3D Microscopy Research Articles

Label-Free Intracellular Multi-Specificity in Yeast Cells by Phase-Contrast Tomographic Flow Cytometry.

In-flow phase-contrast tomography provides a 3D refractive index of label-free cells in cytometry systems. Its major limitation, as with any quantitative phase imaging approach, is the lack of specificity compared to fluorescence microscopy, thus restraining its huge potentialities in single-cell analysis and diagnostics. Remarkable results in introducing specificity are obtained through artificial intelligence (AI), but only for adherent cells. However, accessing the 3D fluorescence ground truth and obtaining accurate voxel-level co-registration of image pairs for AI training is not viable for high-throughput cytometry. The recent statistical inference approach is a significant step forward for label-free specificity but remains limited to cells' nuclei. Here, a generalized computational strategy based on a self-consistent statistical inference to achieve intracellular multi-specificity is shown. Various subcellular compartments (i.e., nuclei, cytoplasmic vacuoles, the peri-vacuolar membrane area, cytoplasm, vacuole-nucleus contact site) can be identified and characterized quantitatively at different phases of the cells life cycle by using yeast cells as a biological model. Moreover, for the first time, virtual reality is introduced for handling the information content of multi-specificity in single cells. Full fruition is proofed for exploring and interacting with 3D quantitative biophysical parameters of the identified compartments on demand, thus opening the route to a metaverse for 3D microscopy.

Scientific and technical analysis of lead-barium glaze dragonfly eye beads from the Late Warring States period in China

This paper analyzed five dragonfly eye beads excavated from M176 of the Hejia Cemetery in the Late Warring States period (around 3rd c. BC) by using a super depth of field 3D microscope system (OM), scanning electron microscope-energy dispersive spectrum (SEM–EDS) and Raman spectroscopy. The analytical results confirmed that all the beads were glazed pottery and the glaze material belongs to the lead-barium-silicate (PbO-BaO-SiO2) system. The color component of the glaze is Chinese Blue (BaCuSi4O10). Three beads, M176-2, M176-3, and M176-4, were formed with an inner core support and were made in the same batch. Additionally, two weathering products, CuPb4(SO4)2(OH)6 and PbCO3, were detected on the glaze layer surface. The results of scientific and technological analysis show that these beads have differences in the composition of the body and glaze, and the color composition in the glaze layer is relatively rare in previous studies. The discovery of lead-barium glazed pottery beads from the Late Warring States period in northern China provides new evidence for further exploration into the origins and evolution of early glazed pottery. The identification of weathering products formed on the beads’ surface within an alkaline burial environment holds valuable implications for the study of weathering and deterioration in silicate artifacts.

3D Biological/Biomedical Image Registration with enhanced Feature Extraction and Outlier Detection.

In various applications, such as computer vision, medical imaging and robotics, three-dimensional (3D) image registration is a significant step. It enables the alignment of various datasets into a single coordinate system, consequently providing a consistent perspective that allows further analysis. By precisely aligning images we can compare, analyze, and combine data collected in different situations. This paper presents a novel approach for 3D or z-stack microscopy and medical image registration, utilizing a combination of conventional and deep learning techniques for feature extraction and adaptive likelihood-based methods for outlier detection. The proposed method uses the Scale-invariant Feature Transform (SIFT) and the Residual Network (ResNet50) deep neural learning network to extract effective features and obtain precise and exhaustive representations of image contents. The registration approach also employs the adaptive Maximum Likelihood Estimation SAmple Consensus (MLESAC) method that optimizes outlier detection and increases noise and distortion resistance to improve the efficacy of these combined extracted features. This integrated approach demonstrates robustness, flexibility, and adaptability across a variety of imaging modalities, enabling the registration of complex images with higher precision. Experimental results show that the proposed algorithm outperforms state-of-the-art image registration methods, including conventional SIFT, SIFT with Random Sample Consensus (RANSAC), and Oriented FAST and Rotated BRIEF (ORB) methods, as well as registration software packages such as bUnwrapJ and TurboReg, in terms of Mutual Information (MI), Phase Congruency-Based (PCB) metrics, and Gradiant-based metrics (GBM), using 3D MRI and 3D serial sections of multiplex microscopy images.

