Single-channel response of hamster oocytes to fertilization with homologous spermatozoa.

Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved. Oocyte diameter was 70.2 +/- 2.2 microm; their resting parameters were: membrane potential 23.8 +/- 0.8 mV; total membrane specific resistance 519.1 +/- 94.6 ohms.cm2, and specific capacity 0.99 +/- 0.03 microF.cm(-2). Total membrane current was decreased by 42 % by 4-aminopyridine. Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in control oocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ entry.     

Low friction coefficient estimation for model material experiments

A previously suggested method of estimating low friction coefficients from the bulk deformation of long rectangular laminae during plastic compression, was used for wax specimens with various lubricants to enable the choice of the best lubricant for experiments with model materials. This method was also used for aluminium specimens with liquid and solid lubricants. Existing theory was extended to cover both the Amonton friction model and that of constant friction stress.Friction coefficients for certain waxes lubricated with vaseline were found to be as low as 0.01. With molybdenum disulphide grease on aluminium, the friction coefficient was found to decrease as compression proceeded. Friction coefficients with lead foil were found to agree with their independently known values. Lubrication breakdown at the periphery of specimens was observed in some tests, causing overestimation of the friction coefficient.     

用二维单侧容限系数公式测定压力容器钢疲劳裂纹扩展速率的小样本方法

应用二维单侧容限系数公式,提出了一种测定金属材料疲劳裂纹扩展速率的小样本方法。该方法可以综合利用以往历史数据和当前试验数据来测定金属材料疲劳裂纹扩展速率曲线。与仅能利用当前试验数据的传统方法相比,在精度相同的情况下,可以节省大量试件;在试件数量相同的情况下,可以大大提高测试精度。文中还给出了一个钢Q235A的计算实例。     

Histological control of specimen selection for electron microscopy

This technique enables small specimens for electron microscopy to be obtained from specific zones of large pieces of tissue. The method depends on the use of frozen sections, for localization of histological features, but circumvents the risk of ice-crystal damage to the specimens selected.     

基于最弱环理论的缺口件概率疲劳寿命预测方法

基于最弱环理论和光滑试样疲劳寿命的Weibull分布,建立了一种缺口件概率疲劳寿命预测方法。该方法首先基于最弱环理论和光滑试样的疲劳强度分布,通过定义缺口件的Weibull有效应力,建立了缺口件在给定循环载荷下的疲劳失效概率计算公式。基于Weibull有效应力和光滑试样的疲劳应力-特征寿命方程,可计算得到给定循环载荷时缺口件的特征疲劳寿命,进一步根据光滑试样的Weibull疲劳寿命分布可最终获得缺口件在给定循环载荷下的疲劳寿命分布。采用上述方法对TC4缺口试样进行了概率疲劳寿命预测,并与局部应力应变法预测结果进行了对比。结果表明:局部应力应变法预测结果过于保守,本文方法预测精度较高,50%失效概率时的疲劳寿命预测结果与缺口试样试验均值寿命吻合很好,10%和90%失效概率时的疲劳寿命预测结果基本分布在试验均值寿命的两倍分散带之内。     

Four-dimensional visualization and quantitative analysis of meiotic spindle movements in live mouse oocytes

This paper made a different attempt of real-time observation of the meiotic spindle movements in living mouse oocyte using a convenient method. This method was based on an experimental phenomenon discovered in our work. In living mouse oocytes, a high concentration of calcium ions (Ca(2+) ) was observed throughout the region occupied by the initial meiotic spindle. After Ca(2+) labelling with Fura-2, a weakly fluorescent area (WFA) appeared on each side of the chromosomes. The activities of the WFAs changed during spindle development. By real-time tracking of WFAs, we were able to indirectly observe the meiotic spindle movements. Occasionally, it was observed that the first meiotic spindle rotated from an orientation parallel to the cortex to become perpendicular, instead of migrating from the oocyte centre to the cortex along its axis. Moreover, we analysed this uncommon rotation of the first meiotic spindle and found that the whole rotation process can be divided into two phases: the early slow-speed rotation and the subsequent rapid-speed rotation. We further characterized this rotation with respect to rotational speed and acceleration at all the stages of development. By using a two-photon laser-scanning microscope in combination with Fura-2 dye that is nondamaging to oocytes, we provide a convenient method for indirect visualization and quantitative analysis of spindle movements by real-time tracking of WFAs. This method is easy to operate and master, and economical with time and effort.     

A balanced technique for preparation of specimens from pathogenicity studies for scanning electron microscopy

This paper reports our experiences with preparing delicate biological specimens for scanning electron microscopy. Three different washing methods were evaluated: One method allowed the analysis of the location of the bacterium Mycoplasma mobile on piscine gill epithelium and the optimal evaluation of histopathologic changes caused by this microbe. These results were achieved when specimens were washed three times in a cacodylic acid buffer after completion of the in vitro infection experiment in gill explant cultures. We also found that of three different concentrations of glutaraldehyde, a fixation with a 1.5% solution was sufficient to achieve excellent structural preservation, even without using post fixation in osmium tetroxide. Furthermore, this study showed that the use of acetone-carbon dioxide in the critical point drying procedure resulted in well-preserved piscine gill epithelium and mycoplasmas. Finally, long-term storage of tissue specimens in 0.1 M cacodylic acid buffer is possible if the buffer is changed on a monthly basis to avoid growth of unwanted microorganisms, such as fungi.     

