Bright-field Microscopy Research Articles

Identification of significant imaging features for sensing oocyte viability.

The evaluation of oocyte viability in the laboratory is limited to the morphological assessment by naked eyes, but the realization that most normal-appearing oocytes may conceal abnormalities prompts the search for automated approaches that can detect the abnormalities imperceptible to naked eyes. In this study, we developed an image processing pipeline applicable to bright-field microscope images to quantify the causal relationship between the quantitative imaging features and the developmental potential of oocytes. We acquired 19 imaging features of approximately 700 oocytes and determined two imaging subtypes, namely viable and nonviable subtypes that correlated closely with a viability fluorescence indicator and cleavage rates. The causal relationship between these imaging features and oocyte viability was derived from a viability-oriented Bayesian network that was developed based on the Bayesian information criterion and Tabu search. Our experimental results revealed that entropy with mean Gray Level Co-Occurrence Matrix energy describing the uniformity and texture roughness of cytoplasm were salient features for the automated selection of promising oocytes that exhibited excellent developmental potential.

Low-energy scanning transmission electron microscopy applied to ice-embedded biological macromolecules.

In this report, we applied annular bright-field and annular dark-field low-energy (30 keV) scanning transmission electron microscopy imaging to a vitreous ice-embedded biological macromolecule, T4 phage, to investigate the applicability of these methods for morphological investigation and sample screening. Multiple camera lengths were examined to find the optimal acceptance angle for both modes. Image clarity differed substantially between the modes, with the presence of ice also strongly influencing the quality of acquired micrographs. In annular dark-field mode, the proper discrimination of electrons scattered by the specimen from those scattered by the background ice was found to be difficult due to the severe overlap of the scattered electrons. The resulting micrographs lacked clarity, and the ice-embedded phage particles could only be discerned after post-processing image adjustment. However, in annular bright-field mode, despite similar overlapping of the scattered electrons, it was possible to assess the morphology and intactness of the specimen in the embedding ice, suggesting that this mode may find utility in low-energy cryo-scanning transmission electron microscopy imaging methods.

Phase Separation of Rubisco by the Folded SSUL Domains of CcmM in Beta-Carboxysome Biogenesis.

Carboxysomes are large, cytosolic bodies present in all cyanobacteria and many proteobacteria that function as the sites of photosynthetic CO2 fixation by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The carboxysome lumen is enriched with Rubisco and carbonic anhydrase (CA). The polyhedral proteinaceous shell allows the passage of HCO3- ions into the carboxysome, where they are converted to CO2 by CA. Thus, the carboxysome functions as a CO2-concentrating mechanism (CCM), enhancing the efficiency of Rubisco in CO2 fixation. In β-cyanobacteria, carboxysome biogenesis first involves the aggregation of Rubisco by CcmM, a scaffolding protein that exists in two isoforms. Both isoforms contain a minimum of three Rubisco small subunit-like (SSUL) domains, connected by flexible linkers. Multivalent interaction between these linked SSUL domains with Rubisco results in phase separation and condensate formation. Here, we use Rubisco and the short isoform of CcmM (M35) of the β-cyanobacterium Synechococcus elongatus PCC7942 to describe the methods used for in vitro analysis of the mechanism of condensate formation driven by the SSUL domains. The methods include turbidity assays, bright-field and fluorescence microscopy, as well as transmission electron microscopy (TEM) in both negative staining and cryo-conditions.

Quantitative rheo-microscopy of soft matter

Direct observation of the microscopic material structure and dynamics during rheological shear tests is the goal of rheo-microscopy experiments. Microscopically, they shed light on the many mechanisms and processes that determine the mechanical properties at the macroscopic scale. Moreover, they permit for the determination of the actual deformation field, which is particularly relevant to assess shear banding or wall slip. While microscopic observation of the sample during mechanical probing is achieved by a variety of custom and commercial instruments, the possibility of performing quantitative rheology is not commonly available. Here, we describe a flexible rheo-microscopy setup that is built around a parallