Differential Interference Contrast Research Articles

High-quality InAs homoepitaxial layers grown by molecular beam epitaxy

The growth conditions for InAs homoepitaxy by molecular beam epitaxy were comprehensively studied across a broad spectrum of substrate temperatures, As2/In flux ratios, and growth rates. It was found that the surface morphology and overall quality of the InAs layers were significantly influenced by these parameters. Optimal conditions, including a lower growth temperature, reduced As2 flux, and slower growth rate, were pivotal in achieving high-quality InAs layers. Two primary characterization techniques, differential interference contrast microscopy and atomic force microscopy, were employed to evaluate the material quality. High-quality InAs homoepitaxial layers were successfully grown at a substrate temperature of 455 °C and a growth rate of 0.33 monolayers per second (ML/s). These layers exhibited a remarkably low defect density of approximately 300 defects per square centimeter, which is over an order of magnitude lower than previously reported, and a notably low root-mean-square roughness of 0.116 nm. At a growth rate of 0.33 ML/s, the growth temperature range for InAs homoepitaxial layers was found to be quite broad, whereas the As2/In flux ratio remained within a narrow range. This study underscores the critical role of precise control over growth parameters in the molecular beam epitaxy process for producing high-quality InAs homoepitaxial layers.

MTCSNet: One-Stage Learning and Two-Point Labeling are Sufficient for Cell Segmentation.

Deep convolution neural networks have been widely used in medical image analysis, such as lesion identification in whole-slide images, cancer detection, and cell segmentation, etc. However, it is often inevitable that researchers try their best to refine annotations so as to enhance the model performance, especially for cell segmentation task. Weakly supervised learning can greatly reduce the workload of annotations, while there is still a huge performance gap between the weakly and fully supervised learning approaches. In this work, we propose a weakly-supervised cell segmentation method, namely Multi-Task Cell Segmentation Network (MTCSNet), for multi-modal medical images, including pathological, brightfield, fluorescent, phase-contrast and differential interference contrast images. MTCSNet is learnt in a single-stage training manner, where only two annotated points for each cell provide supervision information, and the first one is the centroid, the second one is its boundary. Additionally, five auxiliary tasks are elaborately designed to train the network, including two pixel-level classifications, a pixel-level regression, a local temperature scaling and an instance-level distance regression task, which is proposed to regress the distances between the cell centroid and its boundaries in eight orientations. The experimental results indicate that our method outperforms all state-of-the-art weakly-supervised cell segmentation approaches on public multi-modal medical image datasets. The promising performance also shows that a single-stage learning with two-point labeling approach are sufficient for cell segmentation, instead of fine contour delineation. The codes are available at: https://github.com/binging512/MTCSNet.

Live-cell imaging under centrifugation characterized the cellular force for nuclear centration in the Caenorhabditis elegans embryo

Organelles in cells are appropriately positioned, despite crowding in the cytoplasm. However, our understanding of the force required to move large organelles, such as the nucleus, inside the cytoplasm is limited, in part owing to a lack of accurate methods for measurement. We devised a method to apply forces to the nucleus of living Caenorhabditis elegans embryos to measure the force generated inside the cell. We used a centrifuge polarizing microscope to apply centrifugal force and orientation-independent differential interference contrast microscopy to characterize the mass density of the nucleus and cytoplasm. The cellular forces moving the nucleus toward the cell center increased linearly at ~12 pN/μm depending on the distance from the center. The frictional coefficient was ~980 pN s/μm. The measured values were smaller than the previously reported estimates for sea urchin embryos. The forces were consistent with the centrosome-organelle mutual pulling model for nuclear centration. The frictional coefficient was reduced when microtubules were shorter or detached from nuclei in mutant embryos, demonstrating the contribution of astral microtubules. Finally, the frictional coefficient was higher than a theoretical estimate, indicating the contribution of uncharacterized properties of the cytoplasm.

Geometric characteristics of stromal collagen fibres in breast cancer using differential interference contrast microscopy.

