Differential contrast imaging of secondary electron signals in cryo-field-emission scanning electron microscopy

Low-temperature scanning electron microscopy (LTSEM) is limited in resolution and image quality by charging of frozen hydrated samples and collection deficiencies of secondary electron signal contrasts. We measured and corrected both effects using differential hysteresis processing (DHP) of LTSEM images, scanned at 15-bit from 5×4 inch Polaroid negatives. Bulk charging produced a major contrast component equal to 44–87% of the intensity range of the image. The strong charging contrast reduced the local high-resolution signal contrasts to an unrecognizable level. Segmentation and imaging of the unaffected surface contrasts produced high-quality images of high contrast from metal-coated samples as well as from uncoated samples. The differential contrast imaging can be used for control of the sequential etching of ice from the non metal-coated sample as well as improved LTSEM imaging of the finally coated sample.     

On the estimation variance for the specific Euler–Poincaré characteristic of random networks

The factors that determine the local magnetic properties of FeCo/SiO₂ nanocomposite powders and films have been analysed by electron energy‐loss spectroscopy (EELS) and transmission electron microscopy (TEM). Attention has been given to the chemical composition, the local electronic structure and the atomic arrangement. The results show that the nanoparticles from sol‐gel prepared powders are generally Fe‐rich, whereas they are Co‐rich in sol‐gel prepared films. In addition, a subnanometre oxide layer at the surface of the FeCo nanoparticles has been clearly observed in the powder sample. It is found that the magnetic moment should be partly governed by alloying effects. Numerical values of the near‐surface magnetic moment have been obtained using the ab‐initio layer‐KKR method. These values should be helpful in understanding the layer‐by‐layer changes of the white line ratio close to the surface of the nanoparticles.     

Exploring dynamic surface processes during silicate mineral (wollastonite) dissolution with liquid cell TEM

Most liquid cell transmission electron microscopy (LC TEM) studies focus on nanoparticles or nanowires, in large part because the preparation and study of materials in this size range is straightforward. By contrast, this is not true for samples in the micrometre size range, in large part because of the difficulties associated with sample preparation starting from a ‘bulk’ material. There are also many advantages inherent to the study of micrometre‐sized samples compared to their nanometre‐sized counterparts. Here, we present a liquid cell transmission electron study that employed an innovative sample preparation technique using focused ion beam (FIB) milling to fabricate micrometre‐sized electron transparent lamellae that were then welded to the liquid cell substrate. This technique, for which we have described in detail all of the fabrication steps, allows for samples having dimensions of several square micrometres to be observed by TEM in situ in a liquid. We applied this technique to test whether we could observe and measure in situ dissolution of a crystalline material called wollastonite, a calcium silicate mineral. More specifically, this study was used to observe and record surface dynamics associated with step and terrace edge movement, which are ultimately linked to the overall rate of dissolution. The wollastonite lamella underwent chemical reactions in pure deionized water at ambient temperature in a liquid cell with a 5‐m‐spacer thickness. The movement of surface steps and terraces was measured periodically over a period of almost 5 h. Quite unexpectedly, the one‐dimensional rates of retreat of these surface features were not constant, but changed over time. In addition, there were noticeable quantitative differences in retreat rates as a function crystallographic orientation, indicating that surface retreat is anisotropic. Several bulk rates of dissolution were also determined (1.6–4.2 ? 10^?7 mol m^?2 s^?1) using the rates of retreat of representative terraces and steps, and were found to be within one order of magnitude of dissolution rates in the literature based on aqueous chemistry data.     

Use of low-temperature scanning electron microscopy to compare and characterize three classes of snow cover

