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In this study, we quantitatively investigate the impact of 1.4 wt.% chromium and 1.4 wt.% molybdenum additions on pearlitic microstructure characteristics in 1 wt.% carbon steels. The study was carried out using a combination of experimental methods and phase field simulations. We utilized MatCalc v5.51 and JMatPro v12 to predict transformation behaviors, and electron microscopy for microstructural examination, focusing on pearlite morphology under varying thermal conditions. Phase field simulations were carried out using MICRESS v7.2 software and, informed by thermodynamic data from MatCalc v5.51 and the literature, were conducted to replicate pearlite formation, demonstrating a good agreement with the experimental observations. In this work, we introduced a semi-automatic reliable microstructural analysis method, quantifying features like lamella dimensions and spacing through image processing by Fiji ImageJ v1.54f. The introduction of Cr resulted in longer, thinner, and more homogeneously distributed cementite lamellae, while Mo led to shorter, thicker lamellae. Phase field simulations accurately predicted these trends and showed that alloying with Cr or Mo increases the density and circularity of the lamellae. Our results demonstrate that Cr stabilizes pearlite formation, promoting a uniform microstructure, whereas Mo affects the morphology without enhancing homogeneity. The phase field model, validated by experimental data, provides insights into the morphological changes induced by these alloying elements, supporting the optimization of steel processing conditions. [ABSTRACT FROM AUTHOR]

Few single‐molecule experiments have enabled the direct imaging of functional biomacromolecules in real‐time in their native liquid environments, resolving their conformational adaptations, transient interactions, and intermediate states. Liquid phase electron microscopy (LP‐EM), due to its unique combination of spatial and temporal resolution, has shown to be a promising tool. Recent experiments have enabled successful imaging of intact structures of organic molecules and biological systems with an ordinary electron microscope. Adapting image processing methods and quantitative data analysis from single particle experiments based on the optical microscope, quantifying motion and relaxation of these interacting molecules allows the experimental observations of pathways, to test theoretical predictions, and discovery of new mechanisms. Combining LP‐EM with tomography, fluorescence, and mass spectroscopy allows for probing multi‐dimensional structural and dynamic information. Challenges remain in obtaining high‐quality data in large quantities, which can be improved by developing new liquid cell platforms and machine learning‐based data analysis. [ABSTRACT FROM AUTHOR]

Detailed knowledge of biological structure has been key in understanding biology at several levels of organisation, from organs to cells and proteins. Volume electron microscopy (volume EM) provides high resolution 3D structural information about tissues on the nanometre scale. However, the throughput rate of conventional electron microscopes has limited the volume size and number of samples that can be imaged. Recent improvements in methodology are currently driving a revolution in volume EM, making possible the structural imaging of whole organs and small organisms. In turn, these recent developments in image acquisition have created or stressed bottlenecks in other parts of the pipeline, like sample preparation, image analysis and data management. While the progress in image analysis is stunning due to the advent of automatic segmentation and server‐based annotation tools, several challenges remain. Here we discuss recent trends in volume EM, emerging methods for increasing throughput and implications for sample preparation, image analysis and data management. [ABSTRACT FROM AUTHOR]

The wear state of an abrasive belt is one of the important factors affecting the grinding precision of belt grinding processes. At present, there are two problems associated with the monitoring method of the wear condition of abrasive belts: (1) there are no uniform wear criteria to classify the wear condition of abrasive belts, and the segmentation threshold of the wear condition is affected by the change in the grinding parameters; and (2) an abrasive belt wear model based on indirect sensor monitoring of signals is affected by the change in the grinding parameters of abrasive belts; therefore, it is only suitable for abrasive belt wear monitoring under specific grinding parameters. This paper introduces a method of belt wear state monitoring based on machine vision and image processing. Surface images of an abrasive belt during its entire life are captured using a noncontact electron microscope. Three image features related to the wear state are selected: first-order distance of color component R, entropy of the horizontal subgraph, and vertical subgraph of the texture feature. Moreover, the wear state is classified into three categories based on the selected features. Using the selected features and the random forest classification algorithm, an abrasive belt wear state classifier is established. The performance of the classifier is verified and evaluated using a data subset of different images. The results show that the proposed method has high recognition accuracy for belt wear state, which can reach 99% in the accelerated wear stage. The proposed method solves the problem of the dependence and sensitivity of the monitoring model on the variation in the grinding parameters in the process of abrasive belt wear monitoring, and it improves the adaptability and versatility of the monitoring method. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Forensic Medicine / Fayixue Zazhi is the property of Journal of Forensic Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

