Improving FIB-SEM Reconstructions by Using Epoxy Resin Embedding

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Accurate characterization of wood microstructure requires high-quality sample preparation, especially for degraded, mineralized, or embedded samples. Through a detailed and systematic investigation, we suggest a simple, fast, and cost-effective method for preparing transverse surfaces of wood for scanning electron microscopy. The methodology used is easily accessible and simple. We compare traditional microtome cutting using both disposable blades and a fixed knife with sanding and polishing techniques. The choice of method is determined by the physical condition and structural characteristics of the sample. Disposable blades, when used with continuous moistening, yielded the best results for reference wood, while the fixed knife proved to be more effective for mineralized or epoxy-embedded samples due to their rigidity and resistance to deflection. Maintaining blade sharpness and surface moisture were critical in all cutting techniques. Polishing proved to be a particularly effective technique for the preparation of degraded, mineral-rich, or heterogeneous samples. In combination with epoxy embedding, it offers a practical alternative to microtome cutting, ensuring the protection of fragile structures, the long-term stability of samples, and the possibility of re-polishing. Epoxy impregnation was easy for degraded wood, while sprucewood required vacuum-pressure treatment. Care must be taken when polishing to avoid reaching non-embedded regions beneath the surface.

Low-field NMR (LF-NMR) is a widely applied technique for evaluating pore size distribution (PSD) in porous materials. Conventional approaches typically assume surface-controlled spin-spin relaxation and negligible diffusion contributions under the fast-diffusion regime, which introduces systematic errors when applied to nano- and microporous systems. In this work, we present the Effective Diffusion Cubic (EDC) model, a new framework for LF-NMR-based PSD estimation in tight rocks. The EDC method incorporates pore-size dependence of both the effective diffusion coefficient and the induced internal magnetic field gradient. Crucially, the effective diffusion coefficient, D(d), is parameterized by a logistic function that faithfully approximates the Padé form, enabling a precise quantification of diffusion-related effects on T2 relaxation. Applied to nine siliciclastic core samples, the EDC approach produced PSDs corrected for diffusion-induced distortions and in closer agreement with independent reference data compared to conventional models. These results demonstrate that the EDC methodology provides a physically consistent and more accurate means of quantifying pore systems, thereby enhancing NMR-based petrophysical characterization of tight rock formations.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-20379-x.

An effective way to reduce the large quantities of Pu currently accumulated worldwide would be to use uranium-free fuel in light water reactors (LWRs) so that no new Pu is produced. Such a possibility could be provided by an LWR fuel consisting of Pu in a neutronically inert matrix. It may be necessary to add a burnable absorber or thorium to reduce the reactivity swing during burnup. The methods and data currently used for LWR analyses have not been tested in conjunction with such exotic fuel materials. An international exercise has accordingly been launched to compare the relative performance of different code systems and the accuracy of the basic data. Comparison of the results of cell calculations done with fixed isotopic densities against reference Monte Carlo results shows fairly small but systematic differences in the multiplication factors. A sensitivity analysis done with different basic cross section libraries and the same code system allows one to distinguish between the effects of the codes and those of the databases.The results of the burnup calculations indicate a fair agreement in k∞ both at beginning of life (BOL) and after 1200 days of irradiation [end of life (EOL)] under conditions representative of a present-day pressurized water reactor. At BOL, the fuel temperature coefficients agree fairly well among the different contributions, but unacceptably large differences are observed at EOL. The void coefficients agree well for low voidage, but for void fractions >90%, there are significant effects mostly due to the databases used. The agreement in the calculated boron worths is good.

Correlative light and electron microscopy (CLEM) is an analytical technique used to evaluate the correlation between fluorescence images of identical samples and regions of fluorescent-labeled organelles captured using light microscopy and electron microscopy. By utilizing various methods during sample preparation, the fluorescence images of fluorescent-labeled organelles are juxtaposed with ultra-structures of the same region obtained using scanning electron microscopy (SEM) or transmission electron microscopy. Unlike animal tissues and adherent cells, visualizing the organelles detected in plant samples via CLEM analysis is difficult because of the non-adherence of plant tissue and cultured cells to glass plates and the size of the cells. Therefore, we combine the serial section SEM method (Array tomography) based on ultra-thin sections and CLEM to identify fluorescent-labeled organelles in plant cells and reconstruct them in three dimensions (3D). In particular, as osmium (Os) epoxy-tolerant fluorescent proteins have recently been reported to be resistant to Os fixation and epoxy resin embedding in animal adherent cells, we applied these fluorescent tags to plant samples. In this paper, we introduce the sample preparation method for the CLEM analysis of organelles labeled with Os epoxy-tolerant fluorescent proteins in plant tissues, alongside the method for obtaining CLEM images and the combined 3D-CLEM analysis method comprising CLEM and array tomography.

