Analytical transmission electron microscopy and electron diffraction for characterization of multiphase Ni-P-Ti surface layer on Ti-6Al-4V alloy

The microstructure, chemical and phase composition of the hard Ni‐P‐Ti layer formed on the Ti‐6Al‐4V alloy after duplex surface treatment were investigated by light microscopy, X‐ray diffraction, scanning electron microscopy and analytical/high‐resolution transmission electron microscopy. These investigations showed that the improved mechanical and tribological properties of the surface‐treated alloy were related to the presence of a multilayered microstructure containing several phases from the Ni‐Ti‐P‐Al system.     

Specimen‐thickness effects on transmission Kikuchi patterns in the scanning electron microscope

We report the effects of varying specimen thickness on the generation of transmission Kikuchi patterns in the scanning electron microscope. Diffraction patterns sufficient for automated indexing were observed from films spanning nearly three orders of magnitude in thickness in several materials, from 5 nm of hafnium dioxide to 3 μm of aluminum, corresponding to a mass‐thickness range of ～5 to 810 μg cm^–2. The scattering events that are most likely to be detected in transmission are shown to be very near the exit surface of the films. The energies, spatial distribution and trajectories of the electrons that are transmitted through the film and are collected by the detector are predicted using Monte Carlo simulations.     

Local rotations due to mixed‐type misfit dislocation at α‐Fe2O3/α‐Al2O3 heterostructure interface

The misfit dislocations at α‐Fe₂O₃/α‐Al₂O₃ heterostructure interfaces were investigated by high‐resolution transmission electron microscopy (HRTEM), geometric phase analysis (GPA) and dislocation density tensor analysis. When imaged along the [110] direction, the misfit dislocation core is a mixed‐type, which can be characterised by one extra (102) plane and one extra (104) plane of α‐Al₂O₃. Dislocation density tensor analysis gave a very high accuracy in determining the corresponding Burgers vectors of two extra half‐planes. By comparing the measured Burgers vectors with theoretical ones, we are able to determine local rotations in the dislocation core region: the (102) plane is rotated clockwise 6.25° and the (104) plane is rotated anticlockwise 4.81°. Such a local rotation is favourable from the viewpoint of both energy and function to relax lattice misfit.     

Scanning electron microscopy and transmission electron microscopy study of hot-deformed γ-TiAl-based alloy microstructure

The aim of this work was to assess the changes in the microstructure of hot‐deformed specimens made of alloys containing 46–50 at.% Al, 2 at.% Cr and 2 at.% Nb (and alloying additions such as carbon and boron) with the aid of scanning electron microscopy and transmission electron microscopy techniques. After homogenization and heat treatment performed in order to make diverse lamellae thickness, the specimens were compressed at 1000 °C. Transmission electron microscopy examinations of specimens after the compression test revealed the presence of heavily deformed areas with a high density of dislocation. Deformation twins were also observed. Dynamically recrystallized grains were revealed. For alloys no. 2 and no. 3, the recovery and recrystallization processes were more extensive than for alloy no. 1.     

Effects of TGF‐β1 on mineralization mediated by rat calvaria‐derived osteogenic cells

In this study, we have analyzed the viability and cell growth, as well as, the mineralization of extracellular matrix (ECM) by alizarin red and von Kossa staining of calvaria‐derived osteogenic cultures, treated with TGF‐β1 alone or associated with Dex comparing with acid ascorbic (AA) + β‐glicerophosphate (βGP) (positive mineralization control). The expression of the noncollagenous proteins bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN) were evaluated by indirect immunofluorescence. In addition, the main ultrastructural morphological findings were assessed by transmission electron microscopy. Osteogenic cells were isolated of calvaria bone from newborn (2‐day‐old) Wistar rats were treated with TGF‐β1 alone or with dexamethasone for 7, 10, and 14 days. As positive mineralization control, the cells were supplemented only with AA+ βGP. As negative control, the cells were cultured with basal medium (α‐MEM + 10%FBS + 1%gentamicin). The treatment with TGF‐β1, even when combined with Dex, decreased the viability and cell growth when compared with the positive control. Osteoblastic cell cultures were positive to alizarin red and von Kossa stainings after AA + βGP and Dex alone treatments. Positive immunoreaction was found for BSP, OPN and FN in all studied treatments. Otherwise, when the cell cultures were supplemented with TGF‐β1 and TGF‐β1 + Dex, no mineralization was observed in any of the studied periods. These present findings suggest that TGF‐β1, in the studied in vitro doses, inhibits the proliferation and differentiation of osteoblastic cells by impairment of nodule formation.     

