Spectrum imaging with energy-dispersive X-ray spectroscopy (EDS) has become ubiquitous in material characterization using electron microscopy. Multivariate statistical methods, commonly principal component analysis (PCA), are often used to aid analysis of the resulting multidimensional datasets; PCA can provide denoising prior to further analysis or grouping of pixels into distinct phases with similar signals. However, it is well known that PCA can introduce artifacts at low signal-to-noise ratios. Unfortunately, when evaluating the benefits and risks with PCA, it is often compared only against raw data, where it tends to shine; alternative data analysis methods providing a fair point of comparison are often lacking. Here, we directly compare PCA with a strategy based on (the conceptually and computationally simpler) weighted least squares (WLS). We show that for four representative cases, model fitting of the sum spectrum followed by WLS (mfWLS) consistently outperforms PCA in terms of finding and accurately describing compositional gradients and inclusions and as a preprocessing step to clustering. Additionally, we demonstrate that some common artifacts and biases displayed by PCA are avoided with the mfWLS approach. In summary, mfWLS can provide a superior option to PCA for analysis of EDS spectrum images as the signal is simply and accurately modeled.

A new method for dark field imaging is introduced, which uses scanned electron diffraction (or 4DSTEM-4-dimensional scanning transmission electron microscopy) datasets as its input. Instead of working on simple summation of intensity, it works on a sparse representation of the diffraction patterns in terms of a list of their diffraction peaks. This is tested on a thin perovskite film containing structural ordering resulting in additional superlattice spots that reveal details of domain structures, and is shown to give much better selectivity and contrast than conventional virtual dark field imaging. It is also shown to work well in polycrystalline aggregates of CuO nanoparticles. In view of the higher contrast and selectivity, and the complete exclusion of diffuse scattering from the image formation, it is expected to be of significant benefit for characterization of a wide variety of crystalline materials.

In this work, samples of chromia (Cr2O3) scale have been prepared for atom probe tomography and field evaporated with deep ultraviolet laser light (258 nm wavelength). The investigated range of laser energies spans more than three orders of magnitude between 0.03 and 90 pJ. Furthermore, the effects of detection rate and temperature were investigated. Simultaneous voltage and laser pulses were employed on additional needle specimens to reduce the standing voltage and minimize background noise during the measurement. Smooth evaporation with minimal mass spectrum peak tails was maintained over the whole range of measurement parameters. High laser energies result in significant underestimation of the oxygen content. Only laser energies below 1 pJ resulted in measured values near the expected oxygen content of 60 at%, the closest being about 58 at%.

Barleria is a palaeotropical genus of herbs, shrubs, and rarely climbers or trees. We investigated the karyotypes and male meiosis of 12 and 13 species, respectively, for the first time. Mitotic metaphases revealed two chromosome counts, 2n = 40 and 2n = 44. Chromosomes had median (m), submedian (sm), and subterminal (st) region centromeres. The total haploid chromosome length (TCL) ranged from 78.95 µm (Barleria sahyadrica) to 37.80 µm (B. nitida). Dispersion index differentiated the species into two groups, one with lower (3.40-4.79) and the other with higher (6.63-12.87) values. Principal component analysis based on six karyological parameters, namely base number (x), 2n, TCL, coefficient of variation of chromosome length, coefficient of variation of centromeric index, and mean centromeric asymmetry, exhibited three clusters. Cluster I included species of the subgenus Barleria. Cluster III had species of the subgenus Prionitis section Somalia. Cluster II comprised species of the subgenus Barleria and the subgenus Prionitis section Prionitis (B. sahyadrica). Pollen grains were oblate spheroidal or distinctly three-lobed, tri-brevicolporate with honey-combed tectum. Our analyses revealed karyological relationships among the investigated species and also provide raw data to breeders interested in horticultural applications of Barleria for accomplishing interspecific hybridization.

The production of plutonium-238 through irradiation of neptunium-237 (237Np) target materials for the use in radioisotope thermoelectric generators is paramount for continued deep space exploration. This work employs scanning electron microscopy to analyze 237Np materials coupled with a well-developed image analysis framework (Morphological Analysis for Material Attribution, or MAMA) to determine the degree of micron-scale homogeneity in the materials. This work demonstrated how the quantification of particle characteristics can validate production materials and affirm the qualitative similarities observed in micrographs. The 237Np oxide particle analysis determined that the materials from five production runs were quantitatively homogenous (significant at α = 0.05) in particle area, circularity, equivalent circular diameter, and ellipse aspect ratio, with two of the sampling dates having statistically significant different means for one of the four characteristics. These metrics not only confirm general homogeneity of the material but also expand the application of MAMA workflows to 237Np materials, demonstrating the utility of MAMA analysis for a wider breadth of nuclear materials than previously reported. In the open literature, this study is the first time that these microanalytical techniques were applied to 237Np materials to this degree.