Autofluorescence imaging, an excellent tool for comparative morphology

Here we present a set of methods for documenting (exo-)morphology by applying autofluorescence imaging. For arthropods, but also for other taxa, autofluorescence imaging combined with composite imaging is a fast documentation method with high-resolution capacities. Compared to conventional micro- and macrophotography, the illumination is much more homogenous, and structures are often better contrasted. Applying different wavelengths to the same object can additionally be used to enhance distinct structures. Autofluorescence imaging can be applied to dried and embedded specimens, but also directly on specimens within their storage liquid. This has an enormous potential for the documentation of rare specimens and especially type specimens without the need of preparation. Also for various fossils, autofluorescence can be used to enhance the contrast between the fossil and the matrix significantly, making even smallest details visible. 'Life-colour' fluorescence especially is identified as a technique with great potential. It provides additional information for which otherwise more complex methods would have to be applied. The complete range of differences and variations between fluorescence macrophotography and different types of fluorescence microscopy techniques are here explored and evaluated in detail. Also future improvements are suggested. In summary, autofluorescence imaging is a powerful, easy and fast-to-apply tool for morphological studies.     

A computational and experimental method for plotting multicycle fatigue curves for structural elements with concentrated stress

A theoretical method is considered for the theoretical plotting of the fatigue curves for specimens with stress concentration using the results of tests for smooth samples. The method is based on the results of a large number of experimental data for each material (from 100 to 200 specimens for each level of stress concentration). The computed and experimental data proved to be in reasonable agreement.     

The use of pattern recognition for classification of stochastic specïmens by a set of standard specimens

This paper describes an empirical method for the experimental assigning of specimens to a set of standard specimens. The procedure was developed especially for stochastic scenes. Typical applications of such a procedure are found in material sciences in assigning specimens to a standard series. The assignment is based on feature vectors obtained from analyses of the specimen texture by means of erosion or opening. In cases where the arrangement of particles is characteristic of the specimen, dilation or closing are used to obtain a feature vector. To increase the sensitivity of the method described the use of more than one structuring element is recommended in the analysis. Besides the classification of an unknown specimen the procedure provides information on the separability of the standard specimen and the significance of assignment.     

Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells

Critical point drying (CPD) is a common method of drying biological specimens for scanning electron microscopy (SEM). Drying by evaporation of hexamethyldisilazane (HMDS) has been described as a good alternative. This method, however, is infrequently used. Therefore, we reassessed HMDS drying. Cultured rat hepatic sinusoidal endothelial cells (LEC), possessing fragile fenestrae and sieve plates, were subjected to CPD and HMDS drying and evaluated in the scanning electron microscope, atomic force microscope (AFM) and transmission electron microscope (TEM). We observed no differences between the two methods regarding cellular ultrastructure. In contrast with CPD, HMDS drying takes only a few minutes, less effort, low costs for chemicals and requires no equipment. We conclude that HMDS-dried specimens have equal quality to CPD ones. Furthermore, the method also proved useful for drying whole-mount cells for TEM and AFM.     

Image analysis for 3D micro-features: A new hybrid measurement method

Measuring 3D micro-features is a challenging task that is usually performed using high-cost systems generally based on technologies with narrow working ranges and very accurate control of the sensor positions. Well-known image analysis methods, such as Photogrammetry, would likely lower the costs of 3D inspection of micro-features and add texture information to the 3D models; however, the behaviour of Photogrammetry is strongly affected by the scaling method because it retrieves a model that must be scaled after its computation. In this paper, an experimental study of the validity of a hybrid 3D image processing method for measuring micro-features is presented. This method exploits the Depth-from-Focus method to retrieve the correct scale for a photogrammetric model. The measurement of properly-designed and manufactured specimens was performed by following and adapting the German guideline VDI/VDE 2634, Part 3 to validate the method using calibrated specimens. The proposed system has been demonstrated to be very promising can achieve an error of less than 10 μm.     

Application of a NDI method using magneto-optical film for micro-cracks

Leakage magnetic flux is occurred in the cracked area of magnetized specimens, and also it changes the magnetic domain area of the magneto-optical film positioned on the specimen. It causes the change of the optical permeability of the magnetic domain on the crack area. So crack images can be obtained easily using this principle. On the other hand, utilizing a laser in this method makes possible to perform a remote sensing by detecting the light intensity contrast between cracked area and normal area. This paper shows the application of non-destructive inspection system taking advantage of magneto-optical method for micro-cracks and presents examples applied to the several types of specimens having fatigue cracks and fabricated cracks using this method. Also the authors prove the possibility of this method as a remote sensing system under the oscillation load considering application to real fields.     

