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.     

Optical characterization of temperature‐ and composition‐dependent microstructure in asphalt binders

We introduce noncontact optical microscopy and optical scattering to characterize asphalt binder microstructure at temperatures ranging from 15°C to 85°C for two compositionally different asphalt binders. We benchmark optical measurements against rheometric measurements of the magnitude of the temperature‐dependent bulk complex shear modulus . The main findings are: (1) Elongated (～5 × 1 μm), striped microstructures (known from AFM studies as ‘bees’ because they resemble bumble‐bees) are resolved optically, found to reside primarily at the surface and do not reappear immediately after a single heating–cooling cycle. (2) Smaller (～1 μm²) microstructures with no observable internal structure (hereafter dubbed ‘ants’), are found to reside primarily in the bulk, to persist after multiple thermal cycles and to scatter light strongly. Optical scattering from ‘ants’ decreases to zero with heating from 15°C to 65°C, but recovers completely upon cooling back to 15°C, albeit with distinct hysteresis. (3) Rheometric measurements of reveal hysteresis that closely resembles that observed by optical scatter, suggesting that thermally driven changes in microstructure volume fraction cause corresponding changes in .     

Geometry and spatial orientation of a quadruple node of triple junctions in a europium‐doped KI crystal as determined by epifluorescence microscopy

The geometry and spatial orientation of a typical arrangement of four triple junctions and six grain boundaries sharing a common quadruple node in a Eu²⁺‐doped KI crystal are investigated by epifluorescence microscopy using the proper doping ion as a fluorochrome. To achieve this, an electronic three‐dimensional reconstruction of the studied arrangement of crystal defects was built from microscopy images of different optical cross‐sections of this arrangement. Previously, the doping ions were induced, by subjecting the crystal to a long annealing treatment, to form europium precipitates into the crystal grain boundaries. The optical properties of these precipitates were characterized by fluorescence spectrophotometry and used to tailor properly the microscope fluorescence mirror unit, whereas the single‐crystal character of the microscope samples was tested by X‐ray diffraction. By inspecting the reconstruction under handling, the dihedral angles between the grain boundaries that meet at a common triple junction as well as the angles between the triple junctions sharing the quadruple node were successfully measured at the quadruple node site. The measuring procedures are carefully described. The resulting values (132º, 109º, 119º, 125º, 111º, 124º, 124º, 111º, 125º, 129º, 109º and 122º ± 2º) for the dihedral angles depart for some few degrees from the characteristic angle (120º) of a 3‐fold symmetry rotation, whereas the resulting values (104º, 111º, 117º, 103º, 100º and 121º ± 2º) for the triple junction angles are not far from the characteristic angle (109.47º) between the legs of a tetrahedron. These results, indicating that in the close neighbourhood of the quadruple node the studied arrangement of crystal defects deviates from a state of full structural stability, allow this arrangement to be fairly modelled in such a neighbourhood by a distorted tetrahedron. The angles between the studied triple junctions and the host lattice directions [11], [11], [11] and [] were also measured at the quadruple node site, and the resulting values (8º, 7º, 6º and 8º ± 2º, respectively) indicate that a symmetry mismatching exists between the tetrahedral model of the studied Eu²⁺‐decorated arrangement of crystal defects and the KI matrix cubic crystal lattice. This symmetry mismatching is discussed to be responsible for the observed deviation from structural stability.     

Blind colour separation of H&E stained histological images by linearly transforming the colour space

Blind source separation methods aim to split information into the original sources. In histology, each dye component attempts to specifically characterize different microscopic structures. In the case of the hematoxylin–eosin stain, universally used for routine examination, quantitative analysis may often require the inspection of different morphological signatures related mainly to nuclei patterns, but also to stroma distribution. Stain separation is usually a preprocessing operation that is transversal to different applications. This paper presents a novel colour separation method that finds the hematoxylin and eosin clusters by projecting the whole space to a folded surface connecting the distributions of a series of planes that divide the cloud of H&E tones. The proposed method produces density maps closer to those obtained with the colour mixing matrices set by an expert, when comparing with the density maps obtained using nonnegative matrix factorization (NMF), independent component analysis (ICA) and a state‐of‐the‐art method. The method has outperformed three baseline methods, NMF, Macenko and ICA, in about 8%, 12% and 52% for the eosin component, whereas this was about 4%, 8% and 26% for the hematoxylin component.     

