Focused Ion Beam Scanning Electron Microscopy Research Articles

Pore network modeling of a microporous layer for polymer electrolyte fuel cells under wet conditions

The gas diffusion layers (GDLs) of polymer electrolyte fuel cells have been developed with applying microporous layers (MPLs) in their catalyst layer (CL) side to alleviate the accumulation of liquid water in the CL for oxygen transport to the cathode CL. A three-dimensional porous structure of our in-house hydrophobic MPL is numerically modeled with a pore network model (PNM). The convective air permeability and oxygen diffusivity, which depend on liquid saturation, are evaluated. To construct the PNM, focused ion beam scanning electron microscopy (FIB-SEM) is used to derive the pore size distribution (PSD). The model is ex-situ validated through air permeability and oxygen diffusivity tests with controlled saturation of non-volatile wetting liquid that is stable in the hydrophobic MPL. Oxygen diffusivity of the MPL is obtained by identifying the diffusion resistances of the concentration boundary layers and GDL substrate in the tests. The model predicts the effects of liquid water saturation in the MPL on the air and liquid water permeations, and the oxygen diffusion, and thus can be used to design optimal PSDs for practical cells.

Influence of Inclusions in the Corrosion Behavior of Plasma Nitrided Stainless Steel

Plasma nitriding is a well‐established technique to improve hardness and tribological properties of austenitic stainless steel. It is proved that it is also possible to preserve the corrosion resistance after nitriding by controlling the process parameters such as time and temperature, obtaining the so‐called S phase. Herein, the corrosion behavior is evaluated for nitrided layers produced by three different plasma treatments: DC plasma nitriding, plasma immersion ion implantation (PIII), and low‐energy ion implantation (LEII), using different parameters in order to obtain the S‐phase in thickness from 1.2 to about 6 μm without nitride precipitation. The microstructure and chemical composition of the nitrided layers is characterized by means of X‐ray diffraction, secondary‐ion mass spectroscopy, and scanning electron microscopy–focused ion beam. The corrosion behavior is evaluated by means of the cyclic polarization tests in NaCl solution. The morphology of the corrosion attack is studied by optical microscopy and SEM‐FIB, revealing a change from crevice to pitting after the nitriding process. The inclusions are observed to be corrosion initiation sites. Due to this, the thickest nitrided layers (with high N concentration) show better corrosion behavior than the thinner ones.

Large-volume FIB-SEM 3D reconstruction: An effective method for characterizing pore space of lacustrine shales

Focused ion beam scanning electron microscopy (FIB-SEM) is a commonly used three-dimensional (3D) pore-network reconstruction method for shales due to its unique capability in imaging nano-scale pores. However, it has been found that for pore space of lacustrine shales with strongly heterogeneous pore structures, the conventional FIB-SEM 3D models usually with dimensions of 10 μm × 10 μm × 10 μm cannot adequately characterize the pore structures as the representative element volume required is much larger than the FIB models. Here, we propose to utilize large volume FIB-SEM (LV-FIB-SEM) 3D models to resolve this challenge. The LV-FIB-SEM model has a significant enhancement in the model size compared with the commonly used conventional FIB-SEM models and a much higher spatial resolution than non-synchrotron nano X-ray CT models for similar imaging sample sizes. With 75 μm × 65 μm × 60 μm as predesigned reconsruction size, after image processing two LV-FIB-SEM 3D models with sizes of 73.56 μm × 38.13 μm × 52.59 μm and 74.01 μm × 43.05 μm × 42.00 μm and model resolution of 30 nm were reconstructed and quantitatively analyzed. When use the conventional FIB-SEM models of 10 μm × 10 μm × 10 μm, the relative deviations between the porosities derived from 100 stochastic models and the average porosity for the two samples studied are −41.13% ∼ +87.31% and −51.66% ∼ +56.05%, respectively, indicating that such small models are not representative of the actual pore structure of the shales investigated. When the model sizes have been increased by 96 times volumetrically, the probabilities of matching average porosities for the two samples increase from 13% to 86% and from 12% to 100%, respectively. This research demonstrates that the upsizing of the FIB-SEM models enables an effective improvement on the representativeness of shale pore structures characterized. It is recommended that LV-FIB-SEM 3D reconstruction be employed to study pore space of lacustrine shales with strongly heterogeneous pore structures, which would enable a more accurate characterization and evaluation of reservoirs for shale oil exploration and development.

