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Background: The interactions between bacterial pathogens and host cells are characterized by a multitude of complexities, leading to a wide range of heterogeneous outcomes. Despite extensive research, we still have a limited understanding of how bacterial motility in complex environments impacts their ability to tolerate antibiotics and adhere to mammalian cell surfaces. The challenge lies in unraveling the complexity of these interactions and developing quantitative microscopy approaches to predict the behavior of bacterial populations. Results: To address this challenge, we directed our efforts towards Pseudomonas aeruginosa, a pathogenic bacterium known for producing thick films in the lungs of cystic fibrosis patients, and Escherichia coli, used as a proof of concept to develop and demonstrate our single-cell tracking approaches. Our results revealed that P. aeruginosa exhibits diverse and complex interactions on mammalian cell surfaces, such as adhesion, rotational motion, and swimming, unlike the less interactive behavior of Escherichia coli. Our analysis indicated that P. aeruginosa demonstrated lower mean-squared displacement (MSD) values and greater adherence to mammalian cells compared to E. coli, which showed higher MSD slopes and less frequent adherence. Genetic mutations in membrane proteins of P. aeruginosa resulted in altered displacement patterns and reduced adhesion, with the ΔfliD mutant displaying a more Gaussian displacement distribution and significantly less adherence to mammalian cells. Adhesion and tolerance mechanisms are diverse and complex, potentially involving distinct pathways; however, our findings highlight the therapeutic potential of targeting the fliD gene (encoding a critical flagellum protein), as its deletion not only reduced adherence but also antibiotic tolerance. Conclusions: Overall, our findings underscore the importance of single cell tracking in accurately assessing bacterial behavior over short time periods and highlight its significant potential in guiding effective intervention strategies. [ABSTRACT FROM AUTHOR]

In this work, we propose a structured-illumination lensless digital holographic microscopy (SI-LDHM). SI-LDHM illuminates a sample with 24 structured illuminations (8 orientations × 3 phase shifts) and records the defocused interferogram formed by two copies of object waves along the ±1st diffraction orders of each SI. The reconstructed object waves under different illumination orientations are respectively propagated to the sample plane along the +1st diffraction order and then averaged, thus yielding a clean image without the artifact of twin images. Experimental results demonstrated that thanks to the multi-oriented SI strategy, the twin image in SI-LDHM is sevenfold reduced compared to conventional DHM, while the spatial resolution is 1.15 times higher. [ABSTRACT FROM AUTHOR]

Sensing and control of size are critical for cellular function and survival. A striking example of size sensing occurs during meiosis in the nematode Caenorhabditis elegans. C. elegans chromosomes compare the lengths of the two chromosome "arms" demarcated by the position of their single off-center crossover, and they differentially modify these arms to ensure that sister chromatid cohesion is lost specifically on the shorter arm in the first meiotic division, while the longer arm maintains cohesion until the second division. While many of the downstream steps leading to cohesion loss have been characterized, the length-sensing process itself remains poorly understood. Here, we have used cytological visualization of short and long chromosome arms, combined with quantitative microscopy, live imaging, and simulations, to investigate the principles underlying length-sensitive chromosome partitioning. By quantitatively analyzing short-arm designation patterns on fusion chromosomes carrying multiple crossovers, we develop a model in which a short-arm-determining factor originates at crossover designation sites, diffuses within the synaptonemal complex, and accumulates within crossover-bounded chromosome segments. We demonstrate experimental support for a critical assumption of this model: that crossovers act as boundaries to diffusion within the synaptonemal complex. Further, we develop a discrete simulation based on our results that recapitulates a wide variety of observed partitioning outcomes in both wild-type and previously reported mutants. Our results suggest that the concentration of a diffusible factor is used as a proxy for chromosome length, enabling the correct designation of short and long arms and proper segregation of chromosomes. [Display omitted] • CO-delimited chromosome functional arms are established in mid-meiotic prophase • Both phosphorylated and non-phosphorylated SYP-1 proteins partition to short arms • Multiple COs can act additively on domains to establish "short" or "long" identity • CO sites block diffusion along the SC, enabling potential concentration of factors The holocentric chromosomes of C. elegans lose cohesion at meiosis I in the shorter of the two domains defined by the single crossover. Rodriguez-Reza et al. provide evidence that the sensing of short versus long domains relies on comparing the concentration of crossover-derived factors accumulating within these domains as a proxy for physical length. [ABSTRACT FROM AUTHOR]

The intracranial cavity contains vitally important organs. The brain, cerebellum, meninges and the vessels that supply these organs are located in the intracranial cavity. Therefore, it is important to learn about the intracranial cavity and to study it. However, there is limited information about the intracranial cavity in the veterinary field. The aim of this study was to determine the differences between the intracranial cavities of different species of animals by using stereology and tomography methods, volume calculations and morphometric measurements. In addition, the compatibility of the methods used with each other was investigated. In the study, six male adult goats and six male adult sheep were used. In this study, the intracranial cavities of sheep and goats were calculated by using Cavalieri's principle and 3D modelling using tomography sections. Morphometric measurements were taken over the intracranial space, and index calculations were made. In 3D models using computed tomography, the intracranial volume was 153.31 ± 24.06 cm3 in goats and 128.07 ± 7.93 cm3 in sheep. In the calculation using Cavalieri's principle, it was determined to be 152.73 ± 22.73 cm3 in goats and 126.15 ± 8.38 cm3 in sheep. As a result of the study, the MWCC (maximum width of the cranial cavity) parameter was found to be statistically significant between species (p < 0.05). The two methods used in Bland‐Altman analysis were found to be within the limits of agreement, and the methods can be alternative to each other. [ABSTRACT FROM AUTHOR]

The cerebral cortex comprises many distinct regions that differ in structure, function, and patterns of connectivity. Current approaches to parcellating these regions often take advantage of functional neuroimaging approaches that can identify regions involved in a particular process with reasonable spatial resolution. However, neuroanatomical biomarkers are also very useful in identifying distinct cortical regions either in addition to, or in place of functional measures. For example, differences in myelin density are thought to relate to functional differences between regions, are sensitive to individual patterns of experience, and have been shown to vary across functional hierarchies in a predictable manner. Accordingly, the current study provides quantitative stereological estimates of myelin density for each of the 13 regions that make up the feline auditory cortex. We demonstrate that significant differences can be observed between auditory cortical regions, with the highest myelin density observed in the regions that comprise the auditory core (i.e., the primary auditory cortex and anterior auditory field). Moreover, our myeloarchitectonic map suggests that myelin density varies in a hierarchical fashion that conforms to the traditional model of spatial organization in auditory cortex. Taken together, these results establish myelin as a useful biomarker for parcellating auditory cortical regions, and provide detailed estimates against which other, less invasive methods of quantifying cortical myelination may be compared. [ABSTRACT FROM AUTHOR]

The proper function of the placenta is essential for the health and growth of the fetus and the mother. The placenta relies on dynamic gene expression for its correct and timely development and function. Although numerous studies have identified genes vital for placental functions, equine placental molecular research has primarily focused on single placental locations, in sharp contrast with the broader approach in human studies. Here, we hypothesized that the molecular differences across different regions of the equine placenta are negligible because of its diffuse placental type with a macroscopic homogenous distribution of villi across the placental surface. We compared the transcriptome and stereological findings of the body, pregnant horn, and non-pregnant horn within the equine chorioallantois. Our transcriptomic analysis indicates that the variation between regions of the placenta within individuals is less than the variation observed between individuals. A low number of differentially expressed genes (DEGs) (n = 8) was identified when comparing pregnant and non-pregnant horns within the same placenta, suggesting a remarkable molecular uniformity. A higher number of DEGs was identified when comparing each horn to the body (193 DEGs comparing pregnant horn with body and 207 DEGs comparing non-pregnant horn with body). Genes with a higher expression in the body were associated with processes such as extracellular matrix synthesis and remodeling, which is relevant for placental maturation and placenta-endometrial separation at term and implies asynchrony of these processes across locations. The stereological analysis showed no differences in microcotyledonary density, and width between the locations. However, we observed a greater chorioallantoic thickness in the body and pregnant horn compared to the non-pregnant horn. Overall, our findings reveal a uniform transcriptomic profile across the placental horns, alongside a more distinct gene expression pattern between the uterine body and horns. These regional differences in gene expression suggest a different pace in the placental maturation and detachment among the placental locations. [Display omitted] • Gene expression variation observed across different placental locations. • Genes related to ECM degradation vary between the body and horns. • Microscopic features are uniform across the entire placenta. [ABSTRACT FROM AUTHOR]

