New method for the treatment of sperm samples for ultrastructural study based on the use of animal tissues as biological containers

The study of the ultrastructure of spematozoa by means of transmission electron microscopy (TEM) often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa Aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). In order to avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under a bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still-open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved.     

浅谈群钻的手工刃磨方法

群钻作为一种新型的钻头,在生产实践中正日益发挥其重要作用.它是把普通钻头的一条主刃分为外刃、圆弧刃和内刃三段,刃形变化复杂,刃磨步骤较多,刃磨问题一直是一个比较关键性的技术问题.本文从生产实践出发,介绍了群钻的手工刃磨方法.     

Electron microscopic observation of the sagittal structure of Drosophila mature sperm

Observation of sperm development and determination of their morphological characteristics are very important to the understanding of phylogenetic relationships and the study of sperm function during fertilization. Although ultrastructural studies of sperm development in the testes of the fruit fly Drosophila have been performed, there are few reports describing electron microscopic morphology of mature sperm, that is, those released from the testes to the seminal vesicles. Here, we present the first report of the sagittal organization of Drosophila sperm head and neck regions by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The head and tail structures of a mature sperm, for example, the acrosome, nucleus, and flagellum, were easy to distinguish by the morphological characteristics of the sperm surface by SEM. The morphological relationships between the surface and internal structures of mature sperm were confirmed by observing longitudinal sections with TEM. Our approach overcame the technical difficulties involved in sample preparation for electron microscopic observation of the Drosophila mature sperm head, and therefore, this study serves as an important foundation for future genetic dissection of sperm ultrastructure and function in male sterile mutants. Microsc. Res. Tech. 77:661–666, 2014. © 2014 Wiley Periodicals, Inc.     

Electron spectroscopic study (ESI,EELS) of Nanoplast-embedded mammalian lung

The potential of Nanoplast melamine resin embedding for the study of mammalian lung parenchyma was examined by means of electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). Samples were either fixed with glutaralde-hyde-paraformaldehyde or glutaraldehyde-tannic acid, or were directly transferred to the embedding medium without prior fixation. Organic dehydrants, as well as fixatives containing heavy metals and stains, were omitted. A very high level of ultrastructural detail of chromatin, ribosomes, mitochondria and plasma membranes was achieved by ESI from the Nanoplast-embedded samples. The most prominent gain in ultrastructural detail was achieved when moving from an energy loss just below the L_2,3 edge of phosphorus at 132 eV to an energy loss just beyond this edge. This reflects the prominent P L_2,3 edge observed by EELS of Nanoplast-embedded samples in comparison with conventionally processed samples. Thus, taking into account possible sectioning artefacts, excellent heterochromatin images which rely on the phosphorus distribution can be obtained from Nanoplast-embedded samples by computer-assisted analysis of electron spectroscopic images. In this respect glutaraldehyde-paraformaldehyde fixation is preferable to glutaraldehyde-tannic acid fixation because the presence of silicon, revealed by EELS, in tannic-acid-fixed samples may introduce artefacts in phosphorus distribution images obtained by the three-window method because of the close proximity of the L_2,3 edges of silicon and phosphorus.     

Pros and cons: cryo-electron microscopic evaluation of block faces versus cryo-sections from frozen-hydrated skin specimens prepared by different techniques

Over the last two decades, several different preparative techniques have been developed to investigate frozen‐hydrated biological samples by electron microscopy. In this article, we describe an alternative approach that allows either ultrastructural investigations of frozen human skin at a resolution better than 15 nm or sample throughput that is sufficiently high enough for quantitative morphological analysis. The specimen preparation method we describe is fast, reproducible, does not require much user experience or elaborate equipment. We compare high‐pressure freezing with plunge freezing, and block faces with frozen‐hydrated slices (sections), to study variations in cell thickness upon hydration changes. Plunge freezing is optimal for morphological and stereological investigations of structures with low water content. By contrast, high‐pressure freezing proved optimal for high‐resolution studies and provided the best ultrastructural preservation. A combination of these fast‐freezing techniques with cryo‐ultramicrotomy yielded well‐preserved block faces of the original biological material. Here we show that these block faces did not exhibit any of the artefacts normally associated with cryo‐sections, and – after evaporating a heavy metal and carbon onto the surface – are stable enough in the electron beam to provide high‐resolution images of large surface areas for statistical analysis in a cryo‐SEM (scanning electron microscope). Because the individual preparation steps use only standard equipment and do not require much experience from the experimenter, they are generally more usable, making this approach an interesting alternative to other methods for the ultrastructural investigation of frozen‐hydrated material.     