Morphological and confocal laser scanning microscopic investigations of the adductor muscle‐shell interface in scallop

The challenge of joining dissimilar advanced materials has led researchers around the world to search for new and more efficient solutions. This way, we can highlight the muscle‐shell attachment in mollusk, which possessed high strength and toughness. In order to make clear how this “bi‐material interface” derives its superior mechanical properties, the morphological features of the adductor muscle scar in Patinopecten yessoensis was investigated by means of confocal laser scanning microscopy (CLSM). This scar area was found to consist of a myostracum with many evenly distributed pit structures and a fracture section with a parallel arranged prism‐like structure. The measured values of the distribution density, diameter, and depth of those pit structures were 24 , 7.36 2.47 , and 1 0.31 respectively. Profile of each pit wall was arc curve without closed angle. Furthermore, CLSM micrographs showed that considerable micro pits (0.1–0.9 in diameter) distribute round the pit wall and on the pit bottom. This special micromorphology is the first report on the adductor muscle scar in scallop. In addition, the mineral state and mechanical property of the scar surface was analyzed by XRD and nanoindentation test respectively. In general, the study results presented in this work elucidated that the adductor muscle of P. yessoensis was attached to the shell by insertion of collagen fibers and fibril bundles branched from themselves into pits on the myostracum. This specific connection mechanism can increase the strength of the interface without compromising its ductility and toughness. Microsc. Res. Tech. 78:761–770, 2015. © 2015 Wiley Periodicals, Inc.     

Epifluorescence microscopy study of a quadruple node of triple junctions of grain boundaries in a Eu2+‐doped Kcl:Kbr solid solution by using the doping ion as a fluorochrome

A typical quadruple node (QN) of triple junctions (TJs) of grain boundaries (GBs) in a Eu²⁺‐doped KCl_0.52Br_0.48 solid solution is investigated from the geometrical point of view by epifluorescence microscopy using the doping ion as a fluorochrome. The excitation and fluorescence optical properties of the fluorochrome were previously characterised by spectrophotometry whereas the structural nature of the studied material as well as its Bravais lattice type, unit cell size and long‐range translational order degree was determined by X‐ray diffraction. A three‐dimensional reconstruction was built from the microscopy images of different optical cross‐sections of the studied arrangement of crystal defects. In the close vicinity of the QN, the studied arrangement of crystal defects adopts the geometry of a collapsed tristetrahedron which, centred at the QN, has its legs along the TJs and, hence, has its faces as collapsed in pairs into the GBs. The angles defined by different TJ couples as well as the dihedral angles defined by the different GB couples meeting in every TJ were measured at the QN site. All, the image recording and stacking as well as the measuring procedures are carefully described. The measured TJ angles (97°, 117°, 95°, 117°, 99° and 130° ± 2°) depart from the characteristic angle (109.47°) of a tetrahedron whereas the measured GB angles (101°, 119°, 140°; 125°, 127°, 108°; 133°, 109°, 119°; 129°, 99° and 132° ± 2°) depart from the angular argument (120°) of a 3‐fold symmetry rotation indicating that, in the close neighbourhood of the QN, the studied arrangement of crystal defects is structurally unstable. Such an instability is associated with an observed mismatch in orientation (by angles of 20°, 15°, 33° and 30° ± 2°) between the TJs and some <111> zone axis matrix lattice crystallographic directions ([], [11], [11] and [11]), respectively). Local variations in anionic composition, existing within the solid solution matrix, are discussed to be responsible for this mismatching and, therefore, for the observed structural instability.     

European sea bass gill pathology after exposure to cadmium and terbuthylazine: expert versus fractal analysis

