Focused Ion Beam Scanning Electron Microscopy Research Articles

Segmentation of cellular ultrastructures on sparsely labeled 3D electron microscopy images using deep learning.

Focused ion beam-scanning electron microscopy (FIB-SEM) images can provide a detailed view of the cellular ultrastructure of tumor cells. A deeper understanding of their organization and interactions can shed light on cancer mechanisms and progression. However, the bottleneck in the analysis is the delineation of the cellular structures to enable quantitative measurements and analysis. We mitigated this limitation using deep learning to segment cells and subcellular ultrastructure in 3D FIB-SEM images of tumor biopsies obtained from patients with metastatic breast and pancreatic cancers. The ultrastructures, such as nuclei, nucleoli, mitochondria, endosomes, and lysosomes, are relatively better defined than their surroundings and can be segmented with high accuracy using a neural network trained with sparse manual labels. Cell segmentation, on the other hand, is much more challenging due to the lack of clear boundaries separating cells in the tissue. We adopted a multi-pronged approach combining detection, boundary propagation, and tracking for cell segmentation. Specifically, a neural network was employed to detect the intracellular space; optical flow was used to propagate cell boundaries across the z-stack from the nearest ground truth image in order to facilitate the separation of individual cells; finally, the filopodium-like protrusions were tracked to the main cells by calculating the intersection over union measure for all regions detected in consecutive images along z-stack and connecting regions with maximum overlap. The proposed cell segmentation methodology resulted in an average Dice score of 0.93. For nuclei, nucleoli, and mitochondria, the segmentation achieved Dice scores of 0.99, 0.98, and 0.86, respectively. The segmentation of FIB-SEM images will enable interpretative rendering and provide quantitative image features to be associated with relevant clinical variables.

Bundling of collagen fibrils influences osteocyte network formation during bone modeling

Osteocytes form a cellular network by gap junctions between their cell processes. This network is important since intercellular communication via the network is essential for bone metabolism. However, the factors that influence the formation of this osteocyte network remain unknown. As the early stage of osteocyte network formation occurs on the bone surface, we observed a newly formed trabecular bone surface by orthogonal focused ion beam-scanning electron microscopy. The embedding late osteoblast processes tended to avoid bundled collagen fibrils and elongate into sparse collagen fibrils. Then, we examined whether the inhibition of bundling of collagen fibrils using a potent lysyl oxidase inhibitor, β-aminopropionitrile (BAPN) changed the cellular network of the chick calvaria. The osteocyte shape of the control group was spindle-shape, while that of the BAPN group was sphere-shaped. In addition, the osteocyte processes of the control group were elongated vertically to the long axis of the cell body, whereas the osteocyte processes of the BAPN group were elongated radially. Therefore, it was suggested that the bundling of collagen fibrils influences normal osteocyte network formation during bone modeling.

Characterization for 3D pore-fracture systems in Hot-Dry-Rock samples from Gonghe Basin, China

As a new type of renewable energy, Hot-Dry-Rock (HDR) is gaining increasing attention. The physical properties of rock influenced by the pore-fracture structure are vital for the hydraulic and chemical characteristics of HDR reservoirs. However, the natural pore-fracture structure in HDR has not been sufficiently studied. To fulfill this gap, we used focused ion beam-scanning electron microscopy (FIB-SEM) to construct three-dimensional (3D) digital models for four granite samples from the deep HDR reservoir in Gonghe Basin, Qinghai Province, China, and obtained the nanoscale structure of the pores and fractures in HDR reservoir for the first time. Our analysis based on the 3D models indicates that the heat transfer area in the HDR reservoir is mainly from fractures and crack-like pores, but a large amount of small but regular pores could be neglected when heat transfer in HDR is considered. The permeability of HDR reservoirs at nanoscale varies from 9.414 nD to 0.082 mD, and the high permeability in this study is accompanied by throughgoing fractures. The variation of porosity and permeability of deep HDR at nanoscale in this study is much more severe than that of shallow granites previously studied at microscale. The performance of geothermal field is significantly influenced by the size of throats and the average size and tortuosity of the interconnected pore-fracture network in HDR. The hydraulic aperture could be 32-58% lower than the mechanical aperture of the natural fractures in HDR reservoirs, which is a vital clue for the numerical simulation associated with enhanced geothermal systems.