A high-resolution electron microscopic study of defects in sodium β′″-alumina

A high-resolution electron-microscopic study of sodium β′″-alumina, a polytype of the more-widely-studied sodium β- and β″-alumina, has been undertaken using the 600 kV instrument at Cambridge University. Images revealed the loss of sodium-containing planes, which had caused crystals to collapse and shear into defect layers. A model for the structure of these defects is proposed, based on the use of computed images and by comparison with high-resolution images of silver β″-alumina.     

Structural and electron diffraction data for sapphire (α-al2o3)

Crystallographic and electron diffraction data for sapphire (α-Al₂O₃) are presented which enable ready and unique identification of TEM diffraction patterns and facilitate image analysis. Crystallographic data is presented in the form of stereographic projections and in figures listing angles between planes and angles between zones. Electron diffraction data consist of computer-simulated diffraction patterns and tables of extinction distances calculated using atomic and ionic electron scattering factors.     

Fine structure characterization of martensite/austenite constituent in low‐carbon low‐alloy steel by transmission electron forward scatter diffraction

Transmission electron forward scatter diffraction and other characterization techniques were used to investigate the fine structure and the variant relationship of the martensite/austenite (M/A) constituent of the granular bainite in low‐carbon low‐alloy steel. The results demonstrated that the M/A constituents were distributed in clusters throughout the bainitic ferrite. Lath martensite was the main component of the M/A constituent, where the relationship between the martensite variants was consistent with the Nishiyama–Wassermann orientation relationship and only three variants were found in the M/A constituent, suggesting that the variants had formed in the M/A constituent according to a specific mechanism. Furthermore, the Σ3 boundaries in the M/A constituent were much longer than their counterparts in the bainitic ferrite region. The results indicate that transmission electron forward scatter diffraction is an effective method of crystallographic analysis for nanolaths in M/A constituents.     

Backscattered electron imaging and electron backscattered diffraction in the study of bacterial attachment to titanium alloy structure

The application of secondary electron (SE) imaging, backscattered electron imaging (BSE) and electron backscattered diffraction (EBSD) was investigated in this work to study the bacterial adhesion and proliferation on a commercially pure titanium (cp Ti) and a Ti6Al4V alloy (Ti 64) with respect to substrate microstructure and chemical composition. Adherence of Gram‐positive Staphylococcus epidermidis 11047 and Streptococcus sanguinis GW2, and Gram‐negative Serratia sp. NCIMB 40259 and Escherichia coli 10418 was compared on cp Ti, Ti 64, pure aluminium (Al) and vanadium (V). The substrate microstructure and the bacterial distribution on these metals were characterised using SE, BSE and EBSD imaging. It was observed that titanium alloy‐phase structure, grain boundaries and grain orientation did not influence bacterial adherence or proliferation at microscale. Adherence of all four strains was similar on cp Ti and Ti 64 surfaces whilst inhibited on pure Al. This work establishes a nondestructive and straight‐forward statistical method to analyse the relationship between microbial distribution and metal alloy structure.     

User‐independent EBSD parameters to study the progress of recovery and recrystallization in Cu–Zn alloy during in situ heating

Microstructural evolution of cold‐rolled Cu–5%Zn alloy during in situ heating inside field‐emission scanning electron microscope was utilized to obtain user‐independent parameters in order to trace the progress of static recovery and recrystallization. Electron back‐scattered diffraction (EBSD)‐based orientation imaging microscopy was used to obtain micrographs at various stages of in situ heating. It is shown that unlike the pre‐existing methods, additional EBSD‐based parameter can be used to trace the progress of recovery and recrystallization, which is not dependent on user input and hence less prone to error. True strain of 0.3 was imposed during cold rolling of alloy sample. Rolled sample was subjected to in situ heating from room temperature to 500°C (～0.58 Tm) with soaking time of 10 min, at each of the intermediate temperatures viz. 100, 200, 300, 400 and 450°C. After reaching 500°C, the sample was kept at this temperature for a maximum duration of around 15 h. The sample showed clear signs of recovery for temperature up to 450°C, and at 500°C, recrystallization started to take place. Recrystallization kinetics was moderate, and full recrystallization was achieved in approximately 120 min. We found that EBSD parameter, namely, band contrast intensity can be used as an extra handle to map out the progress of recrystallization occurring in the sample. By contrast, mean angular deviation can be used to understand the evolution of recovery in samples. The parameters mentioned in the current study, unlike other pre‐existing methods, can also be used for mapping local microstructural transformations due to recovery and recrystallization. We discuss the benefits and limitations in using these additional handles in understanding the changes taking place in the material during in situ heating.     

