Three-dimensional Confocal Microscopy Research Articles

Non-destructive monitoring and prediction of fouling by organic matters and residual anionic coagulant during membrane process

Physical fouling characteristics on silicon carbide (SiC) membranes induced by various organic matter compounds vary depending on the presence of calcium ions (Ca2+). Both destructive techniques (morphological surface analysis) and non-destructive techniques (fouling properties monitoring) were used to determine the fouling mechanisms and behavior during the membrane filtration systems. Destructive analysis and a modified Hermia model were employed to assess the fouling mechanisms. Fouling behavior was also analyzed through non-destructive monitoring techniques including optical coherence tomography (OCT) and three-dimensional laser scanning confocal microscopy (3D-LSM). At concentrations of 10, 30, and 100 mg/L without Ca2+, the flux decreased by 57–95% for humic acid (HA) and anionic polyacrylamide (APAM). APAM exhibited a notable removal rate of up to 56% without Ca2+. At concentration of 10, 30, and 100 mg/L in the absence of Ca2+, the flux decreased by 6–8% for sodium alginate (SA). However, the addition of Ca2+ led to a reduction in the flux for SA by up to 91% and resulted in a removal rate of 40%. Furthermore, addition of Ca2+ led to an alteration of the fouling characteristics of HA and SA. In the case of HA, higher concentrations resulted in elevated thickness and roughness with correlation coefficients of 0.991 and 0.992, respectively. For SA, increased SA concentration led to a thicker (correlation coefficient of 0.999) but smoother surfaces (correlation coefficients of 0.502). Monitoring of these physical characteristics of the fouling layer through non-destructive analysis is crucial for effective fouling management, optimization of the system performance and extending the lifespan of the membrane. By continuously assessing the fouling layer thickness and surface roughness, we expect to be able to provide insights on the fouling behavior, identify trends, that can help scientists and engineers to make informed decisions regarding fouling control strategies in future.

Tumor-specific migration routes of xenotransplanted human glioblastoma cells in mouse brain

The migration of neural progenitor cells (NPCs) to their final destination during development follows well-defined pathways, such as along blood vessels. Cells originating from the highly malignant tumor glioblastoma (GBM) seem to exploit similar routes for infiltrating the brain parenchyma. In this report, we have examined the migration of GBM cells using three-dimensional high-resolution confocal microscopy in brain tumors derived from eight different human GBM cell lines xenografted into immunodeficient mice. The primary invasion routes identified were long-distance migration along white matter tracts and local migration along blood vessels. We found that GBM cells in the majority of tumors (6 out of 8) did not exhibit association with blood vessels. These tumors, derived from low lamin A/C expressing GBM cells, were comparatively highly diffusive and invasive. Conversely, in 2 out of 8 tumors, we noted perivascular invasion and displacement of astrocyte end-feet. These tumors exhibited less diffusive migration, grew as solid tumors, and were distinguished by elevated expression of lamin A/C. We conclude that the migration pattern of glioblastoma is distinctly tumor cell-specific. Furthermore, the ability to invade the confined spaces within white matter tracts may necessitate low expression of lamin A/C, contributing to increased nuclear plasticity. This study highlights the role of GBM heterogeneity in driving the aggressive growth of glioblastoma.

Direct Fabrication of Te-Doped Black Si with an Enhanced Photoelectric Performance by Femtosecond Laser Irradiation under Water.

Tellurium (Te)-doped black silicon (Si) with enhanced absorption and photoelectric performance over a broad wavelength range of 0.2-2.5 μm was obtained using femtosecond (fs) laser irradiation in liquid water. Prior to laser irradiation, the Si sample was covered with a Te thin film (thickness 200 nm) over an adhesion layer of Cr (thickness 5 nm). Surface analyses by scanning electron microscopy and three-dimensional confocal microscopy evidence the presence of hierarchical surface structures combining quasi-periodic stripes with a spatial period of about 5 μm and subwavelength laser-induced periodic surface structures directed in directions parallel and perpendicular to the direction of the laser polarization, respectively. Moreover, the incorporation of Te generates intermediate levels within the Si bandgap. The Te-doped black Si shows a significant enhancement of the absorption, which reaches values of about 48% in the UV and visible (0.2-1.1 μm) and 70% in the near-infrared (1.1-2.5 μm) spectral ranges, respectively, due to the synergistic effects of multiscale surface structures and Te incorporation. Moreover, the surface reflectance is reduced to almost zero across the entire spectrum. The Te-doped black Si sample is used to realize a photodetector which displays an impressive photoelectric capability, being characterized by a responsivity of 328 mA/W, and an external quantum efficiency of 49.27% at a voltage bias of -10 V for 1064 nm light illumination, with rising and falling times of 55 and 67 ms, respectively. These figures remarkably outperform the response of unprocessed Si under the same experimental conditions.

