Fabrication of a new substrate for atomic force microscopic observation of DNA molecules from an ultrasmooth sapphire plate

A new stable substrate applicable to the observation of DNA molecules by atomic force microscopy (AFM) was fabricated from a ultrasmooth sapphire (alpha-Al2O3 single crystal) plate. The atomically ultrasmooth sapphire as obtained by high-temperature annealing has hydrophobic surfaces and could not be used for the AFM observation of DNA. However, sapphire treated with Na3PO4 aqueous solution exhibited a hydrophilic character while maintaining a smooth surface structure. The surface of the wet-treated sapphire was found by x-ray photoelectron spectroscopy and AFM to be approximately 0.3 nm. The hydrophilic surface character of the ultrasmooth sapphire plate made it easy for DNA molecules to adhere to the plate. Circular molecules of the plasmid DNA could be imaged by AFM on the hydrophilic ultrasmooth sapphire plate.     

High-speed AFM of human chromosomes in liquid

Further developments of the previously reported high-speed contact-mode AFM are described. The technique is applied to the imaging of human chromosomes at video rate both in air and in water. These are the largest structures to have been imaged with high-speed AFM and the first imaging in liquid to be reported. A possible mechanism that allows such high-speed contact-mode imaging without significant damage to the sample is discussed in the context of the velocity dependence of the measured lateral force on the AFM tip.     

Purification and properties of rabbit muscle proteasome, and its effect on myofibrillar structure

This paper describes the purification and properties of a multicatalytic proteinase complex, proteasome, from rabbit skeletal muscle, and its effect on myofibrillar structure. The purified proteasome gave a single band on polyacrylamide gel electrophoresis under non-denaturing conditions and gave eight bands under denaturing conditions, indicating that this enzyme comprises multiple hetero-subunits with low molecular mass. The purified proteasome was not activated by ATP and ubiquitin, and was markedly inhibited by Z-Leu-Leu-Leu-H (aldehyde). These data indicate that the purified proteasome is not 26S, but 20S. The proteasome degraded synthetic peptides maximally at pH 8.0. Relative to pH 8.0, activities were gradually decreased with the lowering of pH, but the degree of decrease was substrate-dependent, and the activity at pH 5.0 still retained about 30˜60% of the activity at pH 8.0, indicating the possibility that the proteasome is active in the muscle during conditioning. When the proteasome was heated at 60 °C for 20 min and treated in the presence of 0.005% SDS, the activity increased over 1.5 and 4.5 times, respectively. SDS remarkably increased the V_max value of the enzyme at pH 8.0. The proteasome was also activated by high hydrostatic pressure up to 100˜150 M Pa and gradually decreased at 200 MPa or higher. Electron microscopic observation revealed that obvious gaps between filamentous structure, the complete loss of M-line and partial loss of Z-line structure were caused by proteasome.     

A useful method for observing intracellular structures of free and cultured cells by scanning electron microscopy

Scanning electron microscopy (SEM) using osmium-maceration methods has been used for analyzing the three-dimensional structure of cell organelles in tissue samples, but it has been quite difficult to observe free and cultured cells with this technique. The present study was performed to develop a method that can be applied to free and cultured cells for SEM studies of intracellular structures after osmium maceration. The method was also applied to light microscopy (LM) and to transmission electron microscopy (TEM). HeLa cells and human leukocytes were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde followed by an additional fixation with 1% osmium tetroxide. These cells were embedded in low-melting-point agarose. A temperature-responsive dish was also used for collection of cultured cells before embedding. For LM and TEM, the cell-embedded agarose was further embedded in epoxy resin, and semi- and ultrathin sections were examined conventionally. For SEM, the agarose was freeze-fractured in 50% dimethyl sulfoxide, processed for osmium maceration and observed in a high-resolution SEM. Low-melting-point agarose was useful as an embedding medium for SEM, because it was well preserved during prolonged osmication for SEM. Thus, the fine structure of cell organelles was clearly analyzed by SEM after osmium-maceration treatment. These SEM images could also be compared with those of LM and TEM of the agarose-embedded tissues.     

Tantalum-gate thin-film SOI nMOS and pMOS for low-powerapplications

The threshold voltages of thin-film fully-depleted silicon-on-insulator (FDSOI) nMOS and pMOS have been controlled by employing tantalum (Ta) as the gate materials. Ta-gate FDSOI MOSFET's have excellent threshold voltage control for 1.0 V application on low impurity concentration SOI layers in both nMOS and pMOS. The low-temperature processing after the gate oxidation step leads to good on/off characteristics in Ta-gate SOI MOSFET's because of no reaction between Ta gate electrode and SiO₂ gate insulator. This technology makes it possible to drastically decrease the number of the process steps for CMOS fabrication, because the same gate material is available for both nMOS and pMOS     

Atomic force microscopy in histology and cytology

This review briefly introduces the principles of the atomic force microscope (AFM) and shows our own results of AFM application to biological samples. The AFM, invented in 1986, is an instrument that traces the surface topography of the sample with a sharp probe while monitoring the interaction forces working between the probe and sample surface. Thus, the AFM provides three-dimensional surface images of the sample with high resolution. The advantage of the AFM for biologists is that AFM can visualize non-conductive materials in a non-vacuous (i.e., air or liquid) environment. AFM images of the plasmid DNA are comparable to those by transmission electron microscopy using a rotary shadowing technique, and have the advantage of examining directly the molecule without staining nor coating. The surface structure of human metaphase chromosomes and mouse collagen fibrils demonstrated in air by the non-contact mode AFM is comparable to that obtained by scanning electron microscopy. Quantitative information on the heights of structures is further obtainable from the AFM images. Embedment-free thin tissue-sections are useful for observing intracellular structures by AFM. The present review also shows AFM images of living cultured cells which have been collected in a contact mode in liquid. This technique afforded us three-dimensional observation of the cellular movement with high resolution. Although there are some innate limitations for AFM imaging, the AFM has great potential for providing valuable new information in histology and cytology.     

Simultaneous collection of topographic and fluorescent images of barley chromosomes by scanning near-field optical/atomic force microscopy

The present study investigated the applicability of scanning near-field optical/atomic force microscopy (SNOM/AFM) to observations of YOYO-1 stained barley chromosomes. Using this technique, topographic and fluorescent images in the same portion were obtained simultaneously, thus enabling the precise analysis of fluorescent structures in relation to the morphology of chromosomes. In YOYO-1 stained chromosomes, the fluorescent intensity roughly reflected the local amount and/or density of DNA in chromosomes. R-banding of chromosomes stained with YOYO-1 and methyl green was also observed clearly as bright spots of various sizes by this microscope. Thus, the SNOM/AFM is expected to become a useful tool for analysing the structure and function of chromosomes more precisely than before.     

An EDTA-KOH method to expose bone cells for scanning electron microscopy.

To observe bone cells by scanning electron microscopy (SEM), the mouse parietal bones were processed by decalcification with EDTA and digestion of collagen fibers with KOH to remove the bone matrix, in addition to the conventional preparation for SEM. The critical-point-dried specimens were split into two membranous pieces along the gaps formed by removing the bone matrix. By this method, osteoclasts showing full three-dimensional images of ruffled borders, osteoblasts showing special structures on the surfaces facing the bone matrix, and osteocytes extending many slender processes were clearly demonstrated in SEM. This new method may provide new viewpoints in bone cell biology.