CDK2 inhibition disorders centrosome stoichiometry and alters cellular outcomes in aneuploid cancer cells

ABSTRACT Cyclin-dependent Kinase 2 (CDK2) inhibition prevents supernumerary centrosome clustering. This causes multipolarity, anaphase catastrophe and apoptotic death of aneuploid cancers. This study elucidated how CDK2 antagonism affected centrosome stoichiometry. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent imaging were used. Studies interrogated multipolar mitosis after pharmacologic or genetic repression of CDK2. CDK2/9 antagonism with CYC065 (Fadraciclib)-treatment disordered centrosome stoichiometry in aneuploid cancer cells, preventing centrosome clustering. This caused ring-like chromosomes or multipolar cancer cells to form before onset of cell death. Intriguingly, CDK2 inhibition caused a statistically significant increase in single centrioles rather than intact centrosomes with two centrioles in cancer cells having chromosome rings or multipolarity. Statistically significant alterations in centrosome stoichiometry were undetected in other mitotic cancer cells. To confirm this pharmacodynamic effect, CDK2 but not CDK9 siRNA-mediated knockdown augmented cancer cells with chromosome ring or multipolarity formation. Notably, engineered gain of CDK2, but not CDK9 expression, reversed emergence of cancer cells with chromosome rings or multipolarity, despite CYC065-treatment. In marked contrast, CDK2 inhibition of primary human alveolar epithelial cells did not confer statistically significant increases of cells with ring-like chromosomes or multipolarity. Hence, CDK2 antagonism caused differential effects in malignant versus normal alveolar epithelial cells. Translational relevance was confirmed by CYC065-treatment of syngeneic lung cancers in mice. Mitotic figures in tumors exhibited chromosome rings or multipolarity. Thus, CDK2 inhibition preferentially disorders centrosome stoichiometry in cancer cells. Engaging this disruption is a strategy to explore against aneuploid cancers in future clinical trials.

Visualizing the Structure, Composition and Activity of Single Catalyst Particles for Olefin Polymerization and Polyolefin Decomposition.

The structural and morphological characterization of individual catalyst particles for olefin polymerization, as well as for the reverse process of polyolefin decomposition, can provide an improved understanding for how these catalyst materials operate under relevant reaction conditions. In this review, we discuss an emerging analytical toolbox of 2D and 3D chemical imaging techniques that is suitable for investigating the chemistry and reactivity of related catalyst systems. While synchrotron-based X-ray microscopy still provides unparalleled spatial resolutions in 2D and 3D, a number of laboratory-based techniques, most notably focused ion beam-scanning electron microscopy, confocal fluorescence microscopy, infrared photoinduced force microscopy and laboratory-based X-ray nano-computed tomography, have helped to significantly expand the arsenal of analytical tools available to scientists in heterogeneous catalysis and polymer science. In terms of future research, the review outlines the role and impact of in situ and operando (spectro-)microscopy experiments, involving sophisticated reactors as well as online reactant and product analysis, to obtain real-time information on the formation, decomposition, and mobility of polymer phases within single catalyst particles. Furthermore, the potential of fluorescence microscopy, X-ray microscopy and optical microscopy is highlighted for the high-throughput characterization of olefin polymerization and polyolefin decomposition catalysts. By combining these chemical imaging techniques with, for example, chemical staining methodologies, selective probe molecules as well as particle sorting approaches, representative structure-activity relationships can be derived at the level of single catalyst particles.

Local Structural Modifications in Metallic Micropillars Induced by Plasma Focused Ion Beam Processing.

A focused ion beam scanning electron microscope (FIB-SEM) is a powerful tool that is routinely used for scale imaging from the micro- to nanometer scales, micromachining, prototyping, and metrology. In spite of the significant capabilities of a FIB-SEM, there are inherent artefacts (e.g., structural defects, chemical interactions and phase changes, ion implantation, and material redeposition) that are produced due to the interaction of Ga+ or other types of ions (e.g., Xe+, Ar+, O+, etc.) with the sample. In this study, we analyzed lattice distortion and ion implantation and subsequent material redeposition in metallic micropillars which were prepared using plasma focus ion beam (PFIB) milling. We utilized non-destructive synchrotron techniques such as X-ray fluorescence (XRF) and X-ray nanodiffraction to examine the micropillars prepared using Xe+ ion energies of 10 keV and 30 keV. Our results demonstrate that higher Xe ion energy leads to higher density of implanted ions within the redeposited and milled material. The mixing of ions in the redeposited material significantly influences the lattice structure, causing deformation in regions with higher ion concentrations. Through an X-ray nanodiffraction analysis, we obtained numerical measurements of the strain fields induced in the regions, which revealed up to 0.2% lattice distortion in the ion bombardment direction.

