An easy method for orientated embedding tissue culture cell monolayers in LR white resin for postembedding immunocytochemistry

Partial polymerization of LR White resin blocks is a frequently encountered problem if oxygen is present during the polymerization reaction. Instructions are given for a simple method to embed cell culture monolayer in LR White acrylic resin, which is suitable for immunocytochemistry. The advantage of this method is that it is easy and reliable. It also ensures accurate orientation of the cell monolayer in relation to the desired plane of sectioning.     

A novel technique for flat-embedding cryofixed plant specimens in LR white resin

Acrylic LR White is used preferentially for many research applications because it possesses unique advantages over other commercially available resin types. LR White has low toxicity; its low viscosity and hydrophilic properties enable it to infiltrate specimens with comparative ease, and specimens embedded in LR White take up stains for light microscopy well and tend to retain superior antigenicity relative to other resins. These qualities make LR White a good choice for immunohistochemistry, light microscopy, and transmission electron microscopy. However, LR White does not heat-polymerize in the presence of oxygen, which precludes its use with many commercially available embedding molds. Furthermore, flat-embedding specimens in LR White is often an arduous task. Therefore, in this paper, we report on the development of novel flat-embedding chambers for use with LR White. Another goal of our studies was the rapid fixation of larger specimens. Chemical fixation was inadequate because of the time required for tissue infiltration, but cryofixation quickly and effectively immobilized intracellular organelles. Mean amyloplast positions differed in vertically oriented versus control specimens after chemical fixation, whereas no significant difference was observed after cryofixation. Furthermore, cryofixation provided acceptable preservation at the light microscopy level, even though our specimens were relatively larger. This underscores the utility of cryofixation for light microscopy determination of organelle positioning within plant cells.     

A synchrotron X-ray study of the changes occurring in the corneal stroma during processing for electron microscopy

Using a high-intensity synchrotron X-ray source, the structural changes occurring in the corneal stroma were monitored during each stage of several different processing runs for the transmission electron microscope (TEM) and scanning electron microscope (SEM). The parameters studied were interfibrillar spacing, intermolecular spacing, D-periodicity and fibril diameter. The processing schedule that produced the least changes in spacings for TEM specimens involved extended fixation in glutaraldehyde followed by low-temperature embedding in Lowicryl K4M resin. However, interfibrillar material was better preserved after embedding in LR White resin or Nanoplast. Almost every processing stage for electron microscopy produced significant changes in one or more structural parameters in the cornea. Glutaraldehyde fixation significantly increased the intermolecular spacings, while resin infiltration and resin polymerization each resulted in shrinkage of all the spacings monitored. Critical-point drying for SEM specimens resulted in considerable shrinkage in all three spacings, but was still preferable to air drying, which caused reduction in the order of the fibril packing, resulting in loss of the interfibrillar X-ray pattern. Perhaps the most drastic effect was caused by post-fixation in osmium tetroxide, which resulted in loss of the intermolecular pattern, and also increased the amount of shrinkage in the interfibrillar spacings and the D-periodicity which occurred during later stages of processing.     

Heating of TEM specimens during ion milling

Sample heating during preparation of electron-thin specimens for observation in transmission electron microscopy (TEM) can produce artefacts which invalidate observations. This is particularly true of two-phase materials such as metal matrix composites, for which sample cooling with liquid nitrogen cannot be used to preserve the substructure during milling. A series of experiments is conducted using an age-hardenable aluminium alloy which produces a trace of peak temperature attained by TEM specimens during ion milling. It is shown that peak temperatures of the order of 650 K can be attained using conventional milling parameters; the technique must therefore be used with caution with materials such as metal matrix composites. A simplified one-dimensional heat transfer analysis of the problem is conducted to show that the most critical parameter is heat transfer along the sample holder legs and across interfaces along the heat path. Temperature differentials within the TEM specimen are shown to be less significant, yet these alone are capable of reaching 60 K within a dimpled specimen under usual milling conditions.     

