Automated spatial drift correction for EFTEM image series

Energy filtering transmission electron microscopy (EFTEM) is a widely used technique in many areas of scientific research. Image contrast in energy-filtered images arises from specific scattering events such as the ionization of atoms. By combining a set of two or more images, relative sample thickness maps or elemental distribution maps can be easily created. It is also possible to acquire a whole series of energy-filtered images to do more complex data analysis. However, whenever several images are combined to extract certain information, problems are introduced due to sample drift between the exposures. In order to obtain artifact-free information, this spatial drift has to be taken care of. Manual alignment by overlaying and shifting the images to find the best overlap is usually very accurate but extremely time consuming for larger data sets. When large amounts of images are recorded in an EFTEM series, manual correction is no longer a reasonable option. Hence, automatic routines have been developed that are mostly based on the cross-correlation algorithm. Existing routines, however, sometimes fail and again make time consuming manual adjustments necessary. In this paper we describe a new approach to the drift correction problem by incorporating a statistical treatment of the data and we present our statistically determined spatial drift (SDSD) correction program. We show its improved performance by applying it to a typical EFTEM series data block.     

Elemental maps from EFTEM images using two different background subtraction models

Acquisition of a great number of energy-filtered images in a TEM (EFTEM) around the characteristic signal with a low energy-selecting slit allows display of the electron energy loss (EEL)-spectrum of regions of interest (ROIs) of a sample. These EEL-spectra can be submitted to the different treatments already in use for electron energy loss spectroscopy (EELS). In particular, it is possible to fit the experimental background with different mathematical models, using images acquired below and above a characteristic ionization edge. After this fitting, elemental maps can be computed by subtraction of the extrapolated/interpolated background from the characteristic images. In this work, we compared two mathematical models for background fitting-the Egerton power law and the log-polynomial law. We studied the low-energy region (40-150 eV) and a higher-energy region (350-600 eV) with the aid of software for interactive processing of EFTEM image series that we developed. The analyzed elements were the constitutive elements: iron, phosphorus, nitrogen, and oxygen in several biological materials. Two analytical TEMs, one equipped with a post-column and the other with an in-column spectrometer, were used. Our experimental results confirm that the power law is very sensitive to the value of the energy loss of the pre-edge images when the background is computed by extrapolation. The log-polynomial model is less sensitive than the power law model to the value of the energy loss of the pre-edge images in the low energy region. For the oxygen K edge at 535 eV, it gives the best fit when it is combined with the interpolation method. The use of programs that facilitate the handling of EFTEM image series, and the controlled calculation of the background under the characteristic images, represent a step forward in the generation of elemental maps.     

Optimization of EFTEM image acquisition by using elastically filtered images for drift correction

Because of its high spatial resolution, energy-filtering transmission electron microscopy (EFTEM) has become widely used for the analysis of the chemical composition of nanostructures. To obtain the best spatial resolution, the precise correction of instrumental influences and the optimization of the data acquisition procedure are very important. In this publication, we discuss a modified image acquisition procedure that optimizes the acquisition process of the EFTEM images, especially for long exposure times and measurements that are affected by large spatial drift. To alleviate the blurring of the image caused by the spatial drift, we propose to take several EFTEM images with a shorter exposure time (sub-images) and merge these sub-images afterwards. To correct for the drift between these sub-images, elastically filtered images are acquired between two subsequent sub-images. These elastically filtered images are highly suitable for spatial drift correction based on the cross-correlation method. The use of the drift information between two elastically filtered images permits to merge the drift-corrected sub-images automatically and with high accuracy, resulting in sharper edges and an improved signal intensity in the final EFTEM image. Artefacts that are caused by prominent noise-peaks in the dark reference image have been suppressed by calculating the dark reference image from three images. Furthermore, using the information given by the elastically filtered images, it is possible to drift-correct a set of EFTEM images already during the acquisition. This simplifies the post-processing for elemental mapping and offers the possibility for active drift correction using the image shift function of the microscope, leading to an increased field of view.     