The 3D organization of the mineralized scales of the sturgeon has structures reminiscent of dentin and bone: a FIB-SEM study

Scales are structures composed of mineralized collagen fibrils embedded in the skin of fish. Here we investigate structures contributing to the bulk of the scale material of the sturgeon (Acipencer guldenstatii) at the millimeter, micrometer and nanometer length scales. Polished and fracture surfaces were prepared in each of the three anatomic planes for imaging with light and electron microscopy, as well as focused ion beam – scanning electron microscopy (FIB-SEM). The scale is composed of three layers, upper and lower layers forming the bulk of the scale, as well as a thin surface layer. FTIR shows that the scale is composed mainly of collagen and carbonated hydroxyapatite. Lacunae are present throughout the structure. Fracture surfaces of all three layers are characterized by large diameter collagen fibril bundles (CFBs) emanating from a plane comprising smaller diameter CFBs orientated in different directions. Fine lineations seen in polished surfaces of both major layers are used to define planes called here the striation planes. FIB-SEM image stacks of the upper and lower layers acquired in planes aligned with the striation planes, show that CFBs are oriented in various directions within the striation plane, with larger CFBs emanating from the striation plane. Fibril bundles oriented in different directions in the same plane is reminiscent of a similar organization in orthodentin. The large collagen fibril bundles emanating out of this plane are analogous to von Korff fibrils found in developing dentin with respect to size and orientation. Scales of the sturgeon are unusual in that their mineralized collagen fibril organization contains structural elements of both dentin and bone. The sturgeon scale may be an example of an early evolved mineralized material which is neither bone nor dentin but contains characteristics of both materials, however, the fossil data required to confirm this is missing.

Intraoral low-temperature degradation of monolithic zirconia dental prostheses: 5-year results of a prospective clinical study with ex vivo monitoring

ObjectivesTo investigate the 5-year intraoral evolution and kinetics of low-temperature degradation (LTD) of second-generation monolithic prostheses made of 3% molar yttrium-doped tetragonal zirconia polycrystal (3Y-TZP) and the influence of masticatory mechanical stresses and glaze layer on this evolution. MethodsA total of 101 posterior tooth elements were included in this prospective clinical study, which comprised ex vivo LTD monitoring (at baseline, 6 months, 1 year, 2 years, 3 years, and 5 years) using Raman spectroscopy (n = 2640 monoclinic phase measurement points per evaluation time) and scanning electron microscopy (SEM). Four types of areas (1–2 mm2 surface, six on molars, and four on premolars) were analysed on each element surface: occlusal, axial, glazed, or unglazed. Raman mapping, high-resolution SEM, and focused ion beam-SEM were performed on selected samples. ResultsThe dental prostheses developed a tetragonal-to-monoclinic transformation at the extreme surface of the material after six months in a buccal environment, and this process increased significantly over time. Over the five years of monitoring, the transformation developed nonuniformly with the presence of localised clusters of monoclinic grains. Tribological stresses generate grain pull-out from these clusters, which may raise questions regarding the release of 3Y-TZP nanoparticles into the body. The prosthesis fracture rate was 4.5% after 5 years. SignificanceLTD developed in vivo on the surfaces of 3Y-TZP dental prostheses and progressed slowly but significantly over time, up to 5 years investigation. However, the effects of aging on the failure rate recorded and of zirconia nanoparticles released into the body require further investigation.

Three-dimensional architecture of granulosa cell derived from oocyte cumulus complex, revealed by FIB-SEM

The oocyte cumulus complex is mainly composed of an oocyte, the perivitelline space, zona pellucida and numerous granulosa cells. The cumulus granulosa cells (cGCs) provide a particularly important microenvironment for oocyte development, regulating its growth, maturation and meiosis. In this study, we studied the internal structures and cell-to-cell connections of mouse cGCs using focused ion beam scanning electron microscopy (FIB-SEM). We reconstructed three-dimensional models to display characteristic connections between the oocyte and cGCs, and to illustrate various main organelles in cGCs together with their interaction relationship. A special form of cilium identified in granulosa cell was never reported in previous literature.