Novel Discrete Fracture Network Model for Multiphase Flow in Coal

Multiphase flow intensely affects the movement and accumulation of other fluids in coal and plays an essential role in predicting the permeability of coal during coalbed methane (CBM) production. Fractures have played a decisive role in the transport of CBM after hydraulic fracturing has occurred. In this study, a multiscale pore network model (PNM) was constructed on the basis of focused ion beam scanning electron microscopy (FIB-SEM) image results. Additionally, a novel discrete fracture network model, fracture–pore network model (F–PNM), was proposed to investigate the effect of fracture density, fracture developing direction, and wettability on multiphase flow. The results reveal that the permeability of F–PNM increases with the increase of the fracture density, which could be the result of the predominance of snap off. The permeability decreases as the angle between the fracture and flow direction increases; initially, the permeability decreases steeply and then it tends to remain stable; and for angles between 0° and 15°, the permeability decreased by as much as 61.8%. Moreover, the wettability of coal has limited impact on its water relative permeability; however, it has a measurable effect on gas relative permeability, which could be owing to water accumulation on the coal surface under different wettability conditions. A good wetting performance would have a negative effect on the CBM production and reduce flowback efficiency.

Quantification of plasmodesmata frequency under three-dimensional view using focused ion beam-scanning electron microscopy and image analysis

The quantitative study of plasmodesmata (PD) frequency is routine in plant science for providing information on the potential of intercellular transportation. Here, we report quantification of plasmodesmatal frequency in virus-infected tobacco vascular tissues using serial sectioning and image analysis. The image datasets were collected by focused ion beam-scanning electron microscopy (FIB-SEM), and the measurements of plasmodesmatal frequency were performed after image analysis with commercial computational programs. With a 5-nm step size (less than half the diameter of PD) during FIB sectioning, exhaustive PD sampling was performed in regions of interest. Segmentation of cell wall (CW) and PD from the background densities was performed manually, and PD were assigned automatically to individual CW interfaces by image analysis and then quantified. The PD quantification results were used to compare the plamodesmatal frequencies among different CW interfaces of individual cells and the average frequencies among different cell types were calculated. CWs lacking PD distribution were found in several cellular types, and the PD frequency were used to determine the possible pathways of PD-based symplasmic transportation. The method enables imaging of samples of several cells containing multiple CW interfaces and minimizes PD omission during sectioning and imaging.

Correlative high-resolution imaging of hydrogen in Mg2Ni hydrogen storage thin films

Nanometer scale imaging of hydrogen in solid materials remains an important challenge for the characterization of advanced materials, such as semiconductors, high-strength metallic alloys, and hydrogen storage materials. Within this work, we demonstrate high-resolution imaging of hydrogen and deuterium within Mg2Ni/Mg2NiH4 hydrogen storage thin films using an in-house developed secondary ion mass spectrometer (SIMS) system attached to a commercially available dual-beam focused ion beam - scanning electron microscope (FIB-SEM) instrument. We further demonstrate a novel approach to measure the size, shape, and distribution of the hydride phase in partially transformed films using laser scanning confocal microscopy (LSCM) to measure surface topography changes from the hydride phase volume expansion. Combining these techniques provides new insights on hydride nucleation and growth within the Mg2NiHx system. Finally, we demonstrate the efficacy of tracking deuterium as a hydrogen analog to reduce the background for SIMS imaging of hydrogen in high-vacuum chambers (∼10−6 mbar).

