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Defects in microarchitected materials exhibit a dual nature, capable of both unlocking innovative functionalities and degrading their performance. Specifically, while intentional defects are strategically introduced to customize and enhance mechanical responses, inadvertent defects stemming from manufacturing errors can disrupt the symmetries and intricate interactions within these materials. In this study, we demonstrate a nondestructive optical imaging technique that can precisely locate defects inside microscale metamaterials, as well as provide detailed insights on the specific type of defect.

Summary There is an increasing demand to boost photosynthesis in rice to increase yield potential. Chloroplasts are the site of photosynthesis, and increasing their number and size is a potential route to elevate photosynthetic activity. Notably, bundle sheath cells do not make a significant contribution to overall carbon fixation in rice, and thus, various attempts are being made to increase chloroplast content specifically in this cell type. In this study, we developed and applied a deep learning tool, Chloro‐Count, and used it to quantify chloroplast dimensions in bundle sheath cells of OsHAP3H gain‐ and loss‐of‐function mutants in rice. Loss of OsHAP3H increased chloroplast occupancy in bundle sheath cells by 50%. When grown in the field, mutants exhibited increased numbers of tillers and panicles. The implementation of Chloro‐Count enabled precise quantification of chloroplasts in loss‐ and gain‐of‐function OsHAP3H mutants and facilitated a comparison between 2D and 3D quantification methods. Collectively, our observations revealed that a mechanism operates in bundle sheath cells to restrict chloroplast occupancy as cell dimensions increase. That mechanism is unperturbed in Oshap3H mutants but loss of OsHAP3H function leads to an increase in chloroplast numbers. The use of Chloro‐Count also revealed that 2D quantification is compromised by the positioning of chloroplasts within the cell. [ABSTRACT FROM AUTHOR]

Understanding alterations in structural disorders in tissue/cells/building blocks, such as DNA/chromatin in the human brain, at the nano to submicron level provides us with efficient biomarkers for Alzheimer's detection. Here, we report a dual photonics technique to detect nano- to submicron-scale alterations in brain tissues/cells and DNA/chromatin due to the early to late progression of Alzheimer's disease in humans. Using a recently developed mesoscopic light transport technique, fine-focused nano-sensitive partial wave spectroscopy (PWS), we measure the degree of structural disorder in tissues. Furthermore, the chemical-specific inverse participation ratio technique (IPR) was used to measure the DNA/chromatin structural alterations. The results of the PWS and IPR experiments showed a significant increase in the degree of structural disorder at the nano to submicron scale at different stages of AD relative to their controls for both the tissue/cell and DNA cellular levels. The increase in the structural disorder in cells/tissues and DNA/chromatin in the nuclei can be attributed to higher mass density fluctuations in the tissue and DNA/chromatin damage in the nuclei caused by the rearrangements of macromolecules due to the deposition of the amyloid beta protein and damage in DNA/chromatin with the progress of AD. [ABSTRACT FROM AUTHOR]

Coral reefs are being degraded at alarming rates and decisive intervention actions are urgently needed. One such intervention is coral cryopreservation. Although the cryopreservation of coral sperm and larvae has been achieved, preservation of coral fragments including both its tissue and skeleton, has not. The overarching aim of this study was to understand and assess the physiological stressors that might underlie coral fragment cryopreservation, understand the long-term consequences of these exposures to continued growth, and develop a health metrics scale for future research. Therefore, we assessed small fragments (~1 cm2) from the Hawaiian coral, Porites compressa, examining: (1) chill sensitivity; (2) chemical sensitivity to complex cryoprotectants; (3) methods to safely remove algal symbionts of coral for cryopreservation; (4) continued growth over time of coral fragments exposed to chilling and cryoprotectants; and (5) assessment of health and viability of coral fragments post the applied treatments. Corals were able to withstand chilling to 0 °C for 1 min and after 2 weeks were not significantly different from the live controls, whereas, corals exposed to complex cryoprotectants needed 3 weeks of recovery. Most importantly, it appears that once the coral fragments had surpassed this initial recovery, there was no difference in subsequent growth. Technological advances in cryo-technology promise to support successful coral fragment cryopreservation soon, and its success could help secure much of the genetic and biodiversity of reefs in the next decade. [ABSTRACT FROM AUTHOR]

The development of stable room-temperature bright single-photon emitters using atomic defects in hexagonal boron nitride flakes (h-BN) provides significant promise for quantum technologies. However, an outstanding challenge in h-BN is the creation and detection of isolated, stable single-photon emitters with high emission rates and with very low Huang–Rhys (HR) factor. Here, we discuss the quantum photonic properties of a single, isolated, stable quantum emitter that emits single photons with a high emission rate and a low HR value of 0.6 ± 0.2 at room temperature. A scanning confocal image confirms the presence of a deserted, single-quantum emitter with a prominent zero-phonon line at ∼578 nm with a well-separated phonon sideband at 626 nm. The second-order intensity-intensity correlation measurement shows an anti-bunching dip of ∼0.25 with an emission lifetime of 2.46 ± 0.1 ns, reinforcing distinct features of the single-photon emitter. The importance of low-energy electron beam irradiation and subsequent annealing is emphasized to achieve stable, reproducible single-photon emitters. [ABSTRACT FROM AUTHOR]

Synthetic microwave focusing methods have been widely adopted in qualitative medical imaging to detect and localize anomalies based on their electromagnetic scattering signatures. This paper discusses the principles, challenges, and limitations of synthetic microwave-focusing techniques in medical applications. It is shown that the various focusing techniques, including time reversal, confocal imaging, and delay-and-sum, are all based on the scalar solution of the electromagnetic scattering problem, assuming the imaged object, i.e., the tissue or object, is linear, reciprocal, and time-invariant. They all aim to generate a qualitative image, revealing any strong scatterer within the imaged domain. The differences among these techniques lie only in the assumptions made to derive the solution and create an image of the relevant tissue or object. To get a fast solution using limited computational resources, those methods assume the tissue is homogeneous and non-dispersive, and thus, a simplified far-field Green's function is used. Some focusing methods compensate for dispersive effects and attenuation in lossy tissues. Other approaches replace the simplified Green's function with more representative functions. While these focusing techniques offer benefits like speed and low computational requirements, they face significant ongoing challenges in real-life applications due to their oversimplified linear solutions to the complex problem of non-linear medical microwave imaging. This paper discusses these challenges and potential solutions. [ABSTRACT FROM AUTHOR]

Coral reefs are being degraded at alarming rates and decisive intervention actions are urgently needed. One such intervention is coral cryopreservation. Although the cryopreservation of coral sperm and larvae has been achieved, preservation of coral fragments including both its tissue and skeleton, has not. The overarching aim of this study was to understand and assess the physiological stressors that might underlie coral fragment cryopreservation, understand the long-term consequences of these exposures to continued growth, and develop a health metrics scale for future research. Therefore, we assessed small fragments (~1 cm2) from the Hawaiian coral, Porites compressa, examining: (1) chill sensitivity; (2) chemical sensitivity to complex cryoprotectants; (3) methods to safely remove algal symbionts of coral for cryopreservation; (4) continued growth over time of coral fragments exposed to chilling and cryoprotectants; and (5) assessment of health and viability of coral fragments post the applied treatments. Corals were able to withstand chilling to 0 °C for 1 min and after 2 weeks were not significantly different from the live controls, whereas, corals exposed to complex cryoprotectants needed 3 weeks of recovery. Most importantly, it appears that once the coral fragments had surpassed this initial recovery, there was no difference in subsequent growth. Technological advances in cryo-technology promise to support successful coral fragment cryopreservation soon, and its success could help secure much of the genetic and biodiversity of reefs in the next decade.