A facile method to compare EFTEM maps obtained from materials changing composition over time

Energy Filtered Transmission Electron Microscopy (EFTEM) is an analytical tool that has been successfully and widely employed in the last two decades for obtaining fast elemental maps in TEM mode. Several studies and efforts have been addressed to investigate limitations and advantages of such technique, as well as to improve the spatial resolution of compositional maps. Usually, EFTEM maps undergo post‐acquisition treatments by changing brightness and contrast levels, either via dedicated software or via human elaboration, in order to maximize their signal‐to‐noise ratio and render them as visible as possible. However, elemental maps forming a single set of EFTEM images are usually subjected to independent map‐by‐map image treatment. This post‐acquisition step becomes crucial when analyzing materials that change composition over time as a consequence of an external stimulus, because the map‐by‐map approach doesn't take into account how the chemical features of the imaged materials actually progress, in particular when the investigated elements exhibit very low signals. In this article, we present a facile procedure applicable to whole sets of EFTEM maps acquired on a sample that is evolving over time. The main aim is to find a common method to treat the images features, in order to make them as comparable as possible without affecting the information there contained. Microsc. Res. Tech. 78:1090–1097, 2015. © 2015 Wiley Periodicals, Inc.     

EFTEM assistant: a tool to understand the limitations of EFTEM

The first version of a free tool for Gatan's Digital Micrographtrade mark is presented which aims to aid the energy-filtered TEM (EFTEM) community by predicting and correcting the most common sources of degradation. The software allows selection of either Krivanek's or Egerton's approach to account for the spatial resolution degradation caused by the electron optical aberrations. The effects of aberrations and signal 'delocalization' are combined to simulate the blurring caused in EFTEM elemental maps. Two microstructural features with ideal geometry are used to illustrate use of the software: spherical particles and parallel sided interfaces. The software also allows the simulation of the effects of the noise and drift in the final elemental map, independently or in combination. It can be easily demonstrated that when the dimensions of the feature of interest are comparable in scale to the image degradation factors, the effects of the latter should not be neglected. More importantly, the software can deconvolute the effects of the degradation factors, revealing the true dimensions and signal intensity of the feature of interest.     

一种激光三角测厚中非线性的校正方法

针对单镜头激光三角法测同一物体厚度时图像探测器上两光斑间距与被测物位置之间的非线性关系,提出了一种非线性校正方法,并用实验验证了校正效果。分析了单镜头激光三角法测厚原理,总结了引起非线性的因素,对校正方法的函数模型进行了理论推导,并对校正前后测量数据进行了详细分析。研究结果表明,使用该非线性校正方法后,图像探测器上成像光斑间距值随被测物位置变化的波动程度明显减小,测量厚度的均方差值也变小,系统的测量精度得到提高。     

EFTEM study of surface plasmon resonances in silver nanoholes

Using energy-filtering transmission electron microscopy we observe localized plasmon resonances in nanoholes of silver. We find two resonances that are typical for single, isolated holes. A comparison with calculations based on the discrete dipole approximation shows that these are dipolar and quadrupolar resonances. In case of the close vicinity of holes we observe modes at lower energies that are ascribed to coupling effects between holes. We propose a hybridization scheme for the case of three collinear holes.     

Quantitative EFTEM mapping of near physiological calcium concentrations in biological specimens

Although electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) provides high sensitivity for measuring the important element, calcium, in biological specimens, the technique has been difficult to apply routinely, because of long acquisition times required. Here we describe a refinement of the complementary analytical technique of energy-filtered transmission electron microscopy (EFTEM), which enables rapid imaging of large cellular regions and measurement of calcium concentrations approaching physiological levels. Extraction of precise quantitative information is possible by averaging large numbers of pixels that are contained in organelles of interest. We employ a modified two-window approach in which the behavior of the background signal in the EELS spectrum can be modeled as a function of specimen thickness t expressed in terms of the inelastic mean free path λ. By acquiring pairs of images, one above and one below the Ca L_2,3 edge, together with zero-loss and unfiltered images, which are used to determine a relative thickness (t/λ) map, it is possible to correct the Ca L_2,3 signal for plural scattering. We have evaluated the detection limits of this technique by considering several sources of systematic errors and applied this method to determine mitochondrial total calcium concentrations in freeze-dried cryosections of rapidly frozen stimulated neurons. By analyzing 0.1 μm² areas of specimen regions that do not contain calcium, it was found that the standard deviation in the measurement of Ca concentrations was about 20 mmol/kg dry weight, corresponding to a Ca:C atomic fraction of approximately 2×10⁻⁴. Calcium concentrations in peripheral mitochondria of recently depolarized, and therefore stimulated and Ca loaded, frog sympathetic neurons were in reasonable agreement with previous data.     