This study, which uses low-temperature scanning electron microscopy (LTSEM), systematically sampled and characterized snow crystals that were collected from three unique classes of snow cover: prairie, taiga, and alpine. These classes, which were defined in previous field studies, result from exposure to unique climatic variables relating to wind, precipitation, and air temperature. Snow samples were taken at 10 cm depth intervals from the walls of freshly excavated snow pits. The depth of the snow pits for the prairie, taiga, and alpine covers were 28, 81, and 110 cm, respectively. Visual examination revealed that the prairie snow cover consisted of two distinct layers whereas the taiga and alpine covers had four distinct layers. Visual measurements were able to establish the range of crystal sizes that occurred in each layer, the temperature within the pit, and the snow density. The LTSEM observations revealed the detailed structures of the types of crystals that occurred in the snow covers, and documented the metamorphosis that transpired in the descending layers. Briefly, the top layers from two of the snow covers consisted of freshly fallen snow crystals that could be readily distinguished as plates and columns (prairie) or graupel (taiga). Alternatively, the top layer in the alpine cover consisted of older dendritic crystal fragments that had undergone early metamorphosis, that is, they had lost their sharp edges and had begun to show signs of joining or bonding with neighboring crystals. A unique layer, known as sun crust, was found in the prairie snow cover; however, successive samplings from all three snow covers showed similar stages of metamorphism that led to the formation of depth hoar crystals. These changes included the gradual development of large, three-dimensional crystals having clearly defined flat faces, sharp edges, internal depressions, and facets. The study, which indicates that LTSEM can be used to enhance visual data by systematically characterizing snow crystals that are collected at remote locations, is important for understanding the physics of snowpacks and the metamorphosis that leads to potential avalanche situations. In addition, the metamorphosis of snow crystals must be considered when microwave radiometry is used to estimate the snow water equivalent in the winter snowpack, because large snow crystals more effectively scatter passive microwave radiation than small crystals.     

Erosion of copper single crystals under conditions of 30° incidence

High purity copper single crystals were eroded at 30° to a (111) face using glass spheres 210 μm in diameter travelling at a velocity of 133 m s^?1. Detailed scanning electron microscopy of both eroded surfaces and metallographic sections has been performed together with transmission electron microscopy of erosion debris. It was observed that there was extensive surface shear that led to ripple formation, folding of the surface, subsurface crack propagation and material loss by flaking similar to delamination wear. Direct evidence was obtained for recrystallization of the surface layers.     

Sliding wear behavior of electron beam surface-melted 0–2 tool steel

Studies were carried out on the dry sliding wear behavior of electron beam melted surface layers on a type 0–2 tool steel and on annealed and conventionally hardened 0–2 steel specimens for comparison. Wear tests were conducted in a flowing argon atmosphere at a sliding speed of 20 cm s^?1 and a load of 10 N against a 52100 bearing steel ring. Wear surface morphology was studied along with subsurface structure using optical and electron microscopy methods. The study concentrated on the wear of this steel after different processing treatments. Electron beam surface melting and subsequent rapid solidification in situ of the steel produced a highly refined martensitic microstructure having higher hardness values and better wear resistance than obtained using conventional quench hardening of that steel. Carbide distribution and martensite phase morphology were affected by this surface melting process; those microstructural characteristics influenced the wear behavior. Variations in electron beam power and surface speed during melting were explored in terms of their effect on the resulting surface layer. The wear test system used was computer interfaced and controlled, permitting continuous measurements of wear depth and friction force.     

The design and use of a simple device for rapid quench-freezing of biological samples

The detailed design of a simple device for rapid quench-freezing of biological samples under reproducible conditions is presented. With spring-augmented descent, sample immersion velocity of 10 m s^?1 into a cryogenic liquid is achieved. Biological samples, loaded in Balzers planchets, Denton holders, or a newly designed ‘titanium envelope’, are suitable for rapid-freezing with this device. Using 4 μm titanium foil, light weight (1 mg) streamlined holders can easily be made to enclose cell suspensions or tissue samples. The foil envelope is designed for efficient heat dissipation while protecting the sample from possible impact or flow distortions occurring from spring-augmented immersion. Human erythrocytes, quench-frozen in the titanium envelope, were prepared for electron microscopy by the freeze-substitution technique. Two opposing 25–30 μm surface zones were frozen in the apparent absence of ice. The extended depth of cryofixation is attributed to the advantages of thin foil in the titanium envelope design and the use of rapid-immersion technique.     

Simple procedures for evaluating the cryofixation of biological samples

Ultra-rapid cooling of biological material can be achieved in the absence of cryoprotectants by using thin samples. Three methods now employed to prepare thin samples for freeze-fracture electron microscopy are compared: contacting the sample against a liquid helium-cooled copper surface (Heuser et al., 1979), spraying the sample with a jet of propane (Mueller et al., 1980), and plunging a streamlined copper ‘sandwich’ into liquid propane (Costello, 1980). In the first method a thin surface layer of the sample is ultra-rapidly cooled while in the other methods the entire sample sandwiched between sheets of conducting metals is cooled. The morphology of fracture-faces of dilauryllecithin-water systems is used to evaluate the effectiveness of cooling methods. At optimum cooling rates the initial disordered arrangement of lipid in the lamellar (L_α) phase is preserved, giving smooth fracture faces. At slower cooling rates a worm-like texture appears which signals the formation of molecular ordering characteristic of the P_β, phase. All three methods are capable of cooling these lipid-water phases as well as other more dilute aqueous suspensions without evidence of ice crystal growth or damage. Measurement of cooling rates employing miniature thermocouples embedded in samples indicates that rates for all three methods are in excess of 10,000 K/s. The propane jet (32 times 10³ K/s, slope at 273 K) exposes the sample to coolant more rapidly than the sandwich plunging method (10 times 10³ K/s, slope at 273 K) and therefore produces slightly higher cooling rates for samples of equivalent mass and thickness. Each method has its advantages. The contact method is well suited for tissues; the sandwich method is simple and inexpensive; the jet method can potentially produce the highest cooling rates. The last two methods yield complementary replicas.     