包装机拧丝机构的研制

针对冶金厂矿高温线材(小盘卷)在线快速捆扎,研制出包装机械中打捆机的一种新型拧丝机构,能替代人工包装捆扎时,拧丝难的问题.对该机构工作原理,相关参数进行了分析、计算.通过样机试验和厂矿使用表明:被捆扎的盘卷紧,结头短而美观,能满足包装质量要求.可广泛应用在线、棒等型材和其他相关产品的包装中.     

A simple method for orienting silk and other flexible fibres in transmission electron microscopy specimens

When microstructures are characterized by transmission electron microscopy (TEM), the interpretation of results is facilitated if the material can be sectioned in defined orientations. In the case of fibres, it is especially useful if transverse and longitudinal sections can be obtained reliably. Here we describe a procedure for orienting spider silk and other flexible fibres for TEM investigation. Prior to embedding in epoxy resin, the silk is wound around a notched support made from polyester film. No glue is required. After the silk and its supporting film have been embedded and the resin has been cured the film can be peeled away to reveal nearly perfectly orientated silk threads. Both transverse and longitudinal sections can then be cut with a microtome. The method can be extended to obtain sections at any intermediate orientation.     

Immunogold labelling in environmental scanning electron microscopy: applicative features for complementary cytological interpretation

We have combined environmental scanning electron microscopy (ESEM) and immunogold labelling (IGL) for the analysis of cell morphology and surface protein detection on human fine needle aspiration, which is processed in thin uniform monolayer (a single layer of cells) on a glass slide by Thin Prep technology. Among scanning electron microscopy techniques, we choose the environmental modality (ESEM) because it allows a slight manipulation of biological samples and an operational time comparable with cytological techniques. Moreover, the Thin Prep technology confirmed a reproducible cell monolayer on glass smear, minimizing problems for the determination of appropriate amount of material per slide. The first experimental data in ESEM-IGL on biological samples with fine needle aspiration Thin Prep, in human thyroid nodules, showed that cells retained their morphology and provided a clear IGL. The optimization of conditions (i.e. vacuum pressure, temperature and relative humidity) confirmed the possibility to observe an immunolabelled biological sample and morphological signal, joined with compositional informations, due to peculiar characteristics of gaseous secondary electron detector in ESEM. The ESEM-IGL and fine needle aspiration Thin Prep could be used in combination for the interpretation of cell morphology and cell surface immunolabelling. Our paper suggests this use as a powerful diagnostic tool in a pre-surgical evaluations, opening a new applicative window for electron microscopy.     

Identification and ultrastructural imaging of photodynamic therapy-induced microfilaments by atomic force microscopy

Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.     

Controlled microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography

High‐pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding and final orientation for sectioning and subsequent transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 μm, the tubing protects small and fragile samples within the thickness constraints of high‐pressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.     

High resolution SEM imaging of gold nanoparticles in cells and tissues

The growing demand of gold nanoparticles in medical applications increases the need for simple and efficient characterization methods of the interaction between the nanoparticles and biological systems. Due to its nanometre resolution, modern scanning electron microscopy (SEM) offers straightforward visualization of metallic nanoparticles down to a few nanometre size, almost without any special preparation step. However, visualization of biological materials in SEM requires complicated preparation procedure, which is typically finished by metal coating needed to decrease charging artefacts and quick radiation damage of biomaterials in the course of SEM imaging. The finest conductive metal coating available is usually composed of a few nanometre size clusters, which are almost identical to the metal nanoparticles employed in medical applications. Therefore, SEM monitoring of metal nanoparticles within cells and tissues is incompatible with the conventional preparation methods. In this work, we show that charging artefacts related to non‐conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive substrate. By growing the cells on glass pre‐coated with a chromium layer, we were able to observe the uptake of 10 nm gold nanoparticles inside uncoated and unstained macrophages and keratinocytes cells. Imaging in back scattered electrons allowed observation of gold nanoparticles located inside the cells, while imaging in secondary electron gave information on gold nanoparticles located on the surface of the cells. By mounting a skin cross‐section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals.     