An Analysis of the Morphology Evolution of YG8 Cemented Carbide by Laser Ablation in the Liquid Phase

To explore the influence of the number of laser ablations on the shape, geometry, and taper of the pitting structure by laser ablation in the liquid phase, three-dimensional confocal microscopy was used to quantitatively characterize the shape of the surface dotting texture of YG8 cemented carbide and analyze the evolution of the morphology based on the liquid-assisted laser ablation test. The results show that the surface pitting structure of YG8 cemented carbide evolves from a micro-convexity to a crater with the increase in the number of laser ablations, and the bottom of the crater produces a convexity after 7 ablations, the shape of the crater evolves to a trapezoidal shape after 13 ablations, and the shape is stable. The size of the dot texture increases with the number of laser ablations and reaches a maximum value of 396 μm in diameter and 149 μm in depth at the 10th and 12th ablations, respectively. The taper of the dot texture showed a trend of decreasing, increasing, and then decreasing with the increase in the number of laser ablations, and the taper was stable with more than seven ablations. This study lays a theoretical foundation for the control of the dot texture morphology on the surface of YG8 cemented carbide by laser ablation in a liquid-phase environment.

Visualisation of Bluetongue Virus in the Salivary Apparatus of Culicoides Biting Midges Highlights the Accessory Glands as a Primary Arboviral Infection Site

BackgroundArthropods transmit a wide range of pathogens of importance for the global health of humans, animals, and plants. One group of these arthropod vectors, Culicoides biting midges (Diptera: Ceratopogonidae), is the biological vector of several human and animal pathogens, including economically important livestock viruses like bluetongue virus (BTV). Like other arthropod-borne viruses (arboviruses), Culicoides-borne viruses must reach and replicate in the salivary apparatus, from where they can be transmitted to susceptible hosts through the saliva during subsequent blood feeding. Despite the importance of the salivary gland apparatus for pathogen transmission to susceptible animals from the bite of infected Culicoides, these structures have received relatively little attention, perhaps due to the small size and fragility of these vectors.ResultsIn this study, we developed techniques to visualize the infection of the salivary glands and other soft tissues with BTV, in some of the smallest known arbovirus vectors, Culicoides biting midges, using three-dimensional immunofluorescence confocal microscopy. We showed BTV infection of specific structures of the salivary gland apparatus of female Culicoides vectors following oral virus uptake, related visualisation of viral infection in the salivary apparatus to high viral RNA copies in the body, and demonstrated for the first time, that the accessory glands are a primary site for BTV replication within the salivary apparatus.ConclusionsOur work has revealed a novel site of virus-vector interactions, and a novel role of the accessory glands of Culicoides in arbovirus amplification and transmission. Our approach would also be applicable to a wide range of arbovirus vector groups including sand flies (Diptera: Psychodidae), as well as provide a powerful tool to investigate arbovirus infection and dissemination, particularly where there are practical challenges in the visualization of small size and delicate tissues of arthropods.

Dislocation interactions during plastic relaxation of epitaxial colloidal crystals

The severe difficulty to resolve simultaneously both the macroscopic deformation process and the dislocation dynamics on the atomic scale limits our understanding of crystal plasticity. Here we use colloidal crystals, imaged on the single particle level by high-speed three-dimensional (3D) confocal microscopy, and resolve in real-time both the relaxation of the epitaxial misfit strain and the accompanying evolution of dislocations. We show how dislocation interactions give rise to the formation of complex dislocation networks in 3D and to unexpectedly sharp plastic relaxation. The sharp relaxation is facilitated by attractive interactions that promote the formation of new dislocations that are more efficient in mediating strain. Dislocation networks form fragmented structures, as dislocation growth is blocked by either attractive interactions, which result in the formation of sessile dislocation junctions, or by repulsion from perpendicular segments. The strength of these blocking mechanisms decreases with the thickness of the crystal film. These results reveal the critical role of dislocation interactions in plastic deformation of thin films and can be readily generalized from the colloidal to the atomic scale.

