The Influence of Sample Preparation on Correlative Microscopy with the Use of Artificial Intelligence Methods

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.

<p>Quantitative modal analysis of rock thin sections or liberation analysis of minerals processing plant materials can be very complex as grain sizes can vary by more than 7 orders of magnitude: Thin sections of rocks may contain extremely coarse grains (mm-sized crystals) down to glassy material with no long-range order (ordered domains <1 nm).</p><p>Material characterisation and modal analysis have traditionally been carried out with a combination of solid-state, microscopic and spectroscopic techniques (e.g., optical / scanning electron microscopy, powder X-ray diffraction, X-ray fluorescence spectroscopy). These techniques require different sample preparation routines, data acquisition and evaluation - a time-consuming process that may be considered too complex to implement in mineral processing plants despite requiring the relevant sample preparation equipment. Scanning electron microscopy (SEM) coupled with energy dispersive spectroscopy (EDS provides an opportunity to carry out this characterisation in a more rigorous and, in certain cases, automated way. This process includes image thresholding (setting of grey levels of present phases by the analyst) and X-ray data collection with EDS. EDS is an ideal analytical technique for this work as it offers high acquisition speeds and the collection of the whole energy spectrum with a single detector, not requiring the selection of a fixed element list prior to data acquisition. Characterisation of coarse-grained rocks requires larger areas to be scanned in order to ensure representativity.</p><p>The analytical workflow can be further optimised by combining SEM-based analytical techniques for in situ, non-destructive, and potentially simultaneous bulk analysis. Electron backscatter diffraction (EBSD) is an SEM-based technique which can be used to determine the crystallographic properties and orientation of mineral grains, as well as to perform fabric analyses on polycrystalline materials. EBSD allows for crystallographic data to be collected simultaneously with chemical data and does not require powdered samples. As a result, the texture of the material can be fully preserved. The sample preparation requirements of the technique are similar to those for standard SEM-EDS, with an additional final polishing step, essential for the removal of surface imperfections, as the EBSD signal is generated on the sample surface. The coupling of EDS and EBSD datasets permits the enhanced interpretation of feature analysis data, allowing for a deeper understanding of the compositional, structural and textural properties of the sample. This, highly-efficient, in-situ, bulk material characterisation, is key for the mining industry, as it provides insights for optimising downstream procedures thereby saving time and resources and bolstering throughput and efficiency.</p>

The identification of crystallographic phases in the scanning electron microscope (SEM) has been limited by the lack of a simple way to obtain electron diffraction data of an unknown while observing the microstructure of the specimen. With the development of charge coupled device (CCD)-based detectors, backscattered electron Kikuchi patterns, alternately referred to as electron backscattered diffraction (EBSD) patterns, can be easily collected. Previously, EBSD has been limited to crystallographic orientation studies due to the poor pattern quality collected with video rate detector systems. With CCD detectors, a typical EBSD can now be acquired from a micron or submicron sized crystal using an exposure time of 1–10 s with an accelerating voltage of 10–40 kV and a beam current as low as 0.1 nA. Crystallographic phase analysis using EBSD is unique in that the properly equipped SEM permits high magnification images, EBSDs, and elemental information to be collected from bulk specimens. EBSD in the SEM has numerous advantages over other electron beam-based crystallographic techniques. The large angular view (∼70°) provided by EBSD and the ease of specimen preparation are distinct advantages of the technique. No sample preparation beyond what is commonly used for SEM specimens is required for EBSD.

Twin boundaries in a crystalline material can be defined by a particular rotation angle about a particular access or a mirrored crystal orientation about a particular plane [1]. For example, copper twins are typically defined by a 60 rotation about where the associated twin plane is of the {111} family. One critical area of twin research looks at deformation twinning as the limiting factor for formability of Mg alloys, such as AZ31 [2]. In AZ31 there are two basic twin modes: compression twinning and tension twinning. The latter phenomenon forms fairly large, easy to detect twinned regions within parent grains, whereas the former tends to form extremely thin twins that are on the order of 100 nm wide. Additionally, the copper which is frequently seen in many microelectronics contains twins on the order of 10 nm [3]. In both cases these features are within the detectable limits for a modern scanning electron microscope (SEM). However, identifying these twins via crystal orientation relations with electron backscatter diffraction (EBSD) in the SEM relies on a larger spatial resolution which makes detecting these twins from crystallographic information difficult in the SEM. This study presents a method whereby improved spatial resolution of thin twins can be achieved with EBSD.

Microscopy Research and Technique virtual issue: "Correlative light and electron microscopy".

FIBSIMS: A review of secondary ion mass spectrometry for analytical dual beam focussed ion beam instruments

Since the first automated electron backscatter diffraction (EBSD) results were attained 22 years ago, EBSD has become a common characterization technique in materials laboratories around the world. This has led to advancements and new understanding in not just crystallography but also in microstructural evolution, phase transformations, defects and dislocations, mechanical behavior, failure, and other materials phenomena. As a complement to scanning electron microscopy (SEM), EBSD is accessible and available to many researchers with little additional sample preparation. With improvements to both SEM and EBSD, it is now possible to analyze large areas for statistically significant data, and as precision increases, higher resolutions and smaller length scales have become accessible. EBSD analyses are also being combined with other characterization and analysis techniques to provide new insights. Although EBSD has matured and measurements have become more commonplace, new techniques, applications, and approaches continue to be introduced regularly. This collection of articles focuses on the versatility of EBSD in the range of materials systems presented, as well as on new techniques and applications of EBSD for materials characterization. Examples of the combination of EBSD with other characterization techniques include the supplementation and enhancement of EBSD data by transmission electron microscopy (TEM), atomic force microscopy (AFM), and electron channeling contrast imaging. Novel approaches to strain measurements, and the analysis of stacking faults, defects, and dislocations are also presented. The versatility of EBSD is demonstrated in the range of materials systems represented in these articles; EBSD analyses of conventional polycrystalline metals and alloys, novel lightweight alloys, second phase precipitates, and the complex structures of thin film solar cells are presented. The article from D. Abou-Ras and his co-workers provides an overview of the application of EBSD on thin-film solar cells. Of particular interest is the integration of EBSD results with several scanning probe microscopy techniques for correlating properties critical to photovoltaic performance with the crystallographic orientation aspects of the observed microstructures. I. Gutierrez-Urrutia, S. Zaefferer, and D. Raabe have reviewed a novel application of EBSD for imaging crystal defects, such as dislocations, cells, and stacking faults. EBSD is used to determine the optimal diffraction conditions for electron channeling contrast imaging to elucidate dislocation substructures similar to those observed using conventional TEM diffraction. Z. Chen and C.J. Boehlert combine EBSD with AFM to quantify deformation in an AZ31 magnesium alloy, noting that grain boundary sliding has a significant contribution. In the article by T. Ben Britton and colleagues, elastic strains are measured using highresolution, cross-correlation-based EBSD, presenting three examples of this exciting technique. Finally, Seiichi Suzuki presents a systematic overview of the transmission EBSD technique, which provides very high spatial resolution in orientation mapping. This collection of articles represents just a sampling of the new and exciting data being collected using EBSD, while also covering a breadth of work that illustrates both the utility and the potential of this important technique. As advancements continue to be made, we anticipate EBSD to fuel further innovative studies and bring exciting results in crystallography, microstructural evolution, materials processing, and microstructure quantification for years to come.

Scanning electron microscopy (SEM) is an indispensable characterization technique for materials science. More recently, scanning electron microscopes can be equipped with scanning transmission electron microscopy (STEM) detectors, which considerably extend their capabilities. It is demonstrated in this work that the correlative application of SEM and STEM imaging techniques provides comprehensive sample information on nanomaterials. This is highlighted by the use of a modern scanning electron microscope, which is equipped with in-lens and in-column detectors, a double-tilt holder for electron transparent specimens and a CCD camera for the acquisition of on-axis diffraction patterns. Using multi-walled carbon nanotubes and Pt/Al2O3 powder samples we will show that a complete characterization can be achieved by combining STEM (mass-thickness and diffraction) contrast and SEM (topography and materials) contrast. This is not possible in a standard transmission electron microscope where topography information cannot be routinely obtained. We also exploit the large tilt angle range of the specimen holder to perform 180 degrees STEM tomography on multi-walled carbon nanotubes, which avoids the missing wedge artifacts.

Systems biology in 3D space--enter the morphome.