Comparison of EFTEM and STEM EELS plasmon imaging of gold nanoparticles in a monochromated TEM

We present and compare two different imaging techniques for plasmonic excitations in metallic nanoparticles based on high energy-resolution electron energy-loss spectroscopy in a monochromated transmission electron microscope. We demonstrate that a recently developed monochromated energy-filtering (EFTEM) approach can be used in addition to the well established scanning technique to directly obtain plasmon images in the energy range below 1 eV. The EFTEM technique is described in detail, and a double experiment performed on the same, triangular gold nanoparticle compares equivalent data acquired by both techniques, respectively.     

Electrically conducting polymers: preparation and investigation of oxidized poly(acetylene) by EFTEM

Energy-filtering transmission electron microscopy (EFTEM) was used to demonstrate that the oxidation of poly(acetylene), necessary to produce a highly conducting phase, proceeds homogeneously on a scale of 100 nm to several micrometres. The distribution of the anions BF₄⁻ and PF₆⁻ serves as indicator for the penetration of poly(acetylene) by the oxidizing agent. The sample preparation necessitates prevention of sample degradation by air and moisture. It was possible to synthesize, orientate, oxidize, embed and section poly(acetylene) samples under inert conditions. Only mounting of the grids into the sample holder of the electron microscope was performed under ambient conditions. EFTEM was possible for phosphorus and boron, but not for fluorine owing to irradiation damage leading to loss of fluorine from the sample. Polyvinylbutyral was chosen as the embedding medium because it allows embedding of the sample prior to oxidation. Embedding of a previously oxidized poly(acetylene) sample leads to migration of the anions from the interior of the sample into the embedding medium.     

Chemical mapping of a block copolymer electrolyte by low-loss EFTEM spectrum-imaging and principal component analysis

Energy-filtered transmission electron microscopy spectrum-imaging (EFTEM SI) in the low electron energy-loss range is a valuable technique for probing the chemical structure of a material with nanoscale spatial resolution using a reduced electron dose. By analyzing EFTEM SI datasets using principal component analysis (PCA), the constituent chemical phases of the material can be identified in an efficient manner without prior knowledge of the specimen. We implement low-loss EFTEM SI together with PCA to investigate thin films of the block copolymer electrolyte poly(styrene-block-ethylene oxide) (PS-b-PEO) blended with a sodium salt. PCA identifies three main phases, the first and second phases corresponding to the two blocks of the copolymer and a third phase corresponding to the salt. The low-loss spectra for these phases are extracted from a noise-reduced EFTEM SI dataset and used to generate a chemical map of the material by multiple linear least square fitting. We validate the results of the low-loss EFTEM SI/PCA technique by applying the method to a control PS-b-PEO sample that does not contain the sodium salt, and by conducting spatially resolved X-ray energy-dispersive spectrometry on the salt-containing PS-b-PEO thin film.     

Image-spectroscopy--I. The advantages of increased spectral information for compositional EFTEM analysis.

The acquisition of a series of energy-filtered TEM images over the energy-loss range of interest creates a three-dimensional data set comprising both spatial and spectral information. Such an image-series contains energy-loss information not available with conventional two- or three-window methods, allowing standard techniques for quantitative EELS analysis to be applied to extracted 'image-spectra'. The increase in spectral information enables improved ionisation edge background extrapolation and interactive image-spectrum analysis to be performed. In this paper, the many advantages of the image-spectroscopy approach are outlined by reference to an example of elemental segregation in an AlZnMgCu alloy.     

