Abnormal formation of the glucan network from regenerating protoplasts in Schizosaccharomyces pombe cps8 actin point mutant

To study the close relationship between the actin cytoskeleton and cell wall formation, the process of cell wall formation in reverting protoplasts of the fission yeast, Schizosaccharomyces pombe, cps8 actin point mutant was investigated by ultra-high-resolution low-voltage scanning electron microscopy (UHR-LVSEM) and transmission electron microscopy (TEM). The protoplast of the cps8 mutant began to form a glucan network in a unipolar manner and to secrete alpha-galactomannan. The site of cell wall formation grew in a cylindrical shape in the wild-type protoplast. The alpha-galactomannan did not fill in the intrafibrillar spaces completely, however, and the fibrils were exposed on the cell surface. UHR-LVSEM images indicated that the glucan fibrils were thin and rope-shaped, forming a looser network than the wild-type. TEM images indicated the finest fibrils were approximately 1.5 nm in diameter, the same diameter as the wild-type. These results suggest that the cps8 mutant was insufficient in developing cross-linkage with the glucan fibrils up to the wide ribbon shape as found in the wild-type [Osumi M et al. (1989) J. Electron Microsc. 38: 457-468; Osumi M (1998) Micron 29: 207-233]. These findings appear to indicate that the actin cytoskeleton controls formation of the glucan network and secretion of beta-1,6-glucan, and confirm the close relationship of the actin cytoskeleton and glucan formation.     

High-pressure freezing is a powerful tool for visualization of Schizosaccharomyces pombe cells: ultra-low temperature and low-voltage scanning electron microscopy and immunoelectron microscopy

Yeast cells have a thick cell wall composed of an inner network of glucans and an outer layer of mannoproteins, which is difficult to penetrate with osmium tetroxide. We previously developed the sandwich technique to overcome this problem. Although the freeze-etching method allows the fracturing of cryofixed yeast cells, it has been difficult to fracture cryofixed yeast cells for examination by cryo-scanning electron microscopy (SEM). The development of an alternative method of cryofixation, namely, high-pressure freezing, began in the 1960s and is now available for the electron microscopic analysis of yeast. We show here that when high-pressure freezing is combined with ultra-low temperature and low-voltage SEM using the new cryo-system, the Gatan Alto 2500 Cryo Transfer System, fractured and coated yeast samples could be quickly prepared. These samples yielded a fine fracture plane and revealed the ultrastructure of both external and internal cell components. We used this method to analyze the process of septum formation, one of the final and most important events of mitosis, and cell separation. The images we obtained provide a three-dimensional view of these processes for the first time. We also showed that high-pressure freezing in combination with immunoelectron microscopy made it possible to preserve the antigenicity, in situ localization, and behavior of the cell wall component alpha-1,3-glucan and its synthase during septum formation in Schizosaccharomyces pombe.     

Image quality improvement using helium gas in low voltage variable pressure scanning electron microscopy

An effective combination of the low voltage and variable pressure (VP) scanning electron microscopy (SEM) are discussed. In low voltage VP-SEM, helium gas is utilized for reducing the amount of scatter of the primary electron beam. Most samples can receive various benefits obtained from the combination of low voltage and low vacuum observation. Compared to a back-scattered electron (BSE) image in air, signal-to-noise ratio (SNR) of a BSE image taken with helium gas is 5.4 times under a pressure of 50 Pa and an accelerating voltage of 1.5 kV.     

Backscattered electron imaging by scanning electron microscopy of regenerating peripheral nerve axons immunostained with antineurofilament antibody.

To demonstrate three-dimensional architecture of regenerating axons growing through basal lamina tubes in cryoinjured nerve graft, backscattered electron (BSE) imaging in a scanning electron microscope (SEM) was used to visualize immunostained axons. Regenerating axons immunostained with an antibody against the 200 kD neurofilament protein (RT97) were clearly visualized in BSE images as bright components pursuing an irregular, often spiral course within the basal lamina tubes, and commonly branching within the tubes. The morphology of these structures corresponded closely to that of putative regenerating axons in SEM preparations following application of the potassium hydroxide-collagenase digestion method. The present approach, however, is a considerable improvement on the latter, providing three-dimensional information together with the identification of regenerating axons.     