Substructure and crystallography of lath martensite in as-quenched interstitial-free steel and low-carbon steel

In this study, the substructures and crystallographic features of as-quenched lath martensite in interstitial-free (IF) steel and low-carbon steel (0.2C steel) were investigated using focused ion beam-scanning electron microscopy serial sectioning and advanced transmission electron microscopy. A three-dimensional analysis revealed that the morphology of the so-called lath, the smallest structural unit, exhibited a wedge-shaped plate instead of the ideal lath shape. The habit plane orientations were distributed from (5 7 5)A to (1 1 1)A in the IF steel, whereas they were near to that predicted based on the phenomenological theory of martensite crystallography in the 0.2C steel. In the IF steel, well-developed dislocation cells, tangled dislocations, and curved dislocations could be observed. Conversely, straight dislocations and cementite formation were observed in the 0.2C steel. Moreover, the orientation changed significantly inside a given lath in both steels. The lath boundary comprised regularly spaced steps with (0 1 1)M atomistic terrace planes. Interfacial dislocations corresponding to the discontinuity of the (0 1 1)M lattice planes could be observed at steps, and these dislocations were introduced to compensate for the misorientation between adjacent laths. Additionally, carbon segregations were observed at the lath and block boundaries in the 0.2C steel. Our observation results on the dislocation structures, cementite formation, and segregation of carbon atoms on the lath and block boundaries indicated that the substructure changed significantly during quenching. Based on our experimental results, we discuss the origin of the substructures and crystallographic features of lath martensite.

Biomimetic Nanopillar Silicon Surfaces Rupture Fungal Spores.

The mechano-bactericidal action of nanostructured surfaces is well-documented; however, synthetic nanostructured surfaces have not yet been explored for their antifungal properties toward filamentous fungal species. In this study, we developed a biomimetic nanostructured surface inspired by dragonfly wings. A high-aspect-ratio nanopillar topography was created on silicon (nano-Si) surfaces using inductively coupled plasma reactive ion etching (ICP RIE). To mimic the superhydrophobic nature of insect wings, the nano-Si was further functionalised with trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFTS). The viability of Aspergillus brasiliensis spores, in contact with either hydrophobic or hydrophilic nano-Si surfaces, was determined using a combination of standard microbiological assays, confocal laser scanning microscopy (CLSM), and focused ion beam scanning electron microscopy (FIB-SEM). Results indicated the breakdown of the fungal spore membrane upon contact with the hydrophilic nano-Si surfaces. By contrast, hydrophobised nano-Si surfaces prevented the initial attachment of the fungal conidia. Hydrophilic nano-Si surfaces exhibited both antifungal and fungicidal properties toward attached A. brasisiensis spores via a 4-fold reduction of attached spores and approximately 9-fold reduction of viable conidia from initial solution after 24 h compared to their planar Si counterparts. Thus, we reveal, for the first time, the physical rupturing of attaching fungal spores by biomimetic hydrophilic nanostructured surfaces.

SEM3De: image restoration for FIB-SEM.

FIB-SEM (Focused Ion Beam-Scanning Electron Microscopy) is a technique to generate 3D images of samples up to several microns in depth. The principle is based on the alternate use of SEM to image the surface of the sample (a few nanometers thickness) and of FIB to mill the surface of the sample a few nanometers at the time. In this way, huge stacks of images can thus be acquired.Although this technique has proven useful in imaging biological systems, the presence of some visual artifacts (stripes due to sample milling, detector saturation, charge effects, focus or sample drift, etc.) still raises some challenges for image interpretation and analyses. With the aim of meeting these challenges, we developed a freeware (SEM3De) that either corrects artifacts with state-of-the-art approaches or, when artifacts are impossible to correct, enables the replacement of artifactual slices by an in-painted image created from adjacent non-artifactual slices. Thus, SEM3De improves the overall usability of FIB-SEM acquisitions. SEM3De can be downloaded from https://sourceforge.net/projects/sem3de/ as a plugin for ImageJ.