Scanning thermal microscopy (SThM) is a scanning probe technique aimed at quantitative characterization of local thermal properties at the length scale down to tens of nanometers. With many probe designs and approaches to interpretation of probe responses, there is a need for a universal framework, which would allow probe calibration and comparison of probe performance. Herein, a calibration framework based on an abstracted, formal, probe model for active SThM probes is developed. The calibration can be accomplished through measurements with two or three calibration samples. Requirements for calibration samples are described with examples of structures of suitable samples identified in published literature. A link to a published experimental work indirectly verifying the proposed procedure is provided. The calibration does not require knowledge of internal probe properties and yields a small and universal set of parameters that can be used to quantify thermal resistance presented to the probe by samples as well as to characterize active‐mode SThM probes of any type and at any measurement frequency. How the probe calibration parameters can be used to guide probe design is illustrated. When the calibration approach can be used directly to measure the thermal conductivity of unknown samples is also analyzed. [ABSTRACT FROM AUTHOR]

We propose a method for integrating confocal amplitude and phase images obtained through dual-comb microscopy (DCM). DCM combines the benefits of confocal laser microscopy and quantitative phase microscopy, offering high axial resolution and scan-less imaging. By leveraging the coherence between confocal amplitude and phase images within the same DCM system, we accurately determine the number of phase wrapping iterations, thereby eliminating ambiguity in phase wrapping. We demonstrate this approach using samples with micrometer-range optical thickness and nanometer-scale surface roughness. The results demonstrate an expanded axial range, spanning from micrometers to millimeters, while maintaining nanometer-level axial resolution. This integrated DCM imaging technique enables the simultaneous acquisition of confocal amplitude image and absolute phase image, thus enhancing its potential for wide-axial-dynamic-range imaging across various applications. [ABSTRACT FROM AUTHOR]

Whole‐organ imaging and characterization at a submicron level provide abundant information on development and diseases while remaining a big challenge, especially in the context of time load. Herein, a quantitative light‐sheet microscopy platform that enabled highly time‐efficient assessments of fibrous structures within the intact cleared tissue is developed. Dual‐view inverted selective plane illumination microscopy (diSPIM), followed by improved registration and deconvolution, led to submicron isotropic imaging of mouse upper genital tract with one hundred‐fold speed‐ups than previous efforts. Further, optical metrics quantifying 3D local density and structural complexity of targets based on parallel and vectorized convolution in both spatial and frequency domains are developed. Collectively, ≈400–2000 fold increases in time efficiency counting for imaging, postprocessing, and quantitative characterization compared to the traditional method is gained. Using this platform, automatic identification of medulla and cortex within the mouse ovary at over 90% overlap with manual selection by anatomy experts is achieved. Additionally, heterogeneous distributions of immune cells in the mouse ovary and fallopian tube, offering a unique perspective for understanding the immune microenvironment are discovered. This work paves the way for future whole‐organ study, and exhibits potential with promise for offering mechanistic insights into physiological and pathological alterations of biological tissues. [ABSTRACT FROM AUTHOR]

The mesoscope has emerged as a powerful imaging tool in biomedical research, yet its high cost and low resolution have limited its broader application. Here, we introduce the Omni-Mesoscope, a high-spatial-temporal and multimodal mesoscopic imaging platform built from cost-efficient off-the-shelf components. This system uniquely merges the capabilities of label-free quantitative phase microscopy to capture live-cell morphodynamics across thousands of cells with highly multiplexed fluorescence imaging for comprehensive molecular characterization. This Omni-Mesoscope offers a mesoscale field of view of ~5 square millimeters with a high spatial resolution down to 700 nanometers, enabling the capture of detailed subcellular features. We demonstrate its capability in delineating molecular characteristics underlying rare morphodynamic cellular phenomena, including cancer cell responses to chemotherapy and the emergence of polyploidy in drug-resistant cells. We also integrate expansion technique to enhance three-dimensional volumetric super-resolution imaging of thicker tissues, opening the avenues for biological exploration at unprecedented scales and resolutions. [ABSTRACT FROM AUTHOR]

In 2023, the ImaBio consortium (imabio-cnrs.fr), an interdisciplinary life microscopy research group at the Centre National de la Recherche Scientifique, celebrated its 20th anniversary. ImaBio contributes to the biological imaging community through organization of MiFoBio conferences, which are interdisciplinary conferences featuring lectures and hands-on workshops that attract specialists from around the world. MiFoBio conferences provide the community with an opportunity to reflect on the evolution of the field, and the 2023 event offered retrospective talks discussing the past 20 years of topics in microscopy, including imaging of multicellular assemblies, image analysis, quantification of molecular motions and interactions within cells, advancements in fluorescent labels, and laser technology for multiphoton and label-free imaging of thick biological samples. In this Perspective, we compile summaries of these presentations overviewing 20 years of advancements in a specific area of microscopy, each of which concludes with a brief look towards the future. The full presentations are available on the ImaBio YouTube channel (youtube.com/@gdrimabio5724). [ABSTRACT FROM AUTHOR]

Ever since Robert Hooke's 17th century discovery of the cell using a humble compound microscope, light--matter interactions have continuously redefined our understanding of cell biology. Fluorescence microscopy has been particularly transformative and remains an indispensable tool for many cell biologists. The subcellular localization of biomolecules is now routinely visualized simply by manipulating the wavelength of light. Fluorescence polarization microscopy (FPM) extends these capabilities by exploiting another optical property -- polarization -- allowing researchers to measure not only the location of molecules, but also their organization or alignment within larger cellular structures. With only minor modifications to an existing fluorescence microscope, FPM can reveal the nanoscale architecture, orientational dynamics, conformational changes and interactions of fluorescently labeled molecules in their native cellular environments. Importantly, FPM excels at imaging systems that are challenging to study through traditional structural approaches, such as membranes, membrane proteins, cytoskeletal networks and large macromolecular complexes. In this Review, we discuss key discoveries enabled by FPM, compare and contrast the most common optical setups for FPM, and provide a theoretical and practical framework for researchers to apply this technique to their own research questions. [ABSTRACT FROM AUTHOR]

Background Materials and methods Results Conclusion Global cerebral ischemia (GCI) leads to significant oxidative damage in the cerebellum, mainly affecting Purkinje cells. This study aimed to investigate the neuroprotective effects of berberine chloride (BBR), a compound known for its antioxidant properties against GCI.Fourty‐two adult male Wistar rats were divided into three groups: Sham, GCI, and GCI + BBR. Rats received BBR (50 mg/kg) seven days before and six hours after inducing GCI via bilateral common carotid artery occlusion for 20 min. They were assessed for locomotor activity by the open‐field test, the cerebellar biochemical factors malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), cerebellum volume and Purkinje neuron count by stereological analysis.The BBR treatment reduced the concentration of MDA and increased the activity of antioxidant enzymes SOD, CAT and GPx in the GCI + BBR group compared to the GCI group. Stereological analysis revealed higher Purkinje cell count, cerebellum, white matter, and gray matter volume in the GCI + BBR group compared to the GCI group. Furthermore, GCI + BBR showed enhanced locomotor function compared to the GCI group.BBR showed therapeutic benefits and improved locomotor function. It demonstrated antioxidative effects by lowering MDA levels, boosting enzymatic activities, and significantly mitigating Purkinje cell death and cerebellar volume loss. [ABSTRACT FROM AUTHOR]