Bone Hierarchical Structure: Heterogeneity and Uniformity

AbstractBone has a complex hierarchical structure with structural integration from nm to cm. The understanding of bone structure is developing rapidly due to improvements in available methodologies that allow unravelling structures across several length scales. These methods include advances in electron microscopy, in particular, focused ion beam scanning electron microscopy (FIB‐SEM), confocal laser scanning microscopy techniques, X‐ray imaging, X‐ray diffraction tomography (XRD‐CT), and tensor tomography (small angle X‐ray scattering tensor tomgraphy, SAXS‐TT and wide angle X‐ray scattering tensor tomgraphy, WAXS‐TT). Special emphasis is placed on the latter X‐ray techniques that are emerging into powerful tools. Through a review of selected recent results on the structure of the bone matrix as well as the lacuno‐canalicular network housing the osteocyte cells of bone, it is proposed that bone is more heterogeneous than typically described and that local variation in composition and crystallography may play a significant role in bone biology in health and disease.

Use of Heat-killed Aspergillus ochraceus NRRL 3174 Discs as Biosorbent for petroleum Removal

The purpose of this work was to evaluate the petroleum sorption capacity of heat-killed fungal discs obtained from Aspergillus ochraceus strain. Effect of various parameters such as biosorbent dose (0.5g-2.5g/100mL), petroleum concentration (0.5-5%), pH (4.0-8.0), contact time (1-12h) and re-usability of biosorbent (1-6) were investigated. Accordingly, the highest biosorption capacity was obtained with 1% petroleum concentration, 1.5 g/100mL heat-killed fungal discs, 10h contact time at pH: 5.0 and room temperature. Additionally, each disc was able to actively use for at least 6 more cycles in biosorption experiments. The specific removal rate was calculated as 0.114 day−1, the rate constant and half-life period were also 1.609 day-1, t1/2 = 0.431, respectively. The kinetic study was described by the pseudo-second order model and the equilibrium modeling was found to be well fitted with Langmuir isotherm. The biosorbent(s) were characterized by Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). Over 80% removal of long-chain n-alkanes by the heat-killed fungal discs was confirmed by GC-MS analysis. Since there has been no similar study investigating the sorption of petroleum with heat-killed Aspergillus ochraceous discs, this novel bio-based sorbent with its low cost, environmentally friendly and easy-to-apply properties can be used in advanced biosorption studies.

Revealing the aging process of solid electrolyte interphase on SiOx anode

As one of the most promising alternatives to graphite negative electrodes, silicon oxide (SiOx) has been hindered by its fast capacity fading. Solid electrolyte interphase (SEI) aging on silicon SiOx has been recognized as the most critical yet least understood facet. Herein, leveraging 3D focused ion beam-scanning electron microscopy (FIB-SEM) tomographic imaging, we reveal an exceptionally characteristic SEI microstructure with an incompact inner region and a dense outer region, which overturns the prevailing belief that SEIs are homogeneous structure and reveals the SEI evolution process. Through combining nanoprobe and electron energy loss spectroscopy (EELS), it is also discovered that the electronic conductivity of thick SEI relies on the percolation network within composed of conductive agents (e.g., carbon black particles), which are embedded into the SEI upon its growth. Therefore, the free growth of SEI will gradually attenuate this electron percolation network, thereby causing capacity decay of SiOx. Based on these findings, a proof-of-concept strategy is adopted to mechanically restrict the SEI growth via applying a confining layer on top of the electrode. Through shedding light on the fundamental understanding of SEI aging for SiOx anodes, this work could potentially inspire viable improving strategies in the future.

