Observations of unstained biological specimens using a low-energy, high-resolution STEM

Low-energy, high-resolution scanning transmission electron microscopy (STEM) is introduced as a convenient method for observing unstained biological specimens. By reducing the electron energy, the cross section for light elements becomes comparable to that of conventional electron microscopy observations. The STEM mode exhibited the advantage that the induced energy loss and charge build-up in the sample affected the image to a lesser extent than in the TEM or SEM mode. Furthermore, the efficiency of an STEM detector is high, and the total radiation damage can be reduced if thermal damage due to localized heating at a slow scan operation can be overcome. We applied this method for observations of biological samples that were in the form of thin slices, fine fibers and small particles. When the supporting film for samples is absent, the resolution and the contrast of STEM images can be maintained similar to SEM and TEM images, respectively.     

Variational algorithms to remove stationary noise: applications to microscopy imaging

A framework and an algorithm are presented in order to remove stationary noise from images. This algorithm is called variational stationary noise remover. It can be interpreted both as a restoration method in a Bayesian framework and as a cartoon+texture decomposition method. In numerous denoising applications, the white noise assumption fails. For example, structured patterns such as stripes appear in the images. The model described here addresses these cases. Applications are presented with images acquired using different modalities: scanning electron microscope, FIB-nanotomography, and an emerging fluorescence microscopy technique called selective plane illumination microscopy.     

Cathodoluminescence study of one-dimensional free-standing widegap-semiconductor nanostructures: GaN nanotubes, Si3N4 nanobelts and ZnS/Si nanowires

Luminescence properties of one-dimensional free-standing widegap-semiconductor nanostructures were characterized by means of cathodoluminescence (CL). GaN nanopipes, alpha-Si3N4 nanobelts and ZnS/Si nanowires were fabricated by a catalyst-free method, namely grown in an induction furnace from powders. After the observation of morphology by scanning electron microscopy as well as the confirmation of their crystal structures by transmission electron microscopy, their CL spectra and images were observed. The CL spectra mapping as well as the monochromatic CL imaging revealed the variation of the luminescence spectra of different nanowires as well as that along a single wire. These results revealed the optical features of nanostructures.     

Scanning electron microscopic observation of immunohistochemically specified stromal cells following the NaOH maceration method

The present study introduces a method combining immunohistochemistry and a chemical digestion technique applied to scanning electron microscopy (SEM) observation of chemically characterized stromal cells in the myenteric layer of the monkey small intestine. The whole-mount preparation containing the myenteric layer was treated immunohistochemically using small colloidal gold and silver enhancements for detecting a certain type of stromal cell, followed by an alkali maceration method to remove extracellular matrices covering the immunohistochemically stained cells. This method permitted direct SEM observation of the stained stromal cells. Secondary and backscatter electron images clearly visualized the ultrastructure of the immunohistochemically-stained cells and the spatial distribution of the reaction products, respectively. It is suggested that the present method is useful for demonstrating the three-dimensional organization of the chemically specified stromal cells.     

Nano-area electron diffraction pattern reconstructed from three-dimensional Fourier spectrum.

A method to obtain a nano-area electron diffraction pattern in transmission electron microscopy (TEM) was proposed, based on three-dimensional (3D)) image formation theory. This method allows us to reconstruct an electron diffraction pattern from a 3D Fourier spectrum of high-resolution through-focus images. As a test case, an electron diffraction pattern from a tilted Si single crystal was reconstructed using the proposed method and compared with the conventional selected-area electron diffraction pattern. The intensity distribution of the reconstructed electron diffraction pattern was confirmed to be qualitatively equal to that of the selected-area electron diffraction pattern, though the degree of the equivalency between these patterns reduces at the high frequency region and the reproducibility of the intensity degrades when the number of images used in the image processing was decreased. By selecting areas in a reconstructed exit wave field, some electron diffraction patterns were obtained from the nano-areas without the influence of spherical aberration.     

Cadmium selenide@sulfide nanoparticle composites: Facile precipitation preparation,characterization, and investigation of their photocatalyst activity

We report a two-step synthesis and structural characterization of CdSe/CdS core/shell nanoparticles by the precipitation method. Besides, the effects of preparation parameters such as reaction temperature, the speed of the reaction stirrer, and the rate of adding sulfide source on the size, morphology, and photocatalyst activity of CdSe/CdS core–shell nanoparticles were studied by SEM images and degradation of methyl orange (MO) dye test. The structure and composition of the obtained products were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), high-resolution field-emission transmission electron microscope (HRTEM), photoluminescence spectroscopy (PL), scanning electronic microscopy (SEM), spectra energy dispersive analysis of X-ray (EDS), and ultraviolet–visible (UV–vis) techniques.     