Breast cancer (BC) is characterised by a high level of heterogeneity, which is influenced by the interaction of neoplastic cells with the tumour microenvironment. The diagnostic and prognostic role of the tumour stroma in BC remains to be defined. Differential interference contrast (DIC) microscopy is a label-free imaging technique well suited to visualise weak optical phase objects such as cells and tissue. This study aims to compare stromal collagen fibre characteristics between in situ and invasive breast tumours using DIC microscopy and investigate the prognostic value of collagen parameters in BC. A tissue microarray was generated from 200 cases, comprising ductal carcinoma in situ (DCIS; n=100) and invasive tumours (n=100) with an extra 50 (25 invasive BC and 25 DCIS) cases for validation was utilised. Two sections per case were used: one stained with haematoxylin and eosin (H&E) stain for histological review and one unstained for examination using DIC microscopy. Collagen fibre parameters including orientation angle, fibre alignment, fibre density, fibre width, fibre length and fibre straightness were measured. Collagen fibre density was higher in the stroma of invasive BC (161.68±11.2fibre/µm2) compared to DCIS (p<0.0001). The collagen fibres were thinner (13.78±1.08µm), straighter (0.96±0.006, on a scale of 0-1), more disorganised (95.07°±11.39°) and less aligned (0.20±0.09, on a 0-1 scale) in the invasive BC compared to DCIS (all p<0.0001). A model considering these features was developed that could distinguish between DCIS and invasive tumours with 94% accuracy. There were strong correlations between fibre characteristics and clinicopathological parameters in both groups. A statistically significant association between fibre characteristics and patients' outcomes (breast cancer specific survival, and recurrence free survival) was observed in the invasive group but not in DCIS. Although invasive BC and DCIS were both associated with stromal reaction, the structural features of collagen fibres were significantly different in the two disease stages. Analysis of the stroma fibre characteristics in the preoperative core biopsy specimen may help to differentiate pure DCIS from those associated with invasion.

Mixtures of Intrinsically Disordered Neuronal Protein Tau and Anionic Liposomes Reveal Distinct Anionic Liposome-Tau Complexes Coexisting with Tau Liquid-Liquid Phase-Separated Coacervates.

Tau, an intrinsically disordered neuronal protein and polyampholyte with an overall positive charge, is a microtubule (MT) associated protein that binds to anionic domains of MTs and suppresses their dynamic instability. Aberrant tau-MT interactions are implicated in Alzheimer's and other neurodegenerative diseases. Here, we studied the interactions between full-length human protein tau and other negatively charged binding substrates, as revealed by differential interference contrast (DIC) and fluorescence microscopy. As a binding substrate, we chose anionic liposomes (ALs) containing either 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS, -1e) or 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG, -1e) mixed with zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to mimic anionic plasma membranes of axons where tau resides. At low salt concentrations (0 to 10 mM KCl or NaCl) with minimal charge screening, reaction mixtures of tau and ALs resulted in the formation of distinct states of AL-tau complexes coexisting with liquid-liquid phase-separated tau self-coacervates arising from the polyampholytic nature of tau containing cationic and anionic domains. AL-tau complexes (i.e. tau-lipoplexes) exhibited distinct types of morphologies. This included large ∼20-30 μm tau-decorated giant vesicles with additional smaller liposomes with bound tau attached to the giant vesicles and tau-mediated finite-size assemblies of small liposomes. As the salt concentration was increased to near and above 150 mM for 1:1 electrolytes, AL-tau complexes remained stable, while tau self-coacervate droplets were found to dissolve, indicative of the breaking of (anionic/cationic) electrostatic bonds between tau chains due to increased charge screening. The findings are consistent with the hypothesis that distinct cationic domains of tau may interact with anionic lipid domains of the lumen-facing monolayer of the axon's plasma membrane, suggesting the possibility of transient yet robust interactions near relevant ionic strengths found in neurons.

Visible light potentiates rapid cell destruction and death by curcumin in vitro.

Curcumin, a small molecule derived from the plant Curcuma longa, is a pleiotropic agent with widely varying pharmacological activities attributed to it. In addition to its anti-cancer activity curcumin is also known to be cytotoxic upon photoactivation. Time-lapse DIC and correlative fluorescence microscopy were used to evaluate the effects of curcumin, combined with continuous exposure to visible light, on cellular components of RTG-2 cells. Curcumin combined with visible light resulted in rapid and dramatic destruction of cells. F-actin and microtubule cytoskeletons were drastically altered, both showing fragmentation and overall loss from cells. Nuclei exhibited granulated nucleoplasm, condensed DNA, and physical shrinkage. Mitochondria rapidly fragmented along their length and disappeared from cells. Plasma membrane was breached based on lipophilic dye staining and the entrance of otherwise impermeant small molecules into the cell. Grossly distorted morphology hallmarked by significant swelling and coarse granulation of the cytoplasm was consistently observed. All of these effects were dependent on visible light as the same cellular targets in curcumin-treated cells outside the illuminated area were always unperturbed. The combination of curcumin and continuous exposure to visible light enables rapid and irreversible cellular destruction which can be monitored in real-time. Real-time monitoring of this structural disintegration suggests a new approach to applying curcumin in photodynamic treatments, where the progression of cell and tissue destruction might be simultaneously evaluated through optical means.