Enhanced mechanical and functional properties of chitosan/polyvinyl alcohol/hydroxypropyl methylcellulose/alizarin composite film by incorporating cinnamon essential oil and tea polyphenols

In this study, cinnamon essential oil and tea polyphenols were added to chitosan/ polyvinyl alcohol/ hydroxypropyl methylcellulose/ alizarin composite films to enhance their mechanical and functional properties. Their addition to the composite films enhanced their antibacterial and antioxidant properties and significantly improved its elongation at break (p < 0.05). Cinnamon essential oil reduced the water vapor permeability, water content, and water solubility of composite films and improved their transparency. The composite films with additive exhibited excellent UV-barrier ability and pH responsivity. Fourier Transform infrared spectroscopy and X-Ray Diffraction analyses confirmed hydrogen bond formation between the polymer molecules and additives. The results of Scanning Electron Microscope-Focused Ion Beam revealed improved surface and cross-section morphology of the films, leading to the generation of a cross-linked structure. Thermogravimetric and differential scanning calorimetry analysis indicated enhanced thermal stability of the composite films upon cinnamon essential oil addition. Analysis of storage quality indicators (TBARS value, TVC, and TVB-N) revealed that the composite films could prolong the freshness of surimi. The incorporation of cinnamon essential oil and tea polyphenols into the composite films has demonstrated significant potential as an effective and natural alternative for active food packaging.

Porosity evolution in proton irradiated microfine-grained POCO graphite

This work investigated the porosity evolution of POCO ZXF-5Q graphite that has been irradiated by 340 kW, 120 GeV protons inside NT02 target system in Fermilab's NuMI beamline. This POCO graphite has undergone direct bulk dimensional swelling at low dose irradiation and its local microstructural change is still not well-understood during this process. In this work, the (sub-) micrometre scale porosity from six locations across proton beam fluence and temperature gradients have been studied using focused ion beam-scanning electron microscopy (FIB-SEM) tomography. A deep learning-based tomographic image segmentation technique has been established and implemented for porosity segmentation and quantification. It has been found that there is a decrease in the total volumetric percentage of the porosity at proton beam centre (∼ 8 – 8.4 vol.%), by comparing to un-irradiated POCO (∼ 12 – 13 vol.%) and to beam 2σ and 5σ radii (∼ 12 vol.%). This decrease in porosity volume percentage was found to be caused by the reduction in pores with volumes > 0.1 μm3 induced by material bulk dimensional swelling at proton beam centre area. The porosity reduction in relation to dimensional change and irradiation creep was discussed among with other contributing factors, and further investigations through well-controlled irradiation experiment are still needed.

Three dimensional microstructures of carbon deposition on Ni-YSZ anodes under polarization

In the present study, two Ni/YSZ anodes with different volume ratios of Ni and YSZ, 30: 70 and 45: 55 vol%, are operated in dry methane under open circuit and polarized conditions. Three-dimensional (3D) Ni/YSZ microstructures after carbon deposition are reconstructed by the focused ion beam-scanning electron microscopy (FIB-SEM) with the help of machine learning segmentation. From the reconstructed mircostructures, volume fraction, connectivity, three phase boundary (TPB) density, and tortuosity are quantified. In addition, local carbon microstructures are quantitatively reconstructed, and the effect of polarization on carbon morphology is investigated. It is demonstrated that Ni surface in the vicinity of active TPB near the electrolyte is free from carbon formation, while remaining Ni surface at some distances from TPB exhibits severe carbon deposition. In average, total amount of carbon deposition is larger near the electrolyte. These observations imply complex interplay between the electrochemical steam generation and methane cracking on Ni surface which take place very locally near the active TPB.

3D relationship between hierarchical canal network and gradient mineralization of shark tooth osteodentin.

