Among intravital imaging instruments, the intravital two-photon fluorescence excitation microscope has the advantage of enabling real-time 3D fluorescence imaging deep into cells and tissues, with reduced photobleaching and photodamage compared with conventional intravital confocal microscopes. However, excessive motion of organs due to involuntary movement such as breathing may result in out-of-focus images and severe fluorescence intensity fluctuations, which hinder meaningful imaging and analysis. The clinically approved alpha-2 adrenergic receptor agonist dexmedetomidine was administered to mice during two-photon fluorescence intravital imaging to alleviate this problem. As dexmedetomidine blocks the release of the neurotransmitter norepinephrine, pain is suppressed, blood pressure is reduced, and a sedation effect is observed. By tracking the quality of focus and stability of detected fluorescence in two-photon fluorescence images of fluorescein isothiocyanate-sensitized liver vasculature in vivo, we demonstrated that intravascular dexmedetomidine can reduce fluorescence fluctuations caused by respiration on a timescale of minutes in mice, improving image quality and resolution. The results indicate that short-term dexmedetomidine treatment is suitable for reducing involuntary motion in preclinical intravital imaging studies. This method may be applicable to other animal models.

Multiple myeloma (MM) is a deadly, incurable malignancy in which antibody-secreting plasma cells (PCs) become neoplastic. Previous studies have shown that the PC niche plays a role cancer progression. Bone marrow (BM) cores from MM and a premalignant condition known as monoclonal gammopathy of unknown significance (MGUS) patients were analyzed with confocal and transmission electron microscopy. The BM aspirates from these patients were used to generate 3D PC cultures. These in vitro cultures were then assayed for the molecular, cellular, and ultrastructural hallmarks of dysfunctional PC at days 1 and 5. In vivo, evidence of PC endoplasmic reticulum stress was found in both MM and MGUS BM; however, evidence of PC autophagy was found only in MM BM. Analysis of in vitro cultures found that MM PC can survive and maintain a differentiated phenotype over an unprecedented 5 days, had higher levels of paraprotein production when compared to MGUS-derived cultures, and showed evidence of PC autophagy as well. Increased fibronectin deposition around PC associated with disease severity and autophagy dysregulation was also observed. 3D cultures constructed from BM aspirates from MGUS and MM patients allow for long-term culture of functional PC while maintaining their distinct morphological phenotypes.

The development of biochemical analysis techniques to study heterogeneous biological samples is increasing. These techniques include synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy. This method has been applied to analyze biological tissue with multivariate statistical analysis to classify the components revealed by the spectral data. This study aims to compare the efficiencies of SR-FTIR microspectroscopy and focal plane array (FPA)-FTIR microspectroscopy when classifying rice tissue components. Spectral data were acquired for mapping the same sample areas from both techniques. Principal component analysis and cluster imaging were used to investigate the biochemical variations of the tissue types. The classification was based on the functional groups of pectin, protein, and polysaccharide. Four layers from SR-FTIR microspectroscopy including pericarp, aleurone layer, sub-aleurone layer, and endosperm were classified using cluster imaging, while FPA-FTIR microspectroscopy could classify only three layers of pericarp, aleurone layer, and endosperm. Moreover, SR-FTIR microspectroscopy increased the image contrast of the biochemical distribution in rice tissue more efficiently than FPA-FTIR microspectroscopy. We have demonstrated the capability of the high-resolution synchrotron technique and its ability to clarify small structures in rice tissue. The use of this technique might increase in future studies of tissue characterization.

Lysosomal “mottled appearance”, or uneven electron-dense content related to monoclonal gammopathy (MG), has been mainly described in light chain proximal tubulopathy (LCPT). We aimed to determine the ultrastructural characteristics of lysosomal mottled appearance in kidney biopsies and its association with LCPT and MG. Seventy-seven biopsies were grouped into LCPT (n = 5), MG conditions other than LCPT (n = 43), and non-MG conditions (n = 29). The mottled lysosomes in the renal tubules were evaluated using transmission electron microscopy and morphometric analysis. Mottled lysosomes were more prevalent (% of present cases) and frequent (no. of mottled lysosomes/20,000× ultramicroscopic field) in the LCPT group (100% and 8.20 ± 4.15/field) than in the MG (41.9% and 1.13 ± 2.05/field) and non-MG (37.9% and 0.80 ± 1.44/field) groups. In morphometric analysis of all mottled lysosomes (n = 520) detected from the 34 biopsies (5 LCPT, 18 MG, and 11 non-MG), we found that mottled lysosomes were larger, more irregular, and more electron-dense for the LCPT group than for the MG and non-MG groups. Therefore, mottled lysosomes can be present in disorders other than LCPT or even without MG. The morphological characteristics of mottled lysosomes could provide objective guidance for the diagnosis of LCPT.