Types and microstructures of pores in shales of the Ordovician Wulalike Formation at the western margin of the Ordos Basin, China

Shale samples from the Ordovician Wulalike Formation at the western margin of the Ordos Basin are studied to define the types, microstructures and connectivity of pores as well as the relationships between the pore structures and gas content of the samples by using experimental techniques such as high-resolution field emission scanning electron microscopy (FESEM), mercury injection capillary pressure (MICP), low-temperature nitrogen adsorption (LTNA), CO2 adsorption, and focused ion beam scanning electron microscopy (FIB-SEM). The results show that the shale has 10 different lithofacies, typical mixed sedimentary characteristics, and poorly developed pores. The reservoir space mainly consists of intercrystalline pores, dissolution pores, intergranular pores, and micro-fissures, with organic pores occasionally visible. The pore size is mostly within 0.4–250 nm range but dominated by micropores and mesopores less than 20 nm, with pore numbers peaking at pore sizes of 0.5 nm, 0.6 nm, 0.82 nm, 3 nm, and 10 nm, respectively. The pores are poorly connected and macropores are rarely seen, which may explain the low porosity and low permeability of the samples. Samples with high content of organic matter and felsic minerals are potential reservoirs for oil and gas with their favorable physical properties and high connectivity. The pores less than 5 nm contribute significantly to the specific surface area and serve as important storage space for adsorbed gas.

Three-dimensional architecture and assembly mechanism of the egg-shaped shell in testate amoeba Paulinella micropora.

Unicellular euglyphid testate amoeba Paulinella micropora with filose pseudopodia secrete approximately 50 siliceous scales into the extracellular template-free space to construct a shell isomorphic to that of its mother cell. This shell-constructing behavior is analogous to building a house with bricks, and a complex mechanism is expected to be involved for a single-celled amoeba to achieve such a phenomenon; however, the three-dimensional (3D) structure of the shell and its assembly in P. micropora are still unknown. In this study, we aimed to clarify the positional relationship between the cytoplasmic and extracellular scales and the structure of the egg-shaped shell in P. micropora during shell construction using focused ion beam scanning electron microscopy (FIB-SEM). 3D reconstruction revealed an extensive invasion of the electron-dense cytoplasm between the long sides of the positioned and stacked scales, which was predicted to be mediated by actin filament extension. To investigate the architecture of the shell of P. micropora, each scale was individually segmented, and the position of its centroid was plotted. The scales were arranged in a left-handed, single-circular ellipse in a twisted arrangement. In addition, we 3D printed individual scales and assembled them, revealing new features of the shell assembly mechanism of P. micropora. Our results indicate that the shell of P. micropora forms an egg shape by the regular stacking of precisely designed scales, and that the cytoskeleton is involved in the construction process.

Freestanding Ultrathin Precisely Structured Hierarchical Porous Carbon Blackbody Film for Efficient Solar Interfacial Evaporation

Solar Interfacial Evaporation In article number 2200803, Binyuan Zhao, Weiping Wu, and co-workers demonstrated high performance solar interfacial evaporation with novel porous carbon with precisely regulating internal pores, revealed by synchrotron X-ray ptychography and scanning electron microscope-focused ion beam 3D reconstructions. Large area, ultra-black, porous graphene surface patterned by femtosecond laser led to even faster solar thermal vapor generation (2.12 kg m−2 h−1 under 1 sun).

Automated large volume sample preparation for vEM.

New developments in electron microscopy technology, improved efficiency of detectors, and artificial intelligence applications for data analysis over the past decade have increased the use of volume electron microscopy (vEM) in the life sciences field. Moreover, sample preparation methods are continuously being modified by investigators to improve final sample quality, increase electron density, combine imaging technologies, and minimize the introduction of artifacts into specimens under study. There are a variety of conventional bench protocols that a researcher can utilize, though most of these protocols require several days. In this work, we describe the utilization of an automated specimen processor, the mPrep™ ASP-2000™, to prepare samples for vEM that are compatible with focused ion beam scanning electron microscopy (FIB-SEM), serial block face scanning electron microscopy (SBF-SEM), and array tomography (AT). The protocols described here aimed for methods that are completed in a much shorter period of time while minimizing the exposure of the operator to hazardous and toxic chemicals and improving the reproducibility of the specimens' heavy metal staining, all without compromising the quality of the data acquired using backscattered electrons during SEM imaging. As a control, we have included a widely used sample bench protocol and have utilized it as a comparator for image quality analysis, both qualitatively and using image quality analysis metrics.