Θ-Net: A Deep Neural Network Architecture for the Resolution Enhancement of Phase-Modulated Optical Micrographs In Silico.

Optical microscopy is widely regarded to be an indispensable tool in healthcare and manufacturing quality control processes, although its inability to resolve structures separated by a lateral distance under ~200 nm has culminated in the emergence of a new field named fluorescence nanoscopy, while this too is prone to several caveats (namely phototoxicity, interference caused by exogenous probes and cost). In this regard, we present a triplet string of concatenated O-Net ('bead') architectures (termed 'Θ-Net' in the present study) as a cost-efficient and non-invasive approach to enhancing the resolution of non-fluorescent phase-modulated optical microscopical images in silico. The quality of the afore-mentioned enhanced resolution (ER) images was compared with that obtained via other popular frameworks (such as ANNA-PALM, BSRGAN and 3D RCAN), with the Θ-Net-generated ER images depicting an increased level of detail (unlike previous DNNs). In addition, the use of cross-domain (transfer) learning to enhance the capabilities of models trained on differential interference contrast (DIC) datasets [where phasic variations are not as prominently manifested as amplitude/intensity differences in the individual pixels unlike phase-contrast microscopy (PCM)] has resulted in the Θ-Net-generated images closely approximating that of the expected (ground truth) images for both the DIC and PCM datasets. This thus demonstrates the viability of our current Θ-Net architecture in attaining highly resolved images under poor signal-to-noise ratios while eliminating the need for a priori PSF and OTF information, thereby potentially impacting several engineering fronts (particularly biomedical imaging and sensing, precision engineering and optical metrology).

Andrology laboratory review: evaluation of sperm morphology

Sperm morphology assessment has an important role in male fertility diagnosis and prognosis, both for humans and animals. Thus, it is important that relevant results are comparable and consistent. To achieve these aims, the following procedures are recommended: a. semen sample is suitably ‘fixed’ (e.g. in isotonic buffered formal-saline); b. sperm are examined at 1,000 x (phase or DIC microscopy); c.at least 200 sperm are counted; d. each sperm is placed into 1 category, only (e.g. normal, head, midpiece etc), and e. 70% ‘normal’ sperm is the threshold for a satisfactory sample. In addition, morphologists should be provided with relevant continuing education, upskilling, and monitoring programs. This review provides guidelines for the best performance of this assessment, as well as for avoiding pitfalls.

229 Awardee Talk: The quest for an ideal in-field embryo evaluation tool in cattle: Opportunities and challenges

Abstract Accurately evaluating bovine embryos to select the most viable ones for transfer and successful pregnancy has been a challenge since embryo transfer began in cattle over six decades ago. Despite more than 3,100 peer-reviewed studies on bovine embryo evaluation published in PubMed from 1964 to 2023, there is still no consensus or gold standard method for assessing their developmental potential. The evaluation “problem” in assisted reproduction extends beyond embryos to gametes as well. Numerous microscopic techniques (e.g., differential interference contrast, electron, fluorescent, time-lapse, and artificial intelligence-based microscopy) and non-microscopic methodologies (e.g., genomics, transcriptomics, epigenomics, proteomics, metabolomics, and nuclear magnetic resonance) have been tested to find an embryo evaluation technique superior to morphological evaluation. While many of these research tools can accurately determine embryo quality and viability, most are invasive, expensive, labor-intensive, technically complex, and time-consuming, making them impractical for in-field embryo evaluation. Employing complex methods like RNA sequencing, SNP chips, mass spectrometry, and multiphoton microscopy at thousands of embryo production and collection facilities is unrealistic, no matter how accurate they may be. Therefore, future research should focus on innovating field-friendly, simple benchtop tests using existing findings, particularly from omics-based research methodologies. Time-lapse monitoring and artificial intelligence-based automated image analysis also potentially provide accurate embryo evaluation. However, further research is necessary to develop economically feasible options for in-field applications. Our recent work has examined possible applications of nuclear magnetic resonance (NMR) imaging, label-free microscopy, particularly holographic microscopy techniques like gradient light interference microscopy (GLIM), spatial light interference microscopy (SLIM), and multiphoton holographic microscopy in IVF labs. The ideal embryo evaluation tool should be accurate, objective, non-invasive, affordable, and simple enough for widespread adoption by embryo production and collection facilities worldwide. Under current circumstances, it is doubtful that transcriptomics, proteomics, metabolomics, GLIM, SLIM, NMR, multiphoton holographic microscopy, and similar techniques can be directly used for in-field embryo evaluation. However, biomarkers identified by these methodologies could be used for in-field embryo evaluation by devising simple, affordable benchtop techniques. Further investigation on time-lapse microscopy (TLM) and AI-based automated image processing may make these methods more affordable and potentially adoptable by the masses. There is great potential for these methodologies to become widely used, provided they can be made more economically viable. In conclusion, the next generation of microscopy capable of providing objective markers for gamete and embryo quality is on the horizon.