Erosion of copper single crystals under conditions of 90° impact

High purity copper single crystals were eroded parallel to [111] at 90° to the surface with glass spheres 70 μm in diameter travelling at a velocity of 122 m s^?1. Detailed scanning electron microscopy of eroded surfaces together with transmission electron microscopy of both erosion debris and near-surface layers of the target was performed. It was observed that a hilland-valley surface topology developed ; this has not been reported previously and gave rise to a local low angle cutting component of erosion at 90°. Direct evidence was obtained of a transient heating effect from the particle impact as well as the formation of subsurface voids in heavily deformed regions. In addition it was shown that subsurface cracking produced a flaking mechanism of material loss.     

A portable cryo-storage system for low-temperature scanning electron microscopy,suitable for international transport of cryo-specimens

A cryo-specimen storage system for low-temperature scanning electron microscopy (LTSEM) specimens is described, which: liberates multi-specimen experiments from sampling restrictions imposed by the rate at which LTSEM specimens can be examined in the SEM; provides security against experiment loss resulting from breakdown of the SEM or cryo-system; enables collection of specimens in the field or in laboratories remote from the SEM laboratory; and facilitates international air transport of LTSEM specimens. The components of the system, which has a capacity of 98 stub-mounted specimens, are readily made in a laboratory workshop. The details of the design may be altered to suit particular specimen types or experimental approaches.     

Optimum conditions for cryoquenching of small tissue blocks in liquid coolants

Three approaches were taken with the aim of defining the optimum conditions for rapid cryopreservation in liquid quenchants. In a theoretical approach, two mathematical models were used. The first is of value in defining the absolute maximum rates of cooling which could be achieved at various depths in the tissues. The second highlights the poor thermal properties of liquid coolants and therefore emphasizes the essential requirement for vigorous quenchant mixing and rapid specimen entry. Experimental work with thermocouples showed that fastest cooling rates occur at the leading edge of the object entering coolant. Of five liquid quenchants investigated, cooling rates were in the order, propane> Freon 22> Freon 12> liquid nitrogen slush> liquid nitrogen. Other considerations, however, may affect the choice of quenchant. For a given quenchant, cooling rate is maximal near the equilibrium freezing point. The consequences of quenching in the presence of thermal gradients either within the coolant or in the gas layer above it are shown. Cooling rate was found to be approximately proportional to entry velocity at least up to ～2 m s^?1 in our system. Stereological analysis of rapidly quenched, freeze-substituted tissue samples, of geometry which imposed an approximately unidirectional heat flow, revealed four zones: (i) a narrow surface layer (～10 μm) of low image contrast and apparent absence of ice crystals; (ii) a zone of enhanced contrast with ice crystals whose size increased rapidly with depth from the surface (the ‘slope’); (iii) a sharply defined zone (the ‘ridge’) of maximum ice crystal size beyond which there is (iv) an extensive ‘plateau’ with smaller ice crystals and no marked increase in size with depth. The ‘ridge’ of maximal ice-crystal damage was consistently found but varied considerably in depth from the surface (～25–120 μm) between samples. The existence of the deeper plateau region of relatively uniform ice-crystal-size may be of significance in X-ray microanalytical studies of physiological processes at some depth from the sample surface. In terms of our present understanding of the quenching process, the conditions for optimal cryofixation of small tissue samples are listed.     

Surface Hardness and Abrasive Wear Resistance of Ion-Implanted Steels

The hardnesses of nitrogen-implanted steel surfaces have been measured with an abrasive wear technique capable of characterizing surface layers as thin as 25 nm. Treated steel disks and reference disks were abraded with 1–5 μm diamond, and relative wear resistances were calculated from the mass losses. Surface hardness was obtained from a relationship between wear resistance and hardness.