Data management is a critical component of modern experimental workflows. As data generation rates increase, transferring data from acquisition servers to processing servers via conventional file-based methods is becoming increasingly impractical. The 4D Camera at the National Center for Electron Microscopy generates data at a nominal rate of 480 Gbit s-1 (87,000 frames s-1), producing a 700 GB dataset in 15 s. To address the challenges associated with storing and processing such quantities of data, we developed a streaming workflow that utilizes a high-speed network to connect the 4D Camera's data acquisition system to supercomputing nodes at the National Energy Research Scientific Computing Center, bypassing intermediate file storage entirely. In this work, we demonstrate the effectiveness of our streaming pipeline in a production setting through an hour-long experiment that generated over 10 TB of raw data, yielding high-quality datasets suitable for advanced analyses. Additionally, we compare the efficacy of this streaming workflow against the conventional file-transfer workflow by conducting a postmortem analysis on historical data from experiments performed by real users. Our findings show that the streaming workflow significantly improves data turnaround time, enables real-time decision-making, and minimizes the potential for human error by eliminating manual user interactions.

A detailed analysis of ptychography for three-dimensional (3D) phase reconstructions of thick specimens is performed. We introduce multi-focus ptychography, which incorporates a 4D-STEM defocus series to enhance the quality of 3D reconstructions along the beam direction through a higher overdetermination ratio. This method is compared with established multi-slice ptychography techniques, such as conventional ptychography, regularized ptychography, and multi-mode ptychography. Additionally, we contrast multi-focus ptychography with an alternative method that uses virtual optical sectioning through a reconstructed scattering matrix (S-matrix), which offers more precise 3D structure information compared to conventional ptychography. Our findings from multiple 3D reconstructions based on simulated and experimental data demonstrate that multi-focus ptychography surpasses other techniques, particularly in accurately reconstructing the surfaces and interface regions of thick specimens.

Amorphous thin films grown by magnetron co-sputtering exhibit changes in atomic structure with varying growth and annealing temperatures. Structural variations influence the bulk properties of the films. Scanning nanodiffraction performed in a transmission electron microscope (TEM) is applied to amorphous Tb17Co83 (a-Tb-Co) films deposited over a range of temperatures to measure relative changes in medium-range ordering (MRO). These measurements reveal an increase in MRO with higher growth temperatures and a decrease in MRO with higher annealing temperatures. The trend in MRO indicates a relationship between the growth conditions and local atomic ordering. By tilting select films, the TEM measures variations in the local atomic structure as a function of orientation within the films. The findings support claims that preferential ordering along the growth direction results from temperature-mediated adatom configurations during deposition, and that oriented MRO correlates with increased structural anisotropy, explaining the strong growth-induced perpendicular magnetic anisotropy found in rare earth-transition metal films. Beyond magnetic films, we propose the tilted FEM workflow as a method of extracting anisotropic structural information in a variety of amorphous materials with directionally dependent bulk properties, such as films with inherent bonding asymmetry grown by physical vapor deposition.

Tuneable phase plates for free electrons are a highly active area of research. However, their widespread implementation, similar to that of spatial light modulators in light optics, has been hindered by both conceptual and technical challenges. A specific technical challenge involves the need to minimize obstruction of the electron beam by supporting films and electrodes. Here, we describe numerical and analytical mathematical frameworks for three-dimensional stacks of phase plates that can be used to provide near-arbitrary electron beam shaping with minimal obstruction.

Recent work in ultra-high temperature in situ electron microscopy has presented the need for accurate, contact-free temperature determination at the microscale. Optical measurement based on thermal radiation (pyrometry) is an attractive solution but can be difficult to perform correctly due to effects, such as emissivity and optical transmission, that must be accounted for. Here, we present a practical guide to calibrating and using a spectral pyrometry system, including example code, using a Czerny-Turner spectrometer attached to a transmission electron microscope. Calibration can be accomplished using a thermocouple or commercial heated sample holder, after which arbitrary samples can be reliably measured for temperatures above ∼600∘C. An accuracy of 2% can be expected with the possibility of sub-second temporal resolution and sub-Kelvin temperature resolution. We then demonstrate this capability in conjunction with traditional microscopic techniques, such as diffraction-based strain measurement for thermal expansion coefficient, or live-video sintering evolution.