High-resolution study of the giant viruses presents one of the latest challenges in cryo-electron microscopy (EM) of viruses. Too small for light microscopy but too large for easy study at high resolution by EM, they range in size from ∼0.2 to 2 μm from high-symmetry icosahedral viruses, such as Paramecium burseria Chlorella virus 1, to asymmetric forms like Tupanvirus or Pithovirus. To attain high resolution, two strategies exist to study these large viruses by cryo-EM: first, increasing the acceleration voltage of the electron microscope to improve sample penetration and overcome the limitations imposed by electro-optical physics at lower voltages, and, second, the method of 'block-based reconstruction' pioneered by Michael G. Rossmann and his collaborators, which resolves the latter limitation through an elegant leveraging of high symmetry but cannot overcome sample penetration limitations. In addition, more recent advances in both computational capacity and image processing also yield assistance in studying the giant viruses. Especially, the inclusion of Ewald sphere correction can provide large improvements in attainable resolutions for 300 kV electron microscopes. Despite this, the study of giant viruses remains a significant challenge. [ABSTRACT FROM AUTHOR]

Cryo-electron microscopy using single particle analysis requires the computational averaging of thousands of projection images captured from identical macromolecules. However, macromolecules usually present some degree of flexibility showing different conformations. Computational approaches are then required to classify heterogeneous single particle images into homogeneous sets corresponding to different structural states. Nonetheless, sometimes the attainable resolution of reconstructions obtained from these smaller homogeneous sets is compromised because of reduced number of particles or lack of images at certain macromolecular orientations. In these situations, the current solution to improve map resolution is returning to the electron microscope and collect more data. In this work, we present a fast approach to partially overcome this limitation for heterogeneous data sets. Our method is based on deforming and then moving particles between different conformations using an optical flow approach. Particles are then merged into a unique conformation obtaining reconstructions with improved resolution, contrast and signal-to-noise ratio. We present experimental results that show clear improvements in the quality of obtained 3D maps, however, there are also limits to this approach, i.e., the method is restricted to small deformations and cannot determine local patterns of flexibility of small elements, such as secondary structures, which we discuss in the manuscript. [Display omitted] • Cryo-EM using SPA requires the computational averaging of thousands of projections captured from identical macromolecules. • Computational approaches are required to classify heterogeneous particles into homogeneous sets at different conformations. • We propose a fast and efficient approach that is aimed at "making the best" from relatively limited data sets. • Our method focuses on artificially increasing the number of particles in a class. • In this regard, we use a regularized optical flow approach to move particles between different classes/conformations. [ABSTRACT FROM AUTHOR]

Background: Microscopic imaging is a crucial technology for visualizing neural and tissue structures. Large-area defects inevitably occur during the imaging process of electron microscope (EM) serial slices, which lead to reduced registration and semantic segmentation, and affect the accuracy of 3D reconstruction. The continuity of biological tissue among serial EM images makes it possible to recover missing tissues utilizing inter-slice interpolation. However, large deformation, noise, and blur among EM images remain the task challenging. Existing flow-based and kernel-based methods have to perform frame interpolation on images with little noise and low blur. They also cannot effectively deal with large deformations on EM images. Results: In this paper, we propose a sparse self-attention aggregation network to synthesize pixels following the continuity of biological tissue. First, we develop an attention-aware layer for consecutive EM images interpolation that implicitly adopts global perceptual deformation. Second, we present an adaptive style-balance loss taking the style differences of serial EM images such as blur and noise into consideration. Guided by the attention-aware module, adaptively synthesizing each pixel aggregated from the global domain further improves the performance of pixel synthesis. Quantitative and qualitative experiments show that the proposed method is superior to the state-of-the-art approaches. Conclusions: The proposed method can be considered as an effective strategy to model the relationship between each pixel and other pixels from the global domain. This approach improves the algorithm's robustness to noise and large deformation, and can accurately predict the effective information of the missing region, which will greatly promote the data analysis of neurobiological research. [ABSTRACT FROM AUTHOR]

