Dual-beam Scanning Electron Microscope Research Articles

Erratum: Preparing Lamellae from Vitreous Biological Samples using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography.

An erratum was issued for:Preparing Lamellae from Vitreous Biological Samples using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography. The Authors section was updated from: Claudine Bisson1,2 Corey W. Hecksel3,4 James B. Gilchrist3 Roland A. Fleck1 1Centre for Ultrastructural Imaging, New Hunt's House, Guy's Campus, King's College London 2Department of Biological Science, Birkbeck College, University of London 3Electron Bio-Imaging Centre, Diamond Light Source, Harwell Science and Innovation Campus 4SLAC National Accelerator Laboratory, Stanford University to: Claudine Bisson1,2 Corey W. Hecksel3,4 James B. Gilchrist3 M. Alejandra Carbajal1 Roland A. Fleck1 1Centre for Ultrastructural Imaging, New Hunt's House, Guy's Campus, King's College London 2Department of Biological Science, Birkbeck College, University of London 3Electron Bio-Imaging Centre, Diamond Light Source, Harwell Science and Innovation Campus 4SLAC National Accelerator Laboratory, Stanford University.

ELI trifocal microscope: a precise system to prepare target cryo-lamellae for in situ cryo-ET study

Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo-fluorescence and electron microscopy lacks sufficient accuracy and efficiency to precisely prepare cryo-lamellae of target locations for subsequent cryo-ET. Here we describe a precise cryogenic fabrication system, ELI-TriScope, which sets electron (E), light (L) and ion (I) beams at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella. ELI-TriScope uses a commercial dual-beam scanning electron microscope modified to incorporate a cryo-holder-based transfer system and embed an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule. Using ELI-TriScope, we prepared a batch of cryo-lamellae of HeLa cells targeting the centrosome with a success rate of ~91% and discovered new in situ structural features of the human centrosome by cryo-ET.

Impregnation methods for quantitative analyses of the gas diffusion layer in proton exchange membrane fuel cells

Microstructural analysis is central to understand and predict the performance and degradation of proton exchange membrane fuel cell (PEMFC) materials. Advanced electron and x-ray 3-D microscopy methods are needed to characterize the reticulate structures forming the catalyst, gas diffusion layer/ microporous layers. The high-throughput dual beam scanning electron microscope allows the 3-D imaging of such layers with a spatial resolution in the range of a few nm. However, for accurate quantitative analyses, impregnation of the porous sample is required to mitigate artefacts due to milling and pore-through effect, and provide sufficient contrast between the material phases for image segmentation. This study investigates an impregnation method based upon the addition of Cobalt (II) acetylacetonate nanoparticles in an epoxy polymer resin. The results were compared with conventional methods using commercial resins stereology on sections polished by focused ion beam (FIB). The results indicate the appropriateness of the newly proposed method, providing adequate contrast for accurate image segmentation. An Energy Dispersive X-ray (EDX) analysis was included to obtain an information about the composition, which can be used for further improvements of the resin.

Carbonaceous Deposits in a Fuel-Film Cooled Rocket Combustor: Electron Microscopy

The high gas–side heat transfer coefficients expected in high-pressure oxygen-rich staged combustion kerosene–oxygen engines require fuel–film cooling in parallel with regenerative cooling to keep metal wall temperatures at acceptable levels. In such engines, the fuel–film transitions from a liquid to supercritical state, leaving behind dense and soot layers of carbonaceous deposits. We used a 4.8 MPa kerosene– fuel–film cooled combustor with removable chamber liner samples for posttest analysis of carbonaceous deposits. Dense layer deposit depths were measured in cross-sectioned samples with a dual-beam scanning electron microscope. The varying fuel–film flow rate did not affect deposit depths in the near field. However, higher film flow rates resulted in longer regions of carbonaceous deposits in the far field. The dense and soot layers were hypothesized to be formed by heterogeneous condensation of polycyclic aromatic hydrocarbons and thermophoretic diffusion of soot, respectively.