3D electron microscopy in the physical sciences: the development of Z-contrast and EFTEM tomography

The rapid advances in nanotechnology and the ever decreasing size of features in the microelectronics industry brings with it the need for advanced characterisation with high spatial resolution in two and three dimensions. Stereo microscopy allows some insight into the three-dimensional nature of an object but for true quantitative analysis, one has to turn to tomography as a way to reconstruct a three-dimensional object from a series of two-dimensional projections (images). X-ray tomography allow structures to be imaged at relatively large length scales, atom probe tomography at the atomic level. Electron tomography offers an intermediate resolution (of about 1nm) with a field of view of hundreds of nm making it ideal for the characterisation of many nanoscale devices. Whilst electron tomography has been used in the biological sciences for more than 30 years, it is only now being applied to the physical sciences. In this paper, we review the status of electron tomography, describe the basis behind the technique and some of the practicalities of recording and analysing data for tomographic reconstruction, particularly in regard to solving three-dimensional problems that are encountered in materials science at the nanometre level. We present examples of how STEM dark-field imaging and energy-filtered TEM can be used successfully to examine nearly all types of specimens likely to be encountered by the physical scientist.     

Quantification and visualization of anisotropy in trabecular bone

A number of methods for measuring anisotropy in trabecular bone using high‐resolution X‐ray computed tomography exist, which give different answers but have not been compared in detail. In this study, we examine the mean‐intercept length (MIL), star volume distribution (SVD) and star length distribution (SLD) methods, their algorithmic implementation for three‐dimensional (3D) data, and how their results relate to each other. A uniform ordered sampling scheme for determining which orientations to sample during analysis enhances the reproducibility of anisotropy and principal component direction determinations, with no evident introduction of biasing. This scheme also facilitates the creation of a 3D rose diagram that can be used to gain additional insights from the data. The directed secant algorithm that is frequently used for traversing pixel and voxel grids for these calculations is prone to bias unless a previously unreported normalization is used. This normalization ameliorates the bias present when using cubic voxels, and also permits calculations on data sets in which the slice spacing is not equal to the pixel spacing. Overall, the three methods for quantification of anisotropy give broadly similar results, but there are systematic divergences that can be traced to their differences in data and processing, and which may impact on their relative utility in estimating mechanical properties. Although discussed in the context of computed tomography of trabecular bone, the methods described here may be applied to any 3D data set from which fabric information is desired.     

分光光度计的线性校正

介绍了采用光叠加法校正分光光度计光度线性的基本原理及测量方法 ;给出了折半法线性校正误差的计算公式 ;分析了多系列高精度线性校正的措施及定量计算的相互关系     

Real space maps of atomic transitions

Considering the rapid technical development of transmission electron microscopes, we investigate the possibility to map electronic transitions in real space on the atomic scale. To this purpose, we analyse the information carried by the scatterer's initial and final state wave functions and the role of the different atomic transition channels for the inelastic scattering cross section. It is shown that the change in the magnetic quantum number in the transition can be mapped. Two experimental set-ups are proposed, one blocking half the diffraction plane, the other one using a cylinder lens for imaging. Both methods break the conventional circular symmetry in the electron microscope making it possible to detect the handedness of electronic transitions as an asymmetry in the image intensity. This finding is of important for atomic resolution energy-loss magnetic chiral dichroism (EMCD), allowing to obtain the magnetic moments of single atoms.     

Analysis of engine oil and phosphorus volatility

An apparatus has been designed that allows the measurement of phosphorus volatility, which is important in, among other things, controlling the degradation of exhaust catalysers in automobiles. Following on from the original Noack volatility test, the Selby—Noack apparatus can measure up to 99.9% of volatiles. The apparatus has been used in the present work to assess the relationship between engine oil volatility and engine oil phosphorus volatility (from additives), some reflections on which are presented.