It is well known that two DNA molecules are wrapped around histone octamers and folded together to form a single chromosome. However, the nucleosome fiber folding within a chromosome remains an enigma, and the higher-order structure of chromosomes also is not understood. In this study, we employed electron diffraction which provides a noninvasive analysis to characterize the internal structure of chromosomes. The results revealed the presence of structures with 100–200 nm periodic features directionally perpendicular to the chromosome axis in unlabeled isolated human chromosomes. We also visualized the 100–200 nm periodic features perpendicular to the chromosome axis in an isolated chromosome whose DNA molecules were specifically labeled with OsO4 using electron tomography in 300 keV and 1 MeV transmission electron microscopes.

Accurate predetermination of the quantum yield ratio (QA/QD) and the extinction coefficient ratio (KA/KD) between acceptor and donor is a prerequisite for quantitative fluorescence resonance energy transfer (FRET) imaging. We here propose a method to measure KA/KD and QA/QD by measuring the excitation–emission spectra (ExEm-spectra) of one dish of cells expressing m (≥3) kinds of FRET constructs. The ExEm-spectra images are unmixed to obtain the weight maps of donor (WD), acceptor (WA), and acceptor sensitization (WS). For each cell, the frequency distribution plots of the WS/WD and WS/WA images are fitted by using a single-Gaussian function to obtain the peak values of WS/WD (SD) and WS/WA (SA). The statistical frequency-SD/SA plots from all cells are fitted by using a multi-Gaussian function to obtain the peak values of both SD and SA, and then the ranges of WS/WD (RSD) and WS/WA (RSA) for each FRET construct are predetermined. Based on the predetermined RSD and RSA values of FRET constructs, our method is capable of automatically classifying cells expressing different FRET constructs. Finally, the WS/WD–WA/WD plot from different kinds of cells is linearly fitted to obtain KA/KD and QA/QD values.

Scanning transmission electron microscopy (STEM) is an extremely versatile method for studying materials on the atomic scale. Many STEM experiments are supported or validated with electron scattering simulations. However, using the conventional multislice algorithm to perform these simulations can require extremely large calculation times, particularly for experiments with millions of probe positions as each probe position must be simulated independently. Recently, the plane-wave reciprocal-space interpolated scattering matrix (PRISM) algorithm was developed to reduce calculation times for large STEM simulations. Here, we introduce a new method for STEM simulation: partitioning of the STEM probe into “beamlets,” given by a natural neighbor interpolation of the parent beams. This idea is compatible with PRISM simulations and can lead to even larger improvements in simulation time, as well requiring significantly less computer random access memory (RAM). We have performed various simulations to demonstrate the advantages and disadvantages of partitioned PRISM STEM simulations. We find that this new algorithm is particularly useful for 4D-STEM simulations of large fields of view. We also provide a reference implementation of the multislice, PRISM, and partitioned PRISM algorithms.

Tramadol is used worldwide and is listed in many medical guidelines to treat both acute and chronic pains. There is a growing evidence of abuse of tramadol in some African and West Asian countries. Tramadol has some side effects. The present study designed to follow up the treatment of the cellular responses which might be induced in the kidney of tramadol mice. Treated mice received daily injection of tramadol dose (125 μg/100 g b.wt) for 20 and 40 days. Other mice received tramadol for 40 days and then were divided into three groups: the first received distilled water, the second received Lagenaria siceraria, and the third received melatonin daily for 40 days. Both the daily injection of tramadol for 20 and 40 days resulted in radical, extensive, and severe alterations in the normal histological architecture of the kidney. Treatment with Lagenaria siceraria or melatonin after tramadol administration for a long-term, markedly changed the collagen content and other chemical components, that may reach nearly normal levels. Such findings propose that although tramadol has many cytological and histopathological side effects on the kidneys of male mice, the treatments via Lagenaria siceraria and melatonin have effective therapeutic impacts on the tramadol side effects.