Aim Methodology Results Conclusions Fluoride is widely used in dentistry for its caries prevention. To reduce dental caries, the optimal fluoride concentration of public water supplies in the United States is 0.7 ppm. However, excessive systemic fluoride consumption can lead to dental/enamel fluorosis. Numerous studies have explored the effects of fluoride on enamel and enamel‐forming cells. However, research on systemic fluoride's impact on dentine is limited, particularly the effect of fluoride on the structure of the dentine–pulp complex. Therefore, this study aimed to identify how excessive fluoride affects dentine microstructure using an experimental mouse model.C57BL6/J male mice (6–9 weeks old) were randomized into four groups (Fluoride at 0, 50, 100, or 125 ppm in drinking water) (n = 4/group). Mice were provided water ad libitum for 6 weeks along with fluoride‐free food. Thereafter, mandibular incisors were analysed. Enamel phenotypes were evaluated using light microscopy and quantitative light‐induced fluorescence (QLF) to measure fluorosis levels. Dentine morphology was evaluated using micro‐CT, scanning electron microscopy (SEM), SEM–EDX (energy‐dispersive X‐ray), microhardness test and histological imaging. Data were analysed using one‐way ANOVA with Dunnett's multiple comparisons as a post hoc test and the Kruskal–Wallis test with Dunn's multiple comparisons post hoc test (p < .05).Mice treated with fluoride at 50–125 ppm developed enamel hypoplasia in their erupting incisors and micro‐CT imaging revealed that fluoride 125 ppm caused external resorption of the growing incisor. Dentine mineral density, dentine volume decreased compared with the 0 ppm control, while pulp volume increased compared with the 0 ppm control group. SEM showed wider predentine layer and abnormalities in calcified matrix vesicles derived from odontoblasts in fluoride 100 and 125 ppm groups. Vickers microhardness of dentine significantly decreased in the high‐dose group. Fluoride‐induced dentine hypoplasia in a dose‐dependent manner. Histological evaluation showed excessive fluoride 125 ppm induced micro abscess formation and inflammatory cell infiltration. Fluoride induced dentine dysplasia with a dentine microstructure resembling hypophosphatasia.High doses of systemic fluoride can cause dentine dysplasia. Both three‐dimensional and microstructural analyses showed the structural, chemical and mechanical changes in the dentine and the mineralized tissue components, along with external resorption and pulp inflammation. [ABSTRACT FROM AUTHOR]

In this work, we propose a structured-illumination lensless digital holographic microscopy (SI-LDHM). SI-LDHM illuminates a sample with 24 structured illuminations (8 orientations χ 3 phase shifts) and records the defocused interferogram formed by two copies of object waves along the ±1st diffraction orders of each SI. The reconstructed object waves under different illumination orientations are respectively propagated to the sample plane along the +1st diffraction order and then averaged, thus yielding a clean image without the artifact of twin images. Experimental results demonstrated that thanks to the multi-oriented SI strategy, the twin image in SI-LDHM is sevenfold reduced compared to conventional DHM, while the spatial resolution is 1.15 times higher. [ABSTRACT FROM AUTHOR]

Aims: Spinal muscular atrophy (SMA) is a life‐limiting paediatric motor neuron disease characterised by lower motor neuron loss, skeletal muscle atrophy and respiratory failure, if untreated. Revolutionary treatments now extend patient survival. However, a limited understanding of the foundational neuropathology challenges the evaluation of therapeutic success. As opportunities to study treatment‐naïve tissue decrease, we have characterised spinal cord pathology in severe infantile SMA using gold‐standard techniques, providing a baseline to measure treatment success and therapeutic limitations. Methods: Detailed histological analysis, stereology and transmission electron microscopy were applied to post‐mortem spinal cord from severe infantile SMA patients to estimate neuron number at the end of life; characterise the morphology of ventral horn, lateral horn and Clarke's column neuron populations; assess cross‐sectional spinal cord area; and observe myelinated white matter tracts in the clinically relevant thoracic spinal cord. Results: Ventral horn neuron loss was substantial in all patients, even the youngest cases. The remaining ventral horn neurons were small with abnormal, occasionally chromatolytic morphology, indicating cellular damage. In addition to ventral horn pathology, Clarke's column sensory–associated neurons displayed morphological features of cellular injury, in contrast to the preserved sympathetic lateral horn neurons. Cellular changes were associated with aberrant development of grey and white matter structures that affected the overall dimensions of the spinal cord. Conclusions: We provide robust quantification of the neuronal deficit found at the end of life in SMA spinal cord. We question long‐accepted dogmas of SMA pathogenesis and shed new light on SMA neuropathology out with the ventral horn, which must be considered in future therapeutic design. [ABSTRACT FROM AUTHOR]

Irreproducibility in molecular optical sectioning microscopy has hindered the transformation of acquired digital images from qualitative descriptions to quantitative data. Although numerous tools, metrics, and phantoms have been developed, accurate quantitative comparisons of data from different microscopy systems with diverse acquisition conditions remains a challenge. Here, they develop a simple tool based on an absolute measurement of bulk fluorophore solutions with related Poisson photon statistics, to overcome this obstacle is developed. Demonstrated in a prototypical multiphoton microscope, this tool unifies the unit of pixelated measurement to enable objective comparison of imaging performance across different modalities, microscopes, components/settings, and molecular targets. The application of this tool in live specimens identifies an attractive methodology for quantitative imaging, which rapidly acquires low signal‐to‐noise frames with either gentle illumination or low‐concentration fluorescence labeling. [ABSTRACT FROM AUTHOR]

A 12-week feeding trial with common carp (Cyprinus carpio) was conducted to test the hypothesis that compound diets formulated on the basis of different soybean cultivars will have effects on growth parameters, feed utilization, and digestive system histology. Soybean grains were from the following cultivars: Alisa (SB-A), Balkan (SB-B), and Galeb (SB-G). The fourth tested diet contained grains from a mixture of different SB cultivars (SB-M). The results confirmed the tested hypothesis, as the fish from the group SB-A showed higher average weight gain, specific growth rates, and feed efficiency, as well as lower feed intake and feed conversion ratio, compared to the groups SB-B and SB-M. On the other hand, diets were expected to cause inflammation in the distal intestine of the fish, but this did not occur. Histological examination of the intestine and liver, performed at the end of weeks 1, 3, 6 and 12, showed no pathological changes. Most of the differences between the groups were found at the end of the trial. The group SB-A had a higher surface area of different intestinal layers compared to the groups SB-B and SB-G. The surface area of the goblet cells was greatest at most of the time points in SB-M. In the liver, the evaluation of the surface area of hepatocytes and their nuclei showed no significant differences between the groups. The differences in final body mass, which showed a maximum value of 18% between the groups, could be of significant importance for culture of this freshwater species. [ABSTRACT FROM AUTHOR]

Background: Sodium butyrate is a potential antibiotic growth promoter and has had advantageous effects on the poultry industry. Methods: Evaluating the effect of sodium butyrate on the intestinal villi and the humoral part of innate immunity of the male Cobb 500 broiler using scanning electron microscopy and quantitative real-time PCR analysis, the control group and treated group of Cobb 500 with SB supplemented received water containing 0.98 mg sodium butyrate. Results: The administration of sodium butyrate changed the villi characters, as the shape changed from tongue to long tongue. They were mainly parallel to each other and long finger-like at the duodenum. The tips of the villi in the control group appeared thin-slight curved with a prominent center in the duodenum, thin rectangular in the jejunum, and ileum in the control group. In contrast, in the treatment group, they changed to thick rectangular in the duodenum and ileum zigzag shape in the jejunum. The epithelium lining of the duodenal villi showed a dome shape, the jejunal villi showed a polygonal shape, and the ileal villi appeared scales-like. The epithelium lining showed irregular microfolds and many different-sized pores, and the treatment group showed islands of long microvilli in the duodenum and solitary long microvilli in the ileum. Real-time PCR of AvBD 1, 2, 10, and 12 significantly (P < 0.01). The better expression of AvBD 1, 2, and 12 was determined in the duodenum, while AvBD 10 was in the jejunum. Conclusion: Sodium butyrate enhanced the chicks' growth and small intestine parameters, modified the morphology of the intestinal villi, and improved the humoral part of innate immunity. [ABSTRACT FROM AUTHOR]