Quantitative Analysis of Differentiation Activity for Mouse Embryonic Stem Cells by Deep Learning for Cell Center Detection using Three-Dimensional Confocal Fluorescence Microscopy Images.

Accurate single cell segmentation provides means to monitor the behavior of single cell within a population of cells. Time-lapse fluorescence images are used to reveal heterogeneous nature of single mouse embryonic stem cell (ESC) colony and monitor fluctuations of the cell states. Automatic quantification of speed and status shifts of the ESCs depends on accurate single cell segmentation that is used to calculate the 3D center of every cell and track this cell for the quantification. This study proposes a new 3D U-net to accurately detect center of each single cell in 3D confocal images. The dimension of input 3D images to the U-net is flexible so that multiple center detections from different image directions can be implemented simultaneously to improve the center detection accuracy. This study showed that our method can improve accuracy for cell center detection and thus the quantification for ESC speeds and status shifts.

Study on the Wear Performance of Polyethylene Inner Lining Pipe under Different Load and Mineralization Conditions

This study conducted pin disc friction and wear performance tests on polyethylene-lined oil pipes and four types of centralizing materials (45# steel, nylon, polytetrafluoroethylene (PTFE), and surface alloy coating) in oil fields. The friction coefficient and wear rate were tested, and the wear mechanism was analyzed using scanning electron microscopy (SEM) and three-dimensional confocal microscopy. Using a combination of experimental testing analysis and theoretical research, a comprehensive evaluation of the current wellbore centering and anti-wear technology for oil was conducted. The experimental results indicate that the usage limit of polyethylene-lined oil pipes is 400 N, and compared to metal oil pipe materials, the wear rate of both stabilizing material and tubing material is lower, indicating that it has a certain service life. From the perspective of testing load, taking into account the factors of friction coefficient and wear rate, the recommended sequence of straightening material for polyethylene lined oil pipes is (1) surface alloy coating, (2) nylon, (3) PTFE, and (4) 45# steel.

Mechanically manipulating glymphatic transport by ultrasound combined with microbubbles

The glymphatic system is a perivascular fluid transport system for waste clearance. Glymphatic transport is believed to be driven by the perivascular pumping effect created by the pulsation of the arterial wall caused by the cardiac cycle. Ultrasound sonication of circulating microbubbles (MBs) in the cerebral vasculature induces volumetric expansion and contraction of MBs that push and pull on the vessel wall to generate a MB pumping effect. The objective of this study was to evaluate whether glymphatic transport can be mechanically manipulated by focused ultrasound (FUS) sonication of MBs. The glymphatic pathway in intact mouse brains was studied using intranasal administration of fluorescently labeled albumin as fluid tracers, followed by FUS sonication at a deep brain target (thalamus) in the presence of intravenously injected MBs. Intracisternal magna injection, the conventional technique used in studying glymphatic transport, was employed to provide a comparative reference. Three-dimensional confocal microscopy imaging of optically cleared brain tissue revealed that FUS sonication enhanced the transport of fluorescently labeled albumin tracer in the perivascular space (PVS) along microvessels, primarily the arterioles. We also obtained evidence of FUS-enhanced penetration of the albumin tracer from the PVS into the interstitial space. This study revealed that ultrasound combined with circulating MBs could mechanically enhance glymphatic transport in the brain.

Characterization and Optimization of Fluorescent Organosilica Colloids for 3D Confocal Microscopy Prepared Under "Zero-Flow" Conditions.

We optimize and characterize the preparation of 3-trimethoxysilyl propyl methacrylate (TPM) colloidal suspensions for three-dimensional confocal microscopy. We revisit a simple synthesis of TPM microspheres by nucleation of droplets from prehydrolyzed TPM oil in a "zero-flow" regime and demonstrate how precise and reproducible control of particle size may be achieved via single-step nucleation with a focus on how the reagents are mixed. We also revamp the conventional dyeing method for TPM particles to achieve uniform transfer of a fluorophore to the organosilica droplets, improving particle identification. Finally, we illustrate how a ternary mixture of tetralin, trichloroethylene, and tetrachloroethylene may be used as a suspension medium which matches the refractive index of these particles while allowing independent control of the density mismatch between particle and solvent.