Independent trafficking of flavocytochrome b558 and myeloperoxidase to phagosomes during phagocytosis visualised by energy‐filtering and energy‐dispersive spectroscopy‐scanning transmission electron microscopy

When polymorphonuclear leukocytes (PMNs) phagocytose opsonised zymosan particles (OPZ), free radicals and reactive oxygen species (ROS) are formed in the phagosomes. ROS production is mediated by NADPH oxidase (Nox), which transfers electrons in converting oxygen to superoxide (O₂^?). Nox‐generated O₂^? is rapidly converted to other ROS. Free radical‐forming secretory vesicles containing the Nox redox center flavocytochrome b558, a membrane protein, and azurophil granules with packaged myeloperoxidase (MPO) have been described. Presuming the probable fusion of these vesicular and granular organelles with phagosomes, the translation process of the enzymes was investigated using energy‐filtering and energy‐dispersive spectroscopy‐scanning transmission electron microscopy. In this work, the primary method for imaging cerium (Ce) ions demonstrated the localisation of H₂O₂ generated by phagocytosing PMNs. The MPO activity of the same PMNs was continuously monitored using 0.1% 3,3′‐diaminobenzidine‐tetrahydrochloride (DAB) and 0.01% H₂O₂. A detailed view of these vesicular and granular structures was created by overlaying each electron micrograph with pseudocolors: blue for Ce and green for nitrogen (N).     

TEM and HREM study of Al3Zr precipitates in an Al-Mg-Si-Zr alloy

The structure of Al₃Zr precipitates in Al‐1.0Mg‐0.6Si‐0.5Zr (in wt.%) alloy was investigated using conventional transmission electron microscopy (TEM) and high‐resolution TEM (HREM). After annealing of the alloy in the temperature range 450–540 °C, spherical precipitates of metastable L1₂‐Al₃Zr phase appeared nearly homogeneously within the matrix, and elongated particles were found at grain boundaries. L1₂‐structured Al₃Zr were about 20–30 nm in diameter and coherent with the matrix. Inside some of them, planar faults parallel to {100} planes were revealed by use of HREM. Most probably, these faults are an indication of the transition stage of transformation to the stable D0₂₃‐type Al₃Zr phase. The elongated precipitates (about 100 nm) were identified as D0₂₂‐type Al₃Zr. Energy‐dispersive X‐ray analysis showed that they contain, apart from Al, mainly Zr with small amounts of Si. The substitution of Al by Si increased the stability of the D0₂₂‐Al₃Zr as compared with D0₂₃‐Al₃Zr.     

3D reconstruction of prior β grains in friction stir–processed Ti–6Al–4V

The prior β grain structure and orientations in the central stir zone of friction stir–processed Ti–6Al–4V were reconstructed from measured α phase orientations obtained by three‐dimensional serial sectioning in a dual‐beam focused ion beam scanning electron microscope. The data were processed to obtain the α colony and β grain size distributions in the volume. Several β grains were individually analysed to determine the total number of unique α variants and the respective volume fractions of each. The analysis revealed that some grains experienced overwhelming variant selection (i.e. one variant dominated) whereas other β grains contained a more evenly distributed mixture of all 12 variants.     

A new experimental procedure to quantify annular dark field images in scanning transmission electron microscopy

A new procedure to quantify the contrast in annular dark field images recorded without lattice resolution in a scanning transmission electron microscope is proposed. The method relies on the use of an in‐column energy filter prior to the annular dark field detector and the acquisition of a series of energy‐filtered images as a function of the inner detection angle. When the image contrast of an interface between two materials in such energy‐filtered annular dark field images is plotted vs. camera length and extrapolated to zero (i.e. infinite scattering angle), the contrast is shown to behave exactly as predicted by Rutherford's scattering formula (i.e. intensity scales ∝Z²). This can then be used to determine the local chemistry at and the effective chemical widths of interfaces or thin films without any additional spectroscopy method for calibration, provided the global chemical composition is known. As examples, the systems SiGe/Si and InGaAs/Ge are considered in detail.     

Frictional behaviour of oxygen diffusion hardened titanium in dry sliding against Co–28Cr–5W–4Fe–3Ni–1Si cobalt alloy

The results of conformal pin-on-disc tribological tests of a hard layer of the solid solution of oxygen in α-titanium sliding against a Co–28Cr–5W–4Fe–3Ni–1Si cobalt alloy counterspecimen are presented. The α-Ti(O) layer was diffusely produced over 2–8 h of oxidising in the superficial zone of a technical quality titanium specimen.The friction and wear responses of the system were recorded and the wear mechanisms were studied. Investigations of the material structure and chemical constitution in micro-areas of the titanium specimen, cobalt alloy counterspecimen and wear debris formed in dry sliding were performed with a Philips XL20 microscope equipped with an EDAX analyser. Crushing of the α-Ti(O) layer, lowering of the wear rate after comminution of the hard α-Ti(O) layer, local tack spots and fine powder wear particles, mostly Ti oxides, were detected at the beginning of each test. Gradual brittle fracture and decay by pulverising of the α-Ti(O) particles embedded in both mating surfaces, which occurred during the test, led to the increase of the wear rate of the couple and domination of microcutting and tack spots spalling after their partial oxidation. Finally, after the disappearance of the α-Ti(O) loose particles, adhesive junctions, metal transfer and smearing become leading wear mechanisms.     