In the medical field, microwave imaging technology has experienced rapid development due to its non-invasive and non-radioactive nature. The confocal algorithm is a method commonly used for microwave breast cancer imaging, with the key objective of removing clutter in images to achieve high-quality results. However, the current methods are facing challenges in removing clutter. In order to reduce the clutter in images, a multiplicative improved coherence factor delay multiply and sum algorithm based on the maximum interclass differencing method is proposed. The algorithm compares the starting and ending moments of tumor signals in different channels to determine whether the tumor-scattered signals in different channels overlap in time. An improved coherence coefficient is obtained by summing the non-overlapping signals and multiplying the time window. The multiplicative improved coherence factor, which is obtained by multiplying the coherence coefficients of the improved multi-pair signals, is then multiplied by the focal point intensity obtained using the delay multiply and sum algorithm to reduce clutter in an image. To evaluate the performance of the proposed algorithm, several low-cost uniform and non-uniform models of human breast and tumor tissue with dielectric properties were prepared for testing. The experimental results show that, compared to the existing algorithm, the proposed algorithm can greatly reduce the clutter in images, with a signal-to-clutter ratio of at least 4 dB higher as well as contrast at least six-fold higher. [ABSTRACT FROM AUTHOR]

In adult olfactory epithelium (OE), ATP plays a role in constant cell turnover and post-injury neuroregeneration. We previously demonstrated that constitutive and ATP-evoked ATP release are present in neonatal mouse OE and underlie continuous cell turn-over and post-injury neuroregeneration, and that activation of purinergic P2X7 receptors is involved in the evoked release. We hypothesized that both releases are present in adult mouse OE. To study the putative contribution of olfactory sensory neurons to ATP release, we used olfactory sensory neuronal-like OP6 cells derived from the embryonic olfactory placode cells. Calcium imaging showed that OP6 cells and primary adult OE cell cultures express functional purinergic receptors. We monitored ATP release from OP6 cells and whole adult OE turbinates using HEK cells as biosensors and luciferin–luciferase assays. Constitutive ATP release occurs in OP6 cells and whole adult mouse OE turbinates, and P2X7 receptors mediated evoked ATP release occurs only in turbinates. The mechanisms of ATP release described in the present study might underlie the constant cell turn-over and post-injury neuroregeneration present in adult OE and thus, further studies of these mechanisms are warranted as it will improve our knowledge of OE tissue homeostasis and post-injury regeneration.

Synthetic microwave focusing methods have been widely adopted in qualitative medical imaging to detect and localize anomalies based on their electromagnetic scattering signatures. This paper discusses the principles, challenges, and limitations of synthetic microwave-focusing techniques in medical applications. It is shown that the various focusing techniques, including time reversal, confocal imaging, and delay-and-sum, are all based on the scalar solution of the electromagnetic scattering problem, assuming the imaged object, i.e., the tissue or object, is linear, reciprocal, and time-invariant. They all aim to generate a qualitative image, revealing any strong scatterer within the imaged domain. The differences among these techniques lie only in the assumptions made to derive the solution and create an image of the relevant tissue or object. To get a fast solution using limited computational resources, those methods assume the tissue is homogeneous and non-dispersive, and thus, a simplified far-field Green’s function is used. Some focusing methods compensate for dispersive effects and attenuation in lossy tissues. Other approaches replace the simplified Green’s function with more representative functions. While these focusing techniques offer benefits like speed and low computational requirements, they face significant ongoing challenges in real-life applications due to their oversimplified linear solutions to the complex problem of non-linear medical microwave imaging. This paper discusses these challenges and potential solutions.

Exercise and physical activity exert mechanical loading on the bones which induces bone formation. However, the relationship between the osteocyte lacunar-canalicular morphology and mechanical stress experienced locally by osteocytes transducing signals for bone formation is not fully understood. In this study, we used computational modeling to predict the effect of canalicular density, the number of fluid inlets, and load direction on fluid flow shear stress (FFSS) and bone strains and how these might change following the microstructural deterioration of the lacunar-canalicular network that occurs with aging. Four distinct computational models were initially generated of osteocytes with either ten or eighteen dendrites using a fluid–structure interaction method with idealized geometries. Next, a young and a simulated aged osteocyte were developed from confocal images after FITC staining of the femur of a 4-month-old C57BL/6 mouse to estimate FFSS using a computational fluid dynamics approach. The models predicted higher fluid velocities in the canaliculi versus the lacunae. Comparison of idealized models with five versus one fluid inlet indicated that with four more inlets, one-half of the dendrites experienced FFSS greater than 0.8 Pa, which has been associated with osteogenic responses. Confocal image-based models of real osteocytes indicated a six times higher ratio of canalicular to lacunar surface area in the young osteocyte model than the simulated aged model and the average FFSS in the young model (FFSS = 0.46 Pa) was three times greater than the aged model (FFSS = 0.15 Pa). Interestingly, the surface area with FFSS values above 0.8 Pa was 23 times greater in the young versus the simulated aged model. These findings may explain the impaired mechano-responsiveness of osteocytes with aging. [ABSTRACT FROM AUTHOR]

This study develops an innovative cell-based carrier to simultaneously encapsulate multiple phytochemicals from a complex plant source. Muscadine grapes (MG) juice prepared from fresh fruit was used as a model juice. After incubation with inactivated bacterial cells, 66.97% of the total anthocyanins, and 72.67% of the total antioxidant compounds were encapsulated in the cells from MG juice. Confocal images illustrated a uniform localization of the encapsulated material in the cells. The spectral emission scans indicated the presence of a diverse class of phenolic compounds, which was characterized using high-performance liquid chromatography (HPLC). Using HPLC, diverse phytochemical compound classes were analyzed, including flavanols, phenolic acid, hydroxycinnamic acid, flavonols, and polymeric polyphenols. The analysis validated that the cell carrier could encapsulate a complex profile of bioactive compounds from fruit juice, and the encapsulated content and efficiencies varied by the chemical class and compound. In addition, after the heat treatment at 90 °C for 60 min, >87% total antioxidant capacity and 90% anthocyanin content were recovered from the encapsulated MG. In summary, these results highlight the significant potential of a selected bacterial strain for simultaneous encapsulation of diverse phenolic compounds from fruit juice and improving their process stability.

