a stratigraphic analysis which takes advantage of the height information contained in the scanning force microscopy images, it is possible to assign the chirality of the local supercoiling of the individual molecules.

1-Propanol↗
PubMed1993

Atomic force microscopy of oriented linear DNA molecules labeled with 5nm gold spheres.

The atomic force microscope (AFM;1) can image DNA and RNA in air and under solutions at resolution comparable to that obtained by electron microscopy (EM) (2-7). We have developed a method for depositing and imaging linear DNA molecules to which 5nm gold spheres have been attached. The gold spheres facilitate orientation of the DNA molecules on the mica surface to which they are absorbed and are potentially useful as internal height standards and as high resolution gene or sequence specific tags. We show that by modulating their adhesion to the mica surface, the gold spheres can be moved with some degree of control with the scanning tip.

DNA↗
PubMed1993

Colloidal gold particles as an incompressible atomic force microscope imaging standard for assessing the compressibility of biomolecules.

Colloidal gold particles have multiple uses as three-dimensional atomic force microscopy imaging standards because they are incompressible, monodisperse, and spherical. The spherical nature of the particles can be exploited to characterize scanning tip geometry. As uniform spheres, colloidal gold particles may be used to calibrate the vertical dimensions of atomic force microscopy at the nanometer level. The monodisperse and incompressible nature of the gold can be used to characterize the vertical dimensions of coadsorbed biomolecules. Simultaneous measurements of gold with tobacco mosaic virus show that, at the same applied vertical force, the tobacco mosaic virus is undamaged by blunt tips but is compressed or disintegrated under sharper scanning styli, suggesting that specimen degradation is partly a pressure-dependent effect.

Biophysical Phenomena↗
PubMed1993

Humidity effects on atomic force microscopy of gold-labeled DNA on mica.

Recent work in atomic force microscopy (AFM) of deoxyribonucleic acid (DNA) has relied on immobilizing DNA molecules by drying a small volume of buffered DNA solution onto cleaved mica. When imaging in air, relative humidity has been known to affect both the resolution and measured height of the DNA strands. We present data of measured height versus humidity for DNA and attached gold labels, and we propose a model for this data based on swelling of coadsorbed buffer salts upon exposure to moisture. In this model, small particles (e.g., DNA) stay near the top of the swelling salt layer, whereas larger particles (e.g., gold spheres) tend to be anchored down to the substrate until a moderate humidity is reached. At high humidity (around 65%), the salt layer becomes fluid-like and susceptible to tip-induced motion; the salts are either removed from the scan area or aggregate into island structures, depending on initial salt concentration on the surface.

Aluminum Silicates↗
PubMed1992

Circular DNA molecules imaged in air by scanning force microscopy.

Routine and reproducible imaging of DNA molecules in air with the scanning force microscope (SFM) has been accomplished. Circular molecules of plasmid DNA were deposited onto red mica and imaged under various relative humidities. In related experiments, the first images of the Escherichia coli RNA polymerase-DNA complex have also been obtained. This has been possible by (1) the use of specially modified SFM tips with a consistent radius of curvature of 10 nm or less, to minimize the amount of image distortion introduced by the finite dimensions of commercially available tips, (2) the optimization of a method to deposit and bind DNA molecules to the mica surface in a stable fashion, and (3) careful control of the sample humidity, to prevent solvation of the molecules and detachment from the surface by the scanning tip or stylus. Contact forces in the range of a few nanonewtons are routinely possible in air and in the presence of residual humidity. The spatial resolution of the images appears determined by the radius of curvature of the modified styli, which can be estimated directly from the apparent widths of the DNA molecules in the images.

Air↗
PubMed1992

Substrate preparation for reliable imaging of DNA molecules with the scanning force microscope.

A simple method of substrate preparation for imaging circular DNA molecules with the scanning force microscope (SFM) is presented. These biomolecules are adsorbed onto mica that has been soaked in magnesium acetate, sonicated and glow-discharged. The stylus-sample forces that may be endured before sample damage occurs depends on the ambient relative humidity. Images of circular DNA molecules have been obtained routinely using tips specially modified by an electron beam with a radius of curvature, Rc, of about 10 nm [D. Keller and C. Chih-Chung, Surf. Sci. 268 (1992) 333]. The resolution of these adsorbed biomolecules is determined by the Rc. At higher forces individual circular DNA molecules can be manipulated with the SFM stylus. Strategies to develop still sharper probes will be discussed.

Aluminum Silicates↗
PubMed1992

Atomic force microscopy of uncoated plasmid DNA: nanometer resolution with only nanogram amounts of sample.

Reproducible, high-contrast, nanometer-resolution AFM images of uncoated plasmid DNA can be obtained with nanogram quantities of DNA with the help of two advances in sample preparation: (1) Heating a DNA solution at 35 degrees C for 10 to 20 minutes before deposition on mica helps separate and spread the DNA, and (2) Using 5 microliter drops of the heated DNA solution in the concentration range of 2 to 10 nanogram/microliter in contact with a specially prepared mica surface for 5 to 10 minutes gives optimal coverage with only nanograms of DNA.

Aluminum Silicates↗