Comparison of reconstructed spatial microstructure images using different statistical descriptors

Performance and durability of cathodes have been one of the main factors impeding commercialization of fuel cells for automotive applications. This work reports an optimized Rotating Disk Electrode (RDE) technique and a mass activity and durability for a baseline Pt/Graphitized Carbon (Pt/GC). An accelerated stress test (AST) is performed to determine the durability of the catalysts by cycling 30,000 times between 0.6 and 1.0V with a 1 sec hold at each potential. Mass activity and electrochemical surface area are measured at the beginning of life (BOL) and at the end of life (EOL) after 30,000 cycles. The BOL mass activity of the Pt/GC baseline is 218 A/g-Pt. In-house prepared Pt-Ni-Co ternary alloys show a BOL mass activity of 600-900 A/g-Pt for the best performing catalysts not shown in this report. However optimization of post heat and acid treatment of catalysts is necessary to improve the durability. The optimum temperature for the initial and final post heat treatment is found to be 400 oC for the ternary alloy catalysts. The heat-acid-heat treated catalyst is more durable showing a 52% degradation rate (EOL: 292 A/g-Pt) compared to 75% (EOL: 196 A/g-Pt) for the heat-acid treated catalysts.

The high demand on specific capacity and occasionally harsh environment in electric vehicles has detrimental effects on electrochemical, mechanical and thermal properties of NMC/graphite Li-ion batteries. The battery lifetime is reduced at high voltage and high temperature, both in terms of capacity fade and impedance increase. At the graphite electrode, the notion of SEI layer formation and growth leading to battery aging is widely accepted. On the other hand, aging effects on the NMC electrode needs to be investigated more thoroughly. Xiong et al. [1] reported that Li-ion cells could experience rapid failure due to impedance rise at the NMC electrode when cycled at an elevated temperature or a cut-off voltage above 4.2 V. A surface film formation for transition metal oxides, the growth of which accelerates upon high temperature and charge voltages, has been reported [2,3]. Literature also shows that the interaction between the positive and negative electrodes in full lithium-ion cells contribute to declined cell performance [4]. However, these aging mechanisms are not independently distinguishable. Therefore, it is important to be able to quantify each contribution and to decouple different aging mechanisms occurring on each electrode at different temperatures under high voltage condition. This study focuses on impedance behavior of commercial NMC111 (LiNi1/3Mn1/3Co1/3O2) and graphite electrodes in a small laboratory pouch cells. The NMC111/graphite in full-cell configuration are calendar aged and cycle aged between +3.8 V and +4.6 V vs. Li at 25 °C and 40 °C until 50% capacity fade is reached. Capacity and impedance behavior of the full cells are measured periodically in order to track the performance changes with aging. Each electrode is harvested at specific intervals of cycling stages and electrochemically characterized in symmetric cells by means of electrochemical impedance spectroscopy (EIS). A physics-based model is employed to parameterize aging parameters by fitting the EIS results from the symmetric cells using a least square fitting tool in COMSOL Multiphysics. The pseudo two-dimensional (P2D) model is an extension to the work of Doyle et al. [5] incorporating intercalation kinetics, mass and charge transport based on concentrated solution porous electrode theory. From the preliminary EIS results on NMC111 symmetric cells in Fig. 1, impedance increases at a small magnitude at 25 °C at the end of life (EOL) compared to the beginning of life (BOL). At 40 °C, however, large impedance rise at EOL is seen. On the other hand, it had been shown that capacity fade occurs at both temperatures during slow cycling on half-cells. The model optimization is firstly done for cells at BOL to deduce the aging-independent parameters. Towards EOL, the quantification of intrinsic physical aging properties such as the particle sizes, porosity, local contact resistance and surface film resistance which are the causes of impedance rise or capacity fade can be made. The potential effects of calendar aging on cell degradation is separated from the effects of cycle aging. The comparison with experimental capacity loss and impedance rise will further validate the model. The extraction of the parameters from the model enables deduction of different aging-induced mechanisms at each electrode. This quantitative analysis of aging behavior will contribute to improved lifetime predictive model for NMC111/graphite lithium-ion batteries. [1] D. J. Xiong, R. Petibon, M. Nie, L. Ma, J. Xia, J.R. Dahn, J. Electrochem. Soc. 163 (2016), 3, A546-A551 [2] P. Niehoff, M. Winter, Langmuir 29 (2013), 51, 15813-15821 [3] M. Wohlfahrt-Mehrens, C. Vogler, J. Garche, J. Power Sources 127 (2004) 58-64 [4] K. Xu, Chem. Rev. 114 (2014) 11503-11618 [5] M. Doyle, J. Newman, A. C. Gozdz, C. N. Schmutz, J.-M. Tarascon, J. Electrochem. Soc. 143 (1996) 1890–1903. Figure 1