Examining Penetration and Residual Depth in Modern Acrylic Foldable Intraocular Lenses: A Laboratory Study Using Differential Interference Contrast Microscopy to Compare Hydrophilic and Hydrophobic Materials.

Introduction The material of modern intraocular lenses must meet the highest standards and fulfill various requirements. It is crucial that the material shows the best biocompatibility and should be flexible for an uncomplicated implantation process through small corneal incisions but also sufficiently rigid for good stability and centering in the capsular bag. In addition, the optic must remain clear for life and retain the best optical properties. Methods In this laboratory experiment, we performed scratch tests for the mechanical assessment of acrylic intraocular lenses. The aim was to determine differences in the behavior in regard to the manufacturing process and water content of hydrophilic and hydrophobic acrylic intraocular lenses. The scratch tests were performed using a Nano Scratch Tester. A conical indenter with a tip radius of 1 µm and a cone angle of 90° was selected to scratch the samples at three different constant loads of 5, 10, and 15 mN, respectively. The scratch length was set to 100 µm at a scratch speed of 200 µm/min. Hydrophilic and hydrophobic acrylic intraocular lenses (with different water content) were tested. Results The results showed that for sample A (hydrophilic acrylate), the penetration depth increases steadily with increasing force from 25-30 µm (5 mN) to 28-33 µm (10 mN) and 34-37 µm (15 mN). The penetration depths during the scratches seem to be load-dependent. In sample B (hydrophobic acrylate), the same forces lead to steadily increasing penetration depths: 25-30 µm (5 mN), 40-44 µm (10 mN), and 54-57 µm (15 mN). The evaluation of the residual depth showed much lower values for all samples. In the hydrophilic, softer samples (A), the residual depth was between 1 µm and 4 µm. In the hydrophobic, more solid, samples (B), the residual depth was more pronounced with values between 5 µm and 17 µm. The plastic influence and deformation zone seemed to be wider for the hydrophobic samples than for the hydrophilic samples. Conclusion The laboratory experiment confirms that modern, acrylic intraocular lenses are sensitive to scratches/touch, and penetration depths during scratching depend on the load. The remaining depths after the scratches are significantly lower and show a load dependence. The deforming zone was higher in the hydrophobic acrylates than in the hydrophilic acrylates. However, the results confirm that damage can occur with hydrophobic and hydrophilic acrylic materials, depending on the force applied. Therefore, careful handling during the preparation and implantation process is crucial to prevent permanent defects.

Lemon Juice and Peel Constituents Potently Stabilize Rat Peritoneal Mast Cells.