Osteodentin is a dominant mineralized collagenous tissue in the teeth of many fishes, with structural and histological characteristics resembling those of bone. Osteodentin, like bone, comprises osteons as basic structural building blocks, however, it lacks the osteocytes and the lacuno-canalicular network (LCN), which are known to play critical roles in controlling the mineralization of the collagenous matrix in bone. Although numerous vascular canals exist in osteodentin, their role in tooth maturation and the matrix mineralization process remain poorly understood. Here, high resolution micro-computed tomography (micro-CT) and focused ion beam-scanning electron microscopy (FIB-SEM) were used to obtain 3D structural information of osteodentin in shark teeth at multiple scales. We observed a complex 3D network of primary canals with a diameter ranging from ∼10 µm to ∼120 µm, where the canals are surrounded by osteon-like concentric layers of lamella, with 'interosteonal' tissue intervening between neighboring osteons. In addition, numerous hierarchically branched secondary canals extended out radially from the primary canals into the interosteonal tissue, with a decreasing diameter from ∼10 µm to hundreds of nanometers. Interestingly, the mineralization degree increases from the periphery of primary canals to the interosteonal tissue, suggesting that mineralization begins in the interosteonal tissue. Correspondingly, the hardness and elastic modulus of the interosteonal tissue is higher than those of the osteonal tissue. These results demonstrate that the 3D hierarchical canal network is positioned to play a critical role in controlling the gradient mineralization of osteodentin, also providing valuable insight into the formation of mineralized collagenous tissue without osteocytes and LCN. STATEMENT OF SIGNIFICANCE: : Bone is a composite material with versatile mechanical properties. Osteocytes and lacuno-canalicular network (LCN) are kown to play critical roles during formation of human bone. However, the skeleton of many fishes, including bone and osteodentin, although lacking osteocytes and LCN, could exhibit similar osteon-like structure and mechanical functions. Here, using various high resolution 3D characterization techniques, we reveal that the 3D network of primary canals and numerous hierarchically branched secondary canals correlate with the mineralization gradient and micromechanical properties of osteonal and interosteonal tissues of shark tooth osteodentin. This work significantly improves our understanding of the construction of bone-like mineralized tissue without osteocytes and LCN, and provides inspirations for the fabrication of functional materials with hierarchical structure.

An interactive deep learning-based approach reveals mitochondrial cristae topologies.

The convolution of membranes called cristae is a critical structural and functional feature of mitochondria. Crista structure is highly diverse between different cell types, reflecting their role in metabolic adaptation. However, their precise three-dimensional (3D) arrangement requires volumetric analysis of serial electron microscopy and has therefore been limiting for unbiased quantitative assessment. Here, we developed a novel, publicly available, deep learning (DL)-based image analysis platform called Python-based human-in-the-loop workflow (PHILOW) implemented with a human-in-the-loop (HITL) algorithm. Analysis of dense, large, and isotropic volumes of focused ion beam-scanning electron microscopy (FIB-SEM) using PHILOW reveals the complex 3D nanostructure of both inner and outer mitochondrial membranes and provides deep, quantitative, structural features of cristae in a large number of individual mitochondria. This nanometer-scale analysis in micrometer-scale cellular contexts uncovers fundamental parameters of cristae, such as total surface area, orientation, tubular/lamellar cristae ratio, and crista junction density in individual mitochondria. Unbiased clustering analysis of our structural data unraveled a new function for the dynamin-related GTPase Optic Atrophy 1 (OPA1) in regulating the balance between lamellar versus tubular cristae subdomains.

The fate of mitochondria during platelet activation

AbstractBlood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam–scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction.

Fluid Flow in the Gas Diffusion Layer Using Computational Fluid Dynamics and Microscopy Techniques