Veklury®(remdesivir) formulations inhibit initial membrane-coupled events of SARS-CoV-2 infection due to their sulfobutylether-β-cyclodextrin content.

Despite its contradictory clinical performance, remdesivir (Veklury®) has a pivotal role in COVID-19 therapy. Possible contributions of the vehicle, sulfobutylether-β-cyclodextrin (SBECD) toVeklury® effects have been overlooked. The powder and solution formulations of Veklury® are treated equivalently despite their different vehicle content. Our objective was to study Veklury® effects on initial membrane-coupled events of SARS-CoV-2 infection focusing on the cholesterol depletion-mediated role of SBECD. Using time-correlated flow cytometry and quantitative three-dimensional confocal microscopy, we studied early molecular events of SARS-CoV-2-host cell membrane interactions. Veklury® and different cholesterol-depleting cyclodextrins (CDs) reduced binding of the spike receptor-binding domain (RBD) to ACE2 and spike trimer internalization for Wuhan-Hu-1, Delta and Omicron variants. Correlations of these effects with cholesterol-dependent changes in membrane structure and decreased lipid raft-dependent ACE2-TMPRSS2 interaction establish that SBECD is not simply a vehicle but also an effector along with remdesivir due to its cholesterol-depleting potential. Veklury® solution inhibited RBD binding more efficiently due to its twice higher SBECD content. The CD-induced inhibitory effects were more prominent at lower RBD concentrations and in cells with lower endogenous ACE2 expression, indicating that the supportive CD actions can be even more pronounced during in vivo infection when viral load and ACE expression are typically low. Our findings call for the differentiation of Veklury® formulations in meta-analyses of clinical trials, potentially revealing neglected benefits of the solution formulation, and also raise the possibility of adjuvant cyclodextrin (CD) therapy, even at higher doses, in COVID-19.

Fusome topology and inheritance during insect gametogenesis.

From insects to mammals, oocytes and sperm develop within germline cysts comprising cells connected by intercellular bridges (ICBs). In numerous insects, formation of the cyst is accompanied by growth of the fusome-a membranous organelle that permeates the cyst. Fusome composition and function are best understood in Drosophila melanogaster: during oogenesis, the fusome dictates cyst topology and size and facilitates oocyte selection, while during spermatogenesis, the fusome synchronizes the cyst's response to DNA damage. Despite its distinct and sex-specific roles during insect gametogenesis, elucidating fusome growth and inheritance in females and its structure and connectivity in males has remained challenging. Here, we take advantage of advances in three-dimensional (3D) confocal microscopy and computational image processing tools to reconstruct the topology, growth, and distribution of the fusome in both sexes. In females, our experimental findings inform a theoretical model for fusome assembly and inheritance and suggest that oocyte selection proceeds through an 'equivalency with a bias' mechanism. In males, we find that cell divisions can deviate from the maximally branched pattern observed in females, leading to greater topological variability. Our work consolidates existing disjointed experimental observations and contributes a readily generalizable computational approach for quantitative studies of gametogenesis within and across species.

Colec12 and Trail signaling confine cranial neural crest cell trajectories and promote collective cell migration.

Collective and discrete neural crest cell (NCC) migratory streams are crucial to vertebrate head patterning. However, the factors that confine NCC trajectories and promote collective cell migration remain unclear. Computational simulations predicted that confinement is required only along the initial one-third of the cranial NCC migratory pathway. This guided our study of Colec12 (Collectin-12, a transmembrane scavenger receptor C-type lectin) and Trail (tumor necrosis factor-related apoptosis-inducing ligand, CD253) which we show expressed in chick cranial NCC-free zones. NCC trajectories are confined by Colec12 or Trail protein stripes in vitro and show significant and distinct changes in cell morphology and dynamic migratory characteristics when cocultured with either protein. Gain- or loss-of-function of either factor or in combination enhanced NCC confinement or diverted cell trajectories as observed in vivo with three-dimensional confocal microscopy, respectively, resulting in disrupted collective migration. These data provide evidence for Colec12 and Trail as novel NCC microenvironmental factors playing a role to confine cranial NCC trajectories and promote collective cell migration.