Acephate (Ac) is an organophosphate (OPP) that is widely used as an insecticide. It is highly toxic, thus simple, and rapid detection method of Ac is in urgent demand. Citrate capped gold nanoparticles (Cit- AuNPs) are excellent materials for colorimetric detection of OPPs. The effect of Cit-AuNPs and Ac volume ratio (Cit-AuNPs: Ac) on the colorimetric sensor capability was evaluated for detection of Ac in this study. Cit-AuNPs were synthesised via reduction of chloroauric acid (HAuCl4) using tri-sodium citrate dihydrate and used to detect Ac. Colour images of all complexes formed were captured and digitised using ImageJ software to obtain red values (RVs) as the response of the detection. Analysis using Ultraviolet-Visible (UV-Vis) spectrophotometer and High Resolution-Transition Electron Microscope (HRTEM) were employed to examine the red shift and Cit-AuNPs aggregation, respectively, whereas Fourier Transform Infra-red (FTIR) was used to investigate chemical interaction between Cit- AuNPs and Ac after the detection. The results of this study demonstrated that the lowest volume ratio of Cit-AuNPs: Ac (1:9) was sufficient to produce obvious colour change for the detection of Ac. [ABSTRACT FROM AUTHOR]

Thanks to their high brightness, field emitters are the electron sources of choice in most high-end electron microscopes. Under typical operating conditions, the available emission current from these emitters is largely limited by practical considerations, and extracting significantly larger currents is usually not possible without reducing the lifetime of the emitter or even damaging it. Such limitations may, however, not apply if the emitter is only briefly subjected to extreme operating conditions so that damage can be outrun. Here, we demonstrate that it is possible to temporarily operate a Schottky emitter far outside its stable operating regime and significantly increase its emission current. We do so by locally heating the tip of the emitter with a microsecond laser pulse, which boosts the emission current by a factor of 3.7 to nearly 450 μA. We believe that the generation of intense microsecond electron pulses from a field emitter will particularly benefit the atomic-resolution imaging of fast processes that occur on the microsecond timescale. [ABSTRACT FROM AUTHOR]

Fringe analysis is a commonly used method to quantify soot nanostructures. However, the settings of the involved filters and their impact on the results are rarely addressed. In this study, the influence of the three filter parameters as well as two aspects of the image acquisition was assessed experimentally. For the analysis, a carbon black as well as one diesel engine and one gasoline direct injection (GDI) engine soot sample were used. Gaussian low-pass filter standard deviations larger 1.5 yielded only minor differences in fringe metrics. Standard deviations between 2.0 and 3.0 enabled realistic representation of fringes. A linear correlation was found between the white top-hat transformation disk size and all fringe analysis metrics. For realistic nanostructure representation, disk sizes of 5 px and 7 px are most suitable. Threshold values as calculated by Otsu's method generally yielded the best nanostructure representation. Any deviation distorted the extracted fringes and noticeably reduced their total number. Thus, consistent use of Otsu thresholds without alterations is advised. Deviating from the neutral electron microscope focus point by under- and over-focusing resulted in distinctive drops in both fringe lengths and Otsu thresholds. Consistent focusing with the help of fast Fourier transformations of the respective particles is vital for reliable results. The effect of reduced noise levels by repeated averaged images was found to be minor beyond the model of the camera used. The region of interest size correlated linearly with the number of extracted fringes, however, it did not affect the fringe metrics. For statistically reliable analysis, a minimum of 4000 fringes is suggested. The GDI sample exhibited the shortest fringes and the highest tortuosity. For diesel soot and carbon black, similar fringe lengths could be observed. The highest tortuosity was found for GDI soot, followed by diesel soot and carbon black. [ABSTRACT FROM AUTHOR]