Methods for specimen thickness determination in electron microscopy

A new method is described for specimen thickness determination in transmission electron microscopy. This is carried out by marking specimens with gold particles and analysing the images of a tilt series by computer. The method makes it possible to distinguish populations of particles on different planes and calculate the distance between the planes with statistical variation. We have applied it to carbon films as test objects and compared the results with those obtained by transverse sectioning, STEM mass measurement, optical density and frequency change of a quartz crystal oscillator. We have then used the method for thickness measurement of multilayered protein crystals and thin sectioned cells.     

Improved staining of microfilament bundles in plant cells for high voltage electron microscopy

A post-polymerization en bloc staining method for high voltage electron microscopy is described. Embedded specimens were initially trimmed to an area close to the point of interest. Trimmed blocks were stained in 5% uranyl acetate/75% ethanol solutions for 36 h at 333 K. This procedure allowed specimens to be stained without the necessity of exposing Formvar films to damaging solutions. After such staining, both the contrast and fine detail of structures such as microfilament bundles were superior to that seen in material stained with aqueous solutions of uranyl salts.     

Note: direct measurement of the point-to-point resolution for microns-thick specimens in the ultrahigh-voltage electron microscope

We report on a direct measurement method and results of the point-to-point resolution for microns-thick amorphous specimens in the ultrahigh-voltage electron microscope (ultra-HVEM). We first obtain the ultra-HVEM images of nanometer gold particles with different sizes on the top surfaces of the thick epoxy-resin specimens. Based on the Rayleigh criterion, the point-to-point resolution is then determined as the minimum distance between centers of two resolvable tangent gold particles. Some values of resolution are accordingly acquired for the specimens with different thicknesses at the accelerating voltage of 2 MV, for example, 18.5 nm and 28.4 nm for the 5 μm and 8 μm thick epoxy-resin specimens, respectively. The presented method and results provide a reliable and useful approach to quantifying and comparing the achievable spatial resolution for the thick specimens imaged in the mode of transmission electron including the scanning transmission electron microscope.     

A comparative study of mechanical behavior of ABS material based on UVC sterilization for medical usage

This study aims to examine the mechanical properties of acrylonitrile butadiene styrene specimens using ASTM 638, 695, and 790. UVC radiation was also used as a sterilizing method. The fused deposition modeling of 3D-printed polymerize with 30% filling was used to manufacture 30 specimens for tensile, compression, and bending. Half of the specimens were treated with UVC, whereas the other half were not. The chosen dosage of 13.5 J/cm² with an exposure time of 48 min corresponds to 3650 sterilization treatments or 10 years of sterilization. The average ultimate stress in the tensile test, compression test, and bending test was 34.5 ± 7.4, 25.4 ± 0.5, and 24.5 ± 2.1 Mpa, respectively. The analysis of variance test shows that UVC radiation has a demonstrable influence on tensile specimens, with a P-value of 0.012, which is less than the significance threshold of 0.05. Thus, the null hypothesis is rejected.

     

Synaptic process in the rat (Rattus norvegicus): Influence of methodology on results

Synaptonemal complex (SC) analysis is a widely used method for assessing the effects of genotoxic agents in germ cells. Although the evolution of the SCs and their related annexed structures, such as nucleoli, has been well established, sometimes it is difficult to assess whether the abnormal features observed correspond to genotoxic effects or to an artefact related to the method used to obtain the SC preparations. In this article, we describe a new method of obtaining SC preparations for electron microscopy, as well as the results of a study of the first meiotic prophase in oocytes and spermatocytes of the rat (Rattus norvegicus Sprague Dawley) in which we analysed how the methodology used can influence the results. Besides important sex-specific differences, mainly during desynapsis (diplotene), a relationship between several bivalents and nucleolar structures, that in some cases could disturb the synaptic process, was observed in oocytes. At the same time, the characteristic SC fragmentation in oocytes was verified, but this fragmentation, in addition to a sex-specific component, was influenced by the method itself. By reducing to a minimum the artefacts produced by the method, it is possible to optimise the analysis of SCs as a method of testing genotoxic effects in the germ line.     

混凝土和水泥砂浆二维定位取样方法试验研究

通过研发一种混凝土和水泥砂浆二维定位取样装置,提出一种可以对混凝土和水泥砂浆进行精确二维定位取样的方法。该方法可以将混凝土和砂浆试块进行有效固定,避免取样过程中试块发生移动,通过周边加固,避免取样过程中出现开裂和崩落问题,并通过加装高分子薄板减少粉末样本流失,从而达到对混凝土和水泥砂浆进行二维精确取样的目的。     

Tannin-osmium conductive staining of biological specimens for non-coated scanning electron microscopy

Osmication of biological specimens with a mixture of tannic acid, guanidine hydrochloride, arginine hydrochloride and glycine imparted sufficient electron conductivity to permit SEM observations of non-coated samples. No charging was noted at 25 kV acceleration voltage and 1 × 10^?10 A specimen current in rat kidney specimens. The foot-processes of the glomerular podocytes were clearly visible without metal coating. This tannin-osmium method even allowed block staining and enabled continuous observations during and after dissection in the SEM.