The objective of this study was to compare expert versus fractal analysis as new methods to evaluate branchial lamellar pathology in European sea bass Dicentrarchus labrax (Linnaeus, 1758) experimentally exposed to cadmium and to terbuthylazine. In particular, guided expert quantitative and fractal analysis were performed on selected images from semithin sections to test possible differences according to exposure class (unexposed, cadmium exposed, or terbuthylazine exposed) and the discrimination power of the two methods. With respect to guided expert quantitative analysis, the following elementary pathological features were assessed according to pre‐determined cover classes: ‘epithelial lifting’, ‘epithelial shrinkage’, ‘epithelial swelling’, ‘pillar cells coarctation’, ‘pillar cells detachment’, ‘channels fusion’, ‘chloride cells swelling’ and ‘chloride cells invasion’. Considering fractal analysis, D_B (box dimension), D_M (mass dimension), (mean fractal dimension) as fractal dimensions and lacunarity from D_M and scan types were calculated both from the outlined and skeletonized (one pixel wide lines) images. Despite significant differences among experimental classes, only expert analysis provided good discrimination with correct classification of 91.7 % of the original cases, and of 87.5 % of the cross‐validated cases, with a sensitivity of 95.45 % and 91.3 %, respectively, and a specificity of 75 % in both cases. Guided expert quantitative analysis appears to be a reliable method to objectively characterize fish gill pathology and may represent a powerful tool in environmental biomonitoring to ensure proper standardization and reproducibility. Though fractal analysis did not equal the discrimination power of the expert method, it certainly warrants further study to evaluate local variations in complexity or possible multiple scaling rules.     

HRTEMFringeAnalyzer a free python module for an automated analysis of fringe pattern in transmission electron micrographs

A python module (HRTEMFringeAnalyzer ) is reported to evaluate the local crystallinity of samples from high‐resolution transmission electron microscopy images in a mostly automated fashion. The user only selects the size of a square analyser window and a step size which translates the window in the micrograph. Together they define the resolution of the results obtained. Regions where fringe patterns are visible are identified and their lattice spacing d and direction ? as well as the corresponding mean errors σ determined. is proportional to the coherence length of the structure, whereas is a measure of how well the direction of the fringes is defined. Maps of these four indicators are computed. The performance of the program is demonstrated on two very different samples: ill‐crystalline carbon deposits on a coked Ni/LFNO (reduced LaFe_0.8Ni_0.2O) catalyst and well‐crystallized nanoparticles of zinc doped ceria. In the latter case, the automatic segmentation of large aggregates into individual crystalline domains is achieved by ? maps.     

Online interferometric study of viscoelastic rupture and necking deformation of as‐spun (iPP) fibres due to creep process

Creep deformation under constant load leads to rupture when the polymer chains can no longer separate and accommodate the load. This fracture phenomenon is investigated interferometrically. The creep behaviour of as‐spun isotactic Polypropylene (iPP) fibres is studied at different stresses, different initial lengths and different radii. The creep rate, which defines the velocity of the creep deformation and the dimensional stability of the material, is studied. The failure time and stress of iPP due to creep process is determined. The necking deformation was in situ detected during creep process. The mean refractive indices (n^P and) profiles of iPP fibres were determined at different positions along the fibre axis before and after necking. The relation between the creep behaviour and different optical and structural parameters is investigated. Microinterferograms are given for illustration.     

Electron backscattered diffraction analysis of friction stir processed nanocomposites produced via spark plasma sintering

In the present study, Spark Plasma Sintered (SPSed) aluminium matrix composites were severely deformed through Friction stir processing (FSP). Pure aluminium powders and bimodal sized Al₂O₃ particles (80 nm and 25 m) were firstly mixed by ball milling and then consolidated by spark plasma sintering. The effect of the heat input as well the bimodal particle size of the alumina on the materials’ microstructure and texture development was evaluated by electron back scattered diffraction (EBSD) analysis. The EBSD analysis clearly showed that the SPSed nanocomposites possessed bimodal aluminium matrix grain structure as well as a crystallography characterised by random texture. In addition, microstructural examination revealed that the partial recrystallisation occurred during SPS for all the nanocomposites. Also, it is revealed that the Zener pinning effect of Al₂O₃ nanoparticles retarded recrystallised grain growth following recrystallisation during FSP and then leading to grain refinement of the aluminium. The results revealed that the heat generated during FSP has a remarkable effect on the grain distribution as well as on the crystallographic orientation. Also, a mixture of {112} <110> shear elements and an ideal strong B/ component were observed. The microstructural changes, occurred during FSP in the stir zone region for Al‐Al₂O₃ nanocomposites, were attributed to both the discontinuous along with the continuous recrystallisation (DDRX/CDRX). It should be pointed out that with increasing the heat input, recrystallised grains portion increased.     