Pancreatic β cells secrete insulin in response to elevated levels of glucose. Stem cell derived β (SCβ) cells aim to replicate this glucose-stimulated insulin secretion (GSIS) function, but current preparations cannot provide the same level of insulin as natural β cells. Here, we develop an assay to measure GSIS at the single cell level to investigate the functional heterogeneity of SCβ cells and donor-derived islet cells. Our assay involves randomly depositing single cells and insulin capture microbeads in opentop nanowells (40 × 40 × 55 μm³ ) fabricated on glass-bottom imaging microwell plates. Insulin secreted from single cells is captured on microbeads and then stained using a detection antibody. The nanowell microstructure limits diffusion of secreted insulin. The glass substrate provides an optically flat surface for quantitative microscopy to measure the concentration of secreted insulin. We used this approach to measure GSIS from SCβ cells and donor-derived islet cells after 15 minutes exposure to 3.3 mM and 16.7 mM glucose. Both cell types exhibited significant GSIS heterogeneity, where elite cells (<20%) produced the majority of the secreted insulin (55–78%). This assay provides an immediate readout of single cell glucose-stimulated insulin secretion in a flexible well plate-based format. [ABSTRACT FROM AUTHOR]

Macroautophagy is often quantified by live imaging of autophagosomes labeled with fluorescently tagged ATG8 protein (FP–ATG8) in Arabidopsis thaliana. The labeled particles are then counted in single focal planes. This approach may lead to inaccurate results as the actual 3D distribution of autophagosomes is not taken into account and appropriate sampling in the Z -direction is not performed. To overcome this issue, we developed a workflow consisting of immunolabeling of autophagosomes with an anti-ATG8 antibody followed by stereological image analysis using the optical disector and the Cavalieri principle. Our protocol specifically recognized autophagosomes in epidermal cells of Arabidopsis root. Since the anti-ATG8 antibody recognizes multiple At ATG8 isoforms, we were able to detect a higher number of immunolabeled autophagosomes than with the FP– At ATG8e marker, that most probably does not recognize all autophagosomes in a cell. The number of autophagosomes per tissue volume positively correlated with the intensity of autophagy induction. Compared with the quantification of autophagosomes in maximum intensity projections, stereological methods were able to detect the autophagosomes present in a given volume with higher accuracy. Our novel workflow provides a powerful toolkit for unbiased and reproducible quantification of autophagosomes and offers a convenient alternative to the standard of live imaging with FP–ATG8 markers. [ABSTRACT FROM AUTHOR]

Soil-transmitted helminth (STH) infections account for a significant global health burden, necessitating mass drug administration with benzimidazole-class anthelmintics, such as albendazole (ALB), for morbidity control. However, ALB efficacy shows substantial variability, presenting challenges for achieving consistent treatment outcomes. We have explored the potential impact of the baseline gut microbiota on ALB efficacy in hookworm-infected individuals through microbiota profiling and machine learning (ML) techniques. Our investigation included 89 stool samples collected from hookworm-infected individuals that were analyzed by microscopy and quantitative PCR (qPCR). Of these, 44 were negative by microscopy for STH infection using the Kato-Katz method and qPCR 21 days after treatment, which entails a cure rate of 49.4%. Microbiota characterization was based on amplicon sequencing of the V3–V4 16S ribosomal RNA gene region. Alpha and beta diversity analyses revealed no significant differences between participants who were cured and those who were not cured, suggesting that baseline microbiota diversity does not influence ALB treatment outcomes. Furthermore, differential abundance analysis at the phylum, family and genus levels yielded no statistically significant associations between bacterial communities and ALB efficacy. Utilizing supervised ML models failed to predict treatment response accurately. Our investigation did not provide conclusive insights into the relationship between gut microbiota and ALB efficacy. However, the results highlight the need for future research to incorporate longitudinal studies that monitor changes in the gut microbiota related to the infection and the cure with ALB, as well as functional metagenomics to better understand the interaction of the microbiome with the drug, and its role, if there is any, in modulating anthelmintic treatment outcomes in STH infections. Interdisciplinary approaches integrating microbiology, pharmacology, genetics and data science will be pivotal in advancing our understanding of STH infections and optimizing treatment strategies globally. [ABSTRACT FROM AUTHOR]

A parameter study of ultrashort pulse laser‐induced modifications in the bulk of ZnS crystals is reported. Experimental results on these modifications and their dependence on the pulse energy, writing speed, and depth are presented, with an emphasis on cross‐sectional morphology and induced refractive index changes. Localized permanent material modifications have been inscribed in the bulk of the crystal using a laser with a center emission wavelength of 2.09 μm and a pulse duration of ≈4 ps. The morphology strongly depends on the laser and optical focusing parameters, in particular, on the pulse energy and processing speed, with a significant shift in depth dependence for short pulse‐to‐pulse separations. Depending on the applied pulse energy, distortions of the lateral profile of the refractive index changes appear in the form of oscillatory features in the transverse plane relative to the inducing laser beam. The true extent of the modified material is revealed by the alternating lateral profile with largest induced negative refractive index change, Δn, of −3.88 × 10−2 ± 0.18. Such parametric insight is of critical importance for the understanding and optimization of the fabrication process and for realizing compact 3D photonic devices in the bulk of materials in a reproducible manner. [ABSTRACT FROM AUTHOR]

The objective of this study is to recognize and characterize the nanoscale phase modulus mapping of the asphalt film in pavement mixture cores using atomic force microscopy quantitative nanomechanical technology. The pavement core samples from the upper and middle layers of four highways and laboratory samples were taken as the research object. The phase modulus–macro property correlation of recovered asphalt was analyzed using mathematical statistics. The results showed that the pavement core samples had more significant multi-phase and diversified phase characteristics compared to lab samples. This indicated that the asphalt in the pavement core had an obvious phase separation phenomenon due to aging. The phase modulus of each sample was distributed across a relatively wide numerical range, and there were also many numerical points with large fluctuations. Especially for the mixture sample containing SBS (Styrene-Butadiene-Styrene)-modified asphalt, the phase modulus distribution mappings presented a multi-peak phenomenon. Hence, considering the distribution characteristics of the data, the box plot method was introduced. Compared with quantified results from laboratory samples, the phase modulus of SBS-modified asphalt increased by 0.96 times, 1.18 times and 1.15 times, and that of base asphalt increased by 0.59 times, 0.56 times, 0.42 times, 1.24 times and 0.39 times, respectively. This indicates that the aging degree of asphalt in the upper layer was generally greater than that of the asphalt in the middle layer and that there was an aging gradient in the direction of pavement depth. All points were within the 95% confidence band in terms of correlation fitting, indicating a better fitting effect between phase modulus and complex shear modulus, as well as between phase modulus and penetration. This research provides innovative ideas for future multi-scale numerical simulation and cross-scale performance model development of asphalt binders. [ABSTRACT FROM AUTHOR]