Single‐particle analysis by electron microscopy is a well established technique for analyzing the three‐dimensional structures of biological macromolecules. Besides its ability to produce high‐resolution structures, it also provides insights into the dynamic behavior of the structures by elucidating their conformational variability. Here, the different image‐processing methods currently available to study continuous conformational changes are reviewed. In this paper, the existing image‐processing approaches in electron microscopy for the analysis of continuous conformational changes of biological macromolecules are reviewed. [ABSTRACT FROM AUTHOR]

[Display omitted] Recent technological advances in cryo electron microscopy (cryo-EM) have led to new opportunities in the structural biology field. Here we benchmark the performance of two 300 kV latest-generation cryo electron microscopes, Titan Krios G4 from Thermofisher Scientific and CRYO ARM 300 from Jeol, with regards to achieving high resolution single particle reconstructions on a real case sample. We compare potentially limiting factors such as drift rates, astigmatism & coma aberrations and performance during image processing and show that both microscopes, while comprising rather different technical setups & parameter settings and equipped with different types of energy filters & cameras, achieve a resolution of around 2 Å on the human ribosome, a non-symmetric object which constitutes a key drug target. Astigmatism correction, CTF refinement and correction of higher order aberrations through refinement in separate optics groups helped to account for astigmatism/coma caused by beam tilting during multi-spot and multi-hole acquisition in neighbouring holes without stage movement. The obtained maps resolve Mg2+ ions, water molecules, inhibitors and side-chains including chemical modifications. The fact that both instruments can resolve such detailed features will greatly facilitate understanding molecular mechanisms of various targets and helps in cryo-EM structure based drug design. The methods and analysis tools used here will be useful also to characterize existing instruments and optimize data acquisition settings and are applicable broadly to other drug targets in structural biology. [ABSTRACT FROM AUTHOR]

Many pharmaceutical products are obtained via freeze-drying of liquid solutions to obtain stable long lasting preparations. The freeze-dry process produces porous cakes whose structure strongly depends on the freezing phase, so that monitoring and optimizing this phase can help both reducing the product cost and insuring its constant quality. Nowadays the optimization is usually performed by determining the cake mass transfer coefficient via a costly process in pilot plants, while the quality is assured only by controlling the process conditions. This paper describes an alternative way of approximately estimating the mass transfer coefficient, which is based on the observation of the product structure by a simple electron microscope followed by a frequency domain imaging process. While the process has been designed and characterized specifically for pharmaceutical products, the proposed approach can be used in several other fields where the characteristics of porous material have to be monitored. [ABSTRACT FROM AUTHOR]

[Display omitted] • Tool to simulate tomograms of membrane-embedded proteins. • Detangles influence of data acquisition parameters from sample quality issues. • Rapid evaluation and optimization of cryo-ET data acquisition parameters. • Proof-of-concept provided with integrins and SARS-CoV-2 spike simulations. The throughput and fidelity of cryogenic cellular electron tomography (cryo-ET) is constantly increasing through advances in cryogenic electron microscope hardware, direct electron detection devices, and powerful image processing algorithms. However, the need for careful optimization of sample preparations and for access to expensive, high-end equipment, make cryo-ET a costly and time-consuming technique. Generally, only after the last step of the cryo-ET workflow, when reconstructed tomograms are available, it becomes clear whether the chosen imaging parameters were suitable for a specific type of sample in order to answer a specific biological question. Tools for a-priory assessment of the feasibility of samples to answer biological questions and how to optimize imaging parameters to do so would be a major advantage. Here we describe MEPSi (M embrane E mbedded P rotein Si mulator), a simulation tool aimed at rapid and convenient evaluation and optimization of cryo-ET data acquisition parameters for studies of transmembrane proteins in their native environment. We demonstrate the utility of MEPSi by showing how to detangle the influence of different data collection parameters and different orientations in respect to tilt axis and electron beam for two examples: (1) simulated plasma membranes with embedded single-pass transmembrane αIIbβ3 integrin receptors and (2) simulated virus membranes with embedded SARS-CoV-2 spike proteins. [ABSTRACT FROM AUTHOR]