Reflection electron energy loss spectroscopy (REELS) has played a pivotal role in allowing researchers to explore the characteristics of various bulk materials. This study presents results for the low-loss region of REELS with a new cylindrical lens spectrometer integrated into a low-voltage scanning electron microscope. The operational principles and implementation of the spectrometer are explained through comparisons between electron optical simulations and experimental results. Notably, the analysis shows the ability to distinguish samples in film and bulk forms. Graphene and graphite, despite their identical elemental composition and crystalline structure, are found to have distinct energy spectra as indicated by plasmon peaks. Furthermore, the study explores the bandgap measurement of SiO2 at low-energy conditions of 2.5 keV, highlighting the proposed instrument's advantages in the measurement without the harmful effect of Cherenkov loss. Additionally, this method reaffirms the capability to measure multiple plasmon peaks from the energy spectra of bulk gold samples, thus introducing a pioneering avenue in energy spectrum measurement. Leveraging the compact size and simple experimental setup of the spectrometer for REELS, the method enables the measurement of energy spectra of both bulk- and film-formed samples under low electron energy conditions, marking a significant advancement in the field.

Stacking faults (SFs) are important structural defects that play an essential role in the deformation of engineering alloys. However, direct observation of SFs at the atomic scale can be challenging. Here, we use the analytical field ion microscopy, including density functional theory-informed contrast estimation, to image local elemental segregation at SFs in a creep-deformed solid-solution single-crystal alloy of Ni-2 at% W. The segregated atoms are imaged brightly, and time-of-flight spectrometry allows for their identification as W. We also provide the first quantitative analysis of trajectory aberration, with a deviation of approximately 0.4 nm, explaining why atom probe tomography could not resolve these segregations. Atomistic simulations of substitutional W atoms at an edge dislocation in face-centered cubic Ni using an analytic bond-order potential indicate that the experimentally observed segregation is due to the energetic preference of W for the center of the SF, contrasting with, for example, Re segregating to partial dislocations. Solute segregation to SF can hinder dislocation motion, increasing the strength of Ni-based superalloys. Yet, direct substitution of Re by W, envisaged to lower the superalloys' costs, requires extra consideration in alloy design since these two solutes do not have comparable interactions with structural defects during deformation.

Intensities in high-resolution phase-contrast images from electron microscopes build up discretely in time by detecting single electrons. A wave description of pulse-like coherent-inelastic interaction of an electron with matter implies a time-dependent coexistence of coherent partial waves. Their superposition forms a wave package by phase decoherence of 0.5 - 1 radian with Heisenbergs energy uncertainty ΔEH = ħ/2 Δt-1 matching the energy loss ΔE of a coherent-inelastic interaction and sets the interaction time Δt. In these circumstances, the product of Planck's constant and the speed of light hc is given by the product of the expression for temporal coherence λ2/Δλ and the energy loss ΔE. Experimentally, the self-coherence length was measured by detecting the energy-dependent localization of scattered, plane matter waves in surface proximity exploiting the Goos-Hänchen shift. Chromatic-aberration Cc-corrected electron microscopy on boron nitride (BN) proves that the coherent crystal illumination and phase contrast are lost if the self-coherence length shrinks below the size of the crystal unit cell at ΔE > 200 eV. In perspective, the interaction time of any matter wave compares with the lifetime of a virtual particle of any elemental interaction, suggesting the present concept of coherent-inelastic interactions of matter waves might be generalizable.

The present communication aims at demonstrating the wealth of information accessible by 1D-atom probe experiments using pulsed field desorption mass spectrometry (PFDMS), ultimately combined with video-field ion microscopy, while subjecting metallic samples to elevated gas pressures and studying surface reaction kinetics. Two case studies are being presented here: (a) the microkinetics of nickel tetracarbonyl (Ni(CO)4) formation through reaction of carbon monoxide with nickel and (b) the nitric oxide decomposition and reaction with hydrogen on platinum at variable steady electric fields mimicking electrocatalytic conditions. In both cases, surface areas with 140-150 atomic sites of the stepped Ni (001) and Pt (111) sample surfaces were probed. Under (a), we demonstrate variable repetition frequencies of field pulses to inform kinetic and mechanistic details of the surface reaction while under (b), we reveal the occurrence of field-induced processes impacting the surface reaction mechanism of nitric oxide with hydrogen and therefore opening new pathways not available under purely thermal conditions (in the absence of electric fields). Some aspects of PFDMS technical achievements will be discussed as they may provide clues for designing dynamic atom probe tomography instrumentation.