The morphological and molecular characterization of Baylisascaris devosi Sprent, 1952 (Ascaridoidea, Nematoda), collected from Pine marten (Martes martes) in Iran

BackgroundBaylisascaris devosi is an intestinal nematode found in several carnivores including fisher, wolverine, Beech marten, American marten and sable in different parts of the world, but this nematode has not been reported from Pine marten. Therefore, this study aimed to identify Baylisascaris isolated from a Pine marten in Iran using morphological and molecular approaches.MethodsSpecimens of B. devosi were collected from one road-killed Pine marten in northern Iran. Morphological features were evaluated using scanning electron microscopy, energy dispersive x-ray analysis and ion sectioning. The molecular characterization was carried out using partial Cox1, LSU rDNA and ITS-rDNA genes.ResultsThe nematodes isolated from the Pine marten were confirmed to be B. devosi based on the morphological features and the sequence of ribosomal and mitochondrial loci. X-ray scans (EDAX) were completed on gallium cut structures (papillae, eggs, male spike and mouth denticles) of B. devosi using a dual-beam scanning electron microscope. The male spike and mouth denticles had a high level of hardening elements (Ca, P, S), helping to explain the chemical nature and morphology of the worm. Based on these genetic marker analyses, our sequence had the greatest similarity with Russian B. devosi isolated from sable.ConclusionsIn this study, to our knowledge, the occurrence of B. devosi infection in Pine marten is reported for the first time. Molecular analysis showed that these three genes are suitable molecular markers for identification and inferring phylogenetic relationships of Baylisascaris species. Furthermore, the high divergence of Cox1 between Baylisascaris species indicates that Cox1 could be used for their phylogenetic and taxonomic studies.Graphical

The study of the silica clay dust elemental composition in building materials’ production

The paper discusses the physical properties of the silica clay used as building material and mineral. During crushing, pouring, packing and other processes the dust which enters the storage bunkers of dust removal systems, is formed; it is emitted from the dust collecting devices into the atmosphere, and a part of the dust enters the working area. The paper compares the elemental composition of the silica clay before the crushing operation; the dust collected in bunkers of dust removal systems; in the air emissions; the dust after using the dust collectors and working area air dust. The study was conducted using a Versa 3D Dual Beam scanning electron microscope. The dust characteristics’ comparative studies were carried out in three intake zones for three types of dust collectors used in dust removal systems: fabric filters, cyclones and swirling approach flows (SAF). The homogeneity hypothesis was tested using the Kolmogorov-Smirnov criterion. Based on the measurement results, it was concluded that the dust from the dust bunkers for all the dust collector options has a close elemental composition and can be returned to the additives’ production process. It is shown that the dust entering the working area air and in emissions into the atmosphere are close in their characteristics. The results of measuring the random distribution functions of the elemental composition and molecular weight are obtained, which allow calculating particle weight and other characteristics with 95% certainty, as well as evaluating the aerodynamic characteristics of dust particles, etc.

Application of 3D EBSD Technique to Study Crystallographic Texture in Heavily Cold-Rolled and Recrystallized Modified 9Cr–1Mo Steel

Automated electron backscatter diffraction (EBSD) technique in a dual-beam field emission gun scanning electron microscope has been successfully used to obtain three-dimensional (3D) orientation mapping of grains in modified 9Cr–1Mo after severe plastic deformation and recrystallization. In this technique, the microstructure and micro-texture across several sections of the material were studied by means of the state-of-the-art “slice and view” methodology using grazing incidence high-energy Ga+ focused ion beam for slicing and electron beam for viewing and EBSD analysis. By combining the data from each slice, a 3D texture map could be generated by means of image reconstruction technique. The orientation map thus generated provided volumetric microstructural and micro-textural information. The 3D EBSD studies on the heavily deformed mod-9Cr–1Mo steel (cold-rolled 88%) revealed that rolled grains were elongated like plates with thickness ≤ 200 nm. Analysis of the fiber texture components in rolled specimen across the sections showed near equal preference for all fiber texture components with some enhancement of the α-fiber texture. However, by recrystallizing at 1023 K for 1 h, elongated grains along rolling direction with large diameters (~ 40 to 100 µm) were observed together with finer (size ~ 0.5 to 2 µm) polygonal grains and γ-fiber texture component dominated over other texture components.

Smallest microhouse in the world, assembled on the facet of an optical fiber by origami and welded in the μRobotex nanofactory

