We demonstrate a fabrication of an atomically controlled single-crystal heart-shaped nanostructure using a convergent electron beam in a scanning transmission electron microscope. The delicately controlled e-beam enable epitaxial crystallization of perovskite oxide LaAlO₃ grown out of the relative conductive interface (i.e. 2 dimensional electron gas) between amorphous LaAlO₃/crystalline SrTiO₃.

Synaptic vesicles, which are endogenous to neurotransmitters, are involved in exocytosis by active potentials and release neurotransmitters. Synaptic vesicles used in neurotransmitter release are reused via endocytosis to maintain a pool of synaptic vesicles. Synaptic vesicles show different types of exo- and endocytosis depending on animal species, type of nerve cell, and electrical activity. To accurately understand the dynamics of synaptic vesicles, direct observation of synaptic vesicles is required; however, it was difficult to observe synaptic vesicles of size 40-50 nm in living neurons. The exo-and endocytosis of synaptic vesicles was confirmed by labeling the vesicles with a fluorescent agent and measuring the changes in fluorescence intensity. To date, various methods of labeling synaptic vesicles have been proposed, and each method has its own characteristics, strength, and drawbacks. In this study, we introduce methods that can measure presynaptic activity and describe the characteristics of each technique.

We examined the morphology of fertilized egg and ultrastructures of fertilized egg envelopes of dwarf rainbowfish (Melanotaenia praecox) belong to Melanotaeniidae using light and electron microscopes. The fertilized eggs were spherical with adhesive filament, transparent, demersal, and had a narrow perivitelline space and small oil droplets. The size of fertilized egg was 1.02 ± 0.18 mm (n = 30), and there were two kinds of adhesive filament on the fertilized eggs. The long and thick (diameter 12.22 ± 0.52 ㎛, n = 20) adhesive filaments were only at the area of animal pole, and short and thin (diameter 1.99 ± 0.23 ㎛, n = 20) adhesive filaments were around the long filaments. A micropyle was conical shaped with adhesive filament and located near the animal pole of egg. The outer surface of fertilized egg was rough side. Also, the total thickness of the fertilized egg envelope was about 7.46 ± 0.41 ㎛ (n = 20), the fertilized egg envelope consisted of two layers, an inner lamellae layer and an outer layer with high electron-density. And the inner layer was 8 layers. Collectively, these morphological characteristics and adhesive property of fertilized egg with adhesive filaments, and ultrastructures of micropyle, outer surface, and section of fertilized egg envelope are showed species specificity.

Fluorescence in situ hybridization (FISH) is a technique to visualize specific DNA/RNA sequences within the cell nuclei and provide the presence, location and structural integrity of genes on chromosomes. A confocal Whole Slide Imaging (WSI) scanner technology has superior depth resolution compared to wide-field fluorescence imaging. Confocal WSI has the ability to perform serial optical sections with specimen imaging, which is critical for 3D tissue reconstruction for volumetric spatial analysis. The standard clinical manual scoring for FISH is labor-intensive, time-consuming and subjective. Application of multi-gene FISH analysis alongside 3D imaging, significantly increase the level of complexity required for an accurate 3D analysis. Therefore, the purpose of this study is to establish automated 3D FISH scoring for z-stack images from confocal WSI scanner. The algorithm and the application we developed, SHIMARIS PAFQ, successfully employs 3D calculations for clear individual cell nuclei segmentation, gene signals detection and distribution of break-apart probes signal patterns, including standard break-apart, and variant patterns due to truncation, and deletion, etc. The analysis was accurate and precise when compared with ground truth clinical manual counting and scoring reported in ten lymphoma and solid tumors cases. The algorithm and the application we developed, SHIMARIS PAFQ, is objective and more efficient than the conventional procedure. It enables the automated counting of more nuclei, precisely detecting additional abnormal signal variations in nuclei patterns and analyzes gigabyte multi-layer stacking imaging data of tissue samples from patients. Currently, we are developing a deep learning algorithm for automated tumor area detection to be integrated with SHIMARIS PAFQ.