The biological reality of the interlacunar network in the embryonic,cartilaginous, skeleton: A thiazine dye/absolute ethanol/LR White resin protocol for visualizing the network with minimal tissue shrinkage

Third toe phalanges of chicks aged 8–13 days in ovo and 7-day post-natal rat femoral growth plate were examined to determine whether the interlacunar network (IN), a structure with no lipoprotein membrane component or cytoplasmic organelles, is a genuine component of young growth cartilage. In chick phalanges dehydrated by 70% (v/v) ethanol and LR White resin, variable metachromatic staining of the interlacunar network by toluidine blue and red staining by picro-Sirius red indicate the presence of glycosaminoglycans and collagen. The network in phalanges dehydrated by 80% (v/v) ethanol appears little different; however, the network is much less widely detectable in phalanges dehydrated by 90% (v/v) ethanol and, after dehydration by absolute ethanol, is almost completely undetectable. In contrast, when the young cartilage is permeated by a thiazine dye such as toluidine blue, using a solution of dye in the aldehyde fixative, the network is widely detectable, following dehydration by absolute ethanol, both in chick phalanges and in rat growth plate. Comparison of projected areas shows that the extent to which whole chick feet are found to have shrunk, by the time that they are photographed under LR White resin, is determined principally by the extent of dehydration, by 70% (v/v) or absolute ethanol; post-shrinkage areas are 33% or 35% of areas measured in buffer for 70% (v/v) ethanol/LR White resin and 71% or 75% for absolute ethanol/LR White resin (the higher value in each is for the toluidine blue treatment). The network is thus present in radically shrunk tissue, but, significantly, is also fully represented in tissue shrunk by only a conventional margin and is therefore not produced as an artefact by exceptional tissue shrinkage as has been suggested.     

Flat embedding and immunolabelling of SW 1116 colon carcinoma cells in LR white: an improved technique in light and electron microscopy.

Human SW 1116 colon carcinoma cells were grown on matrix-covered coverslips and flat embedded in specially prepared gelatin capsules in the hydrophylic resin LR White. Dehydration and polymerization were carried out so as to maximize preservation of antigenicity. Sections were cut perpendicular to the substratum. To visualize mucin, semithin sections of SW 1116 cells were stained with periodic acid Schiff (PAS) reagent for light microscopy, and ultrathin sections were labelled with a monoclonal mucin antibody (Mab 19-9) and immunogold for electron microscopy. Immunofluorescence was carried out on whole cultured cells using Mab 19-9. The morphological preservation of SW 1116 cells embedded in LR White was comparable to that of Epon-embedded cells. Mucin was localized on the microvillar surface of the apical plasma membrane and occasionally in intercellular spaces between adjacent cells. Mucin was also present in vesicles in the apical and lateral part, and to a lesser extent in the basal part of the cells. We conclude that this new technology significantly improves the morphological preservation of cells and tissues in LR White, while also serving to sustain the antigenicity of cellular antigens.     

复合材料冲击损伤高分辨率超声成像检测与损伤行为分析

冲击损伤是复合材料结构易产生的一种危害性缺陷,针对碳纤维复合材料层压结构特点,分析脉冲超声波在复合材料中的反射特性,当能减小入射声波脉冲的时域占宽,提高其品质,可显著改善超声检测表面盲区和纵向分辨率。利用入射声波在冲击损伤区形成的反射信号及其渡越时间可确定冲击损伤的深度和大小。研究采用高分辨率脉冲超声B扫描和层深C扫描(T扫描)成像方法,可揭示复合材料内部冲击损伤及其大小与沿厚度方向的分布特征,进行复合材料冲击损伤量化评估与损伤行为可视化分析。试验结果表明,在入射脉冲单周脉冲特性条件下,当来自复合材料缺陷的回波信号的渡越时间达到入射声波脉冲时域占宽的一半左右时,即可清晰地提取到时域可分辨的缺陷回波信号,使超声检测表面盲区和纵向分辨率达到单个复合材料预浸料铺层的厚度,约0.13 mm。利用超声断面B扫描成像,可以形象地再现复合材料中冲击损伤的断面分布与扩展情况以及损伤的深度等确切信息。采用超声T扫描成像,则可以直观地再现冲击损伤在复合材料铺层方向的分布及其损伤区域(面积)等定量信息。二者的结合则为揭示复合材料冲击损伤的3D分布及其特征提供一种超声成像检测量化评估和损伤行为可视化分析方法。     

Enhancement of contrast in living and fixed specimens by the use of fibre optics

A method is described which enhances the contrast of living and fixed specimens examined with the stereomicroscope. It consists of immersing the ends of flexible fibre optic light sources together with the specimen in the fluid used for examination. It is reported that not only does this method increase the contrast of living specimens but that it may also be applied to specimens being prepared as thin sections or freeze fracture surfaces for examination with the transmission electron microscope. A further method of enhancement of contrast is suggested which involves the fitting of light filters of complementary colours, one to each of the fibre optic light sources, before immersion with the specimen.     

