Michael J. Allen

Tip-radius-induced artifacts in AFM images of protamine-complexed DNA fibers

Isolated DNA fibers complexed with protamine (the chromosomal protein that packages DNA in mammalian sperm) have been produced by partially decondensing the highly compacted mouse sperm chromatin particle on a glass coverslip. These DNA fibers were then scanned with the atomic force microscope (AFM). While the smallest of the fibers appear in AFM images as ribbon-like structures 250-350 A wide and 10-25 A high, experiments indicate that these images are the result of a convolution of the imaging-tips shape with the objects actual shape. In such convolutions the height of the object is affected only by the compressibility of the object, while the width is affected in addition by the sharpness of the tip. Images of polyamidoamine particles also appear to show this artifact. We have also deduced the tips radius of curvature from images of sharp steps and attempt to demonstrate the artifacts associated with a relatively large imaging tip.

Atomic force microscopy of mammalian sperm chromatin

We have used the atomic force microscope (AFM) to image the surfaces of intact bull, mouse and rat sperm chromatin and partially decondensed mouse sperm chromatin attached to coverglass. High resolution AFM imaging was performed in air and saline using uncoated, unfixed and unstained chromatin. Images of the surfaces of intact chromatin from all three species and of an AFM-dissected bull sperm nucleus have revealed that the DNA is organized into large nodular subunits, which vary in diameter between 50 and 100 nm. Other images of partially decondensed mouse sperm chromatin show that the nodules are arranged along thick fibers that loop out away from the nucleus upon decondensation. These fibers appear to stretch or unravel, generating narrow smooth fibers with thicknesses equivalent to a single DNA-protamine complex. High resolution AFM images of the nodular subunits suggest that they are discrete, clipsoid-shaped DNA packaging units possibly only one level of packaging above the protamine-DNA complex.

Deposition of SiC films by pulsed excimer laser ablation

Thin films of β‐SiC were grown on Si substrates by excimer laser pulse ablation of bulk SiC. The films were examined by Auger electron, x‐ray, and photoelectron spectroscopies and laser ionization mass analysis techniques. The film was smooth as monitored by scanning electron microscopy. Scanning electron and scanning tunneling microscopy (STM) showed inclusions in the deposited SiC films, and laser ionization mass analysis detected SiC dimers in the vapor plume emitted from the target.

Direct visualization of surface charge in aqueous solution

Tapping mode atomic force microscopy operated in the phase shift mode is used to directly visualize the surface charge on biological macromolecules under solution. A simple theory is presented which is qualitatively consistent with experimental observations, although a more complicated theoretical analysis is required for a quantitative comparison.

Extent of sperm chromatin hydration determined by atomic force microscopy

Volume measurements were performed on intact bull and mouse sperm heads and amembranous sperm nuclei, both in the fully hydrated (fluid cell) and dehydrated (air‐dried on glass coverslips) states by atomic force microscopy (AFM). Data were obtained by analyzing a small population of cells/nuclei, as well as by performing repeated measurements on single cells imaged following the addition of increasing concentrations of propanol. Results show that the volume of fully hydrated, intact sperm heads and amembranous sperm chromatin particles are at least twice the volume of their air‐dried counterparts. Dehydration occurs rapidly in air, and the reduction in volume of chromatin induced by water loss appears to be completely reversible. These studies demonstrate that both mouse and bull sperm chromatin are extensively hydrated in the native state, and are not as compact as previous studies have suggested.

Clusters formed in laser‐induced ablation of Si, SiC, Pt, UO2 and evaporation of UO2 observed by laser ionization time‐of‐flight mass spectrometry and scanning tunneling microscopy

Cluster formation is traditionally observed by mass spectrometry, which has the disadvantage that the detection sensitivity often decreases with increasing mass. Alternatively, one may collect the clusters onto an atomically flat substrate and identify them by scanning tunneling microscopy (STM). Both techniques were used here. For the first technique, a Nd:YAG laser (frequency quadrupled to 266 nm, 5 ns pulse width) focused onto spots of 4–100 μm diameter was used to ablate refractory materials, and a reflectron time‐of‐flight tube served to mass‐analyze the plumes. The observed mass spectra for Si, Pt, SiC, and UO2 varied in the distribution of ablation products among atoms, molecules, and clusters, depending on laser power density and target material. For the second technique, cleaved surfaces of highly oriented pyrolytic graphite were positioned either 10 cm away from materials ablated at 10−5 Torr by 1–3 excimer laser (308 nm) pulses of 20 ns duration, or 1 m away from materials vaporized at 10−8 Tor...