Ordered cubic mesoporous silicas with large pore sizes (LP-SBA-16) have been successfully synthesized at high temperatures (180-220 degrees C) using polymer surfactant of F127 as a template. Compared with conventional SBA-16 with entrance size at 3.6 nm, LP-SBA-16 samples synthesized at high temperatures show large entrance sizes at 11.8-24.7 nm, confirmed by the pore size distribution and TEM images. The formation of these large pore sizes in the samples is attributed to the increase of surfactant hydrophobicity and the merging of mesopores at high temperatures. The results obtained from NMR and XRD techniques show that ordered mesostructure in LP-SBA-16 samples basically remained even if the surfactant (F127) is fully decomposed at high temperatures, indicating that F127 surfactant becomes unimportant when the mesoporous walls of silica have been condensed. Furthermore, we have compared the adsorption capacity of myoglobin over conventional SBA-16 and LP-SBA-16 samples, and it is found that LP-SBA-16 samples exhibit much higher adsorption capacity than conventional SBA-16, which is potentially important for immobilization of enzymes on ordered mesoporous materials.

Polymer electrolyte membrane (PEM) water electrolysis is a promising green hydrogen generation technology to mitigate the effects of anthropogenic climate change. To achieve stable cell voltages, a conditioning procedure is typically required, where current or voltage cycling is applied until stable performance is achieved. Previous studies have demonstrated that a decrease in voltage occurs within the first few hours of operation [1], but this conditioning period has not been extensively studied for PEM electrolyzers, despite a variety of standard conditioning protocols available for PEM fuel cells in the literature. Previous works additionally suggest that changes in performance may be induced due to structural changes in the anode catalyst layer (CL) caused by large gas bubble evolution during the first few hours of operation [2]. However, to establish and optimize a conditioning procedure for PEMWEs, the specific structural changes caused by conditioning on the anode CL must be elucidated and correlated with the electrochemical performance parameters.In this work, we applied scanning transmission X-ray microscopy (STXM) to PEMWE catalyst layers to reveal pore and ionomer distributions in pristine and conditioned commercial CL samples. Prior to imaging, CL samples were embedded in epoxy and cut to 50 nm thick slices using an ultramicrotome. Using near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, Carbon 1s and Fluorine 1s absorption edges were probed to reveal pore and ionomer distributions, respectively. After image acquisition, the image stacks were processed to obtain the ionomer and epoxy spectra to enable spectral fitting and thresholding. Through this analysis, we quantified various morphological properties of catalyst layers (including CL thickness, porosity, pore size distribution, agglomerate distribution, and ionomer content) and compared these properties to those of pristine CLs to explain changes in cell performance. The insights gained from this work will aid in the development of efficient conditioning protocols and identify optimal post-conditioned anode CL morphology to inform the design of next-generation catalyst materials. A. Weiß et al., J Electrochem Soc, 166, F487–F497 (2019).O. Panchenko et al., Mater Today Energy, 16, 100394 (2020).

A series of electrospun nanofiber mat electrodes with two different commercial Pt/C catalysts and a binder of Nafion and poly(acrylic acid) were fabricated and evaluated. The electrodes were assembled into membrane-electrode-assemblies (MEAs) using Nafion 211 as the membrane. Variations in catalyst type, nanofiber composition (the ratio of Pt/C to Nafion), and fiber diameter had little or no impact on hydrogen/air fuel cell power output. 25 cm2 nanofiber and sprayed gas diffusion electrode MEAs were compared in terms of beginning of life (BoL) and end of life (EoL) performance after automotive-specific load cycling (Pt dissolution) and start-stop cycling (carbon corrosion) cathode durability protocols. Nanofiber electrode MEAs (0.10 mg/cm2 Pt loading for the anode and cathode) were clearly superior to sprayed MEAs; they produced more power at BoL and maintained a higher percentage of their power after the carbon corrosion durability protocol, resulting in much higher EoL fuel cell performance. On the other hand, there was no effect of electrode morphology on MEA durability for the Pt dissolution test. The higher MEA power output after carbon corrosion with electrospun electrodes is attributed to better oxygen and water transport within the nanofiber electrode and a higher electrochemical surface area for the fiber cathode.