The surface of a hardened and tempered carbon steel implanted with nitrogen ions (10¹⁷/cm²) was significantly harder than with other treatments including quench hardening and nitriding. The hardness decreased to the bulk value over a depth corresponding to the initial implantation depth.

Nitorgen-implanted stainless-steel surfaces wore faster than un-implanted ones, possibly due to interference with transformation hardening which normally occurs during wearing. This “softening” effect persisted to depths several times the depth of implantation, and may help to explain the reduction of sliding wear produced by the implantation of stainless steels. Analyses by Auger electron spectroscopy indicated nitrogen migrated toward the bulk during wear.

Titanium implanted in stainless steel (4.6 × 10¹⁷ ions/cm²) produced a very hard surface with more than 10 times the abrasive wear resistance of the bulk metal.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.     

溶胶—凝胶法制备Al_2O_3涂层对工程陶瓷表面改性的研究

利用溶胶—凝胶法在SG4工程陶瓷基体上成功制备了Al2 O3 涂层 ;利用差热分析 (DTA)方法对Al2 O3凝胶粉末的热处理过程进行了研究。对线切割、粗磨、精磨、Al2 O3 一次溶胶涂层和Al2 O3 二次溶胶涂层试样的抗弯强度进行了比较 ,并通过观察Al2 O3 一次溶胶涂层和Al2 O3 二次溶胶涂层试样表面和断面的SEM形貌 ,初步分析了表面改性的原因。分析结果表明 :溶胶涂层弥合基体表面微裂纹是提高抗弯强度的根本原因 ;Al2 O3 溶胶二次涂层试样表面质量好于一次涂层试样 ,可以更好地弥合基体表面微裂纹 ,使基体的抗弯强度更高。因此溶胶—凝胶法是陶瓷表面改性的一种有效方法。     

A spectroscopic study of a copper surface under boundary lubrication: I. Chemisorbed stearic acid multilayers

The thickness of chemisorbed stearic acid films on copper surfaces was deduced by Fourier transform infrared (FTIR) spectroscopy and Auger electron spectroscopy (AES). The chemisorption was detected by FTIR from the carboxyl (COO) asymmetric stretching band at 1585 cm⁻¹. Multilayer growth was observed by monitoring the intensity increase in the asymmetric CH₂ stretching mode at 2915 cm⁻¹. The growth of the chemisorbed layer was found to depend on the concentration of the stearic acid/hexadecane solution. The chemisorption kinetic profile had a step-function due to the change in the number of layers. High carbon concentration in Auger spectroscopic characterisation indicated that the surface was coated with a stearic acid film. AES depth profiling analysis supports this step-function model and agrees well with the FTIR results.     

Visualization and characterization of engineered nanoparticles in complex environmental and food matrices using atmospheric scanning electron microscopy

Imaging and characterization of engineered nanoparticles (ENPs) in water, soils, sediment and food matrices is very important for research into the risks of ENPs to consumers and the environment. However, these analyses pose a significant challenge as most existing techniques require some form of sample manipulation prior to imaging and characterization, which can result in changes in the ENPs in a sample and in the introduction of analytical artefacts. This study therefore explored the application of a newly designed instrument, the atmospheric scanning electron microscope (ASEM), which allows the direct characterization of ENPs in liquid matrices and which therefore overcomes some of the limitations associated with existing imaging methods. ASEM was used to characterize the size distribution of a range of ENPs in a selection of environmental and food matrices, including supernatant of natural sediment, test medium used in ecotoxicology studies, bovine serum albumin and tomato soup under atmospheric conditions. The obtained imaging results were compared to results obtained using conventional imaging by transmission electron microscope (TEM) and SEM as well as to size distribution data derived from nanoparticle tracking analysis (NTA). ASEM analysis was found to be a complementary technique to existing methods that is able to visualize ENPs in complex liquid matrices and to provide ENP size information without extensive sample preparation. ASEM images can detect ENPs in liquids down to 30 nm and to a level of 1 mg L^?1 (9×10⁸ particles mL^?1, 50 nm Au ENPs). The results indicate ASEM is a highly complementary method to existing approaches for analyzing ENPs in complex media and that its use will allow those studying to study ENP behavior in situ, something that is currently extremely challenging to do.     