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The rabbit is widely used as a laboratory animal in experimental models of kidney diseases. This species is also important from a veterinary perspective as a companion animal. Stereology has been accepted as an accurate approach to kidney morphometry. The objective of the present project was to provide normal quantitative stereological parameters for adult rabbit kidneys. The left kidneys of five adult male New Zealand rabbits were used. Isotropic sections were obtained using the orientation method. Total kidney volume was calculated by the Cavalieri principle. The volume fraction of the renal structures was estimated using the point counting system. The lengths of the proximal convoluted tubule (PCT) and distal convoluted tubule (DCT) were calculated using counting frames. The total glomerular number was accounted for using the physical/fractionator technique. The mean glomerular volume was obtained by dividing the total volume of glomeruli by their total number. The total volume of rabbit kidneys calculated was 10.39 ± 1.98 cm3. The fractional volume of the kidney cortex and medulla accounted for 57.79 ± 0.65% and 42.2 ± 0.65%, respectively. The total glomerular volume was 2.18 ± 0.32% of the whole kidney. The total number of glomeruli in the rabbit kidney was estimated as 204.68 ± 12 × 103. The mean glomerular volume measured 1.07 ± 0.12 × 106 μm3. The total length of PCT and DCT was 2.96 ± 0.29 km and 1.38 ± 0.24 km, respectively. These findings can be used as a reference in experimental nephrology research and may help to expand the knowledge of nephrology in mammals by comparing with available data on humans and other species. Research Highlights: Three‐dimensional morphometry of adult rabbit kidney structures was analyzed using quantitative stereology.Total volume of kidney, fractional volume of cortex and medulla, length of renal tubules and number of nephrons were estimated.These three‐dimensional morphometrical data can be used as a reference in experimental nephrology research and may help to expand the knowledge of nephrology in mammals. [ABSTRACT FROM AUTHOR]

Lab-on-a-Chip microfluidic devices present an innovative and cost-effective platform in the current trend of miniaturization and simplification of imaging flow cytometry; they are excellent candidates for high-throughput single-cell analysis. In such microfluidic platforms, cell tracking becomes a fundamental tool for investigating biophysical processes, from intracellular dynamics to the characterization of cell motility and migration. However, high-throughput and long-term cell tracking puts a high demand on the consumption of computing resources. Here, we propose a novel strategy to achieve rapid 3D cell localizations along the microfluidic channel. This method is based on the spatiotemporal manipulation of recorded holographic interference fringes, and it allows fast and precise localization of cells without performing complete holographic reconstruction. Conventional holographic tracking is typically based on the phase contrast obtained by decoupling the calculation of optical axial and transverse coordinates. Computing time and resource consumption may increase because all the frames need to be calculated in the Fourier domain. In our proposed method, the 2D transverse positions are directly located by morphological calculation based on the hologram. The complex-amplitude wavefronts are directly reconstructed by spatiotemporal phase shifting to calculate the axial position by the refocusing criterion. Only spatial calculation is considered in the proposed method. We demonstrate that the computational time of transverse tracking is only one-tenth of the conventional method, while the total computational time of the proposed method decreases up to 54% with respect to the conventional approach. The proposed approach can open the route for analyzing flow cytometry in quantitative phase microscopy assays. [ABSTRACT FROM AUTHOR]

(1) Background: Considering the increasing workload of pathologists, computer-assisted methods have the potential to come to their aid. Considering the prognostic role of myocardial fibrosis, its precise quantification is essential. Currently, the evaluation is performed semi-quantitatively by the pathologist, a method exposed to the issues of subjectivity. The present research proposes validating a semi-automatic algorithm that aims to quantify myocardial fibrosis on microscopic images. (2) Methods: Forty digital images were selected from the slide collection of The Iowa Virtual Slidebox, from which the collagen volume fraction (CVF) was calculated using two semi-automatic methods: CIELAB-MATLAB® and CIELAB-Python. These involve the use of color difference analysis, using Delta E, in a rectangular region for CIELAB-Python and a region with a random geometric shape, determined by the user's cursor movement, for CIELAB-MATLAB®. The comparison was made between the stereological evaluation and ImageJ. (3) Results: A total of 36 images were included in the study (n = 36), demonstrating a high, statistically significant correlation between stereology and ImageJ on the one hand, and the proposed methods on the other (p < 0.001). The mean CVF determined by the two methods shows a mean bias of 1.5% compared with stereology and 0.9% compared with ImageJ. Conclusions: The combined algorithm has a superior performance compared to the proposed methods, considered individually. Despite the relatively small mean bias, the limits of agreement are quite wide, reflecting the variability of the images included in the study. [ABSTRACT FROM AUTHOR]

Exposure to bisphenol A (BP), an environmental pollutant, is potentially harmful to both human health and the environment. The purpose of the current research was to evaluate the effectiveness of β-caryophyllene (CF) (200 mg/kg) on rat cerebellar tissues exposed to BP (250 mg/kg). Thirty-five randomly selected male rats were split into five groups as: control (CON), olive oil (OL), BP, CF, and CF+BP. On day 15 of the experiment, all rats' cerebellar tissues were immediately extracted, followed by stereological and histological examination. Our results revealed that MDA level was significantly elevated in the BP group compared to the CON group (p

Abstract The ultrastructure of human oocytes has been described only qualitatively. To offer a precise organelle spatial distribution and organelle volume during the main maturation stages, we previously conducted stereological studies on prophase-I (GV) and metaphase-I (MI) oocytes, and here we present results on metaphase-II (MII) oocytes. Five donor oocytes from different donors were processed for transmission electron microscopy, and quantification of organelle distribution was performed using point-counting stereology. Statistical tests compared the means of the relative volumes occupied by organelles among oocyte regions. The most abundant organelles were elements of the smooth endoplasmic reticulum (SER), such as SER small vesicles, SER medium vesicles, SER large vesicles and SER isolated tubules, along with mitochondria, followed by SER tubular aggregates, cortical vesicles and lysosomes. Significant differences between oocyte regions were found for lysosomes, cortical vesicles and SER large vesicles. Comparisons of MII oocytes to previous findings in GV and MI oocytes evidenced specific patterns of organelle distribution and relative volumes. This final evaluation thus enables to track organelle spatial reorganization across oocyte stages, which, in addition to gathered knowledge, may be useful to assist in improvements of stimulation protocols, in-vitro maturation media and cryopreservation techniques.

[Display omitted] Liquid-liquid phase separation is a key phenomenon in the formation of membrane-less structures within the cell, appearing as liquid biomolecular condensates. Protein condensates are the most studied for their biological relevance, and their tendency to evolve, resulting in the formation of aggregates with a high level of order called amyloid. In this study, it is demonstrated that Human Insulin forms micrometric, round amyloid-like structures at room temperature within sub-microliter scale aqueous compartments. These distinctive particles feature a solid core enveloped by a fluid-like corona and form at the interface between the aqueous compartment and the glass coverslip upon which they are cast. Quantitative fluorescence microscopy is used to study in real-time the formation of amyloid-like superstructures. Their formation results driven by liquid–liquid phase separation process that arises from spatially heterogeneous distribution of nuclei at the glass-water interface. The proposed experimental setup allows modifying the surface-to-volume ratio of the aqueous compartments, which affects the aggregation rate and particle size, while also inducing fine alterations in the molecular structures of the final assemblies. These findings enhance the understanding of the factors governing amyloid structure formation, shedding light on the catalytic role of surfaces in this process. [ABSTRACT FROM AUTHOR]

