Indexing electron backscatter diffraction patterns with a refined template matching approach.

Electron backscatter diffraction (EBSD) is a well-established method of characterisation for crystalline materials. Using this technique, we can rapidly acquire and index diffraction patterns to provide phase and orientation information about the crystals on the material surface. The conventional analysis method uses signal processing based on a Hough/Radon transform to index each diffraction pattern. This method is limited to the analysis of simple geometric features and ignores subtle characteristics of diffraction patterns, such as variations in relative band intensities. A second method, developed to address the shortcomings of the Hough/Radon transform, is based on template matching of a test experimental pattern with a large library of potential patterns. In the present work, the template matching approach has been refined with a new cross correlation function that allows for a smaller library and enables a dramatic speed up in pattern indexing. Refinement of the indexed orientation is performed with a follow-up step to allow for small alterations to the best match from the library search. The refined template matching approach is shown to be comparable in accuracy, precision and sensitivity to the Hough based method, even exceeding it in some cases, via the use of simulations and experimental data collected from a silicon single crystal and a deformed α-iron sample. The speed up and pattern refinement approaches should increase the widespread utility of pattern matching approaches.

Validation of an algorithm to reveal the U wave in atrial fibrillation.

Major cardiac organisations recommend U wave abnormalities should be reported during ECG interpretation. However, U waves cannot be measured in patients with atrial fibrillation (AF) due to the obscuring fibrillatory wave. The aim was to validate a U wave measurement algorithm for AF patients. Multi-beat averaging was applied to ECGs of 25 patients during paroxysms of AF and the presence of U waves compared to those from the same patients during sinus rhythm (SR). In a further database of 10 long-term AF recordings, the number of beats for effective U wave extraction by the algorithm was calculated. U waves were revealed in all AF recordings and there was no significant difference between the presence of U waves in AF and SR (p = 0.88). U wave amplitude was significantly increased in AF (mean (s.d.) amplitude 55 (39) AF vs 37 (28) µV SR, p = 0.005). The presence of U waves could easily be discerned when as few as 10 beats were used in the algorithm. The study demonstrates the validity of the algorithm to reveal U waves in AF recordings. The algorithm offers the potential to detect U wave abnormalities in patients with AF.

The dissolvable bead: A novel in vitro biofilm model for evaluating antimicrobial resistance.

In vitro biofilm assays are a vital first step in the assessment of therapeutic effectiveness. Current biofilm models have been found to be limited by throughput, reproducibility, and cost. We present a novel in vitro biofilm model, utilising a sodium alginate substratum for surface biofilm colony formation, which can be readily dissolved for accurate evaluation of viable organisms. The dissolving bead biofilm assay was evaluated using a range of clinically relevant strains. The reproducibility and responsiveness of the assay to an antimicrobial challenge was assessed using standardised methods. Cryo-scanning electron microscopy was used to image biofilm colonies. Biofilms were grown for 20h prior to testing. The model provides a reproducible and responsive assay to clinically-relevant antimicrobial challenges, as defined by established guidelines. Moreover cryo-scanning electron microscopy demonstrates that biofilm formation is localised exclusively to the alginate bead surface. Our results suggest that this simple model provides a robust and adaptable assay for the investigation of bacterial biofilms.

Quantitative imaging of anti-phase domains by polarity sensitive orientation mapping using electron backscatter diffraction.

Advanced structural characterisation techniques which are rapid to use, non-destructive and structurally definitive on the nanoscale are in demand, especially for a detailed understanding of extended-defects and their influence on the properties of materials. We have applied the electron backscatter diffraction (EBSD) technique in a scanning electron microscope to non-destructively characterise and quantify antiphase domains (APDs) in GaP thin films grown on different (001) Si substrates with different offcuts. We were able to image and quantify APDs by relating the asymmetrical intensity distributions observed in the EBSD patterns acquired experimentally and comparing the same with the dynamical electron diffraction simulations. Additionally mean angular error maps were also plotted using automated cross-correlation based approaches to image APDs. Samples grown on substrates with a 4° offcut from the [110] do not show any APDs, whereas samples grown on the exactly oriented substrates contain APDs. The procedures described in our work can be adopted for characterising a wide range of other material systems possessing non-centrosymmetric point groups.