Characteristic and Mechanistic Investigation of Stress-Assisted Microbiologically Influenced Corrosion of X80 Steel in Near-Neutral Solutions.

The behavior and mechanisms of the stress-assisted microbiologically influenced corrosion (MIC) of X80 pipeline steel induced by sulfate-reducing bacteria (SRB) were investigated using focused ion beam-scanning electron microscopy (FIB). Electrochemical results show that SRB and stress have a synergistic effect on the corrosion of X80 steel. SRB accelerated the transformation of Fe3O4 into iron-sulfur compounds and may have caused the film breakage of X80 steel products. The obtained FIB results provide direct evidence that SRB promotes the corrosion of X80 steel.

IMAGE-IN: Interactive web-based multidimensional 3D visualizer for multi-modal microscopy images.

Advances in microscopy hardware and storage capabilities lead to increasingly larger multidimensional datasets. The multiple dimensions are commonly associated with space, time, and color channels. Since "seeing is believing", it is important to have easy access to user-friendly visualization software. Here we present IMAGE-IN, an interactive web-based multidimensional (N-D) viewer designed specifically for confocal laser scanning microscopy (CLSM) and focused ion beam scanning electron microscopy (FIB-SEM) data, with the goal of assisting biologists in their visualization and analysis tasks and promoting digital workflows. This new visualization platform includes intuitive multidimensional opacity fine-tuning, shading on/off, multiple blending modes for volume viewers, and the ability to handle multichannel volumetric data in volume and surface views. The software accepts a sequence of image files or stacked 3D images as input and offers a variety of viewing options ranging from 3D volume/surface rendering to multiplanar reconstruction approaches. We evaluate the performance by comparing the loading and rendering timings of a heterogeneous dataset of multichannel CLSM and FIB-SEM images on two devices with installed graphic cards, as well as comparing rendered image quality between ClearVolume (the ImageJ open-source desktop viewer), Napari (the Python desktop viewer), Imaris (the closed-source desktop viewer), and our proposed IMAGE-IN web viewer.

Structure-preserving Gaussian denoising of FIB-SEM volumes

FIB-SEM (Focused Ion Beam-Scanning Electron Microscopy) is an imaging technique that allows 3D ultrastructural analysis of cells and tissues at the nanoscale. The acquired FIB-SEM data are highly noisy, which makes denoising an essential step prior to volume interpretation. Gaussian filtering is a standard method in the field because it is fast and straightforward. However, it tends to blur the biological features due to its linear nature that ignores the rapid changes of the structures throughout the volume. To address this issue, we have developed a new approach to structure-preserving noise reduction for FIB-SEM. It has abilities to locally adapt the filtering to the biological structures while taking advantage of the simplicity of Gaussian filtering. It uses the Optical Flow (OF) to estimate the variations of the structural features across the volume, so that they are compensated before the subsequent filtering with a Gaussian function. As demonstrated qualitatively and objectively with datasets from different samples and acquired under different conditions, our denoising approach outperforms the standard Gaussian filtering and is competitive with state-of-the-art methods in terms of noise reduction and preservation of the sharpness of the structures.

CLEMSite, a software for automated phenotypic screens using light microscopy and FIB-SEM.

In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume ultrastructural imaging. We present a toolset for adherent cells that enables tracking and finding cells, previously identified in light microscopy (LM), in the FIB-SEM, along with the automatic acquisition of high-resolution volume datasets. We detect the underlying grid pattern in both modalities (LM and EM), to identify common reference points. A combination of computer vision techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscope's field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5 µm accuracy while iterating between different targets and implementing unattended data acquisition. Our results demonstrate that executing volume acquisition in multiple locations autonomously is possible in EM.

Three-dimensional analysis of membrane structures associated with tomato spotted wilt virus infection.