A preliminary study evaluating the relationship between force and incised trauma on pig rib bones

In forensic contexts, understanding of the complicated relationships between level of force, type of knife blade, and dimensions of incisions remains limited. The purpose of this research was to explore how incisions on pig rib bones vary depending on the type of knife blade and quantity of perpendicular force inflicted. A cutting rig (designed to position a bone, knife, and weights) facilitated the creation of incisions on fleshed rib bones (n = 59), defleshed rib bones (n = 77), and synthetic materials (n = 36). Five different masses of weights (measuring 4381, 8861, 13515, 18267, and 23343 g) were applied to four knife blades (two serrated and two non-serrated blades), with each combination of variables repeated thrice. 3D digital microscopy was utilized to model and measure each incision. The two-way ANOVA found significant differences in depth and length between knife types and levels of force across all sample sets (p < 0.05), with greater perpendicular forces and serrated blades creating longer and deeper cuts. These findings demonstrate that there is a complex relationship between force, type of knife blade, and the related dimensions of incisions.

Sialylation of EGFR by ST6GAL1 induces receptor activation and modulates trafficking dynamics

Aberrant glycosylation is a hallmark of a cancer cell. One prevalent alteration is an enrichment in α2,6-linked sialylation of N-glycosylated proteins, a modification directed by the ST6GAL1 sialyltransferase. ST6GAL1 is upregulated in many malignancies including ovarian cancer. Prior studies have shown that the addition of α2,6 sialic acid to the epidermal growth factor receptor (EGFR) activates this receptor, although the mechanism was largely unknown. To investigate the role of ST6GAL1 in EGFR activation, ST6GAL1 was overexpressed in the OV4 ovarian cancer line, which lacks endogenous ST6GAL1, or knocked-down in the OVCAR-3 and OVCAR-5 ovarian cancer lines, which have robust ST6GAL1 expression. Cells with high expression of ST6GAL1 displayed increased activation of EGFR and its downstream signaling targets, AKT and NFκB. Using biochemical and microscopy approaches, including total internal reflection fluorescence microscopy, we determined that the α2,6 sialylation of EGFR promoted its dimerization and higher order oligomerization. Additionally, ST6GAL1 activity was found to modulate EGFR trafficking dynamics following EGF-induced receptor activation. Specifically, EGFR sialylation enhanced receptor recycling to the cell surface following activation while simultaneously inhibiting lysosomal degradation. 3D widefield deconvolution microscopy confirmed that in cells with high ST6GAL1 expression, EGFR exhibited greater colocalization with Rab11 recycling endosomes and reduced colocalization with LAMP1-positive lysosomes. Collectively, our findings highlight a novel mechanism by which α2,6 sialylation promotes EGFR signaling by facilitating receptor oligomerization and recycling.

Evaluation of A New Approach to Enhance Visualization Performance in Cataract Surgery: 3D Visualization System with Coaxial Illumination.

To evaluate the visualization performance of different approaches, including the 3D Visualization system with coaxial illumination and the 3D system or microscope with standard illumination. Fuzhou Ophthalmic Hospital, Fuzhou City, China. Cross-sectional study. This two-part performance assessment for visualization comprised of an objective analysis utilizing surgical video images and a subjective survey collecting feedback from surgeons. Data of each eye were obtained with 3 approaches: standard surgical microscope with standard illumination (SOM-S), 3D Visualization system with standard illumination (3D-S) and 3D Visualization system with coaxial illumination (3D-C). Totally 112 eyes (107 cases) and 6 cataract surgeons were involved. Red reflex value was markedly greater in the 3D-C approach compared with other 2 approaches (P<0.001). Compared with the SOM-S approach, red reflex increased by 55%, 57%, and 53% in the 3D-C approach corresponding to nuclear grades of II, III, and IV, respectively. In the questionnaire survey, red reflex scores were consistently significantly higher in the 3D-C approach than in the others (P<0.001). Depth of field was enhanced in both 3D approaches compared with the SOM-S approach (P<0.05). The only minor advantage of the SOM system over the 3D-C approach was in the surrounding field clarity score, and the difference was not statistically significant (P=1.000). The 3D-C approach significantly increased the red reflex in both objective and subjective assessments. Surgeons' responses also showed a superior performance for the 3D-C approach.