Diffraction effects and inelastic electron transport in angle-resolved microscopic imaging applications.

We analyse the signal formation process for scanning electron microscopic imaging applications on crystalline specimens. In accordance with previous investigations, we find nontrivial effects of incident beam diffraction on the backscattered electron distribution in energy and momentum. Specifically, incident beam diffraction causes angular changes of the backscattered electron distribution which we identify as the dominant mechanism underlying pseudocolour orientation imaging using multiple, angle-resolving detectors. Consequently, diffraction effects of the incident beam and their impact on the subsequent coherent and incoherent electron transport need to be taken into account for an in-depth theoretical modelling of the energy- and momentum distribution of electrons backscattered from crystalline sample regions. Our findings have implications for the level of theoretical detail that can be necessary for the interpretation of complex imaging modalities such as electron channelling contrast imaging (ECCI) of defects in crystals. If the solid angle of detection is limited to specific regions of the backscattered electron momentum distribution, the image contrast that is observed in ECCI and similar applications can be strongly affected by incident beam diffraction and topographic effects from the sample surface. As an application, we demonstrate characteristic changes in the resulting images if different properties of the backscattered electron distribution are used for the analysis of a GaN thin film sample containing dislocations.

Low birth weight and intelligence in adolescence and early adulthood: a meta-analysis.

BACKGROUND: Research has demonstrated an association between low birth weight (LBW; <2500 g) and adverse intelligence quotient (IQ) outcomes in childhood and early adolescence. We systematically evaluated whether this association persists into late adolescence and early adulthood and also assessed the influence of age of IQ assessment on effect size. METHODS: During Stage 1 (meta-analysis of data on adolescents/adults), we searched for relevant articles in PsychINFO, PubMed, Ovid, CINAHL, ProQuest and ERIC until February 2011 (no lower limit). Studies which assessed full-scale IQ among LBW individuals (<2500 g), aged 13 years and older, with a normal birth weight (NBW; ≥2500 g) comparison group were eligible. A random-effects meta-analysis provided a pooled estimate of the difference in IQ scores between LBW and NBW individuals. Publication bias was assessed using Rosenthal's classic fail-safe N and Duval and Tweedie's Trim and Fill. During Stage 2, we added data from the Kerr-Wilson et al. meta-analysis (which included data from children; in Meta-analysis of the association between preterm delivery and intelligence. Journal Public Health 2011;33:1-8) to our sample from Stage 1 and conducted a meta-regression to evaluate the effect of age of IQ assessment. RESULTS: Using a total of 15 studies in Stage 1, it was demonstrated that NBW individuals scored an average of 7.63 IQ points higher than LBW individuals, CI = 5.95-9.31. After adjusting for publication bias, NBW samples demonstrated an IQ of 4.98 points higher than LBW samples, CI = 3.20-6.77. Furthermore, age at IQ assessment was a significant moderator of the association between birth weight and IQ, in that the effect size decreased from childhood into young adulthood. CONCLUSIONS: Cognitive impairments associated with LBW persist into adolescence and early adulthood; however, the influence of LBW on IQ decreases from childhood to young adulthood. These conclusions must be interpreted with caution due to unmeasured variables and possible influence from publication bias.

Assessing the precision of strain measurements using electron backscatter diffraction--part 1: detector assessment.

We analyse the link between precision of pattern shift measurements and the resolution of the measurement of elastic strain and lattice rotation using high resolution electron backscatter diffraction (HR-EBSD). This study combines analysis of high quality experimentally obtained diffraction patterns from single crystal silicon; high quality dynamical simulations using Bloch wave theory; quantitative measurements of the detector Modulation Transfer Function (MTF) and a numerical model. We have found that increases in exposure time, when 1×1 binning is selected, are the primary reason for the observed increase in sensitivity at greater than 2×2 binning and therefore use of software integration and high bit depth images enables a significant increase in strain resolution. This has been confirmed using simulated diffraction patterns which provide evidence that the ultimate theoretical resolution of the cross correlation based EBSD strain measurement technique with a 1000×1000 pixel image could be as low as 4.2×10(-7) in strain based on a shift precision of 0.001 pixels.