Lemons (Citrus limon ) contain various nutrients and are among the most popular citrus fruit. Besides their antioxidant, anticancer, antibacterial, and anti-inflammatory properties, clinical studies have indicated their anti-allergic properties. Using the differential-interference contrast (DIC) microscopy, we examined the effects of lemon juice and peel constituents, such as citric acid, ascorbic acid, hesperetin and eriodictyol, on the degranulation from rat peritoneal mast cells. Using fluorescence imaging with a water-soluble dye, Lucifer Yellow, we also examined their effects on the deformation of the plasma membrane. Lemon juice dose-dependently decreased the number of degranulated mast cells. At concentrations equal to or higher than 0.25 mM, citric acid, hesperetin, and eriodictyol significantly reduced the number of degranulating mast cells in a dose-dependent manner, while ascorbic acid required much higher doses to exert significant effects. At 1 mM, citric acid, hesperetin, and eriodictyol almost completely inhibited exocytosis and washed out the Lucifer Yellow trapped on the mast cell surface, while ascorbic acid did not. This study provides in vitro evidence for the first time that lemon constituents, such as citric acid, hesperetin, and eriodictyol, potently exert mast cell-stabilizing properties. These properties are attributable to their inhibitory effects on plasma membrane deformation in degranulating mast cells.

Exploring Label-Free Imaging Techniques with Copper Sulfide Microspheres for Observing Breast Cancer Cells.

A single breast cancer is a prevalent form of cancer, affecting over 2.3 million women worldwide, as reported by the World Health Organization. Recently, researchers have extensively explored the utilization of biomaterials in breast cancer theranostics. One notable biomaterial being investigated is various structures of copper sulfide (CuS). In this work, a microsphere (MS) structure composed of CuS was employed for label-free imaging of MCF-7 breast cancer cells and normal Vero cells, respectively. Various label-free imaging techniques, such as bright field, dark field, phase contrast (PC), and differential interference contrast (DIC), were employed to capture images of CuS MSs, cell, and intact CuS MSs within a cell. The study compared the outcomes of each imaging technique and determined that DIC imaging provided the highest resolution for cells incubated with CuS MSs. Furthermore, the combination of PC and DIC techniques proved to be effective for imaging breast cancer cells in conjunction with CuS MSs. This research underscores the potential of CuS MSs for label-free cell detection and emphasizes the significance of selecting appropriate imaging techniques to attain high-quality images in the field of cell observation.

Legionella colonization and 3D spatial location within a Pseudomonas biofilm

Biofilms are known to be critical for Legionella settlement in engineered water systems and are often associated with Legionnaire’s Disease events. One of the key features of biofilms is their heterogeneous three-dimensional structure which supports the establishment of microbial interactions and confers protection to microorganisms. This work addresses the impact of Legionella pneumophila colonization of a Pseudomonas fluorescens biofilm, as information about the interactions between Legionella and biofilm structures is scarce. It combines a set of meso- and microscale biofilm analyses (Optical Coherence Tomography, Episcopic Differential Interference Contrast coupled with Epifluorescence Microscopy and Confocal Laser Scanning Microscopy) with PNA-FISH labelled L. pneumophila to tackle the following questions: (a) does the biofilm structure change upon L. pneumophila biofilm colonization?; (b) what happens to L. pneumophila within the biofilm over time and (c) where is L. pneumophila preferentially located within the biofilm? Results showed that P. fluorescens structure did not significantly change upon L. pneumophila colonization, indicating the competitive advantage of the first colonizer. Imaging of PNA-labelled L. pneumophila showed that compared to standard culture recovery it colonized to a greater extent the 3-day-old P. fluorescens biofilms, presumably entering in VBNC state by the end of the experiment. L. pneumophila was mostly located in the bottom regions of the biofilm, which is consistent with the physiological requirements of both bacteria and confers enhanced Legionella protection against external aggressions. The present study provides an expedited methodological approach to address specific systematic laboratory studies concerning the interactions between L. pneumophila and biofilm structure that can provide, in the future, insights for public health Legionella management of water systems.

Mixtures of Intrinsically Disordered Neuronal Protein Tau and Anionic Liposomes Reveal Distinct Anionic Liposome-Tau Complexes Coexisting with Tau Liquid-Liquid Phase Separated Coacervates.