The study of the sinking phenomenon of diatom cells, which have a slightly larger specific gravity (~1.3) compared to that of water, is an important research topic for understanding photosynthetic efficiency. In this study, we successfully demonstrated the observation of the sinking behaviors of four different species of diatom using a homemade “tumbled” optical microscope. A homemade 1 mm3 microchamber was employed to decrease the effects of convection currents. In the microchamber, diatom cells were basically settled in a linear manner without floating, although some of the cells were rotated during their sinking. Sinking speeds of the four species of diatom cells, Nitzschia sp., Pheodactylum tricornutum, Navicula sp., and Odontella aurita, were 0.81 ± 5.56, 3.03 ± 10.17, 3.29 ± 7.39, and 11.22 ± 21.42 μm/s, respectively, based on the automatic tracking analysis of the centroids of each cell. Manual analysis of a vector between two longitudinal ends of the cells (two-point analysis) was effective for quantitatively characterizing the rotation phenomenon; therefore, angles and angular velocities of rotating cells were well determined as a function of time. The effects of the cell shapes on sinking velocity could be explained by simulation analysis using the modified Stokes’ law proposed by Miklasz et al.

Lysosomes are integration hubs for several signaling pathways, such as autophagy and endocytosis, and also crucial stores of ions, including Zn2+. Lysosomal dysfunction caused by changes in their morphology by fusion and fission processes can result in several pathological disorders. However, the role of Zn2+ in modulating the morphology of lysosomes is unclear. The resolution of conventional epifluorescence microscopy restricts accurate observation of morphological changes of subcellular fluorescence punctum. In this study, we used a modified epifluorescence microscopy to identify the center of a punctum from a series of z-stack images and calculate the morphological changes. We stained primary cultured rat embryonic cortical neurons with FluoZin3, a Zn2+-sensitive fluorescent dye, and Lysotracker, a lysosome-specific marker, to visualize the distribution of Zn2+-enriched vesicles and lysosomes, respectively. Our results revealed that treating neurons with N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine, a cell-permeable Zn2+ chelator, shrank Zn2+-enriched vesicles and lysosomes by up to 25% in an hour. Pretreating the neurons with YM201636, a blocker of lysosome fission, could suppress this shrinkage. These results demonstrate the usefulness of the modified epifluorescence microscopy for investigating the homeostasis of intracellular organelles and related disorders.

The testis of bamboo shark is characterized by diametric development leading to zonation architecture. Here, we investigated the staining pattern of 12 lectins in 6 groups of differential binding specificities within the germ, somatic, and interstitial cells of each zone. The neutral mucopolysaccharides appeared in the interstitial tissue in all the zones and became more significant in the spermatozoal–Sertoli cell junction. The cellular localization of the lectins varies in testicular zones and cell types. There was a gradual increase in glycosylation toward the degenerative zone. The increased intensity of most lectins in the interstitial cells indicates the association of glycoconjugates in their androgen-secreting activity. Statistical analyses showed a significant correlation between the groups of lectins and each lectin used, stronger response to lectins in the interstitial cells (ICs) than other cell types. Moreover, the response to glucosamine (GlcNAc), galactosamine (GalNAc), and fucose tended to be higher than glucose and galactose. Furthermore, the intensity of response was increased toward the degenerative zone. In addition, we can use peanut agglutinin (PNA) as an acrosomal marker in combination with other marker proteins for studying shark spermatogenesis. These findings refer to the crucial role of glycoconjugates in spermatogenesis in the bamboo shark testis.

Immunostaining in clinical routine and research highly depends on standardized staining methods and quantitative image analyses. We qualitatively and quantitatively compared antigen retrieval methods (no pretreatment, pretreatment with pepsin, and heat-induced pretreatment with pH 6 or pH 9) for 17 antibodies relevant for placenta and implantation diagnostics and research. Using our newly established, comprehensive automated quantitative image analysis approach, fluorescent signal intensities were evaluated. Automated quantitative image analysis found that 9 out of 17 antibodies needed antigen retrieval to show positive staining. Heat induction proved to be the most efficient form of antigen retrieval. Eight markers stained positive after pepsin digestion, with β-hCG and vWF showing enhanced staining intensities. To avoid the misinterpretation of quantitative image data, the qualitative aspect should always be considered. Results from native placental tissue were compared with sections of a placental invasion model based on thermo-sensitive scaffolds. Immunostaining on placentas in vitro leads to new insights into fetal development and maternal pathophysiological pathways, as pregnant women are justifiably excluded from clinical studies. Thus, there is a clear need for the assessment of reliable immunofluorescent staining and pretreatment methods. Our evaluation offers a powerful tool for antibody and pretreatment selection in placental research providing objective and precise results.