In this study, the authors have demonstrated that it is possible to realize several three-dimensional (3D) micro- and nanostructures, by the fabrication of the smallest microhouse using a dual beam scanning electron microscope (SEM)/focused ion beam (FIB) Auriga 60 from Zeiss together with a six degree of freedom robot built with SmarAct components. In this new type of nanolab, cutting, etching, folding, assembling, and then welding thin membranes of silica on top of a cleaved optical fiber SMF28, or production of micro- and nanostructures, like the microhouse, are possible. The authors have experimentally shown that FIB can be used, in this new generation of micro/nanofactory, in combination with SEM, and gas injection system, in order to fabricate three-dimensional microstructures: a microhouse in this study, with ultrahigh accuracy assembly down to 10 nm. By using the theory of sputtering, the authors are able to propose a model of folding thin membranes of numerous materials such as metals, polymers, or crystals, i.e., silica, silicon, potassium tantalite, or lithium niobate. This method is usually described as origami in the literature [W. J. Aroa, H. I. Smith, and G. Barbastathis, Microelectron. Eng. 84, 1454 (2007); W. J. Aroa et al., J. Vac. Sci. Technol., B 25, 2184 (2007); and K. Chalapat et al., Adv. Mater. 25, 91 (2013)]. The experimental results indicate that the introduction of a microrobot inside the SEM vacuum chamber will provide the means to enlarge the scope of clean room facilities to build complex and smart 3D microsystems with heterogeneous materials, especially on the facet of an optical fiber in the lab on fiber new field. The authors propose a new way to easily manufacture many kinds of optical functions for light trapping based on nanoantennas, nanophotonic crystal, axicon or lattice, 3D biosensor with origami, and nanopatterning surfaces or carbon nanotubes, etc.

Growth mechanisms of multiscale, mound-like surface structures on titanium by femtosecond laser processing.

Femtosecond laser surface processing (FLSP) can be used to functionalize many surfaces, imparting specialized properties such as increased broadband optical absorption or super-hydrophobicity/-hydrophilicity. In this study, the subsurface microstructure of a series of mound-like FLSP structures formed on commercially pure titanium using five combinations of laser fluence and cumulative pulse counts was studied. Using a dual beam Scanning Electron Microscope with a Focused Ion Beam, the subsurface microstructure for each FLSP structure type was revealed by cross-sectioning. The microstructure of the mounds formed using the lowest fluence value consists of the original Ti grains. This is evidence that preferential laser ablation is the primary formation mechanism. However, the underlying microstructure of mounds produced using higher fluence values was composed of a distinct smaller-grained α-Ti region adjacent to the original larger Ti grains remaining deeper beneath the surface. This layer was attributed to resolidification of molten Ti from the hydrodynamic Marangoni effect driven fluid flow of molten Ti, which is the result of the femtosecond pulse interaction with the material.

Dual-Beam Scanning Electron Microscope (SEM) and Focused Ion Beam (FIB): A Practical Method for Characterization of Small Cultural Heritage Objects

ABSTRACTKnowledge of the composition of many cultural heritage objects is limited, resulting in many unanswered questions in regards to the provenance, composition, and production methods. In this paper, our objective is to show that dual beam scanning electron microscope (SEM) and focused ion beam (FIB) can be used rapidly and non-destructively to determine the surface and bulk metal compositions in small cultural heritage objects. We show, for the first time, that this novel FIB technique can be successfully applied non-destructively to cultural heritage objects by examining three representative silver plated objects (Candelabra, “Century” spoon, and New York World’s Fair spoon) from the Dallas Museum of Art’s unparalleled collection of modern American silver. In each case, we successfully reveal and characterize the bulk metal as well as the Ag-plating, up to ∼80 µm deep and show that there is no visual damage resulting from the milling process of the FIB. This novel characterization technique can be applied, due to its ease of availability and rapid use, to many other problems in addition to silver plated objects, making dual beam SEM/FIB a possible cornerstone technique in the study of cultural heritage objects.

Three-dimensional microstructural characterization of porous cubic zirconia

A set of cubic zirconia samples were investigated using 3-dimensional electron backscatter diffraction (3D EBSD) to analyze the grain structure, grain boundary networks and pore morphology. 3D EBSD is a variation of conventional EBSD, whereby a focused ion beam (FIB) is used in a dual beam scanning electron microscope (SEM) i.e. FIB–SEM to mill away material and to create ‘serial sections’ through the material being analyzed. Each new surface revealed is subject to an EBSD scan, which continues sequentially until a desired volume of material has been removed. In this manner, many consecutive 2D EBSD scans can be rendered in 3D to gain a greater insight of microstructural features and parameters.The three samples were examined in order to determine the effect of differences in the manufacturing process used for each. For each sample, a volume of ca. 15,000μm3 was studied. The analysis of several microstructure parameters revealed a strong dependence on manufacturing conditions. Subsequently, the results of 3D EBSD analysis were compared to conventional 2D EBSD. Significant differences between the values of microstructure parameters determined by 2D and 3D EBSD were observed.