Deep self-learning enables fast, high-fidelity isotropic resolution restoration for volumetric fluorescence microscopy

One intrinsic yet critical issue that troubles the field of fluorescence microscopy ever since its introduction is the unmatched resolution in the lateral and axial directions (i.e., resolution anisotropy), which severely deteriorates the quality, reconstruction, and analysis of 3D volume images. By leveraging the natural anisotropy, we present a deep self-learning method termed Self-Net that significantly improves the resolution of axial images by using the lateral images from the same raw dataset as rational targets. By incorporating unsupervised learning for realistic anisotropic degradation and supervised learning for high-fidelity isotropic recovery, our method can effectively suppress the hallucination with substantially enhanced image quality compared to previously reported methods. In the experiments, we show that Self-Net can reconstruct high-fidelity isotropic 3D images from organelle to tissue levels via raw images from various microscopy platforms, e.g., wide-field, laser-scanning, or super-resolution microscopy. For the first time, Self-Net enables isotropic whole-brain imaging at a voxel resolution of 0.2 × 0.2 × 0.2 μm3, which addresses the last-mile problem of data quality in single-neuron morphology visualization and reconstruction with minimal effort and cost. Overall, Self-Net is a promising approach to overcoming the inherent resolution anisotropy for all classes of 3D fluorescence microscopy.

Research on the Corrosion Resistance of an Epoxy Resin-Based Self-Healing Propylene Glycol-Loaded Ethyl Cellulose Microcapsule Coating

In this work, ethyl cellulose was used as a wall material, propanetriol as a core material, polyvinyl alcohol as a stabilizer and gelatin as an emulsifier. Self-healing microcapsules with a slow-release effect were prepared using the solvent evaporation method. Various analytical techniques, such as 3D confocal microscopy (LCSM), optical microscopy (OM), scanning electron microscopy (SEM), infrared spectroscopy (FT-IR), energy dispersive spectroscopy (EDS), thermal weight loss analysis (TGA), laser particle size tester and electrochemical impedance polarization, are utilized. The morphology, distribution, particle size, corrosion resistance and self-healing ability of the prepared microcapsules and resin-based coatings were characterized and analyzed. The results show that the cross-sectional core–shell structure is clearly seen in the LCSM, showing a smooth, hollow, spherical shape. OM and laser particle size testers have shown that the size of the microcapsules decreases over time. Also, in OM, the microcapsules are uniformly distributed in the emulsion with a smooth and non-adherent surface. In SEM, the microcapsule particle size is about 150 μm, the shell wall thickness is about 18 μm, and the hollow structure of ruptured microcapsules is obvious. FT-IR and TGA confirmed the successful encapsulation of the formulated microcapsules. The results show that when the core-wall mass ratio is 1.2:1 and the amount of microcapsule is 10% of the coating amount, the prepared microcapsule has high thermal stability and certain wear resistance. By electrochemical and immersion experiments, it was found that a 3.5 wt % NaCl solution has the best impedance, the lowest corrosion current density, and good adhesion and tensile toughness. The results showed that glycerol was successfully released from the broken microcapsules and self-healed, forming an anticorrosive coating with excellent corrosion resistance and self-healing ability.

Three-dimensional spatial quantitative analysis of cardiac lymphatics in the mouse heart.

Three-dimensional (3D) microscopy and image data analysis are necessary for studying the morphology of cardiac lymphatic vessels (LyVs) and their association with other cell types. We aimed to develop a methodology for 3D multiplexed lightsheet microscopy and highly sensitive and quantitative image analysis to identify pathological remodeling in the 3D morphology of LyVs in young adult mouse hearts with familial hypertrophic cardiomyopathy (HCM). We developed a 3D lightsheet microscopy workflow providing a quick turn-around (as few as 5-6 days), multiplex fluorescence detection, and preservation of LyV structure and epitope markers. Hearts from non-transgenic and transgenic (TG) HCM mice were arrested in diastole, retrograde perfused, immunolabeled, optically cleared, and imaged. We built an image-processing pipeline to quantify LyV morphological parameters at the chamber and branch levels. Chamber-specific pathological alterations of LyVs were identified, and significant changes were seen in the right atrium (RA). TG hearts had a higher volume percent of ER-TR7+ fibroblasts and reticular fibers. In the RA, we found associations between ER-TR7+ volume percent and both LyV segment density and median diameter. This workflow and study enabled multi-scale analysis of pathological changes in cardiac LyVs of young adult mice, inviting ideas for research on LyVs in cardiac disease.