Towards high-performance polysulfone membrane: The role of PSF-b-PEG copolymer additive

Porosities of porous silicon layers formed on different types of substrates and under different experimental conditions are compared with and related to the pore size distribution determined by gas adsorption experiments. Results show that porous layers formed on lightly P‐doped silicon exhibit a network of very narrow pores, of radii less than 2 nm. Porous films formed on heavily doped silicon present larger radii, ranging between 2 and 9 nm according to the experimental conditions. Larger porosities and larger pore sizes are obtained by increasing the forming current density or by decreasing the concentration. Heavily P‐doped porous silicon layers are homogeneous in depth and generally present a quite sharp pore size distribution. With heavily N‐doped silicon, an increase in porosity with increasing thickness is found, which corresponds to an increase in pore size, leading to a broadening of size distributions. This porosity gradient is attributed to a chemical dissolution of the layer occurring during anodization. In addition, a strong dependence of porosity with small variations in doping level is found.

Simulation of uranium mononitride spent fuel: A crystallographic approach

In polymer electrolyte fuel cells (PEFCs), the catalyst layer (CL) of the cathode needs a large amount of Pt because of the slow oxygen reduction reaction. Since electron, proton, and oxygen are necessary for the cathode reaction, achieving the optimum structure of electrode CL and the efficient transport of the reactants is significantly effective to reduce the usage of Pt catalyst. In this study, the effects of carbon structure on oxygen transport were evaluated using CLs fabricated under several conditions. We compared the performances using fabricated CLs with Ketjen black and Vulcan. Furthermore, the oxygen transport resistance in the CLs was evaluated. Pore size distribution (PSD) of the CL was measured by nitrogen physisorption method, and the CL structural parameters were estimated using the estimation model (1). Using the limiting current density method, we evaluate various oxygen transport resistances in the CL and discuss the contributions of these resistances to the cell performance. Figure 1 shows the estimated schematics of Pt-supported Ketjen black and Vulcan with ionomer. To investigate the structure of the fabricated CLs, we measured the PSDs by nitrogen physisorption method, and the results are shown in Figure 2. In the case of using Ketjen black, there is a small peak around 3-4 nm pore diameter, whereas in the case of Vulcan this peak does not exist. The small peak is considered to correspond to the pores in the carbon particles with a diameter of ~ 10 nm, and the difference in the PSDs for the self-produced CL is caused by the characteristics of the Vulcan with few pores in the particle. From these results, it is expected that most of the platinum particles are located on the outer surface of the Vulcan, and oxygen can easily diffuse to the platinum particles through the ionomer. Figure 3 shows the characteristics of the oxygen transport resistance of different thick CLs. By multiplying the measured oxygen transport resistance in the CL by the thickness of the CL, it is possible to evaluate only the change in the diffusion resistance in the CL pores, eliminating the influence of the oxygen transport resistance on the platinum surface and the dissolution resistance into the ionomer (1). From the gradients of the lines in Fig. 3, the effective diffusion coefficients can be estimated: DCL,eff O2=7.2×10-7 [m2/s] for the CL with Ketjen black and DO2 CL,eff =4.3×10-7 [m2/s] for that with Vulcan. The smaller effective diffusion coefficient with Vulcan is caused by the lower porosity. Figure 4 shows the characteristics of the oxygen transport resistance of the CLs having different platinum surface areas. The vertical axis represents the oxygen transport resistance in the CL, and the horizontal axis represents the inverse of the platinum surface area. Here, Pt-supported carbons with three different Pt densities were used, and the carbon amounts were set similar. Based on the gradients of the lines, the value of the oxygen transfer rate constant kPt at the platinum surface are estimated to be kPt=3.7×10-3 [m/s] for the CL with Ketjen black and kPt=9.3×10-3 [m/s] for that with Vulcan. Figures 5 and 6 show the results calculating the oxygen transport resistance of the fabricated CLs using the parameters estimated from the experiment. The oxygen dissolution rate kdiss=7.0×10-3 [m/s], estimated in the previous study (1), was applied here. With the CL with Ketjen black, the resistance at the platinum surface, R Pt , is a dominant factor, whereas the contribution of the resistance at the platinum surface with Vulcan reduces. This is considered to be because platinum particles are arranged outside the carbon particles. However, the diffusion resistance in the pores with Vulcan increases due to the lower porosity. This suggest that it is effectiveto increase the porosity for further reduction in the oxygen transport resistance in the thick CL with Vulcan. Reference Tabe, et al., ECS Trans., 80(8), 205 (2017). Figure 1