A freeze-fracture replication apparatus for biological specimens.

A freeze-fracture apparatus of original design has been constructed which can be fitted onto a standard vacuum evaporator unit. In it, cell suspensions and organized tissue may be processed by inserting a sample into a cylindrical holder. By leaving a small part of the tissue protruding from the holder, pre-selected and aligned portions of the specimen can subsequently be revealed by fracture under vacuum. After rapid freezing, the specimen remains firmly attached to the inner wall of the sample holder, preventing its possible loss during fracturing. A mechanism, in the form of a double-sided converging wedge, which is operated from outside the vacuum chamber, is used to produce a fracture in the specimen. The device gently induces a fracture in the desired part of the tissue and lifts the protruding part of the specimen out of the way. In this way, reasonably flat fracture faces are produced for subsequent replication. As the fracturing mechanism comes into contact only with the outer edges of the specimen, damage and contamination liable to occur when the entire specimen is traversed by a blade, is avoided. In addition the specimen stage is surrounded by a cold metal shroud which acts as an efficient trap for contaminants. In this way, favourable vacuum conditions are produced in the vicinity of the specimen. Such effective enclosing of the specimen also facilitates controlled sublimation of the sample.     

Transmission electron microscope in situ fatigue experiments: A computer-control approach

A computer-control procedure was developed to facilitate in situ fatigue experiments within an intermediate voltage transmission electron microscope using a goniometer-type straining holder. The procedure was designed to allow sine-wave tension-tension cyclic loading of a microfatigue specimen similar in geometry to a center-crack panel fatigue specimen. Computer control allows greater freedom for the operator to control the experiments while providing better reproducibility from one test to another. Further development of this procedure is possible by coupling this computer-control technique with computer-controlled stage motion and digitized TV imaging.     

In situ transmission electron microscope study of single asperity sliding contacts

Direct observation of events taking place at the contacting interfaces is important to understand many tribological phenomena. Transmission electron microscope (TEM) has the ability to look through materials at very high magnifications. Most of the TEM observations are done long after the deforming loads and stresses have been relaxed and the material state is further disturbed during the specimen preparation. We have developed a specimen holder in which two electron transparent surfaces can be brought in contact and moved relative to each other in JEOL 2000FX microscope. This holder enables visualization of not only the contacting surfaces at nanoscale but also the subsurface deformation resulting from the contact interaction. Sliding experiments have been carried out mimicking a single asperity sliding contact. A sharp tungsten probe is moved laterally against a tip mounted on a cantilever. Magnitude of the contact instability, when the contact is broken is found to be dependent on the local geometry of the contact.     

Crack-tip field characterization of crack-tip opening angle-based crack growth — plane strain single-edge cracked specimen subject to pure extension

The objective of this work is to study the crack-tip field of plane strain fully plastic crack growth of a single-edge cracked specimen subject to pure extension. The nodal release technique [1] was implemented into an FEM model so that the nodes arranged in the crack path are released. The fracture criterion for the crack growth in the model adopts the loading geometry-based crack-tip opening angle-(CTOA) proposed in the work of Ref. [2]. The crack-tip field from the CTOA-based crack growth was investigated and characterized with a slip line field. It was observed that the backface configuration of the specimen, which evolves with the crack growth, is engaged in the formation of the shear band and, as a result, significantly elevates the triaxiality at the crack-tip during the crack growth.     

Increasing the versatility of an EMscope SP2000A sputter cryo system on a Hitachi S-570 scanning electron microscope

The EMscope SP2000A Sputter Cryo System provides biologists with a quick and reproducible procedure to freeze, fracture, etch, and sputter coat biological samples, which are then transferred to a cold stage for scanning electron microscopic (SEM) observations. The standard specimen holder, which is supplied with the unit and referred to as a “stub,” will accommodate most specimens; however, fabricating a variety of additional holders to perform more specialized functions considerably increases the versatility of the cryounit and the resolution that can be achieved. For example, the adaptation of small hinged or hingeless gold holders, which are used in the freeze-fracture technique, produces clean fractures, allows for storage of samples in liquid nitrogen and permits samples to be frozen much more rapidly than the large standard holder. A short working distance holder has been designed, which allows samples to be inserted into the final objective lens, thereby resulting in a negative working distance up to ?3 mm. Use of this holder with the upper detector in the Hitachi S-570 SEM, enhances the secondary electron signal and thereby increases resolution of frozen samples severalfold. Another specimen holder has been designed, which allows conventional SEM stubs to be observed without removal of the cryostage. This modification permits use of the EMscope stage and specimen transfer device at room temperature without any further alteration or adjustment. These and other types of modified holders allow investigators to store, manipulate, fracture, and observe biological samples at resolutions not normally attainable with a standard SEM cryostage.     