The members of the Bacteroidetes phylum move on surfaces by gliding motility in the absence of external motility appendages, leading to the formation of spreading colonies. Here, the structural features of the spreading colony were assessed in a uranium-tolerant Bacteroidetes bacterium, Chryseobacterium sp. strain PMSZPI, by using correlative light and scanning electron microscopy (CLSEM). We developed a simple and convenient workflow for CLSEM using a shuttle and find software module and a correlative sample holding slide designed to transport samples between the light/fluorescence microscope (LM/FM) and the scanning electron microscope (SEM) to image spreading colony edges. The datasets from the CLSEM studies allowed convenient examination of the colonial organization by LM/FM followed by ultrastructural analysis by SEM. The regions of interest (ROIs) of the spreading colony edges that were observed in LM/FM in the absence and presence of uranium could be re-identified in the SEM quickly without prolonged searching. Perfect correlation between LM and SEM could be achieved with minimum preparation steps. Subsequently, imaging of the correlated regions was done at higher resolution in SEM to obtain more comprehensive information. We further showed the association of uranium with the gliding PMSZPI cells by energy-dispersive X-ray spectroscopy (EDS) attached to SEM.

Well-defined reconstruction parameters are essential to quantify the size, shape, and distribution of nanoscale features in atom probe tomography (APT) datasets. However, the reconstruction parameters of many minerals are difficult to estimate because intrinsic spatial markers, such as crystallographic planes, are not usually present within the datasets themselves. Using transmission and/or scanning electron microscopy imaging of needle-shaped specimens before and after atom probe analysis, we test various approaches to provide best-fit reconstruction parameters for voltage-based APT reconstructions. The results demonstrate that the length measurement of evaporated material, constrained by overlaying pre- and post-analysis images, yields more consistent reconstruction parameters than the measurement of final tip radius. Using this approach, we provide standardized parameters that may be used in APT reconstructions of 11 minerals. The adoption of standardized reconstruction parameters by the geoscience APT community will alleviate potential problems in the measurement of nanoscale features (e.g., clusters and interfaces) caused by the use of inappropriate parameters.

In this study, the gut structure and excretory system of Carpocoris mediterraneus which is phytophagous insect, were described with light and electron microscopies and discussed in relation to other Heteroptera species. The salivary system has two principal and accessory salivary glands, two principal and accessory gland ducts. The salivary gland and duct wall have a single layer of cuboidal cells. The duct lumen is surrounded by a thick intima layer. In the cytoplasm of the epithelial cells are seen vesicles. The gut includes fore, mid, and hindguts. The foregut consists of a long narrow tubular pharynx which opens into a slightly wider esophagus. The esophagus is thin walled and in turn opens into the midgut. The midgut has four regions (V1–V4). V1–V4 walls have a monolayered epithelium. V1 epithelium is double-nucleated. V1 cytoplasm contains numerous vesicles, secretory granules, spherocrystals, and cytoplasmic inclusions. Rod-shaped bacteria are seen in V4 lumen. The hindgut has pylorus and rectum. Malpighian tubules were attached in the pylorus. Malpighian tubules have a single-layer cuboidal epithelium. In their lumen, there are spherocrystals. The rectum wall has a monolayer of squamous epithelium and muscle layer. Numerous bacteria and uric acid crystals are seen in its lumen.