Study on the evolution process of corrosion behavior under the sulfate reducing bacteria biofilm in shale gas gathering pipelines

Pipeline corrosion induced by sulfate-reducing bacteria (SRB) is particularly severe during the hydraulic fracturing technology in shale gas exploitation. To investigate the evolution of corrosion beneath the SRB biofilms, 49-day corrosion experiments were performed with and without SRB. The weight loss experiment demonstrated that the corrosion in the Blank group was mitigated by the corrosion product film. In contrast, the SRB group exhibited an initial inhibitory phase followed by an exacerbation. Scanning electron microscopy (SEM) revealed stratification in the Blank group, whereas SRB initially formed colonies and extracellular polymeric substances (EPS), eventually the biofilm evolved into a mixed layer with the corrosion product. 3D microscopy showed a relatively flat surface in the Blank group. Conversely, corrosion pits beneath SRB biofilm were circular and could reach a depth of 29.77 μm, significantly greater than in the Blank group. Over time, SRB-induced pits deepened from the edges to the center and widened to approximately 740 μm, accompanied by an increase in quantity. Energy Dispersive Spectrometer (EDS) and X-ray photoelectron spectroscopy (XPS) analyses detected the presence of elements P, S, and FeS in the SRB group, serving as important indicators of SRB corrosion. Electrochemical experiments revealed significantly higher corrosion currents in the SRB group. The evolution of SRB corrosion consisted of three phases: initial, intermediate, and starvation phases. During the starvation phase, localized corrosion proved to be the most severe, with a corrosion rate of 1.28 mm/a. Therefore, the study of SRB-induced corrosion should be prioritized to study the localization phenomena.

Calibration method for a multi-focus microscopic 3D imaging system.

This Letter presents a novel, to the best of our knowledge, method to calibrate multi-focus microscopic structured-light three-dimensional (3D) imaging systems with an electrically adjustable camera focal length. We first leverage the conventional method to calibrate the system with a reference focal length f0. Then we calibrate the system with other discrete focal lengths fi by determining virtual features on a reconstructed white plane using f0. Finally, we fit the polynomial function model using the discrete calibration results for fi. Experimental results demonstrate that our proposed method can calibrate the system consistently and accurately.

HP1-driven phase separation recapitulates the thermodynamics and kinetics of heterochromatin condensate formation

The spatial segregation of pericentromeric heterochromatin (PCH) into distinct, membrane-less nuclear compartments involves the binding of Heterochromatin Protein 1 (HP1) to H3K9me2/3-rich genomic regions. While HP1 exhibits liquid-liquid phase separation properties in vitro, its mechanistic impact on the structure and dynamics of PCH condensate formation in vivo remains largely unresolved. Here, using a minimal theoretical framework, we systematically investigate the mutual coupling between self-interacting HP1-like molecules and the chromatin polymer. We reveal that the specific affinity of HP1 for H3K9me2/3 loci facilitates coacervation in nucleo and promotes the formation of stable PCH condensates at HP1 levels far below the concentration required to observe phase separation in purified protein assays in vitro. These heterotypic HP1-chromatin interactions give rise to a strong dependence of the nucleoplasmic HP1 density on HP1-H3K9me2/3 stoichiometry, consistent with the thermodynamics of multicomponent phase separation. The dynamical cross talk between HP1 and the viscoelastic chromatin scaffold also leads to anomalously slow equilibration kinetics, which strongly depend on the genomic distribution of H3K9me2/3 domains and result in the coexistence of multiple long-lived, microphase-separated PCH compartments. The morphology of these complex coacervates is further found to be governed by the dynamic establishment of the underlying H3K9me2/3 landscape, which may drive their increasingly abnormal, aspherical shapes during cell development. These findings compare favorably to 4D microscopy measurements of HP1 condensate formation in live Drosophila embryos and suggest a general quantitative model of PCH formation based on the interplay between HP1-based phase separation and chromatin polymer mechanics.

