Application of backscattered electron imaging to enzyme histochemistry of lymphatic capillaries.

A backscattered electron imaging (BEI) in scanning electron microscope (SEM) was applied to the observation of lymphatic capillaries on cryostat-cut tissue blocks of monkey stomachs with enzyme-histochemical method. The differentiation of the lymphatics from blood capillaries was established on cryostat sections using 5'-nucleotidase (5'-Nase)-alkaline phosphatase double-staining method. On the tissue surface, 5'-Nase-positive lymphatic capillary walls were seen as highlights with good contrast and resolution by the BEI. A pair of the backscattered and secondary electron imaging in the same area clearly reveals the correlation between the enzyme activity and surface structure of the tissue. Observation of stereo-pairs of the BEIs increases the information on the three-dimensional architecture of lymphatic capillaries. The present study demonstrates the applicability of the BEI of 5'-Nase reaction to SEM histochemistry of lymphatic capillaries stained with heavy metal.     

47例儿童下丘脑错构瘤免疫细胞的化学及超微病理学研究

用免疫细胞化学及超微结构形态学方法,探讨下丘脑错构瘤导致青春期前性早熟及痴笑样癫痫的发生机制.47例下丘脑错构瘤的手术标本进行促性腺激素释放激素特异性抗体（GnRH）的免疫组织化学和常规超薄切片电镜观察研究.免疫组化结果证实,伴有青春期前性早熟的错构瘤对GnRH抗体呈不同程度的阳性反应,说明其具有分泌促性腺激素释放因子的能力.电镜检查显示下丘脑错构瘤组织内神经细胞形态异常,神经毡内可见突触样结构内含大量清亮圆形的神经递质小泡和少量致密核心的分泌颗粒.研究结果表明,上述超微形态结构异常可能与痴笑性癫痫及青春期前性早熟相关.     

多肽胰岛素正常转运经淋巴而非门脉途径IGMEI胰组织注射SEM观察

探讨胰岛素等多肽激素在细胞外的正常转运途径或规律,对免疫金标记外源性胰岛素（IGMEI）注射的大鼠胰组织冷冻切片,进行了二次电子和背散射电子图像（SEI／BEI）的扫描电镜（SEM）示踪性观察,SEM观察SEI显示胰腺小叶和小叶间结缔组织,血管和淋巴管,胰腺导管和胰岛的结构特点清晰可见,根据管腔内是否存在红细胞和内皮细胞的结构特点,可以区别血管或淋巴管。SEM观察BEI表明,较强的背散射电子出现在胰结缔组织间隙,淋巴管或毛细淋巴管内;然而,胰的血管或毛细血管内,无标记金颗粒的背散射电子或BEI微弱,结果提示,注射或释放入胰组织液中的胰岛素等多肽激素或分泌颗粒,其正常转运途径或规律,可能通过淋巴而非肝门静脉途径转运进入血液。     

The three-dimensional architecture of retinal blood vessels in KK mice, with special reference to the smooth muscle cells and pericytes

In the present study, the three-dimensional architecture of retinal vasculature was studied in KK mice, by combined use of resin injection, chemical treatment and scanning electron microscopy (SEM). In particular, Mercox/methylmethacrylate resin-injected eye tissues were subjected in sequence to NaClO immersion, ultrasonication and HCl treatment. The present technological innovation made possible SEM visualization of deeper retinal vasculature. In KK mice, the tunica media of stem arterioles and of first and second order branches consisted of a single layer of spindle-shaped smooth muscle cells provided with spine-like cytoplasmic processes. In addition, there occurred small triangular smooth muscle cells provided with slender cytoplasmic processes. The processes, giving off tiny secondary processes, overlapped with each other, thus forming assemblages around branching sites. Such a structure was particularly prominent for those branching sites where parent arterioles gave rise to their branches in a side arm-like pattern. The third (and occasionally fourth) order branches were surrounded by atypical smooth muscle cells, with considerable dimension of endothelial surface remaining uncovered. Capillary pericytes consisted of fusiform cell bodies and slender cytoplasmic processes. Smooth muscle cells of retinal venules differed from those of arterioles. They were stellate in shape, exhibiting several cytoplasmic processes.     

A method for visualizing cytoskeletal structures by the scanning electron microscope

The cytoskeleton of intestinal epithelial cells was observed by scanning electron microscope (SEM). Tissues were treated with Triton X-100, stained to enhance conductivity, and freeze-fractured in liquid nitrogen. With these procedures, not only the localization of core filaments (actin-containing microfilaments) and intermediate filaments but also the relations between fibrous structures and cell organellae were clearly revealed. This conventional method is of great value in interpreting the three-dimensional organization of intracellular fine structures.     