Assessing the precision of strain measurements using electron backscatter diffraction--part 2: experimental demonstration.

The residual impression after performing a microhardness indent in silicon has been mapped with high resolution EBSD to reveal residual elastic strain and lattice rotation fields. Mapping of the same area has been performed with variable pattern binning and exposure times to reveal the qualitative and quantitative differences resulting from reducing the pattern size and exposure time. Two dimension 'image' plots of these fields indicate that qualitative assessment of the shape and size of the fields can be performed with as much as 4×4 binning. However, quantitative assessment using line scans reveals that the smoothest profile can be obtained using minimal pattern binning and long exposure times. To compare and contrast with these experimental maps, finite element analysis has been performed using a continuum damage-plasticity material law which has been independently calibrated to Si [9]. The constitutive law incorporates isotropic hardening in compression, and isotropic hardening and damage in tension. To accurately capture the localised damage which develops during indentation via the nucleation and propagation of cracks around the indentation site cohesive elements were assigned along the interfaces between the planes which experience the maximum traction. The residual strain state around the indenter and the size of the cracks agree very well with the experimentally measured value.

Measurement of geometrically necessary dislocation density with high resolution electron backscatter diffraction: effects of detector binning and step size.

Recent advances using cross-correlation analysis of full resolution high quality electron backscatter diffraction (EBSD) patterns have provided a method for quantitatively mapping the stored dislocation density at high spatial resolution. Larger areas could be mapped with image binning or coarser step sizes. We have studied the effects of image binning and step size on the recovery of GND density. Our results suggest that: (i) the measured lower bound GND density noise floor broadly agrees with Wilkinson and Randman's 2009 prediction, where a decrease in step size or an increase in misorientation uncertainty increases the noise floor; (ii) increasing the step size results in a lower GND density being recovered as some dislocations are now considered as statistically stored dislocations (SSDs); (iii) in deformed samples the average GND density stays relatively constant as the degree of pattern binning is increased up to 8×8. Pattern binning thus provides a means of increasing the data acquisition and analysis rate without unduly degrading the data quality.

Rapid nondestructive analysis of threading dislocations in wurtzite materials using the scanning electron microscope.

We describe the use of electron channeling contrast imaging in the scanning electron microscope to rapidly and reliably image and identify threading dislocations (TDs) in materials with the wurtzite crystal structure. In electron channeling contrast imaging, vertical TDs are revealed as spots with black-white contrast. We have developed a simple geometric procedure which exploits the differences observed in the direction of this black-white contrast for screw, edge, and mixed dislocations for two electron channeling contrast images acquired from two symmetrically equivalent crystal planes whose g vectors are at 120° to each other. Our approach allows unambiguous identification of all TDs without the need to compare results with dynamical simulations of channeling contrast.

High resolution electron backscatter diffraction measurements of elastic strain variations in the presence of larger lattice rotations.

In this paper we explore methods of measuring elastic strain variations in the presence of larger lattice rotations (up to -11°) using high resolution electron backscatter diffraction. We have examined the fundamental equations which relate pattern shifts to the elastic strain tensor and modified them to a finite deformation framework from the original infinitesimal deformation one. We incorporate the traction free boundary condition into the minimisation problem for the finite deformation case (i.e. large rotations and small elastic strains). Numerical experiments show that this finite deformation kinematic analysis continues to work well, while the infinitesimal analysis fails, when the misorientation between test and reference pattern is made increasingly high. However, measurements on patterns simulated using dynamical diffraction theory indicated that this formulation is not sufficient to recover elastic strains accurately because the pattern shifts are not determined accurately when large rotations are present. To overcome this issue we remap the test pattern to an orientation that is close to that of reference pattern. This remapping was defined by a finite rotation matrix, which was estimated from the infinitesimal rotation matrix measured using cross-correlation. A second cross-correlation analysis between the reference pattern and the remapped test pattern allows the elastic strains to be recovered using the much simpler infinitesimal deformation theory. We have also demonstrated that accurate recovery of elastic strains requires accurate knowledge of the pattern centre if this remapping algorithm is used.

Measurement of residual elastic strain and lattice rotations with high resolution electron backscatter diffraction.