Three-Dimensional Investigations of Finely Grained Materials/ Trójwymiarowa Analiza Materiałów Drobnokrystalicznych

Abstract Development of new materials requires application of sophisticated techniques to characterize microstructure in a very detailed way. The combination of Electron Backscatter Diffraction (EBSD) and Energy Dispersive Spectrometry (EDS) with Focused Ion Beam (FIB) are excellent examples of such techniques. They are based on serial sectioning of chosen region in the investigated sample followed by data acquisition using the dual-beam scanning electron microscope equipped with both electron and ion columns. Three kinds of samples have been investigated: a) are anticorrosive Ni-Mo coating on ferritic steel; b) the oxidized Crofer22 APU ferritic stainless steel; c) the Al 6013 aluminum alloy after complex plastic deformation. The obtained results allowed to analyze crystalline morphology, distribution of precipitates as well as to reconstruct internal structure of grains and grains boundaries geometry.

Investigation of grain-boundary geometry and pores morphology in dense and porous cubic zirconia polycrystals

Three-dimensional electron backscatter diffraction technique was used for the visualization of grain boundary geometry and pore morphology in cubic zirconia. A set of four samples sintered under different conditions was investigated. Specimens which were characterized by energy dispersive spectroscopy and X-ray diffraction were entirely composed of cubic phase. Investigations of boundaries and pore structures were carried out in a dual-beam scanning electron microscope. For each sample, a volume of 1000μm3 was investigated. The analysis of grain boundary networks reconstructed from inverse pole figure maps revealed a strong dependence between grain boundary density and sample preparation parameters. Sintering also affects the size and distribution of pores. The total number of grains analyzed varied from 17 to 357 and the calculated volume of cavities from 0.01% to 21%. This paper shows the application of three-dimensional crystallographic orientation analysis to characterize the microstructure of yttria stabilized zirconia ceramics.

Correlation of chromatographic performance with morphological features of organic polymer monoliths

Monoliths are considered to be a low pressure alternative to particle packed columns for liquid chromatography (LC). However, the chromatographic performance of organic monoliths, in particular, has still not reached the level of particle packed columns. Since chromatographic performance can be attributed to morphological features of the monoliths, in-situ characterization of the monolith structure in three dimensions would provide valuable information that could be used to help improve performance. In this work, serial sectioning and imaging were performed with a dual-beam scanning electron microscope for reconstruction and quantitative characterization of poly(ethylene glycol) diacrylate (PEGDA) monoliths inside a capillary column. Chord lengths, homogeneity factors, porosities and tortuosities were calculated from three-dimensional (3D) reconstructions of two PEGDA monoliths. Chromatographic efficiency was better for the monolith with smaller mean chord length (i.e., 5.23μm), porosity (i.e., 0.49) and tortuosity (i.e., 1.50) compared to values of 5.90μm, 0.59 and 2.34, respectively, for the other monolithic column. Computational prediction of tortuosity (2.32) was found to be in agreement with the experimentally measured value (2.34) for the same column. The monoliths were found to have significant radial heterogeneity since the homogeneity factor decreased from 5.39 to 4.89 (from center to edge) along the column radius.

A Study of Fracture Mechanisms in RAFM Steel in the Ductile to Brittle Transition Temperature Regime

The fracture behaviour of a Reduced Activation Ferritic Martensitic Steel (RAFM) has been studied within the Ductile to Brittle Transition Temperature (DBTT) regime. The DBTT has been determined by ASTM E 1921 based reference temperature approach under dynamic loading condition. The dynamic reference temperature (T0dy) was found to be − 33.8°C. The fracture mechanism has been studied by extracting TEM specimens precisely at the crack initiation sites using focused ion beam (FIB) technique in a high resolution dual beam scanning electron microscope. Detailed analytical TEM studies revealed that the morphology of carbides play a crucial role in the initiation of a crack. The larger ellipsoidal carbides, which were found to be Cr-rich, have been found to be responsible for dislocation piles ups. The shorter edge of these ellipsoidal carbides are areas of high stress concentration and were found to initiate cracks by decohesion of the particle-matrix interface. On the contrary, the iron rich carbides have been found to be smaller, more spherical, and thus less effective in blocking dislocation movement and therefore formation of pile ups. The results, which reveal an important mechanism towards crack initiation in ferritic-martensitic steels, will be presented in detail.

Electron microscopy of the surfaces of bacillus spores

The surface structures of the spores of Bacillus. cereus, B. thuringiensis, and Brevibacillus laterosporus were studied by transmission and scanning electron microscopy. Platinum deposition and negative staining with uranyl acetate revealed appendages and exosporium in B. thuringiensis and B. cereus. The exosporium structure was visualized by negative staining and ultrathin sectioning. For staining the exosporium polysaccharide, Alcian blue was used during fixation. The results obtained show the differences in structural organization of appendages and exosporium in different strains. Canoe-shaped inclusions were revealed in all Br. laterosporus strains, while strain IGM16-92 had a fibrillar capsule as well. Electron microscopy using a dual beam scanning electron microscope Quanta 200 3D provided the information of the spore surface relief without sample treatment (fixation and dehydration). The spores of Br. laterosporus strains had folded surface, unlike the smooth surface of B. cereus and B. thuringiensis spores. Diversity of external spore structures was shown within a species, which may be used for detection of bacteria at the strain level. Optimized procedures for visualization of spore surface by different electron microscopic techniques were discussed.