The macroscopic properties of particle-filled polymer melts depend sensitively on the state of particle dispersion and the structure and dynamics of the interfacial polymer layer, which, in turn, are governed by factors like polymer molecular weight (Mw), particle concentration (C), and particle-polymer interfacial interactions. However, the combined effect of these factors on the macroscopic properties is far from fully understood, especially for polymers filled with anisotropic particles. In this work, we investigate the combined effect of Mw, C, and polymer end-group (methyl, Me or hydroxyl, OH) on the dynamic viscoelastic behavior of polydimethylsiloxane (PDMS)/clay composites. The linear viscoelastic behavior of these composites follows a non-monotonic dependence on Mw, which varies considerably with a modification in C or the polymer end-group. Furthermore, for both Me-PDMS/clay and OH-PDMS/clay composites, the non-linear tests reveal either strain softening-hardening-softening or sustained softening beyond the linear regime, depending on the combination of C and Mw. The critical strains for the onset of softening and hardening vary differently with Mw for different combinations of C and the polymer end-group. Our results suggest that the morphology and rheological behavior of these composites are dictated by a complex interplay of various competing effects, namely, particle agglomeration, interfacial polymer packing and density, entanglements, and bridging interactions. These findings give insight into tailoring the properties of polymer composites by adjusting the combination of C, Mw, and particle-polymer interactions. [ABSTRACT FROM AUTHOR]

The global health concern posed by age-related visual impairment highlights the need for further research focused on the visual changes that occur during the process of aging. To date, multiple sensory alterations related to aging have been identified, including morphological and functional changes in inner hair cochlear cells, photoreceptors, and retinal ganglion cells. While some age-related morphological changes are known to occur in rod bipolar cells in the retina, their effects on these cells and on their connection to other cells via ribbon synapses remain elusive. To investigate the effects of aging on rod bipolar cells and their ribbon synapses, we compared synaptic calcium currents, calcium dynamics, and exocytosis in zebrafish (Danio rerio) that were middle-aged (MA,18 months) or old-aged (OA, 36 months). The bipolar cell terminal in OA zebrafish exhibited a two-fold reduction in number of synaptic ribbons, an increased ribbon length, and a decrease in local Ca2+ signals at the tested ribbon location, with little change in the overall magnitude of the calcium current or exocytosis in response to brief pulses. Staining of the synaptic ribbons with antibodies specific for PKCa revealed shortening of the inner nuclear and plexiform layers (INL and IPL). These findings shed light on age-related changes in the retina that are related to synaptic ribbons and calcium signals. [ABSTRACT FROM AUTHOR]

In the medical field, microwave imaging technology has experienced rapid development due to its non-invasive and non-radioactive nature. The confocal algorithm is a method commonly used for microwave breast cancer imaging, with the key objective of removing clutter in images to achieve high-quality results. However, the current methods are facing challenges in removing clutter. In order to reduce the clutter in images, a multiplicative improved coherence factor delay multiply and sum algorithm based on the maximum interclass differencing method is proposed. The algorithm compares the starting and ending moments of tumor signals in different channels to determine whether the tumor-scattered signals in different channels overlap in time. An improved coherence coefficient is obtained by summing the non-overlapping signals and multiplying the time window. The multiplicative improved coherence factor, which is obtained by multiplying the coherence coefficients of the improved multi-pair signals, is then multiplied by the focal point intensity obtained using the delay multiply and sum algorithm to reduce clutter in an image. To evaluate the performance of the proposed algorithm, several low-cost uniform and non-uniform models of human breast and tumor tissue with dielectric properties were prepared for testing. The experimental results show that, compared to the existing algorithm, the proposed algorithm can greatly reduce the clutter in images, with a signal-to-clutter ratio of at least 4 dB higher as well as contrast at least six-fold higher.

It is still a challenging work of developing near-infrared (NIR) fluorescence dyes with excellent optical properties. Herein, we synthesized a series of NIR aza-BODIPY fluorescence dyes with electronic push–pull system and further investigated their photophysical properties, photostability, and application in cellular imaging. It was found that introducing electron-withdrawing groups (EWGs) on the para-position of aza-BODIPY 1,7-phenyls not only leads to the significant redshift of absorption and fluorescence emission bands of aza-BODIPY dyes but also increases their fluorescence quantum yield and photostablility. The DFT calculations and cyclic voltammetry measurements proved that introducing EWGs on para-position of 1,7-phenyls can decrease the energy level difference between HOMO and LUMO, resulting in the redshift of maximum absorption and emission wavelengths. On the basis of the feasible experiment results of cellular imaging and cytotoxicity assay, it is expected that the optimized aza-BODIPY dyes have a bright application prospect for in vivo cellular or tissue imaging. [ABSTRACT FROM AUTHOR]

Scanning MEMS mirrors can extend confocal laser microscopy into endoscopic applications, but the practical use of MEMS mirror-based confocal endomicroscopy is hindered partially by various image distortions such as barrel, fan-shaped and nonlinear distortions. In this work, the nonlinear scanning behaviors of an electrothermal MEMS mirror are analyzed and incorporated into an optical scanning model that takes all these three types of distortions into account. The model generates a 2D spatial mapping that can be applied to correct all of the image distortions in one step without a calibration board. To experimentally validate this method, a confocal laser endomicroscope employing a two-axis scanning electrothermal MEMS micromirror is designed and constructed, and confocal fluorescence images of a patterned micro-structure are obtained with the endomicroscope. The results show that the overall image distortion is reduced by at least one order of magnitude in the length direction.