[Display omitted] • New methodology to characterize nanodiscs based on Feret signatures. • Feret signatures distinguish nanodisc morphologies and compositions. • Analysis is highly sensitive to sample quality. • Method selected condition to solve structure of small membrane protein Bcl-xL. Nanodiscs have become a popular tool in structure determination of membrane proteins using cryogenic electron microscopy and single particle analysis. However, the structure determination of small membrane proteins remains challenging. When the embedded protein is in the same size range as the nanodisc, the nanodisc can significantly contribute to the alignment and classification during the structure determination process. In those cases, it is crucial to minimize the heterogeneity in the nanodisc preparations to assure maximum accuracy in the classification and alignment steps of single particle analysis. Here, we introduce a new in-silico method for the characterization of nanodisc samples that is based on analyzing the Feret diameter distribution of their particle projection as imaged in the electron microscope. We validated the method with comprehensive simulation studies and show that Feret signatures can detect subtle differences in nanodisc morphologies and composition that might otherwise go unnoticed. We used the method to identify a specific biochemical nanodisc preparation with low size variations, allowing us to obtain a structure of the 23-kDa single-span membrane protein Bcl-xL while embedded in a nanodisc. Feret signature analysis can steer experimental data collection strategies, allowing more efficient use of high-end data collection hardware, as well as image analysis investments in studies where nanodiscs significantly contribute to the total volume of the full molecular species. [ABSTRACT FROM AUTHOR]

The artist references images captured by electron microscope photography of Dopamine, DNA, and the process of Mitosis (specifically occurring in the human body). Through the use of photoshop the artist was able to manipulate the images of Dopamine, DNA, and Mitosis to create low polygonal artwork. The artist created 15,000-30,000 individual triangles, piecing them together using photoshop to create the images that they were referencing. The body is an amazing spectacle rarely looked at from within by artists. Many find beauty on the surface of the body, but when explored further, the body has much more to offer. The artist is focused on bringing out the beauty from within the by echoing patterns, symmetry, and rhythm, that were used to create breathtaking anatomical structures. Science, Art, and Technology come together in this series to inspire the audience to look deeper than the surface to see beauty. [ABSTRACT FROM AUTHOR]

Extreme ultraviolet lithography (EUVL) patterned mask defect detection is one of the major issues to overcome for realization of EUVL-based device fabrication. We have designed projection electron microscope (PEM) optics that have been integrated into a new inspection system called EBEYE-V30 ("Model EBEYE" is EBARA's model code), and the PEM system performs well in half-pitch (hp) 16-nm node EUVL patterned mask inspection applications. We also discuss the extendibility of this system to 11-nm node defect detection. The progress made in the performance of the PEM optics is not simply about producing an image sensor with higher resolution but is also about improvement of the image processing to enhance the defect signal. A high-speed image sensor, a high-speed image-processing circuit, and a bright and stable electron source are necessary for hp 11-nm defect inspection. We describe the experimental results for EUVL patterned mask inspection using the above system for the hp 11-nm node. Programmed hp 11-nm defects (equivalent to 44 nm on the mask) are used for defect detection sensitivity evaluation. Defects as small as 16 nm on the mask could be detected using the current PEM system configuration. [ABSTRACT FROM AUTHOR]

An improvement to the previously proposed adaptive Canny optimization technique for scanning electron microscope image colorization is reported. The additional feature, called pseudo-mapping technique, is that the grayscale markings are temporarily mapped to a set of pre-defined pseudo-color map as a mean to instill color information for grayscale colors in chrominance channels. This allows the presence of grayscale markings to be identified; hence optimization colorization of grayscale colors is made possible. This additional feature enhances the flexibility of scanning electron microscope image colorization by providing wider range of possible color enhancement. Furthermore, the nature of this technique also allows users to adjust the luminance intensities of selected region from the original image within certain extent. SCANNING 36:530-539, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Intrinsically disordered regions of proteins and conformational flexibility within complexes can be critical for biological function. However, disorder, flexibility, and heterogeneity often hinder structural analyses. CryoEM and single particle image processing techniques offer the possibility of imaging samples with significant flexibility. Division of particle images into more homogenous subsets after data acquisition can help compensate for heterogeneity within the sample. We present the utility of an eigenimage sorting analysis for examining two protein/DNA complexes with significant conformational flexibility and heterogeneity. These complexes are integral to the non-homologous end joining pathway, and are involved in the repair of double strand breaks of DNA. Both complexes include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and biotinylated DNA with bound streptavidin, with one complex containing the Ku heterodimer. Initial 3D reconstructions of the two DNA-PKcs complexes resembled a cryoEM structure of uncomplexed DNA-PKcs without additional density clearly attributable to the remaining components. Application of eigenimage sorting allowed division of the DNA-PKcs complex datasets into more homogeneous subsets. This led to visualization of density near the base of the DNA-PKcs that can be attributed to DNA, streptavidin, and Ku. However, comparison of projections of the subset structures with 2D class averages indicated that a significant level of heterogeneity remained within each subset. In summary, image sorting methods allowed visualization of extra density near the base of DNA-PKcs, suggesting that DNA binds in the vicinity of the base of the molecule and potentially to a flexible region of DNA-PKcs. [Copyright &y& Elsevier]