Microstructure of Heteroepitaxial ZnTe Grown on GaAs(211)B by Molecular Beam Epitaxy

ZnTe was grown on GaAs(211)B by molecular beam epitaxy (MBE). Structural properties and strain relaxation at the ZnTe/GaAs(211)B interface were investigated by high resolution transmission electron microscopy (HRTEM) and scanning transmission electron microscopy (STEM). Application of digital image processing involving a filtered inverse fast Fourier transformation revealed an array of misfit dislocations at the interface and allowed strain relaxation to be estimated. Only one twin defect was observed in the HRTEM images, and details of this twin defect were investigated by STEM.     

高分辨原子像中原子峰位置的精确自动判定与电极化畴的快速可视化?

针对材料原子尺度的量化研究正变得日益重要,而处理高分辨透射电子显微镜（ HRTEM）像耗时耗力的现状,对计算机自动提取HRTEM像原子尺度信息的算法进行了研究,实现了HRTEM像中原子峰位置的自动准确提取;并在此基础上完成了布拉维格子的自动构建。利用前述两项关键图像信息,计算了钙钛矿钴氧化物中原子位置发生的周期性变化和铁电材料中电极化畴翻转等重要物理量,实现了对材料中晶胞尺度晶格形变的量化分析,以及形变和畴结构的快速可视化。当涉及大量图像处理时,其所实现的晶胞尺度晶格形变和畴结构的定量测量与快速可视化方法为分析材料原子及纳米尺度性能提供了一种强有力的分析手段。此外,自动提取HRTEM原子像信息也为后续探索模拟像和实验像的自动匹配及确定精确成像参数与真实原子位置打下了基础。     

肿瘤多细胞球:一种近似实体瘤的体外肿瘤模型

本文介绍了一种简便易行的方法,用以在体外建立近似体内实体瘤的肿瘤模型—肿瘤多细胞球模型(multicellular tumor spheroids,MCTSs).采用液滴重叠法(liquid overlay method)来构建HeLa肿瘤多细胞球模型,并在光镜下对肿瘤多细胞球的生长状况进行观察描述,再应用场发射扫描电...     

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.     

Correlative light microscopy, scanning electron microscopy, and transmission electron microscopy of osmium-macerated biological tissues

A method facilitating correlation of light microscopic (LM), scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images was developed. Rat kidney and heart were initially subjected to the osmium maceration procedure and then embedded in acrylic resin. Semithin sections of the tissue blocks were first provided for LM and then examined by SEM after resin removal. Furthermore, the ultrathin sections adjacent to the semithin sections were observed by TEM. The three-dimensional images of intracellular organelles provided an informative adjunct to LM and TEM.     

A specimen-drift-free EDX mapping system in a STEM for observing two-dimensional profiles of low dose elements in fine semiconductor devices

We developed a specimen-drift-free energy-dispersive X-ray (EDX) mapping system in a scanning transmission electron microscope (STEM) to improve the sensitivity and spatial resolution of EDX elemental mapping images. The amount of specimen drift was analysed from two STEM images before and after specimen drift by using the phase-correlation method, and was compensated for with an image-shift deflector of the STEM by the displacement of the scanning electron beam. We applied this system to observe the two-dimensional distribution of low dose arsenic in silicon semiconductor devices. The sensitivity of the elemental mapping was improved to several tenths atomic % for arsenic atoms while maintaining a spatial resolution of 2 nm.     

Computer-assisted simulation of high-voltage electron microscopy using serial images recorded by conventional transmission electron microscopy

Here we describe a computer-assisted method which, based on conventional transmission electron microscopy, renders simulated high-voltage electron micrographs. We perform arithmetic minimum filtering on stacks of aligned serial transmission electron microscopic images. In this way, the structural information of the separate images is fused into one compound image that highlights organization patterns otherwise easily overlooked or impossible to comprehend. Our method, like high-voltage electron microscopy, offers the possibility to build stereo-pairs for high-resolution three-dimensional analysis of tissue layers 1-2 microm thick. The use of background elimination and the development of a depth enhancement routine improved the three-dimensional effect and facilitated the analysis of the interior of objects. As an example, we use the method to display the distribution of axonal organelles at nodes of Ranvier and the shape and contents of a highly branched hippocampal dendritic spine.     