The objective of this study was to examine the therapeutic efficacy of curcumin (CUR) and α-lipoic acid (ALA) in mitigating UV-A and UV-B-induced damage (UVAB) in rat dorsal skin. This was achieved through the utilisation of immunohistochemical (TUNEL), biochemical and stereological techniques. The rats in the UVAB, UVAB + CUR, and UVAB + ALA groups were subjected to UVAB irradiation for a period of two hours per day over the course of one month. The UVAB + CUR and UVAB + ALA groups were administered 100 mg/kg/day of curcumin and 100 mg/kg/day of α-lipoic acid via gavage 30 min prior to UVAB irradiation. The CUR group was administered 100 mg/kg/day of curcumin via gavage, while the ALA group received the same dose of α-lipoic acid. A significant change in the volume ratio of the dorsal skin epidermis and dermis was observed in the stereological findings of the rats in the UVAB group. These changes exhibited a favourable progression as a consequence of the CUR and ALA applications. In the UVAB group, TOS and OSI were significantly elevated as a consequence of the rise in oxidative stress. Conversely, the treatment groups demonstrated a notable reduction in TOS and OSI levels. The study also revealed a substantial increase in the number of apoptotic cells within the UVAB group. However, the treatment groups exhibited a significant decline in apoptotic cells. In conclusion, the findings suggest that CUR and ALA possess a protective effect against UVAB-induced skin damage. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of minocycline (Mino) on the polarization of types M1/M2 microglia (pro- and anti-inflammatory type) in the spinal dorsal horn of rats with neuropathic pain (NP) induced by spinal nerve ligation (SNL) and its underlying mechanism. Methods A total of 36 adult male SD rats were randomly stratified into Sham-operation (Sham) group, SNL group and Mino+SNL group by stratified random sampling based on body weight. Mechanical pain threshold and cold nociceptive thresholds of rat hind paw were measured in 1 d before and 14 d after modelling. Spinal cord tissue at the lumbar 5 (L5) segment was taken at 14 d after modelling, and the total number of microglia as well as the numbers of M1 and M2 microglia in the spinal dorsal horn were measured with immunohistochemistry and stereology. With aid of bioinformatics techniques, the core target in the spinal cord, Cst7, was selected. Then, the protein levels of microglia marker Iba-1, M1 microglia marker iNOS, M2 microglia marker CD206, Cst7 encoded protein cystatin F (CF) and pathway CatS/CX3CL1/CX3CR1 were detected with Western blotting. The expression levels of TNF-α, IL-6 and IL-10 in the spinal cord tissues were measured with ELISA. Results The mechanical pain and cold nociceptive thresholds were both significantly higher in the M+SNL group than the SNL group at 7~14 d after modelling (P < 0.01). The total number of microglia and the numbers of M1/M2 microglia in the spinal dorsal horn as well as the expression levels of CatS, CX3CL1, CX3CR1, TNF-α, IL-6, and IL-10 in the spinal cord tissues were obviously increased, and the expression level of CF was notably decreased in the SNL model group than the Sham group (P < 0.01). While, Mino treatment remarkably reversed above phenomena, with decreased total number of microglia and number of M1 microglia as well as expression levels of CatS, CX3CL1, CX3CR1, TNF-α and IL-6, and increased number of M2 microglia as well as CF and IL-10 levels when compared with the SNL group (P < 0.05). Conclusion Mino alleviates SNL induced neuropathic pain, probably through up-regulating CF in the microglia, and thus inhibiting the CatS/CX3CL1/CX3CR1 signaling pathway, promoting the conversion of microglia from type M1 to M2 to balance the imbalance in the M1/M2 polarization, and thus reducing neuroinflammation.

Single-particle tracking is a powerful technique to investigate the motion of molecules or particles. Here, we review the methods for analyzing the reconstructed trajectories, a fundamental step for deciphering the underlying mechanisms driving the motion. First, we review the traditional analysis based on the mean squared displacement (MSD), highlighting the sometimes-neglected factors potentially affecting the accuracy of the results. We then report methods that exploit the distribution of parameters other than displacements, e.g., angles, velocities, and times and probabilities of reaching a target, discussing how they are more sensitive in characterizing heterogeneities and transient behaviors masked in the MSD analysis. Hidden Markov Models are also used for this purpose, and these allow for the identification of different states, their populations and the switching kinetics. Finally, we discuss a rapidly expanding field—trajectory analysis based on machine learning. Various approaches, from random forest to deep learning, are used to classify trajectory motions, which can be identified by motion models or by model-free sets of trajectory features, either previously defined or automatically identified by the algorithms. We also review free software available for some of the analysis methods. We emphasize that approaches based on a combination of the different methods, including classical statistics and machine learning, may be the way to obtain the most informative and accurate results. [ABSTRACT FROM AUTHOR]

Here, we provide an overview of the key factors to consider when using fluorescence nanoscopy (FN), including biological questions that can be addressed and aspects that might improve the reliability and effectiveness of FN experiments. We cover the main aspects related to sample preparation, including the selection of appropriate fixation, affinity-based labels, and fluorescent dyes. We discuss current limitations and possible future developments in the field that would facilitate a broader application of FN. We discuss multiplexing possibilities (allowing the simultaneous detection of multiple targets in a single experiment), live cell imaging for the study of cellular and molecular dynamic processes, and quantitative workflows. Recently, biologists have gained access to several far-field fluorescence nanoscopy (FN) technologies that allow the observation of cellular components with ~20 nm resolution. FN is revolutionizing cell biology by enabling the visualization of previously inaccessible subcellular details. While technological advances in microscopy are critical to the field, optimal sample preparation and labeling are equally important and often overlooked in FN experiments. In this review, we provide an overview of the methodological and experimental factors that must be considered when performing FN. We present key concepts related to the selection of affinity-based labels, dyes, multiplexing, live cell imaging approaches, and quantitative microscopy. Consideration of these factors greatly enhances the effectiveness of FN, making it an exquisite tool for numerous biological applications. [ABSTRACT FROM AUTHOR]

ObjectiveMethodsResultsDiscussionPrenatal protein malnutrition produces anatomical and functional changes in the developing brain that persist despite immediate postnatal nutritional rehabilitation. Brain networks of prenatally malnourished animals show diminished activation of prefrontal areas and an increased activation of hippocampal regions during an attentional task [1]. While a reduction in cell number has been documented in hippocampal subfield CA1, nothing is known about changes in neuron numbers in the prefrontal or parahippocampal cortices.In the present study, we used unbiased stereology to investigate the effect of prenatal protein malnutrition on the neuron numbers in the medial prefrontal cortex and the cortices of the parahippocampal region that comprise the larger functional network.Results show that prenatal protein malnutrition does not cause changes in the neuronal population in the medial prefrontal cortex of adult rats, indicating that the decrease in functional activation during attentional tasks is not due to a reduction in the number of neurons. Results also show that prenatal protein malnutrition is associated with a reduction in neuron numbers in specific parahippocampal subregions: the medial entorhinal cortex and presubiculum.The affected regions along with CA1 comprise a tightly interconnected circuit, suggesting that prenatal malnutrition confers a vulnerability to specific hippocampal circuits. These findings are consistent with the idea that prenatal protein malnutrition produces a reorganization of structural and functional networks, which may underlie observed alterations in attentional processes and capabilities. [ABSTRACT FROM AUTHOR]

Momordica charantia (MC) is a traditional plant widely used since ancient times for wound healing. This study evaluated its potential effects on tendon healing. Adult male Wistar albino rats (n = 32, 8 rats in each group) were anesthetized, and their Achilles tendons were prepared for surgical procedures. Group 1 (Cont = control group) was not subjected to any surgery and was used as a control group for baseline values. Group 2 (PR = primary repair group) underwent primary repair (PR) with a monofilament suture after a full-thickness incision of the Achilles tendon. A full-thickness incision was also made to the Achilles tendon of group 3 (CT = collagen tube-administered group), followed by PR and collagen tube insertion. In group 4 (MC = M. charantia-administered group), 1 mL of MC extract was applied locally on the collagen tube in addition to the surgical procedure applied to group 3. The Achilles tendons were excised on the postoperative 40th day and examined stereologically, histologically, and bioinformatically. Data showed that the total volume of the collagen fibers was higher in MC and CT groups than in the PR group. The total volume of the tendon was decreased in MC and CT groups than in the Cont group. The ratios between the volumes of the collagen fibers and total tendon in the MC and CT groups were significantly different from PR, but not different from the Cont group. Additionally, MC improved tenoblastic activity, collagen production, and neovascularization. Bioinformatic interactions showed that the proteases of MC could trigger the signals playing a role on vasculogenesis, reducing inflammation, and contributing to tenoblast activation and collagen remodeling. MC extract ameliorates the healing of injured tendon and can provide satisfactory tendon repair. Further works are recommended to explore the healing capacity of MC. [ABSTRACT FROM AUTHOR]