Freeze-fracturing for conventional and field emission low-temperature scanning electron microscopy: the scanning cryo unit SCU 020

A dedicated cryopreparation system, the SCU 020 (Balzers), is introduced and described in detail for use in low-temperature scanning electron microscopy (LTSEM). The basic unit consists of two parts: (i) a high-vacuum preparation chamber equipped with a cold-stage, motor-driven fracturing microtome, planar magnetron (PM) sputter source, quartz-crystal thin-film monitor, Meissner cold trap, and turbo molecular pump stand; and (ii) a second part (separated from the first by a sliding, high-vacuum valve) residing in the SEM chamber. This is equipped with an anti-contamination cold trap, a fully movable goniometer cold stage (having motor drives for x, y, and rotation) and replaces the SEM's original stage (Raith). The SCU 020 is entirely self contained allowing independence from, and synchroneity with, the SEM of choice. LTSEM micrographs of specimen (that are fully frozen hydrated or partially freeze-dried) surfaces or fracture faces, without or with various metal coatings, can be examined over a broad temperature range (-150 to +50°C). This is made possible by the combined application of the two, independently controlled, cold stages and the on-line, high-vacuum, specimen cryo transfer between them. In-situ etching is simple and straightforward. Intramembranous particles and membrane fracture steps, typically imaged in transmission electron microscopy (TEM), are resolved by PM sputtering with platinum at low specimen temperature and high-resolution LTSEM in a field emission microscope.     

Electrochemical determination of hydrogen peroxide using a novel prussian blue–polythiophene–graphene oxide membrane-modified glassy carbon electrode

A polythiophene–graphene oxide compound membrane and Prussian blue were deposited sequentially on the surface of a glassy carbon electrode by cyclic voltammetry. Due to its excellent electrocatalysis and its analogy with peroxidase enzymes, Prussian blue has been widely used in amperometric biosensors. The polythiophene–graphene oxide compound membrane exhibited good electroconductibility and a large specific surface area. The fabricated Prussian blue/polythiophene/graphene oxide/glassy carbon electrode was characterized by transmission electron microscopy, scanning electron microscopy, and cyclic voltammetry. Under the optimal experimental conditions, the detection of hydrogen peroxide was studied by its amperometric current–time curve. Due to the presence of polythiophene–graphene oxide compound membrane and Prussian blue, the hydrogen peroxide sensor shows a linear calibration range of 1.0?×?10^?6–1.0?×?10^?4?mol?L^?1, detection limits of 3.2?×?10^?7?mol?L^?1 at a signal-to-noise ratio of 3, and recoveries from 95.0 to 105.0%. The results show that the modified glassy carbon electrode has potential practical application for the determination of hydrogen peroxide based on its sensitivity and long-term stability.     

Low-temperature scanning electron microscopy in biology

A review of low-temperature scanning electron microscopy (LTSEM) with regard to preparation protocols, specimen preservation, experimental approaches, and high-resolution studies, is provided. Preparative procedures are described and recent developments in methodologies highlighted. It is now well established that LTSEM, for most biological specimens, provides superior specimen preservation than does ambient-temperature SEM. This is because frozen-hydrated samples retain most or all of their water, are rapidly immobilized and stabilized by cryofixation, and are not exposed to chemical modification or solvent extraction. Nevertheless, artefacts in LTSEM are common and most arise because frozen-hydrated specimens contain water. LTSEM can be used as a powerful experimental tool. Advantages of employing LTSEM for this purpose and ways in which it can be used for novel experimentation are discussed. The most exciting development in recent years has been high-resolution LTSEM. The advantages, problems and requirements for this approach are defined.     

Growing HgTe/Cd<Subscript>0.735</Subscript>Hg<Subscript>0.265</Subscript>Te quantum wells by molecular beam epitaxy

HgTe/Cd_0.735Hg_0.265Te nanostructures with HgTe quantum wells 16.2 and 21.0 nm thick are grown without additional doping on (013)CdTe/ZnTe/GaAs substrates by the method of molecular beam epitaxy. The compositions and thicknesses of the wide-gap layer and quantum well in the course of growth are performed by means of ellipsometry. The accuracy is Δx ? ±0.002 mole fractions of cadmium telluride in determining the composition and Δd ? 0.5 nm in determining the thickness of the wide-gap layer and quantum well. The central fragments of the wide-gap layers ≈ 10 nm thick are additionally doped by indium for a ～ 10¹⁵ cm^?3 volume concentration of charge carriers to be reached. Galvanomagnetic research in a wide range of magnetic field intensities at liquid helium temperatures reveals dimensional quantization levels and the presence of a two-dimensional electron gas in grown nanostructures. High mobility of the two-dimensional electron gas μ _e is obtained: 2 · 10⁵ and 5 · 10⁵ cm²/V · s for electron densities N _s equal to 1.5 · 10¹¹ and 3.5 · 10¹¹ cm^?2, respectively.