Crystallographic Characterization of an Electroplated Zinc Coating Prone to Whiskers

A Zn-electroplated steel prone to Zn whiskers has been investigated in order to obtain information about the microstructure of the Zn coating at the root of a whisker. Indeed, the characterization of the Zn coating is essential to understand the Zn whisker growth mechanism. Care was taken to prepare a sample at a whisker root by using a focused-ion beam in a dual-beam scanning electron microscope. The sample was analyzed using energy dispersive X-ray spectrometry and electron backscattered diffraction. Results show three different regions: 1) an inclusion enriched with Ca, Al, and C; 2) the Zn coating with columnar grains; and 3) the root of the whisker. An important point is that recrystallized grains are found at the whisker root. This observation supports recent whisker growth models based on recrystallization.

Nanorobotic Assembly and Focused Ion Beam Processing of Nanotube-Enhanced AFM Probes

In this paper, a focused ion beam processing technique is presented that facilitates the modification of carbon nanotubes (CNTs) in terms of length, diameter, and orientation. The CNTs are mounted onto an atomic force microscope (AFM) probe by using a nanorobotic microgripper-based pick-and-place handling strategy. Such CNT-enhanced AFM probes are needed for metrology measurements of nanostructures with critical dimensions and high aspect ratios. The complete process of assembly and processing is realized inside a nanorobotic dual beam scanning electron microscope (SEM) and focused ion beam (FIB) machine.

In Situ Mechanical and Electrical Characterization of Individual TiO2 Nanofibers Using a Nanomanipulator System

Young's modulus and electrical resistivity of individual titanium dioxide (TiO(2)) nanofibers were characterized using a nanomanipulator system installed in a focused ion beam-scanning electron microscope (FIB-SEM) dual-beam Scanning Electron Microscope system. Young's modulus of individual nanofibers was deduced from the analysis of their in situ resonance behavior in response to an oscillating electric field. The electrical behavior of a single nanofiber was also analyzed by a two-point method probed by a nanomanipulator. These results will contribute to the design of devices based on single TiO(2) nanofibers, as well as devices based on nanofiber networks. The methods presented here can also be applied to characterize other one-dimensional nanostructures.

Soot and Ash Deposition Characteristics at the Catalyst-Substrate Interface and Intra-Layer Interactions in Aged Diesel Particulate Filters Illustrated using Focused Ion Beam (FIB) Milling

The accumulation of soot and lubrication-derived ash particles in a diesel particulate filter (DPF) increases exhaust flow restriction and negatively impacts engine efficiency. Previous studies have described the macroscopic phenomenon and general effects of soot and ash accumulation on filter pressure drop. In order to enhance the fundamental understanding, this study utilized a novel apparatus, that of a dual beam scanning electron microscope (SEM) and focused ion beam (FIB), to investigate microscopic details of soot and ash accumulation in the DPF. Specifically, FIB provides a minimally invasive technique to analyze the interactions between the soot, ash, catalyst/washcoat, and DPF substrate with a high degree of measurement resolution. The FIB utilizes a gallium liquid metal ion source which produces Ga+ ions of sufficient momentum to directionally mill away material from the soot, ash, and substrate layers on a nm-μm scale. As the FIB cuts into the sample, uncovering intra-layer details, the coupled high resolution SEM imaging and energy dispersive x-ray (EDX) analysis provide both morphological and chemical data. This tool was applied to investigate soot and ash accumulation in the DPF, with a specific focus on characterizing interactions between the soot/ash/DPF interfaces, such as soot penetration into the ash layer, as well as soot and ash accumulation in the DPF surface pores. In particular, ash and soot particle size, layer pore structure, and the extent of penetration or intra-layer mixing, are all parameters directly impacting DPF pressure drop, which may be quantified using this technique. The work in this study leveraged existing databases of aged DPFs containing various levels of soot and ash, originating from field trials and controlled laboratory testing. Results obtained with this technique provide a fresh and complementary perspective, as well as additional details useful to understand the macroscopic observations of the combined ash and soot effects on diesel particulate filter pressure drop.