Nanostructural analysis by atomic force microscopy followed by light microscopy on the same archival slide

Integrated information on ultrastructural surface texture and chemistry increasingly plays a role in the biomedical sciences. Light microscopy provides access to biochemical data by the application of dyes. Ultrastructural representation of the surface structure of tissues, cells, or macromolecules can be obtained by scanning electron microscopy (SEM). However, SEM often requires gold or coal coating of biological samples, which makes a combined examination by light microscopy and SEM difficult. Conventional histochemical staining methods are not easily applicable to biological material subsequent to such treatment. Atomic force microscopy (AFM) gives access to surface textures down to ultrastructural dimensions without previous coating of the sample. A combination of AFM with conventional histochemical staining protocols for light microscopy on a single slide is therefore presented. Unstained cores were examined using AFM (tapping mode) and subsequently stained histochemically. The images obtained by AFM were compared with the results of histochemistry. AFM technology did not interfere with any of the histochemical staining protocols. Ultrastructurally analyzed regions could be identified in light microscopy and histochemical properties of ultrastructurally determined regions could be seen. AFM-generated ultrastructural information with subsequent staining gives way to novel findings in the biomedical sciences. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

A New Method for the Measurement of Real Area of Contact by the Adhesive Transfer of Thin Au film

In this study, a new experimental method is proposed to measure the real area of contact between a ceramic sphere and an Al surface based on the adhesive transfer of the Au film and the scanning electron microscope (SEM) in the back-scattered mode. A thin film of Au is sputtered on the ceramic sphere before the indentation with the Al surface. The success of this method relies on the fundamental assumption that the adhesive transfer of Au only occurs everywhere inside the contact area. A thin polymer (PMMA) film is deposited between gold film and the ceramic surface to further reduce adhesive strength. After indentation, the interfaces of the ceramic sphere and Al surface are observed by SEM. Experimental evidence that the adhesive transfer of the Au film occurs inside the contact area is given. The entire contact regions on the ceramic sphere and the Al surface are captured in the second electron and back-scattered images with a magnification of 220× (resolution: 432 nm, i.e., distance between neighboring pixels). The contact area can be identified based on both the distributions of the ceramic and Au on the ceramic sphere and Al surface, respectively. The back-scattered images with the magnifications of 5000× and 10,000× (resolution: 20 and 4 nm) are captured at four different locations along the radial direction (starting from the contact center), respectively. The real area of contact decreases from the center to the contact edge.     

Atomic force microscopy of histological sections using a chemical etching method

Physiology and pathology have a big deal on tissue morphology, and the intrinsic spatial resolution of an atomic force microscope (AFM) is able to observe ultrastructural details. In order to investigate cellular and subcellular structures in histological sections with the AFM, we used a new simple method for sample preparation, i.e. chemical etching of semithin sections from epoxy resin-embedded specimens: such treatment appears to melt the upper layers of the embedding resin; thus, removing the superficial roughness caused by the edge of the microtome knife and bringing into high relief the biological structures hidden in the bulk. Consecutive ultrathin sections embedded in epoxy resin were observed with a transmission electron microscope (TEM) to compare the different imaging properties on the same specimen sample. In this paper we report, as an example, our AFM and TEM images of two different tissue specimens, rat pancreas and skeletal muscle fibres, showing that most of the inner details are visible with the AFM. These results suggest that chemical etching of histological sections may be a simple, fast and cost-effective method for AFM imaging with ultrastructural resolution.     

Biological ultrastructure as revealed by high resolution cryo-SEM of block faces after cryo-sectioning

Ultrastructural information was obtained by imaging the block face of high-pressure-frozen cryo-sectioned biological samples in a high-resolution cryo-SEM. Cryo-sectioning leads to a well-defined flat artificial surface in contrast to cryo-fracturing. Typical artefacts of cryo-sections such as compression and crevasses were not visible on the block face. The ultrastructural features known from resin sections and from freeze-fractures could also be found on the block faces. The cytoplasms show particles of different size which most likely represent proteins. The effects of radiation damage could be reduced considerably by applying the double layer coating technique and backscattered electron imaging.
High quality cryo-sections are only obtained from vitrified material. Reasonably flat block faces were, however, also obtained from adequately frozen microcrystalline samples, thereby facilitating ultrastructural studies in the frozen hydrated state.     