3D cell segregation geometry and dynamics are governed by tissue surface tension regulation

Tissue morphogenesis and patterning during development involve the segregation of cell types. Segregation is driven by differential tissue surface tensions generated by cell types through controlling cell-cell contact formation by regulating adhesion and actomyosin contractility-based cellular cortical tensions. We use vertebrate tissue cell types and zebrafish germ layer progenitors as in vitro models of 3-dimensional heterotypic segregation and developed a quantitative analysis of their dynamics based on 3D time-lapse microscopy. We show that general inhibition of actomyosin contractility by the Rho kinase inhibitor Y27632 delays segregation. Cell type-specific inhibition of non-muscle myosin2 activity by overexpression of myosin assembly inhibitor S100A4 reduces tissue surface tension, manifested in decreased compaction during aggregation and inverted geometry observed during segregation. The same is observed when we express a constitutively active Rho kinase isoform to ubiquitously keep actomyosin contractility high at cell-cell and cell-medium interfaces and thus overriding the interface-specific regulation of cortical tensions. Tissue surface tension regulation can become an effective tool in tissue engineering.

Collaborative formation pattern of surface topography and residual stress in hard turning of steels

High-performance manufacturing (HPM) is essential for high-quality equipment and parts manufacturing, which requires not only geometric accuracy but also control of surface integrity to satisfy functional performance. Surface topography and residual stress are important elements of surface integrity. However, they are always investigated individually, and their collaborative formation pattern has been ignored for a long time. In this study, surface topography will be measured by a 3D laser scanning microscope and analyzed from the perspective of spectral wavelength (λ) along with its corresponding amplitude. Whereas the residual stress (RS) is going to be analyzed by X-Ray diffraction and finite element (FE) model. Thereafter we introduce the formation pattern of both surface topography and RS. Hereby, simulated points clouds of machined surface topography are generated by superimposing the normally distributed random numbers that take into account the springback of the workpiece material response on tool kinematics and geometry. Furthermore, the RS is divided into mechanical loads induced (RSM) and thermal loads induce (RST) according to the mechanism of formation, which were obtained in cutting speed (Y-direction) and feed direction (Z-direction) by the Mohr circle. Our experimental and numerical results showed that an increase in the feed rate (from 0.05 mm/r to 0.15 mm/r) significantly increases the surface roughness and cutting temperature. The increase in temperature increased the RST by up to 286.1%, resulting in a reduction of compressive RS in Z-direction and an increase of tensile RS in Y-direction. Moreover, the increase in cutting speed (from 40 m/min to 80 m/min) results in a more regular surface and a reduction in surface roughness because of the smaller springback. At the same time, it also leads to a decrease of RSM in Z-direction and an increase of RSM in Y-direction. In addition, the increment in cutting speed leads to a minor increase in temperature and RST (maximum increase of 47.0%). Combining the effects of RSM and RST, RS in both directions increases slightly.

A weak-labelling and deep learning approach for in-focus object segmentation in 3D widefield microscopy

Three-dimensional information is crucial to our understanding of biological phenomena. The vast majority of biological microscopy specimens are inherently three-dimensional. However, conventional light microscopy is largely geared towards 2D images, while 3D microscopy and image reconstruction remain feasible only with specialised equipment and techniques. Inspired by the working principles of one such technique—confocal microscopy, we propose a novel approach to 3D widefield microscopy reconstruction through semantic segmentation of in-focus and out-of-focus pixels. For this, we explore a number of rule-based algorithms commonly used for software-based autofocusing and apply them to a dataset of widefield focal stacks. We propose a computation scheme allowing the calculation of lateral focus score maps of the slices of each stack using these algorithms. Furthermore, we identify algorithms preferable for obtaining such maps. Finally, to ensure the practicality of our approach, we propose a surrogate model based on a deep neural network, capable of segmenting in-focus pixels from the out-of-focus background in a fast and reliable fashion. The deep-neural-network-based approach allows a major speedup for data processing making it usable for online data processing.