In industrial practice, combining grain growth inhibitors (GGIs) with low carbon content effectively restrains WC grain growth in WC-Co cemented carbides. However, the intricate relationship between carbon content, WC grain microstructure, and GGI impact remains unresolved. This study investigates two WC-Co sample sets, maintaining constant VC inhibitor levels but varying carbon content near upper (HC) and lower (LC) limits. SEM analysis revealed distinct morphologies: HC displayed truncated prism shapes, exposing long basal and prismatic terraces, while LC exhibited a saw-tooth morphology with finer grains. EBSD stereology quantified anisotropic WC grain growth, revealing suppressed growth perpendicular to both basal (HC: 0.51 μm vs. LC: 0.38 μm) and prismatic planes (HC: 0.44 μm vs. LC: 0.37 μm) with decreased carbon content. Atomic-resolution EDS showed higher V solute excess in LC at WC(basal)-Co, WC(prismatic)-Co interfaces, and step corners (HC: 4.6, 0.9, and 4.4 atoms/nm2; LC: 5.5, 0.9, and 7.8 atoms/nm2). Our findings elucidate a clear physical understanding that a low carbon content enhances V atom solubility within liquid cobalt, increasing the thickness of complexions to quad-layer on WC (basal)-Co interfaces and forming V-rich nanoprecipitates at step corners. Those inhibitory effects further limited step flow, resulting in pronounced step bunching and a saw-tooth fine-grained morphology in LC sample. This study highlights that complexions housing more inhibitory solute elements exert a stronger drag effect on grain growth front migration. This approach has the potential for studying grain growth in anisotropic materials, highlighting the importance of constructing experimental complexion diagrams in engineering fine-structured ceramics and metals. [ABSTRACT FROM AUTHOR]

Background: Global cerebral ischemia (GCI) is characterized by the hindrance of blood flow to the brain due to obstruction in the carotid arteries. In this study, the neuroprotective effects of berberine chloride (BBR) and cinnamaldehyde (CA) against GCI in male Wistar rats were investigated, and their efficacy was compared. Methods: Twenty‐four rats were randomly assigned to four groups: Sham, GCI, GCI + BBR, and GCI + CA. 100 mg/kg of BBR and CA were administered five minutes and six hours after inducing GCI by occluding two common carotids for 20 minutes. Results: The findings showed that both compounds mitigated GCI‐induced damage to the hippocampus and its subfields, and CA demonstrated better neuroprotective efficacy than BBR. These results was also supported by behavioural tests, which revealed better memory function in GCI + CA compared to GCI + BBR. Conclusion: The study showed the neuroprotective impact of CA and BBR against GCI, and CA emerged as a more effective therapeutic agent against GCI‐induced damage. Further research is necessary to uncover the specific mechanisms behind these differences. [ABSTRACT FROM AUTHOR]

Quantitative characterization of lung structures by morphometrical or stereological analysis of histological sections is a powerful means of elucidating pulmonary structure-function relations. The overwhelming majority of studies, however, fix lungs for histology at pressures outside the physiological/pathophysiological respiratory volume range. Thus, valuable information is being lost. In this perspective article, we argue that investigators performing pulmonary histological studies should consider whether the aims of their studies would benefit from fixation at functional transpulmonary pressures, particularly those of end-inspiration and end-expiration. We survey the pressures at which lungs are typically fixed in preclinical structure-function studies, provide examples of conditions that would benefit from histological evaluation at functional lung volumes, summarize available fixation methods, discuss alternative imaging modalities, and discuss challenges to implementing the suggested approach and means of addressing those challenges. We aim to persuade investigators that modifying or complementing the traditional histological approach by fixing lungs at minimal and maximal functional volumes could enable new understanding of pulmonary structure-function relations. [ABSTRACT FROM AUTHOR]

Stereology, the gold standard of lung morphometry, critically depends on sampling of tissue for analysis. Random sampling approaches guarantee each part of the organ an equal chance of being included in the analysis; hence, they guarantee a representative sample of the whole. However, when biological or pathological structures of interest are rare and/or heterogeneously distributed over the whole lung, the random sampling approach can be inefficient or even result in meaningless data. In such cases, a targeted sampling approach can be useful, which helps to relate the analytical items to an appropriate reference space. Targeted stereology greatly benefits from the increasing availability of multiresolution imaging techniques at macroscopic and microscopic levels as well as digital tools of segmentation. As such, this article outlines two basic sampling scenarios: 1) In the first scenario, computed tomography and microscopy are subsequently used to segment the airway/arterial tree and perform stereological measurements on specific branches of the tree. 2) The second scenario deals with the heterogeneous distribution of pathological lesions. This type of analysis can be divided into two stages: assessment of lesions of interest (LOIs) within the lung and assessment of subcompartments within LOIs. Taken together, targeted stereology has a thorough foundation in stereological theory and is able to not only significantly increase the efficiency of the analysis but also yield new types of information that would be lost with the classical random sampling approach. [ABSTRACT FROM AUTHOR]

Eosinophils are cells of the innate immune system that orchestrate complex inflammatory responses. The study of the cell biology of eosinophils, particularly associated with cell activation, is of great interest to understand their immune responses. From a morphological perspective, activated eosinophils show ultrastructural signatures that have provided critical insights into the comprehension of their functional capabilities. Application of conventional transmission electron microscopy in combination with quantitative assessments (quantitative transmission electron microscopy), molecular imaging (immunoEM), and 3-dimensional electron tomography have generated important insights into mechanisms of eosinophil activation. This review explores a multitude of ultrastructural events taking place in eosinophils activated in vitro and in vivo as key players in allergic and inflammatory diseases, with an emphasis on viral infections. Recent progress in our understanding of biological processes underlying eosinophil activation, including in vivo mitochondrial remodeling, is discussed, and it can bring new thinking to the field. [ABSTRACT FROM AUTHOR]

Large and temperate Lake Peipsi is the fourth largest lake in Europe, where the massive cyanobacterial blooms are composed mostly of Microcystis spp., which have been common for several decades now. The seasonal dynamics of potentially toxic Microcystis were studied using microscopy and quantitative polymerase chain reaction (qPCR) by assessing the microcystin-encoding microcystin synthetase gene E (mcyE) abundances. Water samples were analyzed over the lake areas, varying in depth, trophic level, and cyanobacterial composition during the growing period of 2021. The Microcystis mcyE genes were detected through the growing period (May–October), forming peak abundances in September with decreasing temperatures (8.9–11.1 °C). Total phosphorus (TP) and nitrate (NO3−) were the most relevant environmental variables influencing the Microcystis biomass as well as mcyE abundances. Comparison with previous years (2011, 2012) indicated that the abundance and seasonal dynamics of toxigenic Microcystis can be highly variable between the years and lake areas, varying also in dominant Microcystis species. Contrary to expectations, based on mcyE abundances, the increased risk of toxin-producing Microcystis can occur in Peipsi through the growing period, independently of the water temperature and biomasses of Microcystis. [ABSTRACT FROM AUTHOR]