Fertilization-Induced changes in the vitelline envelope of echinoderm and amphibian eggs: Self-assembly of an extracellular matrix

The surface of the unfertilized sea urchin egg is covered by the vitelline layer (VL), a fibrous extracellular matrix that contains receptors for sperm. At fertilization, cortical granule exocytosis releases enzymes and structural proteins that cause the VL to elevate and become remodelled into the mechanically and chemically tough fertilization envelope. This envelope prevents further penetration of sperm and protects the embryo during early development. A thicker, more complex vitelline envelope surrounds the Xenopus laevis egg. This fibrous coat is also restructured at fertilization to produce an impenetrable barrier to sperm. The biochemical steps that occur during self-assembly of these fertilization envelopes are reviewed, and the ultrastructural changes that occur, as seen in platinum replicas of quick-frozen, deep-etched, and rotaryshadowed eggs, are illustrated.     

ICP-MS法测定添食金属纳米颗粒后家蚕组织中的金属含量

为研究添食金属或金属氧化物纳米颗粒后家蚕体内金属元素含量的变化规律,建立了微波消解结合电感耦合等离子体质谱（ICP-MS）测定家蚕血液、中肠和丝腺中Cu和Ti元素含量的方法。实验中分别给5龄3天家蚕喂食浓度为500.00 mg/L纳米Cu和纳米TiO₂悬浮液;72 h后,剪破家蚕腹足收集家蚕血淋巴,并解剖收集中肠和丝腺。实验发现,Cu和Ti元素的标准曲线呈良好的线性关系,相关系数分别为0.999 95和0.999 89,两种元素的加标回收率均在96.6%～105.4%之间,相对标准偏差RSD小于3.7%。结果表明,对于纳米TiO₂实验组,家蚕血液和丝腺中的Ti元素含量与空白组相比变化不大,而中肠的Ti元素含量明显增加为（181.46±9.58） μg/g,约为空白组含量（63.39±6.44） μg/g的3倍(p＜0.05);在纳米Cu组中,家蚕血液中的Cu元素含量有轻微的变化,而中肠和丝腺中的含量增加较多,分别为（82.73±1.72） μg/g和（3.88±0.17） μg/g,约为空白组含量（11.68±0.46） μg/g和（1.77±0.26） μg/g 的7和2倍(p＜0.05)。该方法可准确地检测喂食纳米颗粒后,家蚕组织中的金属元素含量及其变化,可为进一步研究家蚕的纳米生物效应提供有力支持。     

An experimental study on the ignition and emissions characteristics of wallpapers

The combustion characteristics of wallpaper and the toxicity of gas produced from wallpaper fires were analyzed to evaluate the fire risk of wallpaper used in living spaces. Ash content was measured with a high-temperature electric furnace, and thermal analysis was carried out with thermogravimetric analysis (TGA). Combustion time and smoke concentration were measured with a cone heater and a combustion gas analyzer. The smoke density of samples was measured using the smoke chamber ASTM E 662. Pyrolysis in silk wallpaper began at a lower temperature than the other samples. This means that silk wallpaper can be ignited at a low heat flux and will have a greater fire risk than other kinds of wallpaper. Heat by radiation flux caused silk wallpaper to ignite in the shortest time compared to the other samples, so the time for evacuation in this situation may also be reduced. Silk wallpaper also released the highest carbon monoxide concentration, so the toxicity and harmful effects to consciousness were stronger than any other wallpaper. The smoke densities of silk wallpaper and fire retardant-treated silk wallpaper were very high due to their vinyl coatings.     

A Generic Algorithm for Mesh Optimisation

The method presented in this paper optimises a given triangular mesh surface with respect to prescribed criteria to obtain a unit surface mesh. Two criteria are defined to guide the mesh optimisation scheme. In the procedure, we use three optimis-ation operators (edge split, edge collapse, and edge swap), and a local spherical surface is defined to interpolate the given mesh surface. As a generic algorithm for mesh surface optimisation, our algorithm needs neither parametric surface nor a cloud of points as references during mesh surface optimisation.     

麻花钻后刀面建模方法的优化

在分析和研究麻花钻锥面刃磨法、变导程螺旋面刃磨法及螺旋锥面刃磨法的基础上,提出了横刃及圆柱螺旋线扫掠法,并构建了其数学模型。该方法预先设计横刃,以横刃及螺旋线复合扫掠的方法构建主后刀面。采用该方法得到了麻花钻直线主刀刃和理想的横刃及后角值分布;解决了采用变导程螺旋面刃磨法钻芯强度较弱以及采用锥面刃磨法存在的主后刀面"翘尾"现象;解决了两主后刀面磨削自然形成横刃形状的可控性。