Electron tomography (ET) can be used to determine the shape, and therefore the properties, of an object. It deals with retrieving tridimensional (3D) information from bidimensional (2D) projections of the object under study. While ET has been used widely in biology, its application to the nanoscale world is relatively recent because of the limitations in resolution and aberration of electron microscopes. At atomic scale, classical tomography reconstruction can be replaced by tomography through points reconstruction, that is, the 3D information can be obtained by applying image processing techniques to the projections and by using geometrical models instead of the classical integral approach. In this paper, a methodology for the study of corrugated graphene will be presented. It will be shown how to apply tomographic techniques to the determination of the corrugations in graphene layers with error in 3D location in the range of pm. [ABSTRACT FROM AUTHOR]

Abstract: Cryo electron tomography provides nanometer-scale information on biological matter preserved in a close-to native state. The resolution of tomograms and structures resolved by sub-tomogram averaging is typically limited by the contrast transfer function of the electron microscope, which is especially critical for thick samples. Here, we report a method to increase the attainable resolution by recording tomographic ‘focal pairs’, which are pairs of tilt series of the same object acquired in complementary defocus conditions. Low defocus imaging provides high resolution at low contrast, while high defocus imaging yields high contrast at the price of limited resolution. Quantitative assessment of the quality of lipid bilayer reconstructions in the resulting tomograms demonstrates stable resolution preservation beyond 3nm for cells thicker than 500nm. Further, in computational simulations on synthetic datasets we show the applicability of the method to sub-tomogram averaging, demonstrating its potential for achieving higher resolution. [Copyright &y& Elsevier]

Abstract: To obtain high-resolution images of biological macromolecules, a cryo-electron microscope (cryo-EM) is useful to preserve the hydrated state of the molecules. In addition, the cryo-conditions reduce some of the electron radiation damage, although averaging is necessary to obtain the molecular resolution from biological macromolecules. For the averaging, the three reconstruction methods of electron crystallography, helical reconstruction, and single particle analysis facilitate the determination of the atomic models. Here we describe suitable techniques for high-resolution imaging in these three different methods, based on our experiences using cryo-EM with a liquid-helium cooled stage. Irradiation damage by the electron beam is the most serious problem in the structural analysis of a biological specimen, and we clearly show that further reduction of the specimen temperature, from liquid nitrogen to liquid helium temperature, improves the cryo-protection factor by at least two-fold for both glucose-embedded and frozen-hydrated specimens. We review the image processing, and then discuss the future prospects of cryo-EM. [ABSTRACT FROM AUTHOR]

A computer-controlled procedure is outlined here that first determines the position of the amorphouscrystalline interface in an image. Subsequently, from a time series of these images, the velocity of the crystal growth front is quantified. The procedure presented here can be useful for a wide range of applications, and we apply the new approach to determine growth rates in a so-called fast-growth-type phase-change material. The growth rate (without nucleation) of this material is of interest for comparison with identical material used in phase-change random access memory cells. Crystal growth rates in the amorphous phase-change layers have been measured at various temperatures using in situ heating in a transmission electron microscope. Doped SbTe films (20 nm thick) were deposited on silicon nitride membranes, and samples with and without silicon oxide capping layer were studied. The activation energy for growth was found to be 3.0 eV. The samples without capping layer exhibit a nucleation rate that is an order of magnitude higher than the samples with a silicon oxide capping layer. This difference can be attributed to the partial oxidation of the phase-change layer in air. However, the growth rates of the samples with and without capping are quite comparable. [ABSTRACT FROM AUTHOR]