The utilization of a proton exchange membrane fuel cell (PEMFC) as an energy provider using hydrogen as a fuel has increased drastically. The reasons are the current limitation of fossil fuel-based devices, pollutants-free, high efficiency, and zero-carbon emission. The life-cycle assessment results of this type of fuel cell have also indicated the lowest contribution to global warming, human health, and resource scarcity in comparison to other types of fuel cells. In this regard, improving the performance of PEMFC is of importance.As an important component of the PEMFC, the gas diffusion layer (GDL) transports the gas reactants to the catalyst layer with the least electrical resistance. The GDL is often made of carbon fibers and should have a surface with good electrical contact and hydrophobic properties to facilitate the water removal. Remaining water in the GDL will result in difficulties during the cold-start and enhances the degradation of this layer. It is believed that the water removal of the GDL has a direct relationship with the capillary pressure, which is strongly linked to the GDL’s microstructure and wettability. However, further investigations can be done once a comprehensive simulation model is developed to monitor the changes in the GDL liquid removal by the effective parameters.In this regard, the Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) has been used to perform three-dimensional cross-section imaging of the GDL. The GDL samples are provided by Freudenberg in which the Microporous layer (MPL) is impregnated into the GDL. The average porosity of this sample is in the range of 75% while having the in-plane gas permeability of 2.4 under a compressive stress of 1 MPa. The utilized resin for FIB-SEM imaging is a mixture of Epoxy embedding medium (diglycidyl ether of bisphenol – A) with two different hardeners DDSA (2-Dodecenylsuccinic anhydride) and MNA (Methylnadic anhydride), which will be mixed with DPM – 30 [2,4,6 – Tris(dimethylaminomethyl)phenol] as the accelerator, all provided by Sigma Aldrich. After the preparation of the resin, 0.4 gr of Cobalt (II) acetylacetonate nanoparticles (supported by Sigma Aldrich) were added to 7.14 ml of the resin to improve the contrast of the images. The samples were impregnated with the resin under vacuum to be pressurized (3 MPa) for 20 minutes, and afterward, heated in an oven at 60℃ for 12 hours. The surfaces for analyses were cut by a diamond wire, polished by abrasive plates down to 0.1 and gold coated (20nm).FIB-SEM acquisition consisted of the polishing of cross-sections with a focused ion beam (LMIS Ga+ source) at 30 keV and 1 nA (Zeiss Crossbeam 540), followed by imaging with an electron beam at acceleration voltages of 1.0, 3.0 kV. Milling and imaging were performed at a working distance of 5.2 mm and stage tilt of 54°, i.e., the coincidence point of the electron and ion beams.Once the three-dimensional cross-sections of the GDL are obtained, the segmentation and reconstruction will be made to provide the needed geometry for fluid flow simulation and to calculate the microstructural properties. Fig. 1 shows the generated structure of the GDL after the segmentation and reconstruction of the cross-section images. The geometry will be then used to characterize the effective parameters of the thermal/water management of the PEMFC. This study can also be a valid reference for future computational fluid dynamic analyses in the GDL using the numerical modeling with the conservative equations or the Lattice Boltzmann modeling (LBM) with the kinetic and particle distribution equations. Keywords : Proton exchange membrane fuel cell (PEMFC); Gas Diffusion Layer (GDL); Focused Ion Beam- Scanning Electron Microscopy (FIB-SEM); Three-dimensional simulation; Lattice Boltzmann method (LBM) Figure 1

Synergistic Application of Machine Learning to Microstructural Characterization on Electrode Composites of Solid Oxide Fuel Cells

Solid oxide fuel cells (SOFCs) have been recognized as one of the most potent next-generation energy conversion systems, owing to their higher efficiency, better fuel flexibility, and smaller amount of pollution, unlike their counterpart fuel cells. SOFCs can be analyzed as a highly-complex multi-component materials system consisting of porous electrodes (cathodes and anodes), densified electrolytes, interconnects, and sealing materials, which are sequentially layered and characteristic of heterogeneous and multi-phase materials. The resultant power generation is achieved through the electrochemical reactions involving electronic carriers, ionic carries, and gaseous transports at anodes and cathodes, influencing their electrical, mechanical, and thermal properties. In particular, both cathodes and anodes are typically constructed based on three components: mixed ionic/electronic electrodes, electrolytes, and a porous section. Those three constituents should be optimized at both electrodes with the aim of maximizing the electrochemical reaction sites, i.e., triple-phase boundaries in association with the gaseous transport involved in oxygen reduction reactions at cathodes and hydrogen oxidation reactions at anodes, which leads to high cell performance and guaranteed durability. The electrochemical performances are crucially dependent on their complex microstructures, demanding for highly informative analyses on the electron microscopy images under investigation.This work reports quantitative microstructural analysis methodology incorporating machine learning-assisted microstructural analysis strategy. Quantitative microstructural interpretations were carried out without human involvement through an integrated combination of deep learning and focused ion beam-scanning electron microscopy (FIB-SEM) analytics on Ni/Y2O3-stabilized ZrO2 (Ni/YSZ) cermets. The Ni/YSZ/pore composites were analyzed for the automated extraction of microstructural parameters with the aim to preventing the subjective analysis problems and unavoidable artifacts frequently encountered in lengthy image processing tasks and eliminate biased evaluations. Considering the high volume of image data and future expectations for electron microscopy usage, FIB-SEM was efficiently combined with semantic segmentation targeted at detailed phase recognition on three constituents, i.e, Ni, YSZ, and pores. Traditional image processing analysis tools are in the synergistic manner combined with phase separation predictions by semantic segmentation algorithms, allowing for a quantitative evaluation of microstructural parameters. The combined strategy enables one to significantly enhance poor image quality originating from artifacts in electron microscopy, including charging effects, curtain effects, out-of-focus problems, and unclear phase boundaries encountered in searching for high-efficiency solid oxide fuel cells (SOFCs).