Infectious diseases caused by pathogens are a health burden, but traditional pathogen identification methods are complex and time-consuming. In this work, we have developed well-defined, multifunctional copolymers with rhodamine B dye synthesized by atom transfer radical polymerization (ATRP) using fully oxygen-tolerant photoredox/copper dual catalysis. ATRP enabled the efficient synthesis of copolymers with multiple fluorescent dyes from a biotin-functionalized initiator. Biotinylated dye copolymers were conjugated to antibody (Ab) or cell-wall binding domain (CBD), resulting in a highly fluorescent polymeric dye-binder complex. We showed that the unique combination of multifunctional polymeric dyes and strain-specific Ab or CBD exhibited both enhanced fluorescence and target selectivity for bioimaging of Staphylococcus aureus by flow cytometry and confocal microscopy. The ATRP-derived polymeric dyes have the potential as biosensors for the detection of target DNA, protein, or bacteria, as well as bioimaging. [ABSTRACT FROM AUTHOR]

Upstream advances have led to increased mAb titers above 5 g/L in 14‐day fed‐batch cultures. This is accompanied by higher cell densities and process‐related impurities such as DNA and Host Cell Protein (HCP), which have caused challenges for downstream operations. Depth filtration remains a popular choice for harvesting CHO cell culture, and there is interest in utilizing these to remove process‐related impurities at the harvest stage. Operation of the harvest stage has also been shown to affect the performance of the Protein A chromatography step. In addition, manufacturers are looking to move away from natural materials such as cellulose and Diatomaceous Earth (DE) for better filter consistency and security of supply. Therefore, there is an increased need for further understanding and knowledge of depth filtration. This study investigates the effect of depth filter material and loading on the Protein A resin lifetime with an industrially relevant high cell density feed material (40 million cells/ml). It focuses on the retention of process‐related impurities such as DNA and HCP through breakthrough studies and a novel confocal microscopy method for imaging foulant in‐situ. An increase in loading of the primary‐synthetic filter by a third, led to earlier DNA breakthrough in the secondary filter, with DNA concentration at a throughput of 50 L/m2 being more than double. Confocal imaging of the depth filters showed that the foulant was pushed forward into the filter structure with higher loading. The additional two layers in the primary‐synthetic filter led to better pressure profiles in both primary and secondary filters but did not help to retain HCP or DNA. Reduced filtrate clarity, as measured by OD600, was 1.6 fold lower in the final filtrate where a synthetic filter train was used. This was also associated with precipitation in the Protein A column feed. Confocal imaging of resin after 100 cycles showed that DNA build‐up around the outside of the bead was associated with synthetic filter trains, leading to potential mass transfer problems. [ABSTRACT FROM AUTHOR]

Introduction: Previous studies have illustrated that each physiological change in blood supply following the ionizing beam irradiation is caused by damaging the inner wall of vessels, which is attributed to changes in the cytoskeleton in the endothelium layer. The aim of this study is to investigate the effect of radiotherapy on the fractal dimension parameter of endothelial cells as a biomarker of the cytoskeleton following the single irradiation and fractionation radiotherapy. Methods and Materials: In this study, human umbilical vein endothelial cells (HUVECs) were irradiated by the doses of 0, 2, 4 and 8 Gy by using cobalt-60 therapeutics machine in two situations of single continuous irradiation and fractionation radiotherapy. The cytoskeleton of each group was separately stained within 24 hours after irradiation. Then, the fractal dimension spectrum was performed for each radiation-induced group by processing the confocal microscopy images with the related command in the ImageJ software package. Results: The results showed that the fractal dimension parameter of endothelial cells is significantly changed following the external radiotherapy in two modes of single and fractionation irradiation modalities. Moreover, the reduction of this parameter for the fractionation radiation treatments is statistically higher than that following the continuous irradiation for the same radiation dose. Discussion and Conclusion: Assessing the fractal dimension can be considered as a reliable secondary index for the prognosis of the response of human umbilical vein endothelial cells to radiotherapy with two procedures of single and fractionation modalities. [ABSTRACT FROM AUTHOR]

Abstract Despite the fast progress in our understanding of the complex functions of gut microbiota, it is still challenging to directly investigate the in vivo microbial activities and processes on an individual cell basis. To gain knowledge of the indigenous growth/division patterns of the diverse mouse gut bacteria with a relatively high throughput, here, we propose an integrative strategy, which combines the use of fluorescent probe labeling, confocal imaging with single‐cell sorting, and sequencing. Mouse gut bacteria sequentially labeled by two fluorescent d‐amino acid probes in vivo were first imaged by confocal microscopy to visualize their growth patterns, which can be unveiled by the distribution of the two fluorescence signals on each bacterium. Bacterial cells of interest on the imaging slide were then sorted using a laser ejection equipment, and the collected cells were then sequenced individually to identify their taxa. Our strategy allows integrated acquirement of the growth pattern knowledge of a variety of gut bacteria and their genomic information on a single‐cell basis, which should also have great potential in studying many other complex bacterial systems.

Astrocytes have complex structural, molecular, and physiological properties and form specialized microenvironments that support circuit-specific functions in the CNS. To better understand how astrocytes acquire their unique features, we transplanted immature mouse cortical astrocytes into the developing cortex of male and female mice and assessed their integration, maturation, and survival. Within days, transplanted astrocytes developed morphologies and acquired territories and tiling behavior typical of cortical astrocytes. At 35–47 d post-transplantation, astrocytes appeared morphologically mature and expressed levels of EAAT2/GLT1 similar to nontransplanted astrocytes. Transplanted astrocytes also supported excitatory/inhibitory (E/I) presynaptic terminals within their territories, and displayed normal Ca2+ events. Transplanted astrocytes showed initially reduced expression of aquaporin 4 (AQP4) at endfeet and elevated expression of EAAT1/GLAST, with both proteins showing normalized expression by 110 d and one year post-transplantation, respectively. To understand how specific brain regions support astrocytic integration and maturation, we transplanted cortical astrocytes into the developing cerebellum. Cortical astrocytes interlaced with Bergmann glia (BG) in the cerebellar molecular layer to establish discrete territories. However, transplanted astrocytes retained many cortical astrocytic features including higher levels of EAAT2/GLT1, lower levels of EAAT1/GLAST, and the absence of expression of the AMPAR subunit GluA1. Collectively, our findings demonstrate that immature cortical astrocytes integrate, mature, and survive (more than one year) following transplantation and retain cortical astrocytic properties. Astrocytic transplantation can be useful for investigating cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms contributing to astrocytic development/diversity, and for determining the optimal timing for transplanting astrocytes for cellular delivery or replacement in regenerative medicine. [ABSTRACT FROM AUTHOR]

Metabolic glycoengineering (MGE) has been developed to visualize carbohydrates on live cells. The method allows the fluorescent labeling of sialic acid (Sia) sugar residues on neuronal plasma membranes. For instance, the efficiency of glycosylation along neurite membranes has been characterized as cell health measure in neurotoxicology. Using human dopaminergic neurons as model system, we asked here, whether it was possible to separately label diverse classes of biomolecules and to visualize them selectively on cells. Several approaches suggest that a large proportion of Sia rather incorporated in non‐protein components of cell membranes than into glycoproteins. We made use here of deoxymannojirimycin (dMM), a non‐toxic inhibitor of protein glycosylation, and of N‐butyl‐deoxynojirimycin (NBdNM) a well‐tolerated inhibitor of lipid glycosylation, to develop a method of differential labeling of sialylated membrane lipids (lipid‐Sia) or sialylated N‐glycosylated proteins (protein‐Sia) on live neurons. The time resolution at which Sia modification of lipids/proteins was observable was in the range of few hours. The approach was then extended to several other cell types. Using this technique of target‐specific MGE, we found that in dopaminergic or sensory neurons >60% of Sia is lipid bound, and thus polysialic acid‐neural cell adhesion molecule (PSA‐NCAM) cannot be considered the major sialylated membrane component. Different from neurons, most Sia was bound to protein in HepG2 hepatoma cells or in neural crest cells. Thus, our method allows visualization of cell‐specific sialylation processes for separate classes of membrane constituents. [ABSTRACT FROM AUTHOR]