Compositional images from a SEM (scanning electron microscope) are sometimes complemented by quantitative topographical data from devices such as an AFM (atomic force microscope) or WLI (white light interferometer). Indeed, even if a SEM could provide both kinds of information (composition and topography), the topographical data are incomplete because the SEM does not allow measuring the vertical dimension (i.e., perpendicular to the measurement plane). Thus these two kinds of information are usually measured using two different techniques, and at different locations on the sample. Mean values of surface composition are then linked to mean values of topography, and as a consequence this approach does not allow precisely linking a local topographical peak to its corresponding composition. The present work deals with a SEM/WLI combination methodology, based on the characterization, at the same location, of a nanocomposite (nanoparticles of AlCeO3 dispersed in a plasma-polymerized hexamethyldisiloxane ppHMDSO matrix and deposited by atmospheric plasma on a glass substrate) developed for improving anticorrosion properties. SEM images allow the supposition that the protuberances (peaks) observed on the surface of a specimen are linked to the nanoparticles dispersed in the polymer coating, but this link is not fully convincing. Thanks to a precise localization method, SEM compositional data and WLI topographical data are here measured at the same location. The recombination of both signals to form a 4D image (3D geometry and 1D composition) allows linking protuberances to nanoparticles aggregates without ambiguity. This composite image appears to be an interesting new tool (at the scale of observation, i.e., hundreds of micrometers) for the study of nanostructured coatings. [ABSTRACT FROM AUTHOR]

In bit-patterned media (BPM), media noise is expected to be dominated by fabrication imperfections and to manifest as island size and spacing fluctuations. Previous work has shown that media noise has a significant impact on the error rates of BPM channels. Thus, having a realistic model of media noise is essential to evaluate the performance of the BPM channels. In this work, we apply image processing techniques to a scanning electron microscope image of a candidate BPM nano-mask, produced by a self-assembly technique, to estimate likely size and location fluctuations of the islands. These statistics may also be useful to characterize the samples of candidate BPM masks. We assume that these observed fluctuations are representative of patterning noise that might result from such a mask. The results indicate that these fluctuations are correlated and can be modeled by Gaussian random processes. Using estimated characteristics of islands size and location fluctuations, we model the correlated media noise and investigate the performance of BPM channels in the presence of such correlated media noise. [ABSTRACT FROM AUTHOR]

Abstract: The Contrast Transfer Function (CTF) of an image, which modulates images taken from a Transmission Electron Microscope (TEM), is usually determined from the radial average of the power spectrum of the image (Frank, J., Three-dimensional Electron Microscopy of Macromolecular Assemblies, Oxford University Press, Oxford, 2006). The CTF is primarily defined by the defocus. If the defocus estimate is accurate enough then it is possible to demodulate the image, which is popularly known as the CTF correction. However, it is known that the radial average is somewhat attenuated if the image is astigmatic (see Fernando, K.V., Fuller, S.D., 2007. Determination of astigmatism in TEM images. Journal of Structural Biology 157, 189–200) but this distortion due to astigmatism has not been fully studied or understood up to now. We have discovered the exact mathematical relationship between the radial averages of TEM images with and without astigmatism. This relationship is determined by a zeroth order Bessel function of the first kind and hence we can exactly quantify this distortion in the radial averages of signal and power spectra of astigmatic images. The argument to this Bessel function is similar to an aberration function (without the spherical aberration term) except that the defocus parameter is replaced by the differences of the defoci in the major and minor axes of astigmatism. The ill effects due this Bessel function are twofold. Since the zeroth order Bessel function is a decaying oscillatory function, it introduces additional zeros to the radial average and it also attenuates the CTF signal in the radial averages. Using our analysis, it is possible to simulate the effects of astigmatism in radial averages by imposing Bessel functions on idealized radial averages of images which are not astigmatic. We validate our theory using astigmatic TEM images. [Copyright &y& Elsevier]