Local thickness and composition analysis of TEM lamellae in the FIB

High-resolution TEM image quality is greatly impacted by the thickness of the TEM sample (lamella) and the presence of any surface damage layer created during FIB–SEM sample preparation. Here we present a new technique that enables measurement of the local thickness and composition of TEM lamellae and discuss its application to the failure analysis of semiconductor devices. The local thickness in different device regions is accurately measured based on the X-ray emission excited by the electron beam in the FIB–SEM. Examples using this method to guide FIB–SEM preparation of high quality lamellae and to characterise redeposition are shown for Si and III–V semiconductor devices.     

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.     

泥页岩样品自然断面与氩离子抛光扫描电镜 制样方法的比较与应用

根据不同的研究目的,泥页岩的扫描电镜实验常用自然断面和氩离子抛光两种制样方法.自然断面的样品一般利用二次电子(SE)信号成像,反映样品表面原始形貌,图像立体感强,是最常用的制样方法.自然断面方法适合观察较大的孔隙结构,矿物及有机质形态特征,有机显微组分等.氩离子抛光样品一般利用背散射(BSE)信号成像,反映样品原子序数差异,容易区分有机质与矿物质.氩离子抛光样品表面光滑平整,适合观察泥页岩中纳米级孔隙,能直观地观察到矿物及有机质的颗粒大小及分布情况,但经过切割、研磨和抛光等一系列复杂的处理过程,样品原始形貌受到一定的破坏,会损失掉一些有用信息.本文详细比较了自然断面与氩离子抛光两种制样方法的特点及适用条件,指出如何根据样品特征及研究目的选择合适的制样方法.     

钛合金-不锈钢异种材料激光焊接工艺研究

为了探究钛合金-不锈钢异种金属焊接的特殊性,更好地提升两金属间的焊接性能,采用在钛合金与不锈钢之间加入填充层黄铜进行焊接的新方法,进行了理论分析和实验验证。应用ANSYS有限元分析软件分析得出填充层-黄铜的合理厚度应在0.5mm~0.7mm左右,并基于仿真结果对填充层黄铜厚度为0.5mm~0.7mm的钛钢异种金属焊件进行焊接实验,对焊接试样进行硬度、抗拉性测试及扫描电镜观察。结果表明,填充层黄铜的厚度为0.6mm时,钛合金-不锈钢异种金属激光焊接试样的焊缝形貌和力学性能较好。     

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.     

Nanomanipulation of biological samples using a compact atomic force microscope under scanning electron microscope observation

We introduce a compact nanomanipulator that can be operated inside the sample chamber of a scanning electron microscope (SEM) for biological sample manipulation. The design of the nanomanipulator is based on that of an atomic force microscope (AFM). A self-sensitive cantilever is used to realize the compact body and thus it is possible to put a pair of the standalone AFM units on the sample stage in the SEM chamber. Using this system, we accomplished nanodissection of biological samples as well as AFM imaging under SEM observation. We then fabricated the surface of a rat renal glomerulus by scan-scratching and succeeded in making a small hole on the wall of a blood capillary. As a result, it was possible to observe the internal structure of the capillary, which had been hidden beneath the surface wall. Furthermore, using two AFM units on the sample stage of the SEM, we successfully dissected the lens fiber cells taken from a rat eye in a multi-probe operation using the two cantilevers. This system is expected to become a very useful tool for micro- and nanometer-scale anatomy and engineering applications.     

Histochemistry of food tissue by colour scanning electron microscopy

A low-vacuum scanning electron microscope (SEM) allows microscopy of insulating specimens without metal coating, and so preserves the intact colour information on the specimen surface. We have attempted a new approach to characterize constituent distribution of food tissues by a histochemical method utilizing a colour SEM with an optical microscope and the low-vacuum SEM through digital image processing. To observe food tissues such as brown rice and adzuki bean, a colour SEM image of the specimen that has been stained by a modified method used in optical microscope histochemistry has proved to provide information of both the microscopic structure and constituent distribution on the specimen surface.     