Thyroid C-cells secrete the hormone calcitonin (CT) which acts as an inhibitor of bone resorption. Our aim was to examine the age-related changes in the structure and function of CT-producing C-cells, using histomorphometric, ultrastructural, and biochemical analyses. We used young adult (3-months-old), middle-aged (16-months-old), and old (24-months-old) male rats. The peroxidase-antiperoxidase method was applied for localization of CT. Stereological analysis was performed using the newCAST stereological software package. Serum samples were analyzed for the determination of CT, testosterone (T), calcium (Ca2+), and phosphorus (P). We found a significant increase in the volume density (Vv) of C-cells in both older groups (p < 0.05). The percentage of smaller volume range C-cells increased (p < 0.0001), while the proportion of greater volume range C-cells decreased (p < 0.05) with ageing. Ultrastructural analysis revealed a larger number of secretory granules in older rats. Serum CT increased (p < 0.001), while serum T and P were reduced (p < 0.01) in older rats. Serum Ca2+ was lower (p < 0.0001) in middle-aged rats compared to young adults. We revealed a 20% incidence of C-cell hyperplasia in older rats and one case of medullary thyroid carcinoma in an old rat. Our findings indicate that the ageing process causes significant histomorphometric changes at the thyroid C-cell level.

Telocyte (TC)—a new type of interstitial cell with long telopodes, can form cellular junctions with various tissues or cells to participate in the regulation of multitudes of physiological activities and diseases. This study aimed to characterize the morphology, molecular features, and potential functions of hormone regulation in Chinese soft-shelled turtle (Pelodiscus sinensis) testis TCs at different reproductive stages by histological evaluation, immunohistochemistry (IHC), immunofluorescence (IF), and transmission electron microscopy. During hibernation, TCs were widely distributed in the interstitial tissue. In contrast, during reproductive activity, TCs were noted to be in close proximity with peritubular myoid cells surrounding the seminiferous tubule. Moreover, formed cell–cell junctions were observed between TCs and PTMs. The results of IHC and IF showed that the immunophenotype of testicular TCs in hibernating Chinese soft-shelled turtles is CD34+Vimentin−, while the reproductive telopodes (Tps) show low expression of vimentin. The androgen receptor is expressed in Tps of TCs of testis during hibernation. Our results showed also that TCs in seasonal breeding animals regulate the activity of neighboring cells by releasing extracellular microvesicles (EXMVs), thus influencing the activity of spermatogenesis and steroidogenesis. Consideration of our novel and interesting results indicate that the whole area warrants further research.

We investigated a new method for estimating specimen hydrogen content in atom probe tomography (APT) in experiments where molecular hydrogen ions (${\rm H}_2^ +$) originating from the measurement environment can overlap with deuterium (D+) in the mass-to-charge-state spectrum, thus preventing the direct application of isotopic marking for unambiguous hydrogen analysis. First, we applied an existing method for hydrogen content estimation, using ${\rm H}^ + {/}{\rm H}_2^ +$ ratios obtained from paired deuterated/nondeuterated experiments. These measurements demonstrated sufficient residual uncertainty to motivate exploring an alternative method to accurately estimate hydrogen content. By varying the time between evaporation events, it is then shown that a highly correlated relationship between field evaporation rate and hydrogen content exists and can also be used to predict hydrogen content. This leads to a new method for measuring hydrogen content within the specimen. We combine this extrapolation technique with continuous cycling of the evaporation rate or pulse frequency during an APT experiment. This could enable spatially resolved imaging of hydrogen concentrations despite the presence of a contaminant background hydrogen signal, without the need for deuteration.

Gray-level co-occurrence matrix (GLCM) analysis is a contemporary and innovative computational method for the assessment of textural patterns, applicable in almost any area of microscopy. The aim of our research was to perform the GLCM analysis of cell nuclei in Saccharomyces cerevisiae yeast cells after the induction of sublethal cell damage with ethyl alcohol, and to evaluate the performance of various machine learning (ML) models regarding their ability to separate damaged from intact cells. For each cell nucleus, five GLCM parameters were calculated: angular second moment, inverse difference moment, GLCM contrast, GLCM correlation, and textural variance. Based on the obtained GLCM data, we applied three ML approaches: neural network, random trees, and binomial logistic regression. Statistically significant differences in GLCM features were observed between treated and untreated cells. The multilayer perceptron neural network had the highest classification accuracy. The model also showed a relatively high level of sensitivity and specificity, as well as an excellent discriminatory power in the separation of treated from untreated cells. To the best of our knowledge, this is the first study to demonstrate that it is possible to create a relatively sensitive GLCM-based ML model for the detection of alcohol-induced damage in Saccharomyces cerevisiae cell nuclei.