Autonomic innervation, a peripheral glial cell web, and a novel S100B expressing interstitial cell type impart structural and functional complexity to the sinoatrial node.

The sinoatrial node (SAN) of the heart produces rhythmic action potentials, generated via calcium signaling within and among pacemaker cells. Our previous work has described the SAN as composed of a HCN4+-pacemaker cell meshwork, which merges with a network of connexin 43+/F-actin+ cells. It is also known that sympathetic and parasympathetic innervation create an autonomic plexus in the SAN that modulates heart rate and rhythm. However, the anatomical details of the interaction of this plexus with the pacemaker cell meshwork have yet to be described. The cytoarchitecture of SAN whole-mount preparations was examined by three-dimensional confocal laser scanning microscopy of triple immunolabeled with combinations of antibodies for HCN4, S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), choline acetyltransferase, or vesicular acetylcholine transporter, and tyrosine hydroxylase, and transmission electron microscopy. The SAN exhibited heterogeneous autonomic innervation, which was accompanied by a web of peripheral glial cells and a novel S100B+/GFAP- interstitial cell population, with a unique morphology and a distinct distribution pattern, creating complex interactions with other cell types in the node, particularly with HCN4-expressing cells. Transmission electron microscopy identified a similar population of interstitial cells as telocytes, which appeared to secrete vesicles toward pacemaker cells. Application of S100B to SAN preparations desynchronized Ca2þ signaling in HCN4-expressing cells and increased variability in SAN impulse rate and rhythm. The autonomic plexus, peripheral glial cell web, and a novel S100Bþ/GFAP- interstitial cell type embedded within the HCN4þ cell meshwork increase the structural and functional complexity of the SAN and provide a new regulatory pathway of rhythmogenesis.

Coral holobiont research needs spatial analyses at the microbial scale.

Coral holobiont research needs spatial analyses at the microbial scale.

Differentiation of crescent-forming kidney progenitor cells into podocytes attenuates severe glomerulonephritis in mice.

Crescentic glomerulonephritis is characterized by vascular necrosis and parietal epithelial cell hyperplasia in the space surrounding the glomerulus, resulting in the formation of crescents. Little is known about the molecular mechanisms driving this process. Inducing crescentic glomerulonephritis in two Pax2Cre reporter mouse models revealed that crescents derive from clonal expansion of single immature parietal epithelial cells. Preemptive and delayed histone deacetylase inhibition with panobinostat, a drug used to treat hematopoietic stem cell disorders, attenuated crescentic glomerulonephritis with recovery of kidney function in the two mouse models. Three-dimensional confocal microscopy and stimulated emission depletion superresolution imaging of mouse glomeruli showed that, in addition to exerting an anti-inflammatory and immunosuppressive effect, panobinostat induced differentiation of an immature hyperplastic parietal epithelial cell subset into podocytes, thereby restoring the glomerular filtration barrier. Single-cell RNA sequencing of human renal progenitor cells in vitro identified an immature stratifin-positive cell subset and revealed that expansion of this stratifin-expressing progenitor cell subset was associated with a poor outcome in human crescentic glomerulonephritis. Treatment of human parietal epithelial cells in vitro with panobinostat attenuated stratifin expression in renal progenitor cells, reduced their proliferation, and promoted their differentiation into podocytes. These results offer mechanistic insights into the formation of glomerular crescents and demonstrate that selective targeting of renal progenitor cells can attenuate crescent formation and the deterioration of kidney function in crescentic glomerulonephritis in mice.

U-net structures for segmentation of single mouse embryonic stem cells using three-dimensional confocal microscopy images.

Cell segmentation at a single cell resolution is required to provide insights for basic biology and application study. However, there are issues of low signal-to-noise ratio, weak fluorescence response, and insufficient resolution along the image stacking direction in 3D confocal images (volume). It has been difficult to segment out single cells from close or contacted cells in a cell volume using image processing methods or together with geometric processing methods. Recently, 3D deep learning methods have been used to avoid tedious parameter settings in the image and geometric processing, but still not easy to segment out close or contacted single cells. This paper proposes a 2D U-net to segment cell regions in high accuracy and computing performance. Better 3D cell images and single cell segmentation for close or contacted cells are achieved by combining a 3D U-net to detect the centers of single cells in the volume.