不同品质类型小麦籽粒结构的观察比较

应用扫描电子显微镜观察了3个不同品质类型的小麦品种（强筋小麦济南17号、中筋小麦扬麦12号和弱筋小麦扬麦9号）的籽粒断面结构。结果表明,济南17号胚乳舍有丰富的基质蛋白质,淀粉粒被包埋在基质蛋白质中间,胚乳结构致密;扬麦12号胚乳淀粉粒与基质蛋白质所连接,胚乳结构较疏松;扬麦9号胚乳蛋白质基质含量很少．淀粉粒与蛋白质结合不紧密。采用碱处理和差速沉淀法分离了籽粒胚乳大、小淀粉粒,扫描电镜观察发现品种间淀粉粒的形态无明显差别,但大、小淀粉粒的尺寸和含量在品种间存在很大差异。     

烟草叶片组织结构的扫描电镜观察方法

将烟草叶片经过固定、水解、剥离、降解、浸泡、置换、干燥、喷金等处理 ,成功地制备出了烟草叶片各层组织的扫描电镜样品 ;首次获得烟草叶片各组织层的立体结构图像 ,通过比较 ,发现不同部位叶片内部组织结构存在某些规律差异。为研究烟草叶片各组织层中细胞分布和生长发育规律提供了一条新的途径。     

扫描电子显微镜叔丁醇冷冻干燥快速制样方法的探索

该实验旨在利用扫描电子显微镜(SEM)进行植物叶片微观形态观察,探究一种叔丁醇快速制样新方法.实验以野生型拟南芥叶片为研究对象,通过预设实验步骤对其进行制样处理,结合实验过程及观察结果总结最佳实验处理条件作为新的SEM叔丁醇快速制样方法.实验结果显示,在不同的叔丁醇处理温度、时长,冰冻时间及喷金导电处理条件下,叶片表皮...     

Effective decapsulation of copper wire-bonded microelectronic devices for reliability assessment

The wire bonding industry has made a major shift in wire materials from gold to copper, primarily due to cost concerns. Copper wire-bonds are now present in many commercial off-the-shelf (COTS) devices but minimally used in automotive, industrial, or military-grade applications due to lack of detailed understanding about reliability concerns. A thorough study of wire bond reliability includes performing bond shear and pull strength measurements before and after stress testing. This in turn requires a special decapsulation procedure for copper wire-bonded devices because, unlike gold, copper is chemically potent. Many techniques for copper wire-bonded device decapsulation exist and can be categorized into laser-, plasma-, and acid-based processes. This paper reviews some of these techniques and discusses the decapsulation mechanism, which involves decomposition of the epoxy resin. By understanding the decapsulation mechanism and available techniques, a unique decapsulation method was developed. The effectiveness of this method is presented along with scanning electron microscopy (SEM) images of the results, which indicate minimal etching of copper wire bonds. The critical parameters of this technique are also identified, a suitable range of input for each parameter is analyzed theoretically, and a design of experiment (DOE) is conducted to find optimal values for each parameter. Several SEM images are provided to show both the good and bad results from the DOE. An image method for measuring effectiveness of decapsulation is also presented.     

Comparison of hexamethyldisilazane and critical point drying treatments for SEM analysis of anaerobic biofilms and granular sludge

We present a fast procedure for scanning electron microscopy (SEM) analysis in which hexamethyldisilazane (HMDS) solvent, instead of the critical point drying, is used to remove liquids from a microbiological specimen. The results indicate that the HMDS solvent is suitable for drying samples of anaerobic cells for examination by SEM and does not cause cell structure disruption.     

原生质体扫描电镜观察研究的一种新方法

常规方法制备原生质体扫描电镜样品容易造成原生质体变形和破裂,影响其电镜观测结果。本文工作中,通过选用适宜的固定剂对原生质体进行快速因定,然后将原生质体悬浮液滴在经过硅烷化处理而引入了胺基的玻片上,直接在玻片上进行原生质体洗涤、脱水、置换、临界点干燥、喷镀处理,避免了在制样过程中进行离心操作,从而有效地解决了原生质体变形和破裂的问题。     

Correlative light and electron microscopic observations on ectopic neurons in the cerebellum of dreher mutant mouse

The structure of ectopic neurons in the cerebellum of dreher mutant mouse was investigated by correlative light and electron microscopic observations. Tissue blocks were fixed in buffered aldehyde and embedded in a mixture of 2-hydroxypropyl methacrylate, Quetol 523, and methyl methacrylate. Sections at 0.4-0.5 microns in thickness were examined by electron microscopy after observation under a light microscope. By comparing the electron images with those of light microscopy in the same sites, the structures of ectopic cells were confirmed. Ectopic Purkinje cells were arranged with cell bodies that contained an oval, spherical or wrinkled nucleus without deep invagination and the thin layers of endoplasmic reticulum at the perinuclear regions. Granule cells were ectopically matured in the external granular layer and within the cluster at the cortical region. This method provides a useful procedure for understanding structures of the cerebellar neurons of the mutant.