Epithelial–mesenchymal transition (EMT) is an essential biological process, also implicated in pathological settings such as cancer metastasis, in which epithelial cells transdifferentiate into mesenchymal cells. We devised an image analysis pipeline to distinguish between tissues comprised of epithelial and mesenchymal cells, based on extracted features from immunofluorescence images of differing biochemical markers. Mammary epithelial cells were cultured with 0 (control), 2, 4, or 10 ng/mL TGF-β1, a well-established EMT-inducer. Cells were fixed, stained, and imaged for E-cadherin, actin, fibronectin, and nuclei via immunofluorescence microscopy. Feature selection was performed on different combinations of individual cell markers using a Bag-of-Features extraction. Control and high-dose images comprised the training data set, and the intermediate dose images comprised the testing data set. A feature distance analysis was performed to quantify differences between the treatment groups. The pipeline was successful in distinguishing between control (epithelial) and the high-dose (mesenchymal) groups, as well as demonstrating progress along the EMT process in the intermediate dose groups. Validation using quantitative PCR (qPCR) demonstrated that biomarker expression measurements were well-correlated with the feature distance analysis. Overall, we identified image pipeline characteristics for feature extraction and quantification of immunofluorescence images to distinguish progression of EMT.

Brain circuits are highly interconnected three-dimensional structures fabricated from components ranging vastly in size; from cell bodies to individual synapses. While neuronal activity can be visualized with advanced light microscopy (LM) techniques, the resolution of electron microscopy (EM) is critical for identifying synaptic connections between neurons. Here, we combine these two techniques, affording the advantage of each and allowing for measurements to be made of the same neural features across imaging platforms. We established an EM-label-free workflow utilizing inherent structural features to correlate in vivo two-photon LM and volumetric scanning EM (SEM) in the ferret visual cortex. By optimizing the volume SEM sample preparation protocol, imaging with the OnPoint detector, and utilizing the focal charge compensation device during serial block-face imaging, we achieved sufficient resolution and signal-to-noise ratio to analyze synaptic ultrastructure for hundreds of synapses within sample volumes. Our novel workflow provides a reliable method for quantitatively characterizing synaptic ultrastructure in functionally imaged neurons, providing new insights into neuronal circuit organization.

The mineral composition of eggshells is assumed to be a conserved phylogenetic feature. Avian eggshells are composed of calcite, whereas those of taxa within Chelonia are aragonitic. Yet, the eggshells of a passerine bird were reported to be made of aragonite. Here, we report a new study of the same bird eggshells using a combination of in situ microscopy and chemical techniques. A microstructural analysis finds a similar arrangement to other avian eggshells, despite their very thin and fragile nature. Fourier transform infrared spectrometry (FTIR) and electron backscatter diffraction (EBSD) results also confirm that the eggshells are entirely composed of calcite. Our findings demonstrate that passerine eggshells are not an exception and similar to other birds and reinforce the phylogenetic placement of this bird species.

In insects, the number, cytological and histological structures, and the spherocrystals of the Malpighian tubules (MTs) can vary considerably in different insect groups. These differences are considered important because they can be used as taxonomic characters. For this purpose, the ultrastructure of the MT epithelial cells in Leptophyes albovittata (Kollar, 1833) (Orthoptera, Tettigoniidae) was examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. The wall of each tubule consists of a single layer of cells. These cells have round-shaped nuclei. Two different cell types were demonstrated in the tubule cell. These are cells that have electron-dense cytoplasm and electron-lucent cytoplasm. It was observed that the cytoplasm of these cells has many spherocrystals. The chemical composition of the spherocrystals was found to be high in carbon, phosphorus, and manganese in tubule cells.