Ultrastructure and behavior of actin cytoskeleton during cell wall formation in the fission yeast Schizosaccharomyces pombe

Fluorescence microscopy has shown that F-actin of the fission yeast Schizosaccharomyces pombe forms patch, cable and ring structures. To study the relationship between cell wall formation and the actin cytoskeleton, the process of cell wall regeneration from the protoplast was investigated by transmission electron microscopy (TEM), immunoelectron microscopy (IEM) and three-dimensional reconstruction analysis. During cell wall regeneration from the protoplast, localization of F-actin patches was similar to that of the newly synthesized cell wall materials, as shown by confocal laser scanning microscopy (CLSM). In serial sectioned TEM images, filasomes were spherical, 100-300 nm in diameter and consisted of a single microvesicle (35-70 nm diameter) surrounded by fine filaments. Filasomes were adjacent to the newly formed glucan fibrils in single, cluster or rosary forms. By IEM analysis, we found that colloidal gold particles indicating actin molecules were present in the filamentous area of filasomes. Three-dimensional reconstruction images of serial sections clarified that the distribution of filasomes corresponded to the distribution of F-actin patches revealed by CLSM. Thus, a filasome is one of the F-actin patch structures appearing in the cytoplasm at the site of the initial formation of the cell wall and it may play an important role in this action.     

Evaluation of both image resolution and contrast-to-noise ratio in scanning electron microscopy

The contrast-to-gradient (CG) resolution has been defined as a weighted harmonic mean of the local resolution, i.e. the length defining the object's local fine structure at each pixel position. The newly defined resolution Res is defined as 2 sigma/ sqrt 2, where 2 sigma is a sharpness factor of the image pattern obtained by the conversion 2 sigma = ARCG + B using default constants A and B. In the present study, we have extended the algorithm to change the constants from default values to calibrated ones using standard images that are same in both pattern and contrast-to-noise ratio (CNR) as the original SEM image to be evaluated. The calibrated image resolution with a weak noise dependency is evaluated with the CNR as a given parameter.     

Contrast-to-gradient method for the evaluation of image resolution in scanning electron microscopy

In a previous study, we proposed the contrast-to-gradient (CG) method for evaluating image resolution. Here, the CG resolution is defined as a weighted harmonic mean of the local resolution, which is proportional to the quotient of the threshold contrast divided by the local gradient. The local gradient is calculated from the quadratic function that best fits the local pixel intensities over the region of interest (ROI) of 3 x 3 or 5 x 5 pixels in size. To refine the CG method, some modifications are carried out in the present study. Directional resolutions are employed to evaluate images, including astigmatism or strongly directional patterns as well as isotropic patterns. Here, CG resolution is redefined so as to keep the same value even for the image reversed in black-and-white contrast, because of no difference in the image information during contrast reversing. Besides, CG resolution is characterized to be independent of the brightness/contrast change unless these changes do not bring about both cut-off and saturation in the pixel intensities. Dependencies of the denoising effect and the resolution accuracy on ROI size are demonstrated as a function of image-noise.     

Influence of random noise on the contrast-to-gradient image resolution in scanning electron microscopy

The contrast-to-gradient (CG) method has been proposed previously for the evaluation of image resolution in scanning electron microscopy (SEM). The CG resolution is based on the local resolution, which is defined as the distance to recognize the object's local surface at each pixel position. Then, the CG resolution R is inherently influenced by random noise contained in the image. The present study demonstrates the influence of random noise on the R-values using both personal computer-made dot patterns in various sizes and densities and SEM test micrographs. It is found that the R-values increase with increasing random noise intensity N and their increases are smaller for higher-density patterns, but the R-values are independent of their image sizes under constant pattern density. In addition, the standard CG algorithm is modified especially for straightly fabricated patterns along the x- (or y-) axis, resulting in an improvement in the measurement accuracy of y- (or x-) directional resolutions. The straightness as known information is utilized fully to recognize the local pattern in the random noise.     

Influence of surrounding media on preservation of cell wall ultrastructure of Candida albicans revealed by low temperature scanning electron microscopy.

Influence of surrounding media on the surface structures of the cell wall of Candida albicans was discussed with respect to the preservation of ultrastructure during the specimen preparation for scanning electron microscopy. The fibrillar structure of the cell surface was distinctly identified by the rapid-freezing technique. It was difficult, however, to observe this structure by the conventional specimen preparation technique. The reason for the difference between these two preparation techniques was studied using a low temperature SEM. Through investigating the influence of each step of the conventional technique on the fibrillar structure, it was found that the fibrils were drastically deformed and disappeared during the dehydration step in ethanol above 80% in concentration. In order to study which physicochemical properties participated in this disappearance phenomenon, yeast cells were treated with various media: solutions in different pH ranges and at different salt concentrations, ionic solutions, surfactants, formamide, dimethyl sulfoxide, acetone and Fehling's solution. As a result, the fibrillar structure was found well preserved when the medium had an affinity for the constituent molecules of the fibrils. When without affinity, the fibrils suffered a remarkable deformation. The mechanism of this deformation is discussed in terms of molecular interaction of solute and solvent.     