Laser‐induced reversion of precipitates in an Al‐Li alloy: Study on temperature rise in pulsed laser atom probe

The influence of tuning the laser pulse energy during the analyses on the resulting microstructure in a specimen utilizing an ultra‐fast laser assisted atom probe was demonstrated by a case study of a binary Al‐Li alloy. The decomposition parameters, such as the size, number density, volume fraction, and composition of precipitates, were carefully monitored after each analysis. A simple model was employed to estimate the corresponding specimen temperature for each value of the laser energy. The results indicated that the corresponding temperatures for the laser pulse energy in the range of 10 to 80 pJ are located inside the miscibility gap of the binary Al‐Li phase diagram and fall into the metastable equilibrium field. In addition, the corresponding temperature for a laser pulse energy of 100 pJ was in fairly good agreement with reported range of solvus temperature, suggesting a result of reversion upon heating due to laser pulsing. Microsc. Res. Tech. 79:727–737, 2016. © 2016 Wiley Periodicals, Inc.     

Reflectance difference microscopy for nanometre thickness microstructure measurements

The discontinuity of medium at the boundary produces optically anisotropic response which makes reflectance difference microscopy (RDM) a potential method for nanometre‐thickness microstructure measurements. Here, we present the methodology of RDM for the edge measurement of ultrathin microstructure. The RD signal of microstructure's boundary is mathematically deduced according to boundary condition and polarization optics theory. A normal‐incidence RDM setup was built simply with one linear polarizer, one liquid crystal variable retarder and one 5 × objective. Then, the performance of the developed setup was identified using homogenous reflection mirror and high quality linear polarizer. For demonstration, microstructures array with 100 nm step height was measured. The results show that the RD signal is sensitive to the edge and its sign reflects the change direction of the edge. Furthermore, a height sensitivity of better than 10 nm and a spatial resolution of ～3 m offer this technique a good candidate for characterizing ultrathin microstructures.     

Automated image segmentation of haematoxylin and eosin stained skeletal muscle cross‐sections

The ability to accurately and efficiently quantify muscle morphology is essential to determine the physiological relevance of a variety of muscle conditions including growth, atrophy and repair. There is agreement across the muscle biology community that important morphological characteristics of muscle fibres, such as cross‐sectional area, are critical factors that determine the health and function (e.g. quality) of the muscle. However, at this time, quantification of muscle characteristics, especially from haematoxylin and eosin stained slides, is still a manual or semi‐automatic process. This procedure is labour‐intensive and time‐consuming. In this paper, we have developed and validated an automatic image segmentation algorithm that is not only efficient but also accurate. Our proposed automatic segmentation algorithm for haematoxylin and eosin stained skeletal muscle cross‐sections consists of two major steps: (1) A learning‐based seed detection method to find the geometric centres of the muscle fibres, and (2) a colour gradient repulsive balloon snake deformable model that adopts colour gradient in colour space. Automatic quantification of muscle fibre cross‐sectional areas using the proposed method is accurate and efficient, providing a powerful automatic quantification tool that can increase sensitivity, objectivity and efficiency in measuring the morphometric features of the haematoxylin and eosin stained muscle cross‐sections.     

A 340/380 nm light‐emitting diode illuminator for Fura‐2 AM ratiometric Ca2+ imaging of live cells with better than 5 nM precision

We report the first demonstration of a fast wavelength‐switchable 340/380 nm light‐emitting diode (LED) illuminator for Fura‐2 ratiometric Ca²⁺ imaging of live cells. The LEDs closely match the excitation peaks of bound and free Fura‐2 and enables the precise detection of cytosolic Ca²⁺ concentrations, which is only limited by the Ca²⁺ response of Fura‐2. Using this illuminator, we have shown that Fura‐2 acetoxymethyl ester (AM) concentrations as low as 250 nM can be used to detect induced Ca²⁺ events in tsA‐201 cells and while utilising the 150 s switching speeds available, it was possible to image spontaneous Ca²⁺ transients in hippocampal neurons at a rate of 24.39 Hz that were blunted or absent at typical 0.5 Hz acquisition rates. Overall, the sensitivity and acquisition speeds available using this LED illuminator significantly improves the temporal resolution that can be obtained in comparison to current systems and supports optical imaging of fast Ca²⁺ events using Fura‐2.     