Tau, an intrinsically disordered neuronal protein and polyampholyte with an overall positive charge, is a microtubule (MT) associated protein, which binds to anionic domains of MTs and suppresses their dynamic instability. Aberrant tau-MT interactions are implicated in Alzheimer's and other neurodegenerative diseases. Here, we studied the interactions between full length human protein tau and other negatively charged binding substrates, as revealed by differential-interference-contrast (DIC) and fluorescence microscopy. As a binding substrate, we chose anionic liposomes (ALs) containing either 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS, -1e) or 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG, -1e) mixed with zwitterionic 1,2dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to mimic anionic plasma membranes of axons where tau resides. At low salt concentrations (0 to 10 mM KCl or NaCl) with minimal charge screening, reaction mixtures of tau and ALs resulted in the formation of distinct states of AL-tau complexes coexisting with liquid-liquid phase separated tau self-coacervates arising from the polyampholytic nature of tau containing cationic and anionic domains. AL-tau complexes exhibited distinct types of morphologies. This included, large ≈20-30 micron tau-decorated giant vesicles with additional smaller liposomes with bound tau attached to the giant vesicles, and tau-mediated finite-size assemblies of small liposomes. As the ionic strength of the solution was increased to near and above physiological salt concentrations for 1:1 electrolytes (≈150 mM), AL-tau complexes remained stable while tau self-coacervate droplets were found to dissolve indicative of breaking of (anionic/cationic) electrostatic bonds between tau chains due to increased charge screening. The findings are consistent with the hypothesis that distinct cationic domains of tau may interact with anionic lipid domains of the lumen facing monolayer of the axon plasma membrane suggesting the possibility of transient yet robust interactions at physiologically relevant ionic strengths.

Monitoring of three-dimensional live-cell cultures using a multimode, multiscale imaging system combining confocal fluorescence microscopy and optical coherence microscopy

Live-cell monitoring involves long-term observation and analysis of living cells in tissue cultures, which develop cells or tissues in an artificial environment and are essential for tissue engineering. Fluorescence microscopy (FM) is widely used as a monitoring tool owing to its powerful ability to visualize protein distribution within cells and tissues, thereby providing information on their functions in biological processes. To reduce the photobleaching effect, which is a major limiting factor in FM, FM is generally combined with other imaging modalities, such as phase contrast and differential interference contrast microscopy, which are nondestructive to fluorochromes, to selectively use fluorescence signals only in specific areas of interest within a sample. However, these methods are restricted to thin samples and cannot produce depth-resolved images. Here, we propose a method to determine three-dimensional (3D) positions in volumetric samples for application in high-resolution 3D fluorescence imaging using a multimode and multiscale imaging system that combines optical coherence microscopy (OCM) and line confocal FM (LC–FM). We also demonstrate the benefits of multimodal imaging in 3D cell culture monitoring. To evaluate the performance of the proposed system and method, we rapidly and accurately located the regions of interest in 3D tissue culture models, such as tumor spheroids and microvascular bed, and instantaneously acquired volumetric high-resolution fluorescence images. We also used an integrated system to monitor tumor spheroids mixed with extracellular matrix (ECM) over five days of the culture process, and the FM–OCM integrated technique successfully imaged the overall 3D structures, such as the distribution and boundaries of the spheroids and ECM as well as stained tumor cells. This complementary information is useful for understanding the relationship between cellular behaviors, such as the proliferation and migration of tumor cells, and microenvironments, such as the ECM.

Quantitative measurement of graphitic sp[formula omitted] on single nanodiamonds with sub-monolayer sensitivity using differential interference contrast and photo-thermal microscopy

The surface of nanodiamonds is prone to be decorated with a graphitic layer due to the thermodynamic instability of diamond versus graphite. Notably, this surface layer affects the zeta-potential of nanodiamonds controlling their colloidal stability, modulates the charging of embedded nitrogen vacancy centres used for sensing and quantum technology, and introduces an optical absorption which leads to heating and destruction of nanodiamonds under intense laser irradiation. Physical and chemical treatments to reduce the graphitic carbon are available, but a sensitive method to measure this graphitic layer on single nanodiamonds is lacking. Here, we demonstrate a non-destructive method to quantitatively determine the graphitic carbon correlatively with the nanodiamond size, on individual nanodiamonds. The method allows determining the fraction of graphitic sp2 to diamond sp3 bonded atoms, and estimating the sp2 surface layer thickness. We investigated milled nanodiamonds, both untreated and chemically treated to remove sp2. We found that untreated nanodiamonds were covered by the equivalent of a single sp2 monolayer, which was reduced to 10% of a monolayer for treated nanodiamonds. In both cases, significant variations between individual nanodiamonds were observed. The minimum number of sp2 bonded atoms detectable under the measurement conditions reported here was found to be 2×104.

Abnormal function of EPHA2/p.R957P mutant in congenital cataract.