Application of quenching to create highly triaxial residual stresses in type 316H stainless steels

It has been proposed that highly triaxial residual stress fields may be sufficient to promote creep damage in thermally aged components, even in the absence of in-service loads. To test this proposal, it is necessary to create test specimens containing highly triaxial residual stress fields over a significant volume of the specimen. This paper presents results from an experimental and numerical study on the generation of triaxial residual stresses in stainless steel test specimens. Spray water quenching was used to generate residual stress fields in solid cylinders and spheres made from type 316H stainless steel. A series of finite element simulations and measurements were carried out to determine how process conditions and specimen dimensions influenced the resulting residual stress distributions. The results showed that highly compressive residual stresses occurred around the surfaces of the cylinders and spheres and tensile residual stresses occurred near the centre. Surface residual stresses were measured using the incremental centre hole-drilling technique, while internal residual stresses were measured using neutron diffraction. Overall there was good agreement between the predicted and measured residual stresses. The level of triaxiality was found to be very sensitive to the heat transfer coefficient, and could be controlled by adjusting the cooling conditions and changing the dimensions of the steel samples. This differed from other processes, such as welding and shot-peening, where the magnitudes and distributions of residual stresses are ill-defined and the volume of material subjected to a triaxial residual stress state is relatively small.     

A CAD-CAM methodology to produce bone-remodelled composite femurs for preclinical investigations.

Femoral bone remodelling, following total hip arthroplasty, is a clinically observed phenomenon attributed to the changed stress environment of the postoperative implanted hip. While this process cannot be avoided, there is concern as to its consequences on the long-term survival of hip joint replacements. Previous methods of studying remodelling, such as clinical or animal-based studies, or finite element analyses, have their limitations. The aim of this study is to develop experimental specimens incorporating bone resorption features typical of clinically successful implants. This work describes the use of computer aided design/manufacturing methods (CAD-CAM) to produce these specimens, based on modifying commercially available composite femurs. The procedures are investigated and verified for two different designs of cemented prostheses (Lubinus SPII and Muller Curved). Quantitative clinical data is used to define the remodelled geometry of a CAD model of the femur for each stem design. Composite femur specimens are machined using a three-axis milling machine, where each specimen can be accurately positioned using a custom-designed jig and a digitizer system. The accuracy of the process is assessed by analysing the deviation of the digitized premachined and postmachined surfaces of each specimen in relation to the CAD model. The results demonstrate that the procedure can be used for developing in vitro specimens with bone resorption features. These specimens are proposed as a useful tool for performing preclinical trials, such as load transfer or longevity/stability testing, with the advantage of modelling a long-term clinical situation, rather than solely analysing implanted femurs in an immediate postoperative state.     

Radiation damage in dry and frozen hydrated organic material

Electron-induced radiation damage can cause errors in interpreting electron micrographs. Radiation damage is distinguished from contamination (polymerization of hydrocarbons) and etching (radiolysis in the presence of water), both of which can be controlled by a proper specimen environment in the microscope. While temperature has little effect on the primary interactions of fast electrons with matter, most secondary radiation-damage processes are temperature dependent. Because damage mechanisms differ so greatly among materials, there is no simple factor by which specimen stability is improved as a function of temperature (some cases improve fivefold, others improve 100-fold). While some specimens are stable to almost arbitrarily high doses, some tests reveal damage at 1 e/nm². This paper surveys damage rates and temperature dependencies of various materials as a guide for future electron microscopic studies of organic specimens.     

Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy

A method is described for the cryofixation of biological specimens for ultrastructural analysis and immunocytochemical detection studies. The method employs plunge freezing of specimens in a sealed capillary tube into a cryogen such as liquid propane or liquid nitrogen. Using this method a number of single-cell test specimens were well preserved. Also multicellular organisms, such as Caenorhabditis elegans , could be frozen adequately in low ionic strength media or even in water. The preservation of these unprotected specimens is comparable to that achieved with high-pressure freezing in the presence of cryoprotectant. The results are explained by the fact that cooling of water in a confined space below the melting point gives rise to pressure build-up, which may originate from the conversion of a fraction of the water content into low-density hexagonal ice and/or expansion of water during supercooling. Calculations indicate the pressure may be similar in magnitude to that applied in high-pressure freezing. Because the specimens are plunge cooled, suitable cryogens are not limited to liquid nitrogen. It is shown that a range of cryogens and cryogen temperatures can be used successfully. Because the pressure is generated inside the specimen holders as a result of the cooling rather than applied from an external source as in high-pressure freezing, the technique has been referred to as self-pressurized rapid freezing.     