To study viral infection, the direct structural visualization of the viral life cycle consisting of virus attachment, entry, replication, assemblyand transport is essential. Although conventional electron microscopy (EM) has been extremely helpful in the investigation of virus-host cell interactions, three-dimensional (3D) EM not only provides important information at the nanometer resolution, but can also create 3D maps of large volumes, even entire virus-infected cells. Here, we determined the ultrastructural details of tomato spotted wilt virus (TSWV)-infected plant cells using focused ion beam scanning EM (FIB-SEM). The viral morphogenesis and dynamic transformation of paired parallel membranes (PPMs) were analyzed. The endoplasmic reticulum (ER) membrane network consisting of tubules and sheets was related to viral intracellular trafficking and virion storage. Abundant lipid-like bodies, clustering mitochondria, cell membrane tubules, and myelin-like bodies were likely associated with viral infection. Additionally, connecting structures between neighboring cells were found only in infected plant tissues and showed the characteristics of tubular structure. These novel connections that formed continuously in the cell wall or were wrapped by the cell membranes of neighboring cells appeared frequently in the large-scale 3D model, suggesting additional strategies for viral trafficking that were difficult to distinguish using conventional EM.

Relationship between Reflectivity, Chemical Composition and Mechanical Behaviour of Orthodontic Bonding Nanofiller Resin Materials: A Proposal of an Alternative Method of Investigation

Background: Relationships between reflectivity, hardness and chemical composition of the dispersed phase, included in orthodontic composites Transbond XTTM (Trans), Light-Cure Orthodontic Paste (Leone) and Bisco Ortho Bracket Paste LC (Bisco), were investigated in vitro to evaluate whether reflectivity results can be useful in internal material composition interpretation, thus obtaining information on mechanical behaviours. Methods: Light transmission through 36 resin discs was measured with a UV/Vis spectrophotometer, evaluating the spectral range from 190–1100 nm. To have a benchmark of material hardness and internal composition, Vickers measurements and Cross-Sectional Focus Ion Beam Scanning Electron Microscopy (FIB/SEM) analysis were provided. Results: Bisco has the highest reflectivity, Leone shows an absorption pattern in the UV region similar to Bisco and Transbond has the lowest reflectivity compared to the others. This trend is confirmed by FIB/SEM imaging, showing a more similar induced roughness and internal composition for Bisco and Leone, with respect to Transbond. Higher filler presence in the composition of Bisco and Leone justifies a higher hardness of these two materials, with respect to Transbond, as confirmed by Vickers measurements. Conclusions: Bisco and Leone show similar optical responses and similarities in mechanical performance. This statement is explained by the lower and similar filler content as confirmed also by FIB/SEM analysis. The inner composition of Bisco and Leone provides a higher value of microhardness, as demonstrated by Vickers measurements. Therefore, this study confirms that the UV-Vis analysis can also offer a significant overview on the internal material composition, thus indirectly providing information on the mechanical properties of orthodontic composites.

Characterizing crystalline phase transitions in zeolitization of coal fly ash using focused ion beam-scanning electron microscopy

Coal fly ash (CFA) accounts for a significant portion of coal ash, which is a by-product of thermal power plants. Herein, CFA collected in Korea was used to synthesize a zeolitic material via fusion and hydrothermal synthesis. The crystallographic characteristics of CFA, alkali-fused CFA, and synthesized zeolitic material from CFA (SZC) were investigated via cross-section analysis using focused ion beam-scanning electron microscopy (FIB-SEM) with energy-dispersive X-ray spectroscopy (EDS), which has not been attempted in previous studies. Cross-section milling and EDS line scan were performed using FIB-SEM to develop the existing analysis methods, focusing on the crystalline phase and chemical composition of the surface of synthetic materials. Following the alkali treatment, the amorphous CFA exhibited a biphase comprising both crystalline and amorphous phases. After the synthesis process, both the surface and the interior exhibited a uniform Na-A zeolite crystalline structure with sharp-edged cubes. Furthermore, the SZC comprised crystals of Na-A zeolite with a uniform structure and a Si/Al molar ratio of approximately 1.4.