The olfactory organs of two African lungfishes, Protopterus amphibius and P. dolloi, were investigated using a stereo microscope and a compound light microscope and were described anatomically, histologically, and histochemically. Like other lungfishes, these species present the following general features: i) elongated olfactory chamber (OC), ii) anterior nostril at the ventral tip of the upper lip, iii) posterior nostril on the palate of the oral cavity, iv) lamellae with multiple cell types such as olfactory receptor neurons, supporting cells, basal cells, lymphatic cells, and mucous cells (MC), and vi) vomero-like epithelial crypt (VEC) made of glandular epithelium (GE) and crypt sensory epithelium. Some of these features exhibit differences between species: MCs are abundant in both the lamellar and inner walls of the OC in P. amphibius but occur only in lamellae in P. dolloi. On the other hand, some between feature differences are consistent across species: the GE of both P. amphibius and P. dolloi is strongly positive for Alcian blue (pH 2.5)-periodic acid Schiff (deep violet coloration), and positive with hematoxylin and eosin and with Masson's trichrome (reddish-brown staining), unlike the MCs of the two species which stain dark red with both Alcian blue (pH 2.5)-periodic acid Schiff and Masson's trichrome but respond faintly to hematoxylin and eosin. The differing abundance of MCs in the two lungfishes might reflect different degrees in aerial exposure of the olfactory organ, while the neutral and acid mucopolysaccharide-containing VEC, as indicated by staining properties of the MCs, is evolutionary evidence that P. amphibius and P. dolloi are the closest living relatives to tetrapods, at least in the order Dipnoi.

Histopathology is a well-established standard diagnosis employed for the majority of malignancies, including breast cancer. Nevertheless, despite training and standardization, it is considered operator-dependent and errors are still a concern. Fractal dimension analysis is a computational image processing technique that allows assessing the degree of complexity in patterns. We aimed here at providing a robust and easily attainable method for introducing computer-assisted techniques to histopathology laboratories. Slides from two databases were used: A) Breast Cancer Histopathological; and B) Grand Challenge on Breast Cancer Histology. Set A contained 2480 images from 24 patients with benign alterations, and 5429 images from 58 patients with breast cancer. Set B comprised 100 images of each type: normal tissue, benign alterations, in situ carcinoma, and invasive carcinoma. All images were analyzed with the FracLac algorithm in the ImageJ computational environment to yield the box count fractal dimension (Db) results. Images on set A on 40x magnification were statistically different (p = 0.0003), whereas images on 400x did not present differences in their means. On set B, the mean Db values presented promising statistical differences when comparing. Normal and/or benign images to in situ and/or invasive carcinoma (all p < 0.0001). Interestingly, there was no difference when comparing normal tissue to benign alterations. These data corroborate with previous work in which fractal analysis allowed differentiating malignancies. Computer-aided diagnosis algorithms may beneficiate from using Db data; specific Db cut-off values may yield ~ 99% specificity in diagnosing breast cancer. Furthermore, the fact that it allows assessing tissue complexity, this tool may be used to understand the progression of the histological alterations in cancer.

Neuromorphic systems require integrated structures with high-density memory and selector devices to avoid interference and recognition errors between neighboring memory cells. To improve the performance of a selector device, it is important to understand the characteristics of the switching process. As changes by switching cycle occur at local nanoscale areas, a high-resolution analysis method is needed to investigate this phenomenon. Atomic force microscopy (AFM) is used to analyze the local changes because it offers nanoscale detection with high-resolution capabilities. This review introduces various types of AFM such as conductive AFM (C-AFM), electrostatic force microscopy (EFM), and Kelvin probe force microscopy (KPFM) to study switching behaviors.

Growing demands for comprehending complicated nano-scale phenomena in atomic resolution has attracted in-situ transmission electron microscopy (TEM) techniques for understanding their dynamics. However, simple to safe TEM sample preparation for in-situ observation has been limited. Here, we suggested the optical microscopy based micro-manipulating system for transferring TEM samples. By adopting our manipulator system, several types of samples from nano-wires to plate-like thin samples were transferred on micro-electro mechanical systems (MEMS) chip in a single step. Furthermore, the control of electrostatic force between the sample and the probe tip is found to be a key role in transferring process.

Brain disorders seriously affect life quality. Therefore, non-invasive neuroimaging has received attention to monitoring and early diagnosing neural disorders to prevent their progress to a severe level. This short review briefly describes the current MRI and PET/CT techniques developed for non-invasive neuroimaging and the future direction of optical imaging techniques to achieve higher resolution and specificity using the second near-infrared (NIR-II) region of wavelength with organic molecules.