Numerical analysis of a radiant drying oven for web applications

During the manufacture of prepregs (composite material made of resin with fiber reinforcement), for the electronics industry, solvent evaporation and product heating are important aspects of the production process. A mathematical model is presented to describe convective and infra-red radiant heating and solvent evaporation from a web (product being processed before being finished as prepreg) in a solvent removal oven. Differential equations are used to model the mass and energy transfers occurring within the oven. Radiation enclosure theory is employed to describe the infrared heat transfer within the oven and use is made of a solvent evaporation model from the literature. Results of temperature distributions in the heated plate (oven component), the web and the air/solvent mixture; the convective and radiant heat transfer rates to the web, the web solvent concentration and the effects of varying the web velocity, heater temperature, heater size and inlet air temperature are in agreement with theory. The results yielded good agreement with a similar study in the literature. Careful analysis of the results of computer simulations under different conditions, such as presented in this study, can be expected to lead to better design decisions prior to building an infra-red oven for web applications.     

Atom probe specimen preparation with a dual beam SEM/FIB miller

Dual beam scanning electron microscope/focused ion beam (SEM/FIB) methods complement electropolishing methods and enable specimens to be made from a wider range of materials. Several methods have been developed to fabricate specimens from different forms of materials, including thin ribbons, mechanically ground sheet and fine powders. In addition, FIB-based methods can be used in conjunction with electropolishing methods to improve the shape, surface finish and taper angle of specimens. Several lift-out (LO) methods have been developed for selecting specific microstructural features or other regions of interest such as phases, interfaces, grain boundaries, subsurface or implanted regions and interdendritic regions. These LO methods make use of an in situ nanomanipulator and platinum deposition to transfer and attach the lifted out volume to a post for final annular milling into a needle-shaped specimen. In order to improve the efficiency and to facilitate the LO procedure, some special specimen mounts that hold both the specimen and the support post at the appropriate working distance have been developed.     

Effect of surface etching on condensing heat transfer

This study conducted experiments on humid air condensation during heat transfer in an air preheating exchanger attached to a home condensing boiler to improve thermal efficiency. An etchant composed of sulfuric acid and sodium nitrate was used to create roughness on the heat exchanger surface made from STS430J1L. A counter flow heat exchanger was fabricated to test the performance of heat transfer. Results showed that the overall heat transfer coefficients of all specimens treated with etchant improved with respect to the original specimens (not treated with etchant), and the overall heat transfer coefficient of the 60 s etching specimen increased by up to 15%. However, the increasing rate of the heat transfer coefficient was disproportional to the etching time. When the etching time specifically increased above 60 s, the heat transfer coefficient decreased. This effect was assumed to be caused by surface characteristics such as contact angle. Furthermore, a smaller contact angle or higher hydrophilicity leads to higher heat transfer coefficient.

     

An investigation on the fatigue behavior of DCB specimen bonded with aluminum foam at Mode III

Aluminum foam with its excellent physical and mechanical characteristics is a lightweight metallic material used with good quality in vehicle bumpers, internal shock absorbers on planes, as materials for vessel joints etc. On the contrary, when aluminum foam is used without sufficient investigation, there is the likelihood of damage or destruction of the machine or mechanical structure, and in extreme case it may cause to human casualties. This study aims to analyze the characteristics of adhesive structures with aluminum foam for the closed-type aluminum foam used primarily as shock absorbers. The fatigue analyses of the DCB test specimens at mode III with aluminum foam are verified through a fatigue experiment. As the analysis results, test specimen models with the thicknesses (t) of 35 mm, 45 mm and 55 mm showed the peak load occurrence approximately after the progress from 0 to 50 cycles. And afterwards the load gradually decreased as the cycles increased. The peak loads for each DCB test specimens were ±0.80 kN for the specimen thickness(t) of 35 mm, ±0.98 kN for the specimen thickness(t) of 45 mm and ±1.18 kN for the specimen thickness(t) of 55 mm. It is also shown that the peak load occurring on the specimen increased as specimen thickness increased. These study results are compared with the specimen thickness of 35 mm model as the basis. When the specimen thickness is increased as much as 10 mm, the peak load is increased approximately 1.25 times. When the specimen thickness is also increased as much as 20 mm, the peak load is increased 1.5 times. The analysis data and the real experiment data showed similar results each other. Therefore, it can be thought that the analysis data is applicable in real field. And it is estimated that the mechanical characteristics of the DCB test specimen at mode III during the fatigue load conditions can be systematically and efficiently analyzed.     