Virtual assessment of a possible meningioma in a Roman-period cranium

Diseases have accompanied human populations since prehistoric times. Knowing the paleopathologies and their consequences derived from them can help us to understand their impact and how have been decisive in our ancestors' ways of life. Taphonomic and paleopathological studies are key to understanding how injuries occurred; they can provide information on causes of death, analyzed populations behaviour, such as the existence of interpersonal conflicts or how they took the care of the sick. Those studies also confirm the existence of certain diseases, mentioned in the archaeological record. This paper explains the analysis of four lesions found in a Roman-era cranium from Sima de Marcenejas, located in Northern Spain. An anthropological analysis of this cranium has revealed that it corresponds to an adult male individual. This work focuses on the differential diagnosis of the lesions, to be able to discern the most likely aetiologies. The following techniques have been implemented: classical morphological analysis, forensic taphonomic analysis and virtual analysis. MicroCT and 3D microscopy have been used as essential tools for the virtual analysis of the cranium and its lesions. The results obtained revealed the existence of a tumour and three exocranial traumas, all of them antemortem. The location of the tumour, as well as its morphology together with other aspects, support the meningioma as the most probable tumour type. This possible ancient meningioma represents the first case for these chronologies on the Iberian Peninsula, where there are few documented cases. The three traumatic lesions reveal the existence of injuries produced by both, blunt and sharp objects, related to events of interpersonal violence. By applying virtual 3D analyses, the researchers have demonstrated that it is viable to identify tumours in those internal cranial regions, where the lesion is not visible, thus providing new comparative data for the paleopathological record of past populations. Highlights: Meningiomas are rare in the archaeological record which complicates tracing themin ancient human populations. The use of computerized microtomography (MicroCT) and virtual 3D models makes it possible to identify tumoursin those internal cranial regions where the lesions are not visible. Paleopathological analysis of a Roman cranium has revealed, in addition to cranial trauma, a new possible case of meningioma.

Investigation of Dry Sliding Wear Behavior of CFRP Composite Used in New Generation Aircraft Wings

In this study, it is aimed to examine the effect of reinforcement laying angle on wear resistance of CFRP composites used in the automotive and aerospace industries. Experiments were carried out on Ball-On-Disc wear device under 1, 3 and 5 N loads at room temperature. 3D optical microscope was used to determine the volume losses in the samples. The worn surface morphology of the samples was examined with the help of SEM (Scanning Electron Microscopy). In the test results, the volume losses of the samples increased depending on the increasing loads and shear rate. It has been understood that the change of the laying angle is important in the dry-sliding resistance of the samples. The wear resistance of the samples produced with 45 degree laying was better at both sliding speeds and all loads. It has also been understood that the laying angle is also effective in the coefficient of friction. Delamination, plastic deformation type dominant wear mechanisms have occurred.

Optical single-pixel volumetric imaging by three-dimensional light-field illumination

Three-dimensional single-pixel imaging (3D SPI) has become an attractive imaging modality for both biomedical research and optical sensing. 3D-SPI techniques generally depend on time-of-flight or stereovision principle to extract depth information from backscattered light. However, existing implementations for these two optical schemes are limited to surface mapping of 3D objects at depth resolutions, at best, at the millimeter level. Here, we report 3D light-field illumination single-pixel microscopy (3D-LFI-SPM) that enables volumetric imaging of microscopic objects with a near-diffraction-limit 3D optical resolution. Aimed at 3D space reconstruction, 3D-LFI-SPM optically samples the 3D Fourier spectrum by combining 3D structured light-field illumination with single-element intensity detection. We build a 3D-LFI-SPM prototype that provides an imaging volume of ∼390 × 390 × 3,800 μm3 and achieves 2.7-μm lateral resolution and better than 37-μm axial resolution. Its capability of 3D visualization of label-free optical absorption contrast is demonstrated by imaging single algal cells in vivo. Our approach opens broad perspectives for 3D SPI with potential applications in various fields, such as biomedical functional imaging.

Advanced Cryogenic Light Microscopy Stage to Enable 3D Super-resolved Cryogenic Correlative Light and Electron Microscopy.

Advanced Cryogenic Light Microscopy Stage to Enable 3D Super-resolved Cryogenic Correlative Light and Electron Microscopy.

Optimal Diverse Biological Sample Preparation Methods for 2D and 3D Electron Microscopy Imaging.

Optimal Diverse Biological Sample Preparation Methods for 2D and 3D Electron Microscopy Imaging.