We investigate the sensitivity of symmetry quantification algorithms based on the profile R-factor (R_p) and the normalized cross-correlation (NCC) coefficient (γ). A DM (Digital Micrograph^©) script embedded in the Gatan digital microscopy software is used to develop the symmetry quantification program. Using the Bloch method, a variety of CBED patterns are simulated and used to investigate the sensitivity of symmetry quantification algorithms. The quantification results show that two symmetry quantification coefficients are significantly sensitive to structural changes even for small strain values of < 1%.

Spider capture silk is a natural scaffolding material that outperforms most synthetic materials in terms of its combination of strength and elasticity. Among the various kinds of silk threads, cribellar thread is the most primitive prey-capturing type of spider web material. We analyzed the functional organization of the sieve-like cribellum spigots and specialized calamistral comb bristles for capture thread production by the titanoecid spider Nurscia albofasciata. The outer cribellar surface is covered with thousands of tiny spigots, and the cribellar plate produces non-sticky threads composed of thousands of fine nanofibers. N. albofasciata cribellar spigots are typically about 10 ㎛ long, and each spigot appears as a long individual shaft with a pagoda-like tiered tip. The five distinct segments comprising each spigot is a defining characteristic of this spider. This segmented and flexible structure not only allows for spigots to bend individually and join with adjacent spigots, but it also enables spigots to draw the silk fibrils from their cribella with rows of calamistral leg bristles to form cribellar prey-capture threads.

Intravital video microscopy permits the observation of microcirculatory blood flow. This often requires fluorescent probes to visualize structures and dynamic processes that cannot be observed with conventional bright-field microscopy. Conventional light microscopes do not allow for simultaneous bright-field and fluorescent imaging. Moreover, in conventional microscopes, only one type of fluorescent label can be observed. This study introduces multispectral intravital video microscopy, which combines bright-field and fluorescence microscopy in a standard light microscope. The technique enables simultaneous real-time observation of fluorescently-labeled structures in relation to their direct physical surroundings. The advancement provides context for the orientation, movement, and function of labeled structures in the microcirculation.

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has arisen as a global pandemic affecting the respiratory system showing acute respiratory distress syndrome (ARDS). However, there is no targeted therapeutic agent yet and due to the growing cases of infections and the rising death tolls, discovery of the possible drug is the need of the hour. In general, the study for discovering therapeutic agent for SARS-CoV-2 is largely focused on large-scale screening with fragment-based drug discovery (FBDD). With the recent advancement in cryo-electron microscopy (Cryo-EM), it has become one of the widely used tools in structural biology. It is effective in investigating the structure of numerous proteins in high-resolution and also had an intense influence on drug discovery, determining the binding reaction and regulation of known drugs as well as leading the design and development of new drug candidates. Here, we review the application of cryo-EM in a structure-based drug design (SBDD) and in silico screening of the recently acquired FBDD in SARS-CoV-2. Such insights will help deliver better understanding in the procurement of the effective remedial solution for this pandemic.

Melanin has been a widely researched pigment by scientists for decades as it is undoubtedly the most ubiquitous and ancient pigment found in nature. Melanin plays very signifcant roles in structural plumage colors in birds: it has visible light-absorbing capabilities, and nanoscale structures can be formed by self-assembling melanin granules. Herein, we review recent progress on melanin-based structural coloration research. We hope that this review will provide current understanding of melanin's structural and optical properties, natural coloration mechanisms, and biomimetic methods to implement artifcial melanin-based structural colors.

Puccinia miscanthi teliospores were observed on the leaf surface of Miscanthus sinensis using a field emission scanning electron microscope. Details of teliospore mucilage could be visualized through the axial imaging of secondary electrons for a better understanding of pathogen behavior in rust diseases.

Electron microscopy (EM) is an essential imaging method in biological sciences. Since biological specimens are exposed to radiation and vacuum conditions during EM observations, they die due to chemical bond breakage and desiccation. However, some organisms belonging to the taxa of bacteria, fungi, plants, and animals (including beetles, ticks, and tardigrades) have been reported to survive hostile scanning EM (SEM) conditions since the onset of EM. The surviving organisms were observed (i) without chemical fixation, (ii) after mounting to a precooled cold stage, (iii) using cryo-SEM, or (iv) after coating with a thin polymer layer, respectively. Combined use of these techniques may provide a better condition for preservation and live imaging of multicellular organisms for a long time beyond live-cell EM.