单晶硅表面STM成像理论模型与实验分析

结合扫描隧道显微镜(STM)成像实验和第一性原理原子级模拟计算的方法已经成为材料界面表征的重要手段。超高真空条件下的STM可用于直接观察单原子等微观结构,但其成像原理还未被理解清楚。STM扫描测得的试件表面原子级图像并不直接反映材料原子的形态,实际上是表面形貌和表面电子态局域密度的综合结果。为了解释STM成像,采用第一性原理Siesta方法,研究了Si(001)面STM成像过程的电子结构,对表面粒子的原子轨道和相应的电荷密度进行计算。讨论了等高模式下扫描高度对局域电子云密度分布的影响,并分析了STM针尖几何形状对模拟结果的影响。研究表明,材料表面原子的电子云密度分布可以用来解释STM成像精度和扫描高度对比的变化。     

拓宽激光扫描共聚焦显微技术运用的新探索

利用一些醛类固定液在固定作用中可产生荧光物质的特性，在荧光显微术中选择合适的激发波长和发射波长，使得一些未经荧光标记的生物组织结构也能在激光扫描共聚焦显微镜下成像，并可进行三维图像的构建，这不仅使激光扫描共聚焦显微技术的运用范围大为扩展，而且大大节约了研究费用和时间，提高了效益。此外，该技术还为组织学和细胞学的回顾性研究提供了一个有效手段，并将激光扫描共聚焦显微技术与普通光镜及电镜技术有机地结合起来，使组织和细胞的形态学研究得以全方位进行。     

Structural and optical characterisation of holey fibres usingscanning probe microscopy

An investigation of a holey fibre using scanning probe microscopy techniques, which the authors believe is the first such investigation, is presented. Atomic force microscopy images provide knowledge of the fibre structure without the artefacts associated with scanning electron microscopy imaging. A tapered fibre scanning near-field optical microscopy probe has been used to investigate holey fibre modes at 785 nm     

Si（Ge）MOSFET器件微结构的研究

采用横断面的透射电子显微术，扫描电子显微术和高分辨电子显微学方法，研究了ＧｅＳｉ沟道ｐ－ＭＯＳＦＥＴ的微结构。观察表明，器件是由Ｓｉ基片／ＳｉＯ２非晶层／ＳＯＩＳｉ层／ＧｅＳｉ沟道层／超薄ＳｉＯ２非晶层／Ｓｉ细晶层／ＳｉＯ２非晶层／Ａｌ电极层等组成的；ＳＯＩＳｉ层工作区单晶性良好，很难找到缺陷，缺陷能效地被限制在工作两侧的缺陷聚集区；在ＳＯＳＳｉ层和ＧｅＳｉ沟道层之间存在着一些瓣状衬底，它可能是在     

Joule expansion imaging techniques on microlectronic devices

We have studied the electrically induced off-plane surface displacement on two microelectronic devices using scanning Joule expansion microscopy (SJEM). We present the experimental method and surface displacement results. We show that they can be successfully compared with surface displacement images obtained using an optical interferometry method. We also present thermal images using scanning thermal microscopy (SThM) technique to underline that SJEM is more adapted to higher frequency measurements. The performances of the three methods are compared.     

Fabrication of Organized Porphyrin‐Nanotube‐Attached Heat‐Sensitive Polyelectrolyte Capsules

A facile method of connecting fluorescent meso‐tetrakis(4‐sulfonatophenyl)porphine tetranion nanotubes to polyelectrolyte capsules is developed. Heat‐sensitive robust polyelectrolyte capsules consisting of poly(diallyldimethylammonium chloride) and poly(styrene sulfonate) multilayers have been fabricated using the conventional layer‐by‐layer technique. Supramolecular aggregation of porphyrin monomers to nanotubes is induced in the microenvironment of the capsules by sequential addition of salt and acid. Scanning electron microscopy, transmission electron microscopy, and atomic force microscopy images reveal satellite‐like structures consisting of a central capsule core with porphyrin nanotubes emerging radially from the capsule walls. The growth and the distribution of the porphyrin units have been monitored by UV‐vis spectroscopy, fluorescence spectroscopy, and confocal laser scanning microscopy. Changing the temperature alters the dimensions and the arrangement of the nanotubes on the capsule walls. Such an attachment of porphyrin tubes onto robust functional capsules should help in developing an artificial light‐harvesting system.     

An auto-tuning method for focusing and astigmatism correction in HAADF-STEM, based on the image contrast transfer function

An auto-tuning method for high-angle annular detector dark field scanning transmission electron microscopy (HAADF-STEM) is proposed which corrects the defocus to the optimum Scherzer focus and compensates the astigmatism. Because the method is based on the image contrast transfer function formulated for the HAADF-STEM, the defocus and the astigmatism are accurately measured from input of two different defocus images. The method is designed to work independent of object function in the linear imaging model by analysing the spectral ratio between two Fourier spectra of their images, which is useful for cases where the spectrum of object function is not uniformly spread out over the reciprocal space. The method was preliminarily tested in a Hitachi HD-2000 STEM, and successful results of the auto-tunings from the viewpoint of verification of the algorithm were obtained using general specimens of Au fine particles and a thin section of a semiconductor device.