Abstract: Methods for preserving specimen hydration in protein crystals were pursued in the early 1970s as a prerequisite for protein crystallography using an electron microscope. Three laboratories approached this question from very different directions. One built a differentially pumped hydration chamber that could maintain the crystal in a liquid water environment, a second maintained hydration by rapidly freezing the protein crystal and examining it in a cold stage, and the third replaced the water of hydration by using glucose in the same way as one had previously used “negative stains”. Each of these early efforts succeeded in preserving the structures of protein crystals at high resolution within the vacuum of the electron microscope, as demonstrated by electron diffraction patterns. The next breakthrough came in the early 1980s when a technique was devised to preserve noncrystalline specimens by freezing them within vitreous ice. Since then, with the development of high stability cold stages and transfer mechanisms compatible with many instrument platforms, and by using commercially provided low dose imaging techniques to avoiding radiation damage, there has been an explosion of applications. These now include single particles, helical filaments, 2-D arrays and even whole cells, where the most exciting recent applications involve cryoelectron tomography. These achievements and possibilities generate a new set of research opportunities associated with increasing the reliability and throughput with which specimens can be studied by cryoEM. [Copyright &y& Elsevier]

Abstract: In single particle analysis, two-dimensional (2-D) alignment is a fundamental step intended to put into register various particle projections of biological macromolecules collected at the electron microscope. The efficiency and quality of three-dimensional (3-D) structure reconstruction largely depends on the computational speed and alignment accuracy of this crucial step. In order to improve the performance of alignment, we introduce a new method that takes advantage of the highly accurate interpolation scheme based on the gridding method, a version of the nonuniform fast Fourier transform, and utilizes a multi-dimensional optimization algorithm for the refinement of the orientation parameters. Using simulated data, we demonstrate that by using less than half of the sample points and taking twice the runtime, our new 2-D alignment method achieves dramatically better alignment accuracy than that based on quadratic interpolation. We also apply our method to image to volume registration, the key step in the single particle EM structure refinement protocol. We find that in this case the accuracy of the method not only surpasses the accuracy of the commonly used real-space implementation, but results are achieved in much shorter time, making gridding-based alignment a perfect candidate for efficient structure determination in single particle analysis. [Copyright &y& Elsevier]

Abstract: In electron tomography the sample is tilted in the electron microscope and projections are recorded at different viewing angles. In the correct geometric setting, the tilt-axis of the object under scrutiny is perpendicular to the beam direction. However, we will demonstrate that this does not necessarily apply to all electron microscopes equipped with the default column alignment. The resulting effect is that a conical tilt is performed, which has to be considered in the reconstruction to avoid artifacts and to improve the resolution. A novel solution, with significantly improved convergence properties, will be introduced for calculating the three-dimensional marker model, which is necessary for the alignment of the tilt-series. Thereby, the angle between the beam direction and the tilt-axis is calculated, together with other geometrical distortions, like magnification and rotation changes, and incorporated in the reconstruction. Hereby, artifacts can be eliminated at the image processing basis, and the resolution can be significantly improved at the medium to high range frequencies. Synthetical and real data are used to demonstrate the obstructions caused by this effect and the quality improvement of the reconstructions. Finally, we also present a way to align the hardware of the microscope to correct for the non-perpendicularity between the beam direction and the tilt-axis, which is specifically tailored for tomographic applications. [Copyright &y& Elsevier]

Abstract: Electron cryotomography (cryoET) has the potential to elucidate the structure of complex biological specimens at molecular resolution but technical and computational improvements are still needed. This work addresses the determination and correction of the contrast transfer function (CTF) of the electron microscope in cryoET. Our approach to CTF detection and defocus determination depends on strip-based periodogram averaging, extended throughout the tilt series to overcome the low contrast conditions found in cryoET. A method for CTF correction that deals with the defocus gradient in images of tilted specimens is also proposed. These approaches to CTF determination and correction have been applied here to several examples of cryoET of pleomorphic specimens and of single particles. CTF correction is essential for improving the resolution, particularly in those studies that combine cryoET with single particle averaging techniques. [Copyright &y& Elsevier]