Comparison of surface roughness of root cementum and orthodontically induced root resorption craters from high- and low-fluoridation areas: a 3D confocal microscopy study

BackgroundFluoride has a major role in strengthening the structure of enamel against acids. Despite differences between caries and root resorption processes, both events inherently involve acidic dissolution of dental tissues. The aim of the present study was to investigate the effects of water fluoridation levels on the surface roughness of root cementum and resorption craters. The findings provided more insight into the influence of fluoride on the surfaces of intact cementum surface and resorption craters.MethodsTwenty-eight orthodontic patients were recruited from two cities in Turkey, with high (≥ 2 ppm) and low (≤ 0.05 ppm) water fluoridation. These patients needed bilateral maxillary first premolar extraction as part of their orthodontic treatment and were allocated into two study groups (n = 14 in each group) based on water fluoridation exposure level: the high-fluoride group (HF) and low-fluoride group (LF). 150 g of buccal tipping forces was applied to all maxillary first premolar teeth for 12 weeks with a beta-titanium spring which was reactivated every 4 weeks. All maxillary premolars were removed at the end of the experiment for surface roughness assessment using three-dimensional confocal microscopy and the associated software. The buccal root surface and the largest buccal resorption crater were investigated.ResultsResorption craters were significantly rougher in LF group compared to HF group (p = 0.002). Craters were rougher than the intact root surfaces (p = 0.000). Cervical and apical regions were significantly rougher than the middle region (p = 0.000 and p = 0.024, respectively).ConclusionsHigher water fluoridation level of ≥ 2 ppm resulted in significantly smoother root resorption craters than low water fluoridation level of ≤ 0.05 ppm when the teeth were subjected to 150 g of buccal tipping force. Fluoride seems to have a protective role at the interface of root resorption, and further mineral or histological studies may shed light on the exact protective process against root resorption.

Invasive aspergillosis-on-chip: A quantitative treatment study of human Aspergillus fumigatus infection

Invasive pulmonary aspergillosis is associated with a high mortality rate and poses a direct threat to immunocompromised patients. Here, we present the invasive aspergillosis-on-chip (IAC) model to investigate Aspergillus fumigatus infection in vitro. The model allows the study of the lateral growth and the invasive behaviour of fungal hyphae from the epithelium into the endothelial cell layer in an alveolus-on-chip model. We established an algorithm-based analysis pipeline for three-dimensional confocal microscopy images to visualize and quantify fungal morphology, including hyphal growth and branching. Human macrophages in the IAC model partially inhibited the growth of the fungus, contributed to the release of proinflammatory cytokines (IL-1, IL-6, TNF) and chemokines (IL-8 and MCP-1) associated with an increased number of invasive hyphae. Similar to in vivo, the application of the fungistatic drug caspofungin limited the fungal growth and resulted in morphological changes of the hyphal tree previously described in other studies. The IAC infection model allows the identification and characterization of cellular infection targets and in vitro testing of antifungal drugs in clinically relevant concentrations. It thus represents a promising tool to broaden the understanding of pathogenicity and pathophysiology of invasive aspergillosis.

Enhanced wastewater treatment by modified basalt fiber bio-carriers: Effect of etching and surface functionalization

The application of biological carriers holds a central position in wastewater treatment for biofilm-mediated degradation processes. In this study, the surface of basalt fibers (BF) was modified by combined etching and grouping (surface functionalization via attaching glutamic acid) to produce MBFEG. The etching modification enhanced the surface properties and specific surface area of BF. However, combined etching and functionalization resulted in the successful loading of amino acids on the surface of MBFEG, ultimately improving surface hydrophilicity, adsorption properties, and biophilicity of the bio-carrier. Three-dimensional confocal laser scanning microscopy confirmed that the combined modification was highly suitable in improving microbial activity and biophilicity. The microbial communities on MBFEG bio-carrier were rich and diverse indicating a stronger degree of biogeochemical cycling, indicating enrichment of nitrogen and carbon cycles. Conclusively, etching and grouping modification technology hold a promising future for BF application in biological wastewater treatment schemes.