To identify genetic defects in a Chinese family with congenital posterior polar cataracts and assess the pathogenicity. A four-generation Chinese family affected with autosomal dominant congenital cataract was recruited. Nineteen individuals took part in this study including 5 affected and 14 unaffected individuals. Sanger sequencing targeted hot-spot regions of 27 congenital cataract-causing genes for variant discovery. The pathogenicity of the variant was evaluated by the guidelines of American College of Medical Genetics and InterVar software. Confocal microscopy was applied to detect the subcellular localization of fluorescence-labeled ephrin type-A receptor 2 (EPHA2). Co-immunoprecipitation assay was implemented to estimate the interaction between EphA2 and other lens membrane proteins. The mRNA and protein expression were analyzed by reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay, respectively. The cell migration was analyzed by wound healing assay. Zebrafish model was generated by ectopic expression of human EPHA2/p.R957P mutant to demonstrate whether the mutant could cause lens opacity in vivo. A novel missense and pathogenic variant c.2870G>C was identified in the sterile alpha motif (SAM) domain of EPHA2. Functional studies demonstrated the variant's impact: reduced EPHA2 protein expression, altered subcellular localization, and disrupted interactions with other lens membrane proteins. This mutant notably enhanced human lens epithelial cell migration, and induced a central cloudy region and roughness in zebrafish lenses with ectopic expression of human EPHA2/p.R957P mutant under differential interference contrast (DIC) optics. Novel pathogenic c.2870G>C variant of EPHA2 in a Chinese congenital cataract family contributes to disease pathogenesis.

An enhanced multimode phase imaging method based on the transport of intensity equation.

Label-free biological cell imaging relies on rapid multimode phase imaging of biological samples in natural settings. To improve image contrast, phase is encoded into intensity information using the differential interference contrast (DIC) and Zernike phase contrast (ZPC) techniques. To enable multimode contrast-enhanced observation of unstained specimens, this paper proposes an improved multimode phase imaging method based on the transport of intensity equation (TIE), which combines conventional microscopy with computational imaging. The ZPC imaging module based on adaptive aperture adjustment is applied when the quantitative phase results of biological samples have been obtained by solving the TIE. Simultaneously, a rotationally symmetric shear-based technique is used that can yield isotropic DIC. In this paper, we describe numerical simulation and optical experiments carried out to validate the accuracy and viability of this technology. The calculated Michelson contrast of the ZPC image in the resolution plate experiment increased from 0.196 to 0.394.

Optical, Structural, and Synchrotron X-ray Absorption Studies for GaN Thin Films Grown on Si by Molecular Beam Epitaxy.

GaN on Si plays an important role in the integration and promotion of GaN-based wide-gap materials with Si-based integrated circuits (IC) technology. A series of GaN film materials were grown on Si (111) substrate using a unique plasma assistant molecular beam epitaxy (PA-MBE) technology and investigated using multiple characterization techniques of Nomarski microscopy (NM), high-resolution X-ray diffraction (HR-XRD), variable angular spectroscopic ellipsometry (VASE), Raman scattering, photoluminescence (PL), and synchrotron radiation (SR) near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. NM confirmed crack-free wurtzite (w-) GaN thin films in a large range of 180-1500 nm. XRD identified the w- single crystalline structure for these GaN films with the orientation along the c-axis in the normal growth direction. An optimized 700 °C growth temperature, plus other corresponding parameters, was obtained for the PA-MBE growth of GaN on Si, exhibiting strong PL emission, narrow/strong Raman phonon modes, XRD w-GaN peaks, and high crystalline perfection. VASE studies identified this set of MBE-grown GaN/Si as having very low Urbach energy of about 18 meV. UV (325 nm)-excited Raman spectra of GaN/Si samples exhibited the GaN E2(low) and E2(high) phonon modes clearly without Raman features from the Si substrate, overcoming the difficulties from visible (532 nm) Raman measurements with strong Si Raman features overwhelming the GaN signals. The combined UV excitation Raman-PL spectra revealed multiple LO phonons spread over the GaN fundamental band edge emission PL band due to the outgoing resonance effect. Calculation of the UV Raman spectra determined the carrier concentrations with excellent values. Angular-dependent NEXAFS on Ga K-edge revealed the significant anisotropy of the conduction band of w-GaN and identified the NEXAFS resonances corresponding to different final states in the hexagonal GaN films on Si. Comparative GaN material properties are investigated in depth.

Genomic and morphological characterization of Knufia obscura isolated from the Mars 2020 spacecraft assembly facility

Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.