Nanoparticle (NP) technology holds significant promise to mediate targeted drug delivery to specific organs in the body. Understanding the 3D biodistribution of NPs in heterogeneous environments such as the tumor tissue can provide crucial information on efficacy, safety and potential clinical outcomes. Here we present a novel end-to-end workflow, VIOLA, which makes use of tissue clearing methodology in conjunction with high resolution imaging and advanced 3D image processing to quantify the spatiotemporal 3D biodistribution of fluorescently labeled ACCURIN® NPs. Specifically, we investigate the spatiotemporal biodistribution of NPs in three different murine tumor models (CT26, EMT6, and KPC-GEM) of increasing complexity and translational relevance. We have developed new endpoints to characterize NP biodistribution at multiple length scales. Our observations reveal that the macroscale NP biodistribution is spatially heterogeneous and exhibits a gradient with relatively high accumulation at the tumor periphery that progressively decreases towards the tumor core in all the tumor models. Microscale analysis revealed that NP extravasation from blood vessels increases in a time dependent manner and plateaus at 72 h post injection. Volumetric analysis and pharmacokinetic modeling of NP biodistribution in the vicinity of the blood vessels revealed that the local NP density exhibits a distance dependent spatiotemporal biodistribution which provide insights into the dynamics of NP extravasation in the tumor tissue. Our data represents a comprehensive analysis of NP biodistribution at multiple length scales in different tumor models providing unique insights into their spatiotemporal dynamics. Specifically, our results show that NPs exhibit a dynamic equilibrium with macroscale heterogeneity combined with microscale homogeneity. [Display omitted] • Novel tissue clearing/image analysis pipeline for quantifying NP biodistribution • Comprehensive 3D biodistribution evaluation of PLA-PEG NPs in murine tumor models • Observed 3D heterogeneity in NP and tumor vasculature macroscale distribution • NP extravasation from blood vessels show distinct, time-dependent kinetics • Local NP density around blood vessels exhibit time-dependent equilibration [ABSTRACT FROM AUTHOR]

Dicentrinone (Di), liriodenine (Li) and lysicamine (Ly) are three natural oxoaporphine alkaloids (OAs), which revealed significant biological activity such as anticancer, anti-inflammatory and antimicrobial activities and were considered as potential lead compounds for the development of new clinical chemicals. In the present study, confocal laser scanning fluorescence microscopy observation demonstrated these three natural OAs could traverse inside of the nucleus and get an opportunity to interact with DNA. Their interaction properties with DNA were then investigated simultaneously by two spectral fluorescent probes of ethidium bromide (EB) and methyl green (MG), as well as UV–vis absorption and cyclic voltammetry measurements, and further verified by the molecular docking analysis. Results indicated Di and Li were distinctly classified as the intercalative molecules to DNA, however, Ly was confirmed with a mixed-mode binding of partial intercalation and groove affinity. Their binding ability was revealed as the follows: Di ≥ Li > Ly, which was correlated with their structural changes. Thermodynamic studies revealed the binding process of Li and Ly with ctDNA was all spontaneous, the hydrophobic interaction was the major binding force for Li-ctDNA complex, however, the interaction between Ly and ctDNA relied on both hydrophobic and hydrogen binding force. Molecular docking provided detailed computational interaction of Di, Li and Ly with DNA, which proved the intercalation binding of Li-DNA complex and Di-DNA complex stabilizing mainly by the π-π binding force, however, apart from a small quantity of π-π interaction, another binding force in the Ly-DNA complex mainly was supplied from the weaker Pi-Alkyl, hydrogen bond and Pi-Anion interactions. [ABSTRACT FROM AUTHOR]

Cochlear sound encoding depends on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), but reliance on specific pore-forming subunits is unknown. With 5-week-old male C57BL/6J Gria3-knockout mice (i.e., subunit GluA3KO) we determined cochlear function, synapse ultrastructure, and AMPAR molecular anatomy at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons. GluA3KO and wild-type (GluA3WT) mice reared in ambient sound pressure level (SPL) of 55–75 dB had similar auditory brainstem response (ABR) thresholds, wave-1 amplitudes, and latencies. Postsynaptic densities (PSDs), presynaptic ribbons, and synaptic vesicle sizes were all larger on the modiolar side of the IHCs from GluA3WT, but not GluA3KO, demonstrating GluA3 is required for modiolar–pillar synapse differentiation. Presynaptic ribbons juxtaposed with postsynaptic GluA2/4 subunits were similar in quantity, however, lone ribbons were more frequent in GluA3KO and GluA2-lacking synapses were observed only in GluA3KO. GluA2 and GluA4 immunofluorescence volumes were smaller on the pillar side than the modiolar side in GluA3KO, despite increased pillar-side PSD size. Overall, the fluorescent puncta volumes of GluA2 and GluA4 were smaller in GluA3KO than GluA3WT. However, GluA3KO contained less GluA2 and greater GluA4 immunofluorescence intensity relative to GluA3WT (threefold greater mean GluA4:GluA2 ratio). Thus, GluA3 is essential in development, as germline disruption of Gria3 caused anatomical synapse pathology before cochlear output became symptomatic by ABR. We propose the hearing loss in older male GluA3KO mice results from progressive synaptopathy evident in 5-week-old mice as decreased abundance of GluA2 subunits and an increase in GluA2-lacking, GluA4-monomeric Ca2+-permeable AMPARs.