Nanofabrication of cylindrical STEM specimen of InGaAs/GaAs quantum dots for 3D-STEM observation

We demonstrate the multiazimuth observation (360 degrees in principle) of InGaAs/GaAs quantum dots (QDs) by means of a 300 kV scanning transmission electron microscope (STEM), where both cross-sectional and plan-view observations are performed on a single STEM specimen for the first time. A cylindrical specimen with a diameter of 200-300 nm including the QD layer inside along the rotation axis was fabricated by the focused ion beam (FIB) technique, with the application of a newly developed mesa-cutting method to adjust the position and angle of the QD layer precisely. The 360 degrees STEM observation is realized by mounting the cylindrical specimen on a holder equipped with a specimen-rotation mechanism. High potential of 3D-STEM observation is briefly presented by showing high contrast images of QDs, dark field images, and moire fringes with various incident angles.     

Effect of strength mismatch on fully plastic fields in dissimilar joints under combined loading

Via detailed finite clement limit analyses, plastic limit loads, rotation factors, and crack-tip stress field are investigated for a combined tension and bending of a plance strain single-edge-cracked bimaterial specimen. Limiting bimaterial specimens are considered, consisting of an clastic/perfectly plastic material bonded to an elastic material having the same elastic properties. The limit loads of bimaterial specimens are shown to be very close to those of homogeneous specimens, so that limit load information for homogeneous specimens can be used even for bimaterial specimens. A tractable, approximate elliptical yield locus is proposed, which first the FE. results within 1% for all ranges of tension-to-bending ratios. The plastic rotation factor of bimaterial specimens can be higher than that of homogeneous specimens as much as 25%, when the specimen is subject to small tensile forces. Results from the present analysis is applied to the analysis of typical fracture testing specimens such as compact tension specimens. For both homogeneous and bimaterial specimens, larger tensile forces are associated with substantial loss of crack-tip constrait. Bimaterial specimens have as much as 2 times higher constraint than homogenous specimens, due to plastic strength mismatch. Tractable closed form approximations for crack-tip stresses are proposed in terms of tension-to-bending ratio.     

Freeze-fracturing at low temperatures: I. A device for fracturing biological specimens at 77–10 K under high vacuum

Standard freeze-etching or freeze-cleaving is performed at 173 K in a vacuum of 133 μPa or at 77 K under liquid nitrogen with subsequent transfer of the specimen into a vacuum chamber. It has been suggested that the frequent poor resolution of morphological details, the poor complementarity of innermembrane protein particles and the semi-crystalline substructures in biomembranes are caused by structural distortion or plastic deformation due to sheer forces which occur even at 77 K during fracturing or cleaving. In addition, water contamination and radiant heat damage occurring during replication introduce artefacts to the structural record. These artefacts could be avoided or reduced by lowering the temperature at which fracturing or cleaving and shadowing is carried out, to about 10 K. Therefore, a device for cleaving biological specimens at 15–10 K under high vacuum was constructed. To allow the use of existing equipment, the device was built into a standard Balzers 301 vacuum unit, where the specimen transfer is done via an airlock system which allows hoar frost contamination free transport of the specimen holder onto the specimen table. To reduce or prevent the condensation of water and other residual gases in the vacuum onto the freshly cleaved specimen surface at 10 K, the specimen is surrounded by two cooled surfaces of 6 and 20 K. All condensable gases outside those shielding shrouds will condense on these surfaces before reaching the specimen. This makes it possible to work at a high vacuum of 3 μPa outside the cooled shrouds, which can be reached with standard turbomolecular pumps. The actual vacuum within the cooled shrouds is estimated to be approximately 13 nPa. Residual gas analysis before and during replication reveals equal conditions to ultra high vacuum systems. An analysis of the yeast cell paracrystalline plasmalemma structure shows that the topographic resolution of the crystalline arrays has been improved by working at 12 K. However, plastic deformation still occurs under these conditions. This observation points to the possibility that what is described as plastic deformation, for at least some membrane proteins, may be a loss of resilience at low temperatures.     