Our previous study on the binding activity between Cel5H and clay minerals showed highest binding efficiency among other cellulase enzymes cloned. Here, based on previous studies, we hypothesized that the positive amino acids on the surface of Cel5H protein may play an important role in binding to clay surfaces. To examine this, protein sequences of Bacillus licheniformis Cel5H (BlCel5H) and Paenibacillus polymyxa Cel5A (PpCel5A) were analyzed and then selected amino acids were mutated. These mutated proteins were investigated for binding activity and force measurement via atomic force microscopy (AFM). A total of seven amino acids which are only present in BlCel5H but not in PpCel5A were selected for mutational studies and the positive residues which are present in both were omitted. Of the seven selected surface lysine residues, only three mutants K196A(M2), K54A(M3) and K157T(M4) showed 12%, 7% and 8% less clay mineral binding ability, respectively compared with wild-type. The probable reason why other mutants did not show altered binding efficiency might be due to relative location of amino acids on the protein surface. Meanwhile, measurement of adhesion forces on mica sheets showed a well-defined maximum at 69±19 pN for wild-type, 58±19 pN for M2, 53±19 pN for M3, and 49±19 pN for M4 proteins. Hence, our results demonstrated that relative location of surface amino acids of Cel5H protein especially positive charged amino acids are important in the process of clay mineral-protein binding interaction through electrostatic exchange of charges.

Hair, having distinct stages of growth, is a dynamic component of the integumentary system. Nonetheless, derangement in its structure and growth pattern often provides vital clues for the diagnosis of systemic diseases. Assessment of the hair structure by various microscopy techniques is, hence, a valuable tool for the diagnosis of several systemic and cutaneous disorders. Systemic illnesses like Comel-Netherton syndrome, Griscelli syndrome, Chediak Higashi syndrome, and Menkes disease display pathognomonic findings on hair microscopy which, consequently, provide crucial evidence for disease diagnosis. With minimal training, light microscopy of the hair can easily be performed even by clinicians and other health care providers which can, thus, serve as a useful tool for disease diagnosis at the patient's bedside. This is especially true for resource-constrained settings where access and availability of advanced investigations (like molecular diagnostics) is a major constraint. Despite its immense clinical utility and non-invasive nature, hair microscopy seems to be an underutilized diagnostic modality. Lack of awareness regarding the important findings on hair microscopy may be one of the crucial reasons for its underutilization. Herein, we, therefore, present a comprehensive overview of the available methods for hair microscopy and the pertinent findings that can be observed in various diseases.

The main purpose of this paper is the preparation of transmission electron microscopy (TEM) samples from the microsized powders of lithium-ion secondary batteries. To avoid artefacts during TEM sample preparation, the use of ion slicer milling for thinning and maintaining the intrinsic structure is described. Argon-ion milling techniques have been widely examined to make optimal specimens, thereby making TEM analysis more reliable. In the past few years, the correction of spherical aberration (Cs) in scanning transmission electron microscopy (STEM) has been developing rapidly, which results in direct observation at an atomic level resolution not only at a high acceleration voltage but also at a deaccelerated voltage. In particular, low-kV application has markedly increased, which requires a sufficiently transparent specimen without structural distortion during the sample preparation process. In this study, sample preparation for high-resolution STEM observation is accomplished, and investigations on the crystal integrity are carried out by Cs-corrected STEM.

We explored the link between mitochondrial biogenesis and mitochondrial morphology using transmission electron microscopy (TEM) in lymphoblasts of pediatric acute lymphoblastic leukemia (ALL) patients and compared these characteristics between tumors and control samples. Gene expression of mitochondrial biogenesis markers was analysed in 23 ALL patients and 18 controls and TEM for morphology analysis was done in 15 ALL patients and 9 healthy controls. The area occupied by mitochondria per cell and the cristae cross-sectional area was observed to be significantly higher in patients than in controls (p-value=0.0468 and p-value<0.0001, respectively). The mtDNA copy numbers, TFAM, POLG, and c-myc gene expression were significantly higher in ALL patients than controls (all p-values<0.01). Gene Expression of PGC-1α was higher in tumor samples. The analysis of the correlation between PGC-1α expression and morphology parameters i.e., both M/C ratio and cristae cross-sectional area revealed a positive trend (r=0.3, p=0.1). The increased area occupied by mitochondria and increased cristae area support the occurrence of cristae remodelling in ALL. These changes might reflect alterations in cristae dynamics to support the metabolic state of the cells by forming a more condensed network. Ultrastructural imaging can be useful for affirming changes occurring at a subcellular organellar level.