A set of dynamically simulated electron backscatter patterns (EBSPs) for α-Ti crystals progressively rotated by 1° steps were analysed using cross-correlation to determine image shifts from which strains and rotations were calculated. At larger rotations the cross-correlation fails in certain regions of the EBSP where large shifts are generated. These incorrect shifts prevent standard least square error procedures from obtaining a valid solution for the strain and rotation, where the applied rotation exceeds ∼ 8°. Using a robust iterative fitting routine reliable strains and rotations can be obtained for applied rotations of up to and including ∼ 11° even though some image shifts are measured incorrectly. Finally, high resolution electron backscatter diffraction has been used to analyse the residual elastic strain, lattice rotations and density of stored geometrically necessary dislocations in a sample of copper deformed to 10% total strain. The robust iterative fitting analysis allows reliable analysis of a larger proportion of the map, the difference being most obviously beneficial in regions where significant lattice rotations have been generated.

Factors affecting the accuracy of high resolution electron backscatter diffraction when using simulated patterns.

High resolution EBSD directly compares electron backscattering patterns (EBSPs), generated in a scanning electron microscope, to measure relative strain and rotation to a precision of ∼ 10(-4) in strain and 10(-4)rad (0.006 °) in rotation. However the measurement of absolute strain and rotation requires reference EBSPs of known strain and orientation (or a far field region of known strain). Recent suggestions of using simulated EBSPs with known strain show much promise. However precise measurement of the experimental geometry (pattern centre) is required. Common uncertainties of 0.5% in pattern centre result in uncertainty of ∼ 10(-3) in strain state. Aberrations in the compact lenses used for EBSP capture can also result in image shifts that correspond to strains/rotations of ± 10(-3) between experimental and simulated EBSPs. Simulated EBSPs can be generated using dynamical or kinematic models (or a combination of the two). The choice in simulation model has a significant effect on the measured shifts, particularly at zone axis and high structure factor bands, due to large intensity variations, and for simple kinematic simulations can result in the measurement of rogue shifts and thus erroneous strain measurements. Calibrant samples of known strain provide a method of measuring the experimental geometry but imprecise stage movement combined with the high depth of field in the SEM could also result in uncertainties in strain of ∼ 10(-3).

Ezetimibe monotherapy for cholesterol lowering in 2,722 people: systematic review and meta-analysis of randomized controlled trials.

OBJECTIVES: To study the evidence on the efficacy and safety of ezetimibe monotherapy for the treatment of primary (heterozygous familial and non-familial) hypercholesterolaemia. DESIGN: Systematic review and meta-analysis of randomized controlled trials (RCTs). METHODS: Eleven electronic bibliographic databases covering the biomedical, scientific and grey literature were searched from inception and supplemented by contact with experts in the field. Two reviewers independently determined the eligibility of RCTs, with a minimum treatment duration of 12 weeks, which compared ezetimibe monotherapy (10 mg per day) with placebo. RESULTS: In the absence of data from clinical outcome trials, surrogate endpoints such as changes in lipid concentrations were used as indicators of clinical outcomes. A meta-analysis of eight randomized, double-blind, placebo-controlled trials (all 12 weeks) showed that ezetimibe monotherapy was associated with a statistically significant mean reduction in LDL cholesterol (from baseline to endpoint) of -18.58%, (95% CI: -19.67 to -17.48, P < 0.00001) compared with placebo. Significant (P < 0.00001) changes were also found in total cholesterol (-13.46%, 95% CI: -14.22 to -12.70), HDL cholesterol (3.00%, 95% CI: 2.06-3.94) and triglyceride levels (-8.06%, 95% CI: -10.92 to -5.20). Ezetimibe monotherapy appeared to be well tolerated with a safety profile similar to placebo. CONCLUSIONS: In a meta-analysis restricted to short-term trials in hypercholesterolaemia, significant potentially favourable changes in lipid and lipoprotein levels relative to baseline occurred with ezetimibe monotherapy. Further long-term studies with cardiovascular and other clinical outcome data are needed to assess the efficacy and safety of ezetimibe more fully.

Effects of cigarette smoking upon clinical outcomes of assisted reproduction: a meta-analysis.

