Masatsugu Shigeno

Molecular Visualization of Immunoglobulin Switch Region RNA/DNA Complex by Atomic Force Microscope

Immunoglobulin heavy-chain (IgH) class switch recombination (CSR) is initiated by DNA breakage in the switch (S) region featuring tandem repetitive nucleotide sequences. Various studies have demonstrated that S-region transcription and splicing proceed to genomic recombination and are indispensable for CSR in vivo, although the precise molecular mechanism is largely unknown. Here, we show the novel physical property of the in vitro transcribed S-region RNA by direct visualization using an atomic force microscope (AFM). The S-region sense RNA, but not the antisense RNA, forms a persistent hybrid with the template plasmid DNA and changes the plasmid conformation from supercoil to open circle in the presence of spermidine. In addition, the S-region transcripts generate globular forms and are assembled on the template DNA into a large aggregate that may stall replication and increase the recombinogenicity of the S-region DNA.

Observation of Human Corneal and Scleral Collagen Fibrils by Atomic Force Microscopy

Purpose: We attempted to analyze the three-dimensional ultrastructure of human corneal and scleral collagen fibrils with an atomic force microscope (AFM).Methods: A normal eye removed from a 66-year-old male was used in the study. Suspended corneal and scleral collagen fibrils were individually attached to glass slides by centrifugation. These collagen fibrils were air-dried and observed with a noncontact mode AFM in air.Results: AFM imaging provided information on the surface topography of both corneal and scleral collagen fibrils. The corneal collagen fibrils had a height of 11.9 +/- 1.0 (mean +/- standard deviation) nm and the scleral fibrils of 82.5 +/- 35.6 nm. A periodic banding pattern of grooves and ridges was clearly found in both types of fibrils: the D-periodicity and the groove depth were 65.7 +/- 0.8 nm and 1.46 +/- 0.50 nm in the corneal fibrils, and 67.3 +/- 1.1 nm and 6.16 +/- 1. 23 nm in the scleral fibrils.Conclusions: Surface topographic images of human corneal and scleral collagen fibrils were clearly obtained with the AFM. This technique provides quantitative information on the surface morphology of the collagen fibrils at high resolution.

Observation of Langmuir-Blodgett Films by Scanning Tunneling Microscopy

Langmuir-Blodgett films of cadmium-arachidate have been studied by scanning tunneling microscopy (STM). Substrates of gold and graphite are used to make mono- and bilayer films. The image of the individual molecule cannot be obtained for the films on gold, but the data indicate disturbances on the tunneling current caused by the films. As for the films on graphite, the molecular images with a spacing of 0.4–0.7 nm are recognized in some frames of the variable tunneling current image recorded on videotape. Possible mechanisms of the imaging are discussed.

Atomic force microscopy of living cells

This paper is a review of our results of the application of atomic force microscopy (AFM) to the three-dimensional observation of living cells. First, we showed AFM images of living cultured cells in fluid. Contact mode AFM of living cells provided precise information on the shape of cellular processes (such as spike-like processes or lamellipodia) at the cellular margin. The contour of cytoskeletal elements just beneath the cell membrane was also clearly observable on the upper surface of the cells. Secondly, we showed the data on the discrepancy between the AFM images of living cells and fixed cells. These findings were useful for evaluating AFM images of living cells. Finally, we described the time-lapse AFM of living cells. A fluid chamber system enabled us to obtain AFM images of living cells for over 1 h at time intervals of 2–4 min. A series of these AFM images were useful for examining the movements of cellular processes in relation to subcellular cytoskeletal elements. Time-lapse movies produced by sequential AFM images also gave a realistic view of the cellular dynamics.

Tunneling through a deformed potential

Abstract When molecules exist between electrodes, the barrier shape is deformed. A square one-dimensional potential model was made and the I–V curves were calculated using the free electron and the WKB approximation. The curves show a characteristics change when the adsorbate has the highest occupied molecular orbital (HOMO) or the lowest unoccupied molecular orbital (LUMO) near the Fermi level. It indicates that the LUMOs contribution to the tunneling current is larger than that of the HOMO. The charging effect is also discussed on the basis of the uncertainty principle. Provided an isolated liquid crystal molecule is observed, the charging energy is estimated to be about 1 eV. Since this energy is larger than the thermal energy at room temperature, it should be detected. However, the lifetime of the electron is estimated to be short enough to blur the effect. The lifetime is expected to limit the energy resolution of the tunneling spectroscopy as well.

Measurements of polyphosphoric acid on HOPG

As a sample for initial study on biological materials by using scanning tunnelling microscope (STM), we selected phosphoric acid consisting of phosphorous atoms which play biologically an important role. We measured polyphosphoric acid coated on HOPG by STM and scanning tunnelling spectroscopy (STS) in air. In order to identify molecules on STM images, local I‐V curves were taken simultaneously. The averaged I‐V curves and the normalized conductance spectra of graphite coated with the acid show characteristics different from those of clean graphite surface. Around a step of the substrate, we found domains where normalized conductance spectra were different from those of clean graphite surface and ascribable to that of the adsorbed molecules.

Observation of Liquid Crystal Molecule on Graphite by Scanning Tunneling Microscopy

Abstract Molecules of liquid crystals(LCs) adsorbed on highly-oriented pyrolytic graphite(HOPG) were imaged by scanning tunneling microscopy(STM). Planar layer structures induced by the HOPG surface were obtained. Molecular packing models for the STM images are introduced with the aid of a molecular orbital calculation. Also we discuss the rearrangement of the LC alignment during the continuous STM measurement.

Observation of liquid crystals on graphite by scanning tunneling microscopy

Molecules of liquid crystals adsorbed on graphite were observed by scanning tunneling microscopy (STM). Liquid crystals of both smectic and nematic phases provided periodic STM images of 3-nm-spacing lines, seemingly composed of molecules with their axes vertical to the line. Detailed investigation of the images revealed that several different patterns existed in smectic liquid crystal. On the other hand, nematic liquid crystal exhibited one reproducible pattern. Monolayer, multilayer, and dimer-based molecular arrangements are discussed. Models for the molecular arrangement of the nematic liquid crystal are proposed.

Advanced tip design for liquid phase vibration mode atomic force microscopy.

We have fabricated polymer tips for atomic force microscopy in order to elucidate the effects of tip length and shape on cantilever vibration damping in liquids. The vibration damping is investigated by measuring the vibration amplitude of cantilevers as a function of tip-sample distance. The cantilever with a short tip provides a higher damping effect over long tip-sample distances. When the vibration amplitude was rescaled to show the effect of the cantilever width on oscillation damping, the vibration amplitude of cantilevers with various tip lengths was similarly obtained in a long distance range over 50 microm. This similarity is explained by an acoustic damping model in which an acoustic wave is generated by the cantilever. Finally, the results indicate a cantilever with a sufficiently long tip compared to the cantilever width can dramatically reduce the long-range damping effect in a liquid environment.

Sheet formation of membrane proteins from photosynthetic bacteria

Abstract A sheet of three integral membrane proteins of phosynthetic bacteria was prepared by the detergent dialysis method. The proteins were a photoreaction unit (reaction center-light harvesting protein complex) from Rhodopseudomonas viridis , a reaction center from the same species and a reaction center from Rhodobacter sphaeroides . The photoreaction unit sheet showed linear dichroism in absorption spectra. Hexagonaly-packed particles were observed in the photoreaction unit sheet by scanning tunneling microscopy. The inter-particle distance was 19 nm in the sheet.