A533B-1钢韧脆转变区断裂韧度预测的约束评估法

分析A533B-1压力容器钢韧脆转变温度区的断裂韧度、温度以及裂纹尖端约束效应之间的关系.根据实验数据,给出从浅裂纹到深裂纹,不同裂纹长度时单边裂纹弯曲试件(single edge notched bend specimens)的主曲线(mastercurve).用有限元分析相应试件裂纹尖端区域的应力场,采用J-A2双参数方法对裂纹尖端的三轴约束水平进行评估,并给出约束参数A2与主曲线参考温度T0之间的关系.由此,建立A533B-1钢断裂韧度KJC与温度T和约束参数A2之间的关系.为利用实验室数据对工程中不同裂纹构件的断裂评估提供一种经济、可行的方法.     

Crack-tip opening angle-based numerical implementation for fully plastic crack growth analyses

This study introduces implementation of a nodal release technique into a FEM/continuum model to enable simulation of fully plastic crack growth. The nodal release technique is implemented in the user-defined element form on the symmetry line of a deeply single-edge cracked specimen so that the force at the crack-tip node on the symmetry line is made zero after several steps upon the satisfaction of a chosen fracture criterion, and an incremental crack extension is achieved. The fracture criterion adopts the crack-tip opening angle (CTOA) which is determined from the specimen’s loading geometry [1]. For evaluation of the present model, the crack growth simulation results from the present FEM model were compared to those from the line-spring model of Lee and Parks [2].     

Wechselbiegeapparatur für die in situ-Untersuchung von Zerrüttungsvorgängen im Rasterelektronenmikroskop

An SEM stage (Fig. 1 and 2) is described which allows bending fatigue of metallic specimens in the vacuum of the specimen chamber. With the aid of inductive displacement pick-ups the angle of bending can be measured (Fig. 3) as well as the momentum of bending (Fig. 4). Thus the mechanical hysteresis can be registrated and the fatigue of the specimen can be controlled. Fig. 5 shows the geometry of the fatigue specimen, which has an electrolytically polished surface area of about 6 × 3 mm² on which the development of deformation topography can be studied during the fatigue experiment. The main advantages of the new fatigue apparatus are:

• 1 No interruption of cyclic straining is necessary for the investigation of the specimen surface.
• 2 The specimen is in vacuum during the complete experiment so that environmental influences are reduced.
• 3 The development of surface topography can be monitored continuously.
• 4 Differences of the topography (e. g. fatigue cracks) in the phase of tension and compression can be detected.

In the case of fatigue of polycrystalline metals, fatigue cracks often start at the surface of the specimen in slipbands produced by the cyclic straining. Fig. 6 shows typical fatigue slipbands with extrusions and intrusions for pure Al. A slipband crack in a later stage of fatigue is shown in Fig. 7. For the investigation of the distribution of fatigue cracks at the surface the possibility of watching the surface in different phases of bending is a great help. The cyclic crack opening – which depends on the crack depth – can be seen by eye on the TV-screen and can be recorded on video tape when the frequency of bending is about 1 cycle/s or can be photographed from the record display at very low frequencies of bending. Fig. 8 gives an example of the difference in crack opening in the phase of tension and compression of the specimen surface respectively.     

Modification of a TEM-goniometer specimen holder to enable beam current measurements in a (S)TEM for use in quantitative X-ray microanalysis

In order to have available a specimen holder suited to measure the beam current as is often required in quantitative electron probe X-ray microanalysis, the rod of a low background beryllium specimen holder of a transmission electron microscope was modified. The tip was electrically insulated from the mass of the microscope and connected electrically to the central contact of a BNC connector mounted on the specimen holder handle. With this modified specimen holder the current absorbed by the specimen and/or the specimen holder could be measured easily and accurately. The modified specimen holder has been used to measure the beam current stability of an analytical electron microscope under various conditions. Data were obtained for tungsten as well as lanthanum hexaboride cathodes. Small changes to other types of specimen tips made it possible to exchange these for the low background tip.     

In situ laser heating in an environmental scanning electron microscope

The environmental scanning electron microscope (ESEM) offers improved capabilities for coupling a scanning electron microscope (SEM) with an in situ laser device compared with conventional SEMs. Such coupling generally enables, for example, the observation of laser glazing effects or high-temperature behaviour as well as thermal shock behaviour of materials and devices. In an ESEM, decomposition caused by high-temperature gas reactions can additionally be studied while monitoring the gaseous environment online with a mass spectrometer attached to the specimen chamber. In this work, we demonstrate the capabilities of an in situ laser system suitable for heating specimen in an in situ deformation stage, thus enabling the further study of the mechanical properties of materials far beyond temperatures accomplished by thermal heating stages.     