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Fluorescent probes are highly desirable for accurate diagnosis and play a crucial role in the optical imaging modality. Here, ultrafine (1.2 nm) nanoclusters of undoped gadolinium oxide were synthesized via a simple, one‐pot technique using water as the reaction medium. Prepared nanoclusters exhibit strong tuneable emission spanning from 400‐620 nm (extended visible region) with broad full width at half maximum of (FWHM) ∼140 nm, resulting in white light emission (WLE). Cytotoxicity studies revealed nearly 100 % cell viability encouraging its application in cell imaging. These nanoclusters possessed distinctive properties such as wide‐range pH stability, ionic tolerability, durable photostability, and anomalous colloidal stability (more than 24 months). This brief report uses white light‐emitting gadolinium oxide (Gd2O3) nanoclusters as an optical probe for in‐vitro fluorescence imaging. [ABSTRACT FROM AUTHOR]

Given the established direct correlation that exists among extent of resection and postoperative survival in brain tumors, obtaining complete resections is of primary importance. Apart from the various technological advancements that have been introduced in current clinical practice, histopathological study still remains the gold-standard for definitive diagnosis. Frozen section analysis still represents the most rapid and used intraoperative histopathological method that allows for an intraoperative differential diagnosis. Nevertheless, such technique owes some intrinsic limitations that limit its overall potential in obtaining real-time diagnosis during surgery. In this context, confocal laser technology has been suggested as a promising method to have near real-time intraoperative histological images in neurosurgery, thanks to the results of various studies performed in other non-neurosurgical fields. Still far to be routinely implemented in current neurosurgical practice, pertinent literature is growing quickly, and various reports have recently demonstrated the utility of this technology in both preclinical and clinical settings in identifying brain tumors, microvasculature, and tumor margins, when coupled to the intravenous administration of sodium fluorescein. Specifically in neurosurgery, among different available devices, the ZEISS CONVIVO system probably boasts the most recent and largest number of experimental studies assessing its usefulness, which has been confirmed for identifying brain tumors, offering a diagnosis and distinguishing between healthy and pathologic tissue, and studying brain vessels. The main objective of this systematic review is to present a state-of-the-art summary on sodium fluorescein-based preclinical and clinical applications of the ZEISS CONVIVO in neurosurgery. [ABSTRACT FROM AUTHOR]

In recent years, the zebrafish has become a well-established laboratory model. We describe here the ZeBraInspector (ZBI) platform for high-content 3D imaging (HCI) of 5 days post-fertilization zebrafish eleuthero-embryos (EEs). This platform includes a mounting method based on 3D-printed stamps to create a grid of wells in an agarose cast, facilitating batch acquisitions with a fast-confocal laser scanning microscope. We describe reference labeling in cleared fish with a fluorescent lipophilic dye. Based on this labeling, the ZBI software registers. EE 3D images, making it possible to visualize numerous identically oriented EEs on a single screen, and to compare their morphologies and any fluorescent patterns at a glance. High-resolution 2D snapshots can be extracted. ZBI software is therefore useful for diverse high-content analyses (HCAs). Following automated segmentation of the lipophilic dye signal, the ZBI software performs volumetric analyses on whole EEs and their nervous system white matter. Through two examples, we illustrate the power of these analyses for obtaining statistically significant results from a small number of samples: the characterization of a phenotype associated with a neurodevelopmental mutation, and of the defects caused by treatments with a toxic anti-cancer compound. [Display omitted] • A fast 3D high-content imaging pipeline involving sample clearing protocols. • Lipophilic-dye stain allows brain volume and axis definition by AI. • Power of 3D volumetric analysis demonstrated by two biological validations. • GUI ZBI: Registration simplifies 4-7dpf zebrafish image analysis in batch. • ZBI: analysis of fluorescent lines facilitated by 2D-3D image extractions in batch. [ABSTRACT FROM AUTHOR]

Lipid droplets (LD) are crucial in pathological processes or conditions associated with abnormal lipid metabolism, such as obesity, diabetes, atherosclerosis, fatty liver diseases, and cancers. Cancer cells frequently contain elevated levels of nonpolar lipid droplets (LDs), serving as energy reserves. The proliferation of LDs, accompanied by an increase in viscosity, is a characteristic feature of cancer cells that prompted us to devise a fluorescent sensor for LD detection at physiological pH. However, developing fluorescent LD-specific probes with high polarity sensitivity and deep tissue/cell imaging capability remains challenging. Therefore, we present a TICT probe with strong solvatochromism, superior response to viscosity, microenvironment sensitivity, and a large Stokes shift. Additionally, it offers numerous advantages, including high sensitivity, specificity, high fluorescence quantum yield, and remarkable spatial resolution, which enables precise monitoring of lipid droplets (LD). Thus, this probe can effectively monitor alterations in viscosity and polarity of lipid droplet expression in live cells, thereby offering the potential for visualizing physiological abnormalities or pathological conditions. The probe offers excellent lipid droplet targeting and also sensitively monitors the oleic-acid-mediated lipid droplet accumulation and immunosuppressant/inflammatory drugs in HeLa cells.

Lamins form a proteinaceous meshwork as a major structural component of the nucleus. Lamins, along with their interactors, act as determinants for chromatin organization throughout the nucleus. The major dominant missense mutations responsible for autosomal dominant forms of muscular dystrophies reside in the Ig fold domain of lamin A. However, how lamin A contributes to the distribution of heterochromatin and balances euchromatin, and how it relocates epigenetic marks to shape chromatin states, remains poorly defined, making it difficult to draw conclusions about the prognosis of lamin A-mediated muscular dystrophies. In the first part of this report, we identified the in vitro organization of full-length lamin A proteins due to two well-documented Ig LMNA mutations, R453W and W514R. We further demonstrated that both lamin A/C mutant cells predominantly expressed nucleoplasmic aggregates. Labeling specific markers of epigenetics allowed correlation of lamin A mutations with epigenetic mechanisms. In addition to manipulating epigenetic mechanisms, our proteomic studies traced diverse expressions of transcription regulators, RNA synthesis and processing proteins, protein translation components, and posttranslational modifications. These data suggest severe perturbations in targeting other proteins to the nucleus., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Recent gene expression studies have revealed about 10 different states of microglia, some of which are characteristic for Alzheimer-like amyloid plaque pathology. However, it is not presently known how these translate into morphological features that would reflect microglia interaction with amyloid plaques. With optimized conditions for confocal microscopy in amyloid plaque forming APP/PS1 transgenic mice we reveal new details of how microglia processes interact with amyloid plaques. The microglia contacts differed drastically between purely diffuse plaque and those with a fibrillar core. We identified microglia that extend their enlarged processes through the diffuse shell of the amyloid plaques and cover the fibrillar plaque core with snowplow-like expanded end-feet. These end-feet were filled with the lysosomal marker CD68, while both non-fibrillar and fibrillar amyloid was found in perinuclear vesicles of some "snowplower" microglia. In the organized dense-core plaques, we consistently saw a layer of Apolipoprotein E (ApoE) between the fibrillar core and the microglial end-feet. ApoE covered also loose fibrillar amyloid and diffuse amyloid plaques that were about 10 μm or larger in diameter. These findings are compatible with both amyloid plaque phagocytosis and compaction by microglia. Further, they support a chemotactic role of ApoE for microglia contacts with amyloid plaques., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