神经干细胞克隆内细胞的扫描电镜超微结构研究

近年来，随着神经干细胞体外培养模型的建立，神经干细胞的研究正逐渐成为生命科学领域新的研究热点。为了观察神经干细胞克隆在分化过程中超微结构的变化以及细胞间的相互关系，我们做了单一神经干细胞克隆在poly-orithine上发生分化迁移过程中的扫描电镜观察。     

材料科学中的高分辨电子显微学—发展历史、现状与展望

本文综述了材料科学中的高分辨电子显微学发展历史、现状与展望。重点讨论了在高分辨电子显微学中能直接观察物质中原子排列的直接成像法 ,能区分原子种类的选择成像法、能量过滤选择成像法和 Z-衬度像 ,能研究物质结构变化动态过程的分辨时间高分辨电子显微学 ,能定量地分析物质结构的定量高分辨电子显微学和遥控电子显微术的发展以及其在材料科学中的应用和展望     

Contrast-to-gradient method for the evaluation of image resolution taking account of random noise in scanning electron microscopy

We have taken random image noise into consideration in the contrast-to-gradient (CG) method for the evaluation of image resolution R in scanning electron microscopy (SEM). When looking at the local fine pattern in the SEM micrograph containing much random noise, viewers gradually expand the region of interest (ROI) to improve the signal-to-noise ratio (SNR) and to recognize the pattern. We employed this approach in the CG algorithm to evaluate potential resolution Rpot, which is defined as the minimal/most accurate CG resolution calculated in the ROI expanding process. The image noise or SNR also is evaluated by the parameter deltaR / R, where deltaR is a standard deviation of R. The Rpot values depending on SNR are useful for comparison among images containing different random noise.     

Synthesis of scanning electron microscopy images by high performance computing for the metrology of advanced CMOS processes

Scanning electron microscopy is still the technique of choice for imaging and for in-line measurement of critical dimensions and overlay accuracy in most of the core technology processes. In particular, critical dimension microscopy provides information about design template matching and edge placement errors through links with design having proven beneficial effects on process yield and product reliability. In this paper, the use of high performance computing is demonstrated to simulate linescans and 2D secondary electron images to be used in optical proximity error correction strategies for nanometer scale technologies.     

Detection of glycoconjugates by lectin gold labelling, silver enhancement, and scanning electron microscopy

A method for detecting glycoconjugates on cell surfaces in scanning electron microscopy is described. Terminal saccharides were specifically recognized by a lectin conjugated to biotin, and, after incubation with an anti-biotin antibody conjugated to colloidal gold, silver enhancement was used to produce deposits large enough to be detected in standard scanning electron microscopes. Secondary electron images revealed the ultrastructure of the tissue investigated, while backscattered electron images showed the distribution of lectin binding sites. Using digital recording and processing, the two channels were combined in colour-encoded images. The new method brings together lectin histochemistry and scanning electron microscopy and thus allows the three-dimensional distribution of glycoconjugates to be analysed at an ultrastructural level.     

Er:YAG激光对牙本质超微结构作用的电镜观察

激光照射牙本质被认为是一种改善粘接效果的新方法。此研究的目的是采用电镜观察Er:YAG激光照射后牙本质的超微结构,评价激光处理牙本质以改善牙体组织和修复体之间粘接性能的可行性。共8颗新鲜拔除的人上颌第三磨牙按照牙合贴面的要求进行牙体预备,拔除牙齿进行牙体预备后接受激光照射,采用电镜观察牙本质的超微结构。分为两组,4颗牙齿为对照组,4颗牙齿为实验组。牙体预备后,对实验组牙齿的牙本质进行Er:YAG激光照射。采用电镜观察牙本质小管,熔融和裂隙等情况。对于实验组,观察结果未见玷污层,牙本质小管清晰。同时,对照组可以见到牙本质小管内有明显的玷污层,因此牙本质小管不清晰。该研究提示Er:YAG激光照射牙本质可以清除牙本质小管内的玷污层,这可能会提高牙体组织和修复体之间的粘接性能。     

Static capacitance contrast of LSI covered with an insulator film in low accelerating voltage scanning electron microscope

A new image contrast is reported for LSIs covered with an insulator film in a low accelerating voltage scanning electron microscope. The surface region above the conducting lines is often observed brighter than that without conducting lines. This contrast is quasi-stationarily observed contrary to well-known capacitive-coupled voltage contrast, and is called static capacitance contrast. The optimum irradiation conditions for the maximum image contrast is studied and its mechanism is discussed.