There is a significant gap in cost-effective quantitative phase microscopy (QPM) systems for studying dynamic cellular processes while maintaining accuracy for long-term cellular monitoring. Current QPM systems often rely on complex and expensive voltage-controllable components like Spatial Light Modulators or two-beam interferometry. To address this, we introduce a QPM system optimized for time-varying phase samples using azobenzene liquid crystal as a Zernike filter with a polarization-sensing camera. This system operates without input voltage or moving components, reducing complexity and cost. Optimized for gentle illumination to minimize phototoxicity, it achieves a 1 Hz frame rate for prolonged monitoring. The system demonstrated accuracy with a maximum standard deviation of ±42 nm and low noise fluctuations of ±2.5 nm. Designed for simplicity and single-shot operations, our QPM system is efficient, robust, and precisely calibrated for reliable measurements. Using inexpensive optical components, it offers an economical solution for long-term, noninvasive biological monitoring and research applications. Current quantitative phase microscopy (QPM) systems to study dynamic cellular processes are often expensive and complex. The authors develop a cost-effective QPM system using azobenzene liquid crystals, which operate without input voltage or moving parts. This system achieves accurate, low-noise measurements and is ideal for dynamic and non-invasive biological monitoring. [ABSTRACT FROM AUTHOR]

Fiber Bragg gratings are key components for optical fiber sensing applications in harsh environments. This paper investigates the structural and chemical characteristics of femtosecond laser photo-inscribed microvoids. These voids are at the base of type III fs-gratings consisting of a periodic array of microvoids inscribed at the core of an optical fiber. Using high-resolution techniques such as quantitative phase microscopy, electron transmission microscopy, and scattering-type scanning near-field IR optical microscopy, we examined the structure of the microvoids and the densified shells around them. We also investigated the high-temperature behavior of the voids, revealing their evolution in size and shape under step isochronal annealing conditions up to 1250 °C. [ABSTRACT FROM AUTHOR]

Background/aim: Spindle pole bodies (SPB), the functional equivalent of centrosomes in yeast, duplicate through generation of a new SPB next to the old one. However, SPBs are dynamic structures that can grow and exchange, and mechanisms that regulate SPB size remain largely unknown. This study aims to elucidate the role of Bud14 in SPB size maintenance in Saccharomyces cerevisiae. Materials and methods: We employed quantitative fluorescence microscopy to assess the relative and absolute amounts of SPB structural proteins at SPBs of wildtype cells and in cells lacking BUD14 (bud14Δ). Quantifications were performed using asynchronous cell cultures, as well as cultures synchronously progressing through the cell cycle and upon different cell cycle arrests. We also utilized mutants that allow the separation of Bud14 functions. Results: Our results indicate that higher levels of SPB inner, outer, and central plaque proteins are present at the SPBs of bud14Δ cells compared to wildtype cells during anaphase, as well as during nocodazole-induced M-phase arrest. However, during α-factor mediated G1 arrest, inner and outer plaque proteins responded differently to the absence of BUD14. A Bud14 mutant that cannot interact with the Protein Phosphatase 1 (Glc7) phenocopied bud14Δ in terms of SPB-bound levels of the inner plaque protein Spc110, whereas disruption of Bud14-Kel1-Kel2 complex did not alter Spc110 levels at SPBs. In cells synchronously released from α-factor arrest, lack of Bud14-Glc7 caused increase of Spc110 at the SPBs at early stages of the cell cycle. Conclusion: We identified Bud14 as a critical protein for SPB size maintenance. The interaction of Bud14 with Glc7, but not with the Kelch proteins, is indispensable for restricting levels of Spc110 incorporated into the SPBs. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate receptor-dependent excitotoxicity is one of the most important mechanisms underlying stroke injury and the resulting neuronal death. In the present study, in order to reduce post-stroke brain injury and improve behavioral performance, a new molecule named IC87201, which acts as an inhibitor of PSD95/nNOS interaction in the intracellular signaling pathway of NMDA receptors, was administered. Using the middle cerebral artery occlusion (MCAO) technique, 24 adult male rats were subjected to one hour of cerebral ischemia. Animals were randomly divided into sham, MCAO, MCAO + DXM, and MCAO + IC87201 groups, and in the last two groups, intraperitoneal injection of dextromethorphan hydrobromide monohydrate (DXM), as an NMDA antagonist, and IC87201 was performed after ischemia. Neurobehavioral scores were evaluated for seven days, and on the last two days, the rats' memory performance was appraised using the passive avoidance test. On seventh day, the brain tissue was properly prepared for stereological analysis. Stereological studies of the hippocampus CA1 and CA3 regions revealed that changes in the total and infarcted volumes, total number of neurons, non-neurons, and dead neurons are the consequences of cerebral ischemia. Also, following cerebral ischemia, neurobehavioral and memory function impairments which were assessed by modified neurological severity scores (mNSS) and passive avoidance test, were observed. The aforementioned impairments were recovered after administration of IC87201 significantly and more potently than DXM. Based on our findings, IC87201 successfully attenuated post-ischemia damages. Therefore, this molecule can be considered as a new therapeutic approach in future research. Highlights: • Ischemic stroke severely destroys hippocampus neurons and causes memory impairment. • IC87201 improved hippocampal histological injury following brain ischemia. • IC87201 improved memory impairment resulting from brain ischemia. [ABSTRACT FROM AUTHOR]

Three‐dimensional morphometric data better show the structural and functional characteristics of the brain. The objective of this study was to estimate the volume of the cerebral structures of the sheep using design‐based stereology. The brains of five sheep were used, fixed in formalin 10% and embedded in agar 6%. An average of 10–12 slab was obtained from each brain. All slabs were stained using Mulligan's method and photographs were recorded. The volume of the brain and its structures were estimated using the Cavalieri's estimator and the point counting system. The total volume was 70604.8 ± 132.45 mm3. The volume fractions of the grey and white matters were calculated as 42.55 ± 0.21% and 24.23 ± 0.51% of the whole brain, respectively. The fractional volume of the caudate nucleus and claustrum were estimated at 2.39 ± 0.08% and at 1.008 ± 0.057% of total brain volume. The volumes of corpus callosum, internal capsule and external capsule were 1.24 ± 0.053%, 3.63 ± 0.22% and 0.698 ± 0.049% of total cerebral volume, respectively. These data could help improve the veterinary comparative neuroanatomy knowledge and development of experimental studies in the field. [ABSTRACT FROM AUTHOR]

We developed an automated microregistration method that enables repeated in vivo skin microscopy imaging of the same tissue microlocation and specific cells over a long period of days and weeks with unprecedented precision. Applying this method in conjunction with an in vivo multimodality multiphoton microscope, the behavior of human skin cells such as cell proliferation, melanin upward migration, blood flow dynamics, and epidermal thickness adaptation can be recorded over time, facilitating quantitative cellular dynamics analysis. We demonstrated the usefulness of this method in a skin biology study by successfully monitoring skin cellular responses for a period of two weeks following an acute exposure to ultraviolet light. [ABSTRACT FROM AUTHOR]

The physiological aging process is well known for functional decline in visual abilities. Among the components of the visual system, the dorsal lateral geniculate nucleus (DLG) and superior colliculus (SC) provide a good model for aging investigations, as these structures constitute the main visual pathways for retinal inputs reaching the visual cortex. However, there are limited data available on quantitative morphological and neurochemical aspects in DLG and SC across lifespan. Here, we used optical density to determine immunoexpression of glial fibrillary acidic protein (GFAP) and design‐based stereological probes to estimate the neuronal number, total volume, and layer volume of the DLG and SC in marmosets (Callithrix jacchus), ranging from 36 to 143 months of age. Our results revealed an age‐related increase in total volume and layer volume of the DLG, with an overall stability in SC volume. Furthermore, a stable neuronal number was demonstrated in DLG and superficial layers of SC (SCv). A decrease in GFAP immunoexpression was observed in both visual centers. The results indicate region‐specific variability in volumetric parameter, possibly attributed to structural plastic events in response to inflammation and compensatory mechanisms at the cellular and subcellular level. Additionally, the DLG and SCv seem to be less vulnerable to aging effects in terms of neuronal number. The neuropeptidergic data suggest that reduced GFAP expression may reflect morphological atrophy in the astroglial cells. This study contributes to updating the current understanding of aging effects in the visual system and stablishes a crucial foundation for future research on visual perception throughout the aging process. [ABSTRACT FROM AUTHOR]