Intravital microscopy has emerged as a powerful imaging tool, which allows the visualization and precise understanding of rapid physiological processes at sites of inflammation in vivo, such as vascular permeability and leukocyte migration. Leukocyte interactions with the vascular endothelium can be characterized in the living organism in the murine cremaster muscle. Here, we present a microscopy technique using an Airy Beam Light Sheet microscope that has significant advantages over our previously used confocal microscopy systems. In comparison, the light sheet microscope offers near isotropic optical resolution and faster acquisition speed, while imaging a larger field of view. With less invasive surgery we can significantly reduce side effects such as bleeding, muscle twitching, and surgical inflammation. However, the increased acquisition speed requires exceptional tissue stability to avoid imaging artefacts. Since respiratory motion is transmitted to the tissue under investigation, we have developed a relocation algorithm that removes motion artefacts from our intravital microscopy images. Using these techniques, we are now able to obtain more detailed 3D time-lapse images of the cremaster vascular microcirculation, which allow us to observe the process of leukocyte emigration into the surrounding tissue with increased temporal resolution in comparison to our previous confocal approach.

Volume electron microscopy (VEM) is an essential tool for studying biological structures. Due to the challenges of sample preparation and continuous volumetric imaging, image artifacts are almost inevitable. Such image artifacts complicate further processing both for automated computer vision methods and human experts. Unfortunately, the widely used contrast limited adaptive histogram equalization (CLAHE) can alter the essential relative contrast information about some biological structures. We developed an image-processing pipeline to remove the artifacts and enhance the images without CLAHE. We apply our method to VEM datasets of a Microwasp head. We demonstrate that our method restores the images with high fidelity while preserving the original relative contrast. This pipeline is adaptable to other VEM datasets.

Integrating deep learning into image analysis for transmission electron microscopy (TEM) holds significant promise for advancing materials science and nanotechnology. Deep learning is able to enhance image quality, to automate feature detection, and to accelerate data analysis, addressing the complex nature of TEM datasets. This capability is crucial for precise and efficient characterization of details on the nano-and microscale, e.g., facilitating more accurate and high-throughput analysis of nanoparticle structures. This study investigates the influence of batch normalization (BN) and instance normalization (IN) on the performance of deep learning models for semantic segmentation of high-resolution TEM images. Using U-Net and ResNet architectures, we trained models on two different datasets. Our results demonstrate that IN consistently outperforms BN, yielding higher Dice scores and Intersection over Union metrics. These findings underscore the necessity of selecting appropriate normalization methods to maximize the performance of deep learning models applied to TEM images.

This comprehensive study delved into the detrimental effects of cadmium (Cd), a toxic heavy metal, on the testicular lamina propria (LP), a key player in spermatogenesis, and the maintenance of testicular stem cell niches. Utilizing transmission electron microscopy, immunohistochemistry, and double-labeling immunofluorescence, the research characterized the structural and cellular components of mouse testicular LP under Cd exposure and investigated the protective effects of quercetin. The findings illustrated that Cd exposure results in significant morphological and cellular modifications within the LP, including the apoptosis of peritubular myoid cells, an upsurge in CD34+ stromal cells displaying anti-apoptotic behaviors, and an excessive production of collagen Type I fibers and extracellular matrix. Remarkably, quercetin effectively counteracted these adverse changes by reversing apoptosis, reducing the proliferation of CD34+ stromal cells, and addressing fibrosis markers, thereby mitigating the cellular damage induced by Cd. This study not only highlighted the critical impact of apoptosis and fibrosis in Cd-related testicular damage but also elucidated the protective mechanism of quercetin, laying the groundwork for future clinical applications in addressing testicular damage from heavy metal poisoning through cellular therapeutics and pharmacological interventions.

In this study, we explore the dynamics of grain boundaries in nanocrystalline carbon monolayers, focusing on their variation with electron beam energy and electron dose rate in a spherical and chromatic aberration-corrected transmission electron microscope. We demonstrate that a clean surface, a high-dose rate, and a 60 keV electron beam are essential for precise local control over the dynamics of grain boundaries. The structure of these linear defects has been evaluated using neural network-generated polygon mapping.

Aging is a biological process with gradual decrease of cell function. Kidneys are one of the organs with higher susceptibility to the development of age-dependent tissue damage. Intermittent fasting has several beneficial effects on age-related degenerative changes. The aim of this study was to investigate the possible beneficial effect of intermittent fasting in delaying age-related renal changes and the possible mechanisms of this effect. Thirty male albino rats were classified into three groups: control, adult rats aged 3 months; aged group, 15-month-old rats and maintained until the age of 18 months; and intermittent fasting-aged groups, 15-month-old rats maintained on intermittent fasting for 3 months. Kidneys were processed for histological and immunohistochemical study. Aging resulted in a significant reduction in renal function and significant several degenerative changes in renal corpuscles and tubules which showed abnormal histological structure with increased collagen deposition. Aging caused significant reduction in the expression of autophagic marker light chain 3 with increased expression of active caspase-3 and inducible nitric oxide synthase. Intermittent fasting significantly improved these age-related renal changes. Intermittent fasting effectively prevents age-related renal changes through the reduction of age-related oxidative stress, inflammation, apoptosis, and activation of autophagy.