Given the established direct correlation that exists among extent of resection and postoperative survival in brain tumors, obtaining complete resections is of primary importance. Apart from the various technological advancements that have been introduced in current clinical practice, histopathological study still remains the gold-standard for definitive diagnosis. Frozen section analysis still represents the most rapid and used intraoperative histopathological method that allows for an intraoperative differential diagnosis. Nevertheless, such technique owes some intrinsic limitations that limit its overall potential in obtaining real-time diagnosis during surgery. In this context, confocal laser technology has been suggested as a promising method to have near real-time intraoperative histological images in neurosurgery, thanks to the results of various studies performed in other non-neurosurgical fields. Still far to be routinely implemented in current neurosurgical practice, pertinent literature is growing quickly, and various reports have recently demonstrated the utility of this technology in both preclinical and clinical settings in identifying brain tumors, microvasculature, and tumor margins, when coupled to the intravenous administration of sodium fluorescein. Specifically in neurosurgery, among different available devices, the ZEISS CONVIVO system probably boasts the most recent and largest number of experimental studies assessing its usefulness, which has been confirmed for identifying brain tumors, offering a diagnosis and distinguishing between healthy and pathologic tissue, and studying brain vessels. The main objective of this systematic review is to present a state-of-the-art summary on sodium fluorescein-based preclinical and clinical applications of the ZEISS CONVIVO in neurosurgery.

A lower rim dansyl appended calix[4]-arene, DanC4A has been synthesized and characterized. DanC4A is a promising sensor for selective detection of toxic metal ions, Hg2+, among the twelve separate metal ions studied. The fluorescence titration of DanC4A with Hg2+ shows approximately 80% quenching. Competitive metal ion studies suggest the selective binding of Hg2+ to DanC4A. The biocompatibility of DanC4A has been confirmed by an MTT assay performed using HeLa cells. Confocal microscopy and fluorescence microscopy studies suggest that DanC4A exhibits strong green fluorescence in HeLa cells, and the same is quenched as the concentration of Hg2+ increases in the cells. Apart from sensing, the main concern is the removal of Hg2+ from water and this is not possible with the direct use of DanC4A due to its solubility. Therefore, DanC4A has been embedded into a zeolitic imidazolate framework (ZIF8) to result in an inorganic–organic hybrid nanomaterial, DanC4A_ZIF8_MOFs, by an in situ reaction. Approximately 95% of the Hg2+ ions were removed from water by using the hybrid material, viz., DanC4A_ZIF8_MOFs. Thus, DanC4A is not only a superior sensor for Hg2+ in solution and in mammalian cells but can be converted into an organic–inorganic hybrid material, DanC4A_ZIF8_MOFs, for efficient removal of Hg2+ from water. [ABSTRACT FROM AUTHOR]

Microscopic examination of biopsied and resected prostatic specimens is the mainstay in the diagnosis of prostate cancer. However, conventional analysis of hematoxylin and eosin (H&E)-stained tissue is time-consuming and offers limited two-dimensional (2D) information. In the current study, we devised a method—termed P rostate R apid O ptical examination for cancer STAT us (proSTAT)—for rapid screening of prostate cancer using high-resolution 2D and three-dimensional (3D) confocal images obtained after hydrophilic tissue clearing of 100-µm-thick tissue slices. The results of the proSTAT method were compared with those of traditional H&E stains for the analysis of cores (n =15) obtained from radical prostatectomy specimens (n =5). Gland lumen formation, consistent with Gleason pattern 3, was evident following tracking of multiple optical imaging sections. In addition, 3D rendering allowed visualizing a tubular network of interconnecting branches. Rapid 3D fluorescent labeling of tumor protein p63 accurately distinguished prostate adenocarcinoma from normal tissue and benign lesions. Compared with conventional stains, the 3D spatial and molecular information extracted from proSTAT may significantly increase the amount of available data for pathological assessment of prostate specimens. Our approach is amenable to automation and—subject to independent validation—can find a wide spectrum of clinical and research applications. [ABSTRACT FROM AUTHOR]

Silk fibroin is a well-known biopolymer that is used in several applications in which interactions with biological tissue are required. Fibroin is extremely versatile and can be shaped to form several constructs that are useful in tissue engineering applications. Confocal imaging is usually performed to test cell behavior on a construct, and, in this context, the fibroin intrinsic fluorescence is regarded as a problem. In addition, the intrinsic fluorescence is not intense enough to provide useful morphological images. In fact, to study the construct’s morphology, other techniques are used (i.e., SEM and Micro-CT). In this work, we propose a method based on fluorescence energy transfer (FRET) to suppress the fibroin intrinsic fluorescence and move it to a higher wavelength that is accessible to confocal microscopy for direct imaging. This was done by creating two FRET couples by dispersing two fluorophores (2,5-diphenyloxazole (PPO) and Lumogen F Violet 570 (LV)) into the fibroin matrix and optimizing their percentages to suppress the fibroin intrinsic fluorescence. With the optimized composition, we produced an electrospun mat, and the dimensions of the fibers were accurately determined by confocal microscopy.

Most land plants alternate between generations of sexual gametophytes and asexual sporophytes. Unlike seed plants, fern gametophytes are free living and grow independently of their sporophytes. In homosporous ferns such as Ceratopteris, gametophytes derived from genetically identical spores exhibit sexual dimorphism, developing as either males or hermaphrodites. Males lack meristems and promote cell differentiation into sperm-producing antheridia. In contrast, hermaphrodites initiate multicellular meristems that stay undifferentiated, sustain cell division and prothallus expansion, and drive the formation of egg-producing archegonia. Once initiating the meristem, hermaphrodites secrete the pheromone antheridiogen, which triggers neighboring slower-growing gametophytes to develop as males, while the hermaphrodites themselves remain insensitive to antheridiogen. This strategy promotes outcrossing and prevents all individuals in the colony from becoming males. This study reveals that an evolutionarily conserved GRAS-domain transcriptional regulator (CrHAM), directly repressed by Ceratopteris microRNA171 (CrmiR171), promotes meristem development in Ceratopteris gametophytes and determines the male-to-hermaphrodite ratio in the colony. CrHAM preferentially accumulates within the meristems of hermaphrodites but is excluded from differentiated antheridia. CrHAM sustains meristem proliferation and cell division through conserved hormone pathways. In the meantime, CrHAM inhibits the antheridiogen-induced conversion of hermaphrodites to males by suppressing the male program expression and preventing meristem cells from differentiating into sperm-producing antheridia. This finding establishes a connection between meristem indeterminacy and sex determination in ferns, suggesting both conserved and diversified roles of meristem regulators in land plants. [Display omitted] • CrHAM differentially accumulates in Ceratopteris hermaphrodites and males • CrHAM maintains meristem indeterminacy and cell division in hermaphrodites • CrHAM suppresses antheridiogen-mediated male differentiation, affecting sex ratio • miR171 represses CrHAM during gametophyte development in Ceratopteris The homosporous fern Ceratopteris develops two sex types during the haploid gametophyte phase: the meristem-containing hermaphrodite and the ameristic male. Geng et al. find that the GRAS protein CrHAM maintains hermaphrodite identity and development by sustaining meristem cell proliferation and repressing the male differentiation program. [ABSTRACT FROM AUTHOR]