Surface site specificity on the basal plane of graphite: 1.06 μm laser damage threshold and reactivity with oxygen between 350 and 2300 K

Scanning tunneling microscopy (STM) has been used to document changes in the nanometer‐scale morphology of the basal plane of highly oriented pyrolytic graphite after in situ exposure to 7 ns, 1064 nm Nd:YAG, and to ms Nd:Glass laser pulses producing surface temperatures up to 2300 K, and after ex situ exposure to temperatures as low as 350 K in air. Laser damage produced by the ns pulses was visible by STM at fluences far below those that produce melting and effects visible by other imaging techniques. Damage appears first on step edges and consists of exfoliation of graphite layers and recession of steps through removal of mono‐ or multilayer patches. ms and long‐term heating leads to reaction with oxygen, which proceeds by etching of both crystalline boundaries and step edges. In addition a high concentration of flat‐bottom shallow pits (two monolayers deep) and a smaller number of conical pits (greater than 30 A deep) can clearly be identified. The results give for the reaction at the grain boundaries...

Analysis of adenine and thymine adsorbed on graphite by scanning tunneling and atomic force microscopy

Abstract Analysis by AFM of adenine and thymine adsorbed onto a hot graphite surface shows that these molecules are adsorbed in patches typically 50 nm wide and 5 nm high, but does not provide any discernable atomic structure. In contrast, STM image scans that contain both parts of the graphite substrate and of the adsorbate would mislead one to believe that the adsorbates consist of just one ordered monolayer of either adenine of thymine (like alkylcyanobiphenyl on graphite [D.P.E. Smith et al., Nature 344 (1990) 641]). From these STM images the lattice dimensions, structural periodicities, and the epitaxy of the adsorbed molecules with respect to the basal plane of graphite can be determined. The aromatic regions are strongly detected with near-atomic resolution in both molecules, while the various sidegroups are not well resolved. Thus STM can discriminate between purines and pyrimidines - if they are held in thick patches.

Application of x rays to the analysis of DNA packaging in mammalian sperm

X-ray microscopy has been performed on unlabeled and gold labeled rat sperm nuclei using a tantalum x-ray laser (44.83 angstrom) and an x-ray zone plate lense. Transmission images of nuclei labeled with gold using an antibody to protamine 1 show large clusters of gold as well as individual 400 angstrom gold particles coating the surface of the chromatin. Images of the same nuclei obtained with a scanning x-ray microscope demonstrate that the initial exposure of the sperm nucleus to a photon intensity of 3.0 X 1014 W/cm2 for a duration of 500 ps did not destroy or grossly distort their structure. Other nuclei labeled with an antibody to protamine 2 were partially decondensed during the labeling process and contained large vacuole-like regions. Images were also obtained of unlabeled sperm nuclei. Data obtained from one unlabeled nucleus imaged with both the atomic force and x-ray laser microscopes was used to determine the thickness of the chromatin and estimate the amount of water that must be associated with the DNA-protamine complex in air-dried nuclei. These results suggest that even air-dried sperm nuclei are extensively hydrated and that tightly bound water may comprise as much as one-third of the volume of the dried sperm nucleus.

A pulse‐deposition method for scanning tunneling microscopy of deoxyribonucleic acid on graphite

Images of three different synthetic DNAs have been obtained by scanning tunneling microscopy (STM) following their deposition on graphite using a voltaic pulse. DNA applied to the STM tip is desorbed during a 4 V/10 μs pulse and deposited intact onto the surface of highly oriented pyrolytic graphite. Images of 22, 47, and 100 base‐pair molecules show that deposition occurs in close proximity to the tunneling tip and that the molecules appear to deposit singly or in highly oriented groups.