Toll-like receptor (TLR)-mediated inflammatory processes play a critical role in the innate immune response during the initial interaction between the infecting microorganism and immune cells. This study aimed to investigate the possible microanatomical and histological differences in mandibular and bronchial lymph nodes in Akkaraman and Romanov lambs induced by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and study the gene, protein, and immunoexpression levels of TLR4, myeloid differentiation factor 88 (MyD88), and tumor necrosis factor-α (TNF-α) that are involved in the immune system. Microanatomical examinations demonstrated more intense lymphocyte infiltration in the bronchial lymph nodes of Akkaraman lambs in the LPS and LTA groups compared to Romanov lambs. TLR4, MyD88, and TNF-α immunoreactivities were more intense in the experimental groups of both breeds. Expression levels of MyD88 and TNF-α genes in the bronchial lymph node of Akkaraman lambs were found to increase statistically significantly in the LTA group. TLR4 gene expression level in the mandibular lymph node was found to be statistically significantly higher in the LTA + LPS group. In conclusion, dynamic changes in the immune cell populations involved in response to antigens such as LTA and LPS in the lymph nodes of both breeds can be associated with the difference in the expression level of the TLR4/MyD88/TNF-α genes.

Five compounds 2-(2-hydroxybenzylidene)-N-(prop-2-en-1-yl)hydrazinecarbothioamide (H2L), bis[μ2-2-({2-[(prop-2-en-1-yl)carbamothioyl]hydrazinylidene}methyl)phenolato-S,N,O:O]diaquadicopper(II) nitrate (1), bis[μ2-2-({2-[(prop-2-en-1-yl)carbamothioyl]hydrazinylidene}methyl)phenolato-S,N,O:O]diimidazoldicopper(II) nitrate (2), bis[μ2-2-({2-[(prop-2-en-1-yl)carbamothioyl]-hydrazinylidene}methyl)phenolato-S,N,O:O]bis-(3,5-dibromopyridine)dicopper(II) nitrate (3), bis[μ2-2-({2-[(prop-2-en-1-yl)carbamothioyl]-hydrazinylidene}methyl)phenolato-S,N,O:O]bis(4-methylpyridine)dicopper(II) nitrate hexahydrate (4) were synthesized. The antiproliferative properties of these compounds toward cancer cell lines RD, HeLa, and normal cell line MDCK have been investigated. The tested complexes surpass Doxorubicin (DOXO) in the efficiency of anticancer activity as their IC50 values toward cancer cells are lower than the corresponding values of DOXO and the selectivity indexes exceed the corresponding SI value of DOXO. The tested compounds demonstrated a high antioxidant effect against ABTS•+ radical cations as well as low toxicity on Daphnia magna.

Zirconium alloys are common fuel claddings in nuclear fission reactors and are susceptible to the effects of hydrogen embrittlement. There is a need to be able to detect and image hydrogen at the atomic scale to gain the experimental evidence necessary to fully understand hydrogen embrittlement. Through the use of deuterium tracers, atom probe tomography (APT) is able to detect and spatially locate hydrogen at the atomic scale. Previous works have highlighted issues with quantifying deuterium concentrations using APT due to complex peak overlaps in the mass-to-charge-state ratio spectrum between molecular hydrogen and deuterium (H2 and D). In this work, we use new methods to analyze historic and simulated atom probe data, by applying currently available data analysis tools, to optimize solving peak overlaps to improve the quantification of deuterium. This method has been applied to literature data to quantify the deuterium concentrations in a concentration line profile across an α-Zr/deuteride interface.

This study was conducted to assess the morphology of eggs and histology of the ovaries in female Isophya nervosa Ramme, 1931 (Orthoptera: Tettigoniidae). While the egg morphology of I. nervosa was studied and examined by a stereomicroscope, a light microscope, and a scanning electron microscope, respectively, the morphology and histology of the ovary of this species were studied and examined by a stereomicroscope, a light microscope, a scanning electron microscope, and a transmission electron microscope, respectively. We found that the adult female had two pairs of ovaries, lateral oviduct, common oviduct, and spermatheca. Morphological study of the ovariole revealed that it is categorized under panoistic type of ovariole which is divided into three regions, the terminal filament, the germarium, and the vitellarium. We also observed that the eggs in I. nervosa have an ellipsoidal shape and are brown in color. Three different layers such as extrachorion, exochorion, and endochorion were observed. When the egg morphology is examined, it is understood that the surface pattern of the egg and the features of the micropylar areas may be distinguishing characters at the subfamily level, in addition to known classical taxonomic characters.