预应变对管线钢低温断裂韧度影响研究

油气管线无论是在安装还是在服役过程中,都会不可避免地产生大塑性应变(即预应变).为了研究预应变对材料力学性能和断裂韧度的影响,对X80管线钢原材料和塑性变形材料分别进行不同温度下的拉伸试验和断裂韧度试验,并对试样断口形貌进行分析.试验结果表明,温度对管线钢的断裂韧度具有显著作用,钢材的断裂韧度随着温度的降低显著减小,断裂方式也由延性断裂转变为脆性断裂;拉伸预应变因工作硬化提高了钢材的屈服强度与抗拉强度,而压缩预应变因包申格效应降低了钢材的屈服强度与抗拉强度,但两者都降低了钢材的塑性及断裂韧度,进一步增加脆性断裂发生的概率.因此,在管道设计、选材、安全分析及评定时,应充分考虑温度和预应变对管线钢断裂行为的影响.     

Apparatus for material tests using an internal loading system in high-pressure gas at room temperature

A new type of apparatus for material tests using an internal loading system in high-pressure gas up to 100 MPa at room temperature without conventional material testing equipment was developed. The apparatus consists of a high-pressure control system and a pressure vessel, in which a piston is installed in the cylinder of the pressure vessel. The load caused by the pressure difference between spaces separated by the piston in the vessel cylinder is applied on the specimen connected to the piston in the vessel cylinder. The actual load on the specimen is directly measured by an external load cell and the displacement of the specimen is also measured by an external extensometer. As an example of the application of the apparatus, a tensile test on SUS316 stainless steel the Japanese Industrial Standard (JIS) G4303, which is comparable to the type 316 stainless steel ASTM A276, was conducted in 90 MPa hydrogen and argon. Hydrogen showed a marked effect on the tensile property of the material. The hydrogen gas embrittlement of the material was briefly discussed.     

Methods for additive hydration allowing observation of fully hydrated state of wet samples in environmental SEM

Methods for additive hydration are presented that enable longtime observation of very wet biological specimens in an environmental scanning electron microscope. The changes of structure due to dehydration for specimens placed on a Peltier-cooled holder, put on a special agar base or embedded in it or blown over by water vapor are compared. The degree of dehydration damage of the observed specimen structures is evaluated and compared with the structure of a nondestructively dried specimen, prepared by the critical point drying method.     

An improved transfer module and variable temperature control for a simple commercial cooling holder

A new cold stage transfer module was designed for the commercially available cooling holder of the JEOL JEM 100CX electron microscope. In the new CSTM the entire loading of the specimen is carried out under liquid nitrogen. This gives a frost-free transfer during which the temperature of the sample does not exceed 120 K. Straightforward modifications to the commercial cooling holder permit continuous selection of specimen temperature between 100 and 450 K. The sample can be heated or cooled at rates of up to 7 K/s. These modifications do not impair the resolution of the holder which is better than 1.5 nm. This work illustrates a relatively simple way of modifying a commercial cooling holder into a true cold stage system.     

A controlled atmosphere specimen holder for transmission electron microscopy

A controlled atmosphere specimen holder (CASH) has been developed for transmission electron microscopy (TEM) experiments. It is designed for studying the specimen's microstructure before and after treatments in various gases (H₂, O₂, N₂, Ar, etc.) at temperatures up to 600°C. The experiments are carried out without exposing the specimen to the ambient atmosphere. No modification of the electron microscope itself is needed. The same area of the specimen can be easily located after each gas treatment, thus the changes in the microstructure can be studied directly. Preliminary results on the cyclic oxidation and reduction of a model cobalt catalyst are presented.     

Freeze-fracturing at defined temperatures provides information on temperature rise during fracture,and on membrane complementarity

An apparatus is described which allows freeze-fracturing at defined temperatures, followed by immediate immersion of the specimen in liquid nitrogen. Replication is carried out in a Bullivant & Ames (1966) freeze-fracture device. Using yeast as a test specimen, the following results were obtained with the apparatus: (a) It was confirmed that fracturing at temperatures between 243 K and 223 K gave undeformed volcanoes on the PF of the plasma membrane, as shown originally by Steere et al. (1980). (b) Considerable energy is released by the fracturing process, both as shown by thermocouple readings and by the fact that at relatively high fracture temperatures portions of the specimen surface were melted. A temperature rise of 50–70 K was indicated, (c) Under standard conditions, there is a lack of complementarity between the yeast plasma membrane fracture faces, trigonal point particles not being present opposite the corresponding depressions in the array. Fracturing yeast suspended in salt solutions at 203 K demonstrates these particles. Their absence in normal fractures can be explained by ‘secondary fracture’, a concept based on polymer fracture studies.