BACKGROUND: The aim of this meta-analysis was to investigate whether any difference exists in success rate of clinical outcomes of assisted reproductive technologies (ART) between women who actively smoke cigarettes at the time of treatment and those who do not. METHODS: An intensive computerized search was conducted on published literature from eight databases, using search terms related to smoking, assisted reproduction and outcome measures. Eligible studies compared outcomes of ART between cigarette smoking patients and a control group of non-smoking patients and reported on live birth rate per cycle, clinical pregnancy rate per cycle, ectopic pregnancy rate per pregnancy or spontaneous miscarriage rate per pregnancy, and 21 studies were included in the meta-analyses. Pooled odds ratios (OR) and 95% confidence intervals (CI) were calculated for the data, and statistical heterogeneity was tested for using chi(2) and I(2) values. A systematic review examined the effect of smoking upon fertilization rates across 17 studies. RESULTS: Smoking patients demonstrated significantly lower odds of live birth per cycle (OR 0.54, 95% CI 0.30-0.99), significantly lower odds of clinical pregnancy per cycle (OR 0.56, 95% CI 0.43-0.73), significantly higher odds of spontaneous miscarriage (OR 2.65, 95% CI 1.33-5.30) and significantly higher odds of ectopic pregnancy (OR 15.69, 95% CI 2.87-85.76). A systematic literature review revealed that fertilization rates were not significantly different between smoking and non-smoking groups in most studies. CONCLUSIONS: This meta-analysis provides compelling evidence for a significant negative effect of cigarette smoking upon clinical outcomes of ART and should be presented to infertility patients who smoke cigarettes in order to optimize success rates.

Quasi-cleavage fracture planes in spheroidized A533B steel.

Electron backscattered diffraction (EBSD) has been used to acquire crystal orientation information around unusual microcracks induced by tensile deformation of notched specimens of spheroidized A533B steel. This unusual fracture mode has been called quasi-cleavage and occurs at relatively low temperatures with fracture energies below that of the upper shelf. EBSD measurements on sectioned samples showed that the quasi-cleavage cracks were intragranular. A two-dimensional analysis technique was used in which EBSD measured crystal orientations were combined with secondary electron imaging to obtain the trace of the crack facet on the section plane. The measurements revealed that the observed crack facets were consistent with crack propagation along the {001} and {011} planes.

Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis.

A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.

Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens.

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.

Dimer-induced signal propagation in Spo0A.

Spo0A, the response regulator protein controlling the initiation of sporulation in Bacillus, has two distinct domains, an N-terminal phosphoacceptor (or receiver) domain and a C-terminal DNA-binding (or effector) domain. The phosphoacceptor domain mediates dimerization of Spo0A on phosphorylation. A comparison of the crystal structures of phosphorylated and unphosphorylated response regulators suggests a mechanism of activation in which structural changes originating at the phosphorylatable aspartate extend to the alpha4beta5alpha5 surface of the protein. In particular, the data show an important role in downstream signalling for a conserved aromatic residue (Phe-105 in Spo0A), the conformation of which alters upon phosphorylation. In this study, we have prepared a Phe-105 to Ala mutant to probe the contribution of this residue to Spo0A function. We have also made an alanine substitution of the neighbouring residue Tyr-104 that is absolutely conserved in the Spo0As of spore-forming Bacilli. The spo0A(Y104A) and spo0A(F105A) alleles severely impair sporulation in vivo. In vitro phosphorylation of the purified proteins by phosphoramidate is unaffected, but dimerization and DNA binding are abolished by the mutations. We have identified intragenic suppressor mutations of spo0A(F105A) and shown that these second-site mutations in the purified proteins restore phosphorylation-dependent dimer formation. Our data support a model in which dimerization and signal transduction between the two domains of Spo0A are mediated principally by the alpha4beta5alpha5 signalling surface in the receiver domain.

Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy.

DivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation. During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid-cell position. DivIVA fulfils a quite different role during sporulation in B. subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division. DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin-like alpha-helical coiled-coil. Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images. The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a 'doggy-bone'. The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6- to 8-mers. These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, two-dimensional lattices in a time-dependent manner.