The elusive ettringite under the high‐vacuum SEM – a reflection based on natural samples,the use of Monte Carlo modelling of EDS analyses and an extension to the ettringite group minerals

Ettringite, Ca₆Al₂(SO₄)₃(OH)₁₂.26H₂O, or C₆A₃H₃₂ as it is known in cement chemistry notation, is a major phase of interest in cement science as an hydration product and in polluted soil treatment since its structure can accommodate with many hazardous cations. Beyond those anthropogenic features, ettringite is first of all a naturally occurring mineral (although rare). An example of its behaviour under the scanning electron microscope and during energy dispersive spectroscopy (EDS) qualitative analysis is presented, based on the study of natural ettringite crystals from the N'Chwaning mine in South Africa. Monte Carlo modelling of the electron‐matter interaction zone at various voltages is presented and confronted with actual, observed beam damage on crystals, which burst at the analysis spot. Finally, theoretical energy dispersive spectroscopy spectra for all the ettringite group minerals have been computed as well as Monte Carlo modelling of the electron‐matter interaction zone. The knowledge of the estimation of the size of this zone may thus be helpful for the understanding of energy dispersive spectroscopy analysis in cement pastes or ettringite‐remediated soils.     

Resolution and super‐resolution

Many papers have claimed the attainment of super‐resolution, i.e. resolution beyond that achieved classically, by measurement of the profile of a feature in the image. We argue that measurement of the contrast of the image of a dark bar on a bright background does not give a measure of resolution, but of detection sensitivity. The width of a bar that gives an intensity at the center of the bar of 0.735 that in the bright region (the same ratio as in the Rayleigh resolution criterion) is for the coherent case with central illumination. This figure, which compares with for the Abbe resolution limit with central illumination, holds for the classical case, and so is no indication of super‐resolution. Theoretical images for two points, two lines, arrays of lines, arrays of bars, and grating objects are compared. These results can be used a reference for experimental results, to determine if super‐resolution has indeed been attained. The history of the development of the theory of microscope resolution is outlined.     

FT‐Raman spectroscopy, µ‐EDXRF spectrometry,and microhardness analysis of the dentin of primary and permanent teeth

The chemical compositions (organic and inorganic contents) and mechanical behaviors of the dentin of permanent and deciduous teeth were analyzed and compared using X‐ray fluorescence spectrometry (µ‐EDXRF) Fourier transform Raman spectroscopy (FT‐Raman) and a microhardness test (HD). Healthy fresh human primary and permanent molars (n = 10) were selected, The buccal surfaces facing upwards were stabilized in an acrylic plate, flattened, polished, and submitted to the µ‐EDXRF, FT‐Raman, and HD analysis. The results of the analysis were subjected to ANOVAs and Mann‐Whitney U/Student's t multiple comparisons tests. The data showed similar values for the dentin of the primary and permanent teeth in P content, organic content (amide I peak), inorganic content ( – 430 and 590), and microhardness, Nevertheless, Ca content and Ca/P weight ratio were higher, and the peak was lower in the dentin of the permanent teeth compared to primary teeth. It be concluded that despite permanent teeth showed more Ca element, both substrates showed similar behavior of chemical and physical properties.     

MRT Letter: Two‐photon excitation‐based 2pi Light‐sheet system for nano‐lithography

We propose two‐photon excitation‐based light‐sheet technique for nano‐lithography. The system consists of 2 ‐configured cylindrical lens system with a common geometrical focus. Upon superposition, the phase‐matched counter‐propagating light‐sheets result in the generation of identical and equi spaced nano‐bump pattern. Study shows a feature size of as small as few tens of nanometers with a inter‐bump distance of few hundred nanometers. This technique overcomes some of the limitations of existing nano‐lithography techniques, thereby, may pave the way for mass‐production of nano‐structures. Potential applications can also be found in optical microscopy, plasmonics, and nano‐electronics. Microsc. Res. Tech. 78:1–7, 2015. © 2014 Wiley Periodicals, Inc.     

Image matching between experimental and simulated high‐resolution electron micrographs of sapphire on the orientation

The effects of imaging parameters have been studied on their roles of the severe mismatches between experimental and simulated high‐resolution transmission electron micrographs of sapphire along the direction. Image simulation and convergent‐beam electron diffraction techniques have been performed on misalignments of the electron beam and the crystal specimen. Based on this study, we have introduced an approach to achieve reliable simulation for experimental images of sapphire on the projection by the use of iterative digital image matching.     

Extremely thin layer plastification for focused‐ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells

Since the recent boost in the usage of electron microscopy in life‐science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused‐ion beam scanning electron microscopy (FIB‐SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold‐labelled breast cancer SKBR3 cells was to visualise gold‐labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 m² cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back‐scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB‐SEM. Cross‐sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.