Aims: It is unknown how β‐adrenergic stimulation affects calcium dynamics in individual RyR2 clusters and leads to the induction of spontaneous calcium waves. To address this, we analysed spontaneous calcium release events in green fluorescent protein (GFP)‐tagged RyR2 clusters. Methods: Cardiomyocytes from mice with GFP‐tagged RyR2 or human right atrial tissue were subjected to immunofluorescent labelling or confocal calcium imaging. Results: Spontaneous calcium release from single RyR2 clusters induced 91.4% ± 2.0% of all calcium sparks while 8.0% ± 1.6% were caused by release from two neighbouring clusters. Sparks with two RyR2 clusters had 40% bigger amplitude, were 26% wider, and lasted 35% longer at half maximum. Consequently, the spark mass was larger in two‐ than one‐cluster sparks with a median and interquartile range for the cumulative distribution of 15.7 ± 20.1 vs 7.6 ± 5.7 a.u. (P <.01). β2‐adrenergic stimulation increased RyR2 phosphorylation at s2809 and s2815, tripled the fraction of two‐ and three‐cluster sparks, and significantly increased the spark mass. Interestingly, the amplitude and mass of the calcium released from a RyR2 cluster were proportional to the SR calcium load, but the firing rate was not. The spark mass was also higher in 33 patients with atrial fibrillation than in 36 without (22.9 ± 23.4 a.u. vs 10.7 ± 10.9; P =.015). Conclusions: Most sparks are caused by activation of a single RyR2 cluster at baseline while β‐adrenergic stimulation doubles the mass and the number of clusters per spark. This mimics the shift in the cumulative spark mass distribution observed in myocytes from patients with atrial fibrillation. [ABSTRACT FROM AUTHOR]

Although much is known about the embryo during implantation, the architecture of the uterine environment in which the early embryo develops is not well understood. We employed confocal imaging in combination with 3D analysis to identify and quantify dynamic changes to the luminal structure of murine uterus in preparation for implantation. When applied to mouse mutants with known implantation defects, this method detected striking peri-implantation abnormalities in uterine morphology that cannot be visualized by histology. We revealed 3D organization of uterine glands and found that they undergo a stereotypical reorientation concurrent with implantation. Furthermore, we extended this technique to generate a 3D rendering of the cycling human endometrium. Analyzing the uterine and embryo structure in 3D for different genetic mutants and pathological conditions will help uncover novel molecular pathways and global structural changes that contribute to successful implantation of an embryo.

Background Optical slice microscopy is commonly used to observe cellular morphology in 3D tissue culture, e.g. the formation of cell-derived networks. The morphometric quantification of these networks is essential to study the cellular phenotype. Commonly, the quantitative measurements are performed on 2D projections of the image stack, resulting in the loss of information in the third dimension. Currently available 3D image analysis tools rely on manual interactions with the software and are therefore not feasible for large datasets. Findings Here we present Qiber3D, an open-source image processing toolkit. The software package includes the essential image analysis procedures required for image processing, from the raw image to the quantified data. Optional pre-processing steps can be switched on/off depending on the input data to allow for analyzing networks from a variety of sources. Two reconstruction algorithms are offered to meet the requirements for a wide range of network types. Furthermore, Qiber3D's rendering capabilities enable the user to inspect each step of the image analysis process interactively to ensure the creation of an optimal workflow for each application. Conclusions Qiber3D is implemented as a Python package, and its source code is freely available at https://github.com/theia-dev/Qiber3D. The toolkit was designed using a building block principle to enable the analysis of a variety of structures, such as vascular networks, neuronal structures, or scaffolds from numerous input formats. While Qiber3D can be used interactively in the Python console, it is aimed at unsupervised automation to process large image datasets efficiently. [ABSTRACT FROM AUTHOR]

Sub-cellular mechanics plays a crucial role in a variety of biological functions and dysfunctions. Due to the strong structure-function relationship in cytoskeletal protein networks, light can be shed on their mechanical functionality by investigating their structures. Here, we present a data-driven approach employing a combination of confocal live imaging of fluorescent tagged protein networks, in silico mechanical experiments and machine learning to investigate this relationship. Our designed image processing and nanoFE mechanical simulation framework resolves the structure and mechanical behaviour of cytoskeletal networks and the developed gradient boosting surrogate models linking network structure to its functionality. In this study, for the first time, we perform mechanical simulations of Filamentous Temperature Sensitive Z (FtsZ) complex protein networks with realistic network geometry depicting its skeletal functionality inside organelles (here, chloroplasts) of the moss Physcomitrella patens. Training on synthetically produced simulation data enables predicting the mechanical characteristics of FtsZ network purely based on its structural features (R2⩾0.93), therefore allowing to extract structural principles enabling specific mechanical traits of FtsZ, such as load bearing and resistance to buckling failure in case of large network deformation.

Most of the single point mutations of the LMNA gene are associated with distinct muscular dystrophies, marked by heterogenous phenotypes but primarily the loss and symmetric weakness of skeletal muscle tissue. The molecular mechanism and phenotype-genotype relationships in these muscular dystrophies are poorly understood. An effort has been here to delineating the adaptation of mechanical inputs into biological response by mutant cells of lamin A associated muscular dystrophy. In this study, we implement engineered smooth and pattern surfaces of particular young modulus to mimic muscle physiological range. Using fluorescence and atomic force microscopy, we present distinct architecture of the actin filament along with abnormally distorted cell and nuclear shape in mutants, which showed a tendency to deviate from wild type cells. Topographic features of pattern surface antagonize the binding of the cell with it. Correspondingly, from the analysis of genome wide expression data in wild type and mutant cells, we report differential expression of the gene products of the structural components of cell adhesion as well as LINC (linkers of nucleoskeleton and cytoskeleton) protein complexes. This study also reveals mis expressed downstream signaling processes in mutant cells, which could potentially lead to onset of the disease upon the application of engineered materials to substitute the role of conventional cues in instilling cellular behaviors in muscular dystrophies. Collectively, these data support the notion that lamin A is essential for proper cellular mechanotransduction from extracellular environment to the genome and impairment of the muscle cell differentiation in the pathogenic mechanism for lamin A associated muscular dystrophy., (© 2024 Wiley Periodicals LLC.)