Microscopy and Microanalysis最新文献

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.

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 self-opening transfer shuttle has been designed and fabricated for the transfer of air-sensitive samples to scanning electron microscopy (SEM). Delayed push out of an airtight sample cabin sealed inside the shuttle allows the protection of the sample from air exposure during the pumping of SEM chamber. A compressed spring is employed to automatically drive the push out of the cabin. Once the cabin is fully pushed out, the sample contained inside is revealed for SEM investigation through a hollow window created on the shuttle. The O-rings that are fixed at both ends of the sample cabin not only serve as sealing parts that make the cylinder airtight but also act as resistance elements that provide sufficient friction force to slow down the push out of the sample cabin. With the advantages of self-opening without the need for external control or force, and its reasonably small size, this low-cost and easy-to-use transfer shuttle has wide compatibility with different SEMs and holds a promising application prospect in numerous research areas.

A conduction heat transfer analysis of ex situ lift-out specimen handling under cryogenic conditions (cryo-EXLO) is performed and compared with experimentally determined temperature values using a type K thermocouple. Using a finite-volume solver for heat conduction, the analysis confirms that manipulation of a specimen by a probe above a working surface cooled at liquid nitrogen (LN2) temperatures can remain below the critical vitreous temperature up to several hundreds of micrometers above the working surface, allowing for ample distance for lift out and specimen manipulation. In addition, the temperature above the cryogenic shuttle sample holder working surface remains below the vitreous temperature for several tens of minutes without adding cryogen, yielding sufficient time to complete multiple manipulations. Periodically topping off the cryogen level may allow for unlimited cryo-EXLO manipulations with this hardware and geometry.

To overcome the spatial resolution limit set by aperture-limited diffraction in traditional scanning transmission electron microscopy, microscopists have developed ptychography enabled by iterative phase retrieval algorithms and high-dynamic-range pixel array detectors. Current detector designs are limited by the data rate off chip, so a high-pixel-count detector has a proportionally lower frame rate than the few-segment detectors used for differential phase contrast (DPC) imaging. This slower acquisition speed leads to heightened vulnerability to scan noise, drift, and potential sample damage. This creates opportunities for repurposing fast segmented detectors for ptychography by trading a reduction in reciprocal space pixels for an increase in real space pixels. Here, we explore a strategy of oversampling in real space and instead apply detector pixel upsampling during the reconstruction process. We demonstrate the viability of achieving super-resolution ptychography on thin objects using only 2 × 2 detector pixels, surpassing the resolution of integrated DPC (iDPC) imaging. With optimization using simulated datasets and experiments on MoTe2/WSe2 bilayer moiré superlattices, we achieved super-resolution ptychography reconstructions under rapid acquisition conditions (37.5 pA, 1 μs dwell time), yielding over 50% improvements in contrast and information limit compared to annular dark field and iDPC imaging on the same detectors.

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.

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.

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.

The first attempts of bacteria observation started with the use of glass lenses to generate magnified images of specimens. This technique is constrained by the principal limit to the resolution of any optical system. Besides optical microscopy, other imaging techniques emerged to reveal more levels of details. The more the achievable resolution, the more complex the imaging systems, and at the same time, the more potentially cell-killing or DNA-damaging they may become. This article provides a state of the art of nonconventional sensor techniques that have been used in applications related to bacteria imaging, for the purpose of comparing the information they provide and determine their suitability or find out if their combination can yield new results without compromising the ability to keep the cells alive.

Cerebral organoid cultures from human-induced pluripotent stem cells are widely used to study complex human brain development; however, there is still limited ultrastructural information regarding the development. In this study, we examined the structural details of cerebral organoids using various microscopy techniques. Two protocols were chosen as representative methods for the development of brain organoids: the classic whole-cerebral organoid (Whole-CO) culture technique, and the air-liquid interface-cerebral organoid (ALI-CO) culture technique. Immunostained confocal laser scanning microscopy (CLSM) revealed the formation of the CTIP2- and TBR1-positive cortical deep layer on days 90 and 150, depending on the developmental progress of both methods. Furthermore, the presence of astrocytes and oligodendrocytes was verified through immunostained CLSM utilizing two-dimensional and three-dimensional reconstruction images after a 150-day period. Transmission electron microscopy analysis revealed nanometer-resolution details of the cellular organelles and neuron-specific structures including synapses and myelin. Large-area scanning electron microscopy confirmed the well-developed neuronal connectivity from each culture method on day 150. Using those microscopy techniques, we clearly showed significant details within two representative culture protocols, the Whole-CO and ALI-CO culture methods. These multi-level images provide ultrastructural insight into the features of cerebral organoids depending on the developmental stage.