Electron Backscatter Diffraction (EBSD) in Scanning Electron Microscope (SEM) has been one of the most popular tools for characterizing microstructures of materials and in particular alloys made by additive manufacturing (AM) in recent years. This characterization technique can provide abundant microstructural data, including phase distribution, grain size, grain boundary character, crystallographic orientation, and texture. However, it has recently been noticed that there are several challenges in the characterization of AM alloys by EBSD, causing the misunderstanding of material microstructures. This presentation discusses two challenges associated with the grain size determination by EBSD. Grain size is an important parameter used in understanding the relationship between the mechanical property and microstructure of a 3D-printed alloy, and is usually measured by optical microscope (OM) and EBSD. However, a common issue is that the grain size determined by EBSD sometimes is inconsistent with that from OM. Here We measured the grain size from the same areas in a Ti64 sample by OM and EBSD, respectively, and compared the results, in order to correlate two techniques. The result shows that when the grain size is greater than 10um, the data from OM and EBSD can completely match. When the grain size is in the range of 4~10um, there is a significant deviation between the size distributions. For fine grains, e.g. <4um, the EBSD measurement is more reliable. This indicates that OM and EBSD are equivalent for analyzing grains which sizes are bigger than 10um, but EBSD is more suitable for measuring fine grains due to the higher spatial resolution than OM. Further analysis reveals that the accuracy of grain size measurement especially for the fine grains is strongly related to the threshold angle of grain boundary used in the EBSD data process. Step size is another important parameter to determine the grain size. Another usual challenge in the EBSD analysis is how to choose the step size to obtain a high-quality EBSD map with high spatial resolution and high hit rate [1]. As the typical AM alloy has two different microstructural features in terms of grain size, melt pools with large columnar grains and fine-grained regions between the melt pools, researchers like to choose a small step size to map the sample. This is a conventional EBSD strategy used in the characterization of heterogeneous microstructures in materials. However, the recent characterization in Al printed alloys shows that such a strategy doesn’t work well at low magnification even a very small step size is used. Normally, grains in melt pools are visualized and measured well, but the fine grains at the melt pool boundaries are invisible at the low magnification due to the low hit rate. To improve EBSD map quality and grain size measurement at the fine-grained regions, higher magnification with a smaller step size has been used, but this condition limits the size of the mapping area. Compromising the magnification and grain size determination, a new EBSD strategy that includes two steps of mapping AM samples is recommended, firstly scanning a big area to show the domain microstructures of melt pools, and secondly mapping with higher magnification to show the typical fine-grained region in detail.

Simultaneous Correlative Light and Electron Microscopy of Samples in Liquid

Subject of Research. Methodological aspects of sample preparation and electron backscatter diffraction (EBSD) in the study of microdeformations in zircon grains. Objects and Methods. Fragments of impactites from shock-metamorphosed rocks of the Vredefort (South Africa) and Kara (Pay-Khoy Ridge, Yugorsky Peninsula, Russia) impact craters were investigated using scanning electron microscopy (SEM) and electron backscatter diffraction. Results. The identification of zircon grains with specific microdeformations requires high-spatial-resolution (tens of nanometers) examination of large polished rock surfaces, which demands significant instrument time. To reliably detect microdeformations in zircon, the following methodological challenges were addressed: (1) analyzing the influence of Electron Backscatter Diffraction Pattern (EBSP) imaging conditions at different beam accelerating voltages (10, 20, and 29 kV) on the signal-to-noise ratio, spatial resolution, and Kikuchi band width; (2) comparing zircon grain orientation maps obtained at different voltages; (3) developing an algorithm for mineral identification and microdeformation finding; and (4) validating the methodology on zircon grains from the Vredefort and Kara impact craters. Conclusions. The sample preparation methodology for EBSD analysis was refined, and methods for processing EBSD data to improve Kikuchi diffraction pattern indexing were explored. The efficiency of detecting and analyzing shock-metamorphosed zircon grains using scanning electron microscopy was enhanced through optimized electron imaging and EBSD mapping conditions. An algorithm for mineral identification in thin sections (rock slices) was developed. The methodology was validated on a series of 50 thin sections from the Kara and Vredefort impactites, resulting in the identification of 436 zircon grains, including all known types of zircon microdeformations.

A generic high-throughput microstructure classification and quantification method for regular SEM images of complex steel microstructures combining EBSD labeling and deep learning

Datasets acquired with correlative microscopy method for delineation of prior austenite grain boundaries and characterization of prior austenite grain size in a low-alloy high-performance steel

Correlative microscopy for quantification of prior austenite grain size in AF9628 steel