The crack healing behavior of ZrO2/SiC composite ceramics with TiO2 additive

This paper was carried out to assess the crack-healing behavior of ZrO₂ composite ceramics. The ZrO₂ composite ceramics were made by adding 10 wt.% SiC and TiO₂. The TiO₂ additives were changed to 1, 3 and 5 wt.%. The characteristics of crack-healing behavior were studied as a function of annealing temperature and time. Specimens were mirror polished. The cracks of about 100 ??m were introduced on the specimen surface by Vickers indenter and specimen were heat-treated. The strength of crack-healing was conducted by a three-point bending, and the each specimens was measured by XRD. The results obtained in this study are as follows: The ZST composite ceramics contained crack-healing properties, and the strength of ZST smooth specimens have been shown to be better than ZS specimens. The optimum crack-healing condition of the ZTS3 specimen was 1073 K, 5 hours in the air. Specimens with heat treatment had micro-cracks and large cracks on the surface, but the micro-cracks on the surface did not affect the bending strength. It was found that specimens display phase transformation from tetragonal crystal to monoclinic crystal after heat treatment.     

An approach to an objective background subtraction for elemental mapping with core-edges down to 50 eV: description, evaluation and application

To image the distribution of a specific element in a specimen with an energy filtering TEM, the element-unspecific background under the core-edge has to be subtracted. The most commonly used procedure is the three-window power-law method leading to considerable systematic errors for low-energy core-edges. Here a new method is described which can be considered as a generalized difference method. Characteristic examples for element detection in biological specimens using this method are shown. The background under the core-edge can be described by one or two pre-edge windows as a polynome of third order. This function can be deduced from specimen areas that are not known to contain the element or from a second specimen used as a standard. Control experiments showed that background subtraction for on-overlapping core-edges in the low-loss region (50-200 eV) needs two pre-edge images, whereas at higher-energy losses (> 300 eV) only one pre-edge image is necessary. With the method described, objective elemental mapping becomes possible even for edges at 50-100 eV. This was proven for the M2,3-edge of iron at 60 eV. The detection of phosphorous was possible with a signal-to-noise ratio five times higher than when using the three-window method. Preliminary data showed that it should be possible to detect calcium with only one image before the edge.     

Optimized reflection imaging in laser confocal microscopy and its application to neurobiology: Modificationsa to the biorad MRC-500

Reflection images of biological specimens recorded using laser-scanned confocal microscopes are frequently degraded by low image contrast, poor signal to noise, and the inability to image deeper in the specimen than 10–20 μm. Artifactual internal reflections often are a source of these limitations, but they can be reduced or eliminated by the use of polarization components. Designs for the incorporation and optimum use of these components in the BioRad MRC-500 are presented. The effect of the internal reflections was reduced by optimum rotational alignment of both a quarterwave plate and an analyzer. Absorption of incident and reflected light by both the stained cells and the background tissue of the specimen also seriously degrades image signal to noise, and is a function of specimen preparation and the wavelength of light used. The red line of a helium-neon laser was not as readily absorbed as the blue and green lines of an argon-ion laser when imaging neurobiological specimens contrasted with either peroxidase/diaminobenzidine or Golgi staining. Specimens many times thicker were imaged with red laser light and with superior image quality compared with blue or green laser light.     

LR White sections as slot grid support films for transmission electron microscopy

The utility of LR White sections as slot grid support films for the examination of thin resin‐embedded tissue sections by transmission electron microscopy was investigated and compared with traditional formvar‐carbon films. Throughout a variety of staining procedures, which involved the use of organic solvent, oxidizing agents, strong acid and prolonged incubation, LR White support films remained intact and the attached tissue sections remained adherent. By contrast, complete loss of formvar‐carbon support films occurred in 25% of preparations during routine staining with aqueous reagents. This loss increased to 62% following staining with either alcoholic or oxidizing and acidic stains, and to 66% following prolonged (immunohistochemical) staining. Tissue contrast, ultrastructural detail and immunohistochemical staining intensity were comparable between sections on the two types of support film. The use of LR White sections as support films for slot grids represents a quick, cheap, simple and robust alternative to traditional support films and, furthermore, requires no carbon coating