Defects formed within hardness indenter interaction zone in Al2O3–ZrO2 composite

The Al₂O₃?10 wt% ZrO₂ composites were subjected to hardness tests using a Vickers diamond indenter up to 98.1 N. The microstructure observation using a transmission electron microscopy technique helped to identify up to four zones differing in defect level and character. The densest dislocation tangles, twins accumulation and frequent presence of three slip systems were found in regions that were in contact with the sides of the diamond pyramid. The second zone, characterized by two, or at least one, active slip systems, started at the bottom of the indentation mark and extended up to a distance comparable with the depth of indentation. In the third zone, with a thickness comparable to that above, only some α‐Al₂O₃ crystallites showed the presence of dislocations, whereas other crystallites were defect free. In the last zone the alumina crystals were left unaffected but the ZrO₂ crystallites showed twinning characteristic of strain‐induced transformation.     

F‐actin and α‐actinin reorganization mediates initial fibroblast interaction with CoCr alloy particles in vitro

Asceptic loosening remains the primary cause for failure of joint implant. The active role of fibroblasts in mediating asceptic loosening is however not well documented. In this study the initial interactions of fibroblasts with metal particles was studied by evaluating changes in the cytoskeletal structure and cytokine level. Murine L929 fibroblasts cultured with cobalt chromium particles were observed by phase contrast and scanning electron microscopy (SEM). Changes in the cytoskeletal rearrangement of F‐actin and α‐actinin focal adhesion plaques were studied by confocal microscopy. Expression of the proinflammatory cytokines IL‐6 and IL‐1α were analyzed by ELISA. The role of actin filaments and microtubules in particle uptake were determined at low temperature and in presence of colchicine and cytochalasin B. Phase contrast and SEM studies reveal that the metal particles adhere to the fibroblasts. The cellular cytoplasm was observed to grow over the particles and is suggestive of particle uptake. Confocal microscopy shows the presence of voids within the F‐actin cytoskeletal framework corresponding to areas occupied by the metal particles, indicating the possible uptake of these particles. Aggregates of α‐actinin into patches at the cell surface were also noted. Adherence and uptake of particles did not occur at low temperature and in presence of cytochalasin B, indicating that it is an active energy‐dependent process involving actin filaments. Changes in the levels of cytokine IL‐6 and IL‐1α were not observed suggesting the role of other cytokine molecules in mediating the inflammatory response to wear debri by fibroblasts. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Can scanning near‐field optical microscopy be compared with confocal laser scanning microscopy? A preliminary study on α‐sarcoglycan and β1D‐integrin in human skeletal muscle

The dystrophin–glycoprotein complex and the vinculin–talin–integrin system constitute, together a protein machinery, called costameres. The dystrophin–glycoprotein complex contains, among other proteins, also dystrophin and the sarcoglycans subcomplex, proteins playing a key role in the pathogenesis of many muscular dystrophies and linking the cytoplasmic myofibrillar contractile elements to the signal transducing molecules of the extracellular matrix, also providing structural support to the sarcolemma. The vinculin–talin–integrin system connects some components of the extracellular matrix with intermediate filaments of desmin, forming transverse bridges between Z and M lines. In our previous reports we always studied these systems by confocal laser scanning microscopy (CLSM). In this paper we report on the first applications of optical near‐field fluorescence microscopy to the spatial localization of α‐sarcoglycan and β1D‐integrin in human skeletal muscle fibres in order to better compare and test the images obtained with conventional CLSM and with scanning near‐field optical microscopy (SNOM). In addition, the analysis of the surface morphology, and the comparison with the fluorescence map is put forward and analyzed for the first time on human muscle fibres. In aperture‐SNOM the sample is excited through the nanometre‐scale aperture produced at the apex of an optical fibre after tapering and subsequent metal coating. The acquisition of the topography map, simultaneously to the optical signal, by SNOM, permits to exactly overlap the fluorescence images obtained from the two consecutive scans needed for the double localization. Besides, the differences between the topography and the optical spatial patterns permit to assess the absence of artefacts in the fluorescence maps. Although the SNOM represented a good method of analysis, this technique remains a complementary method to the CLSM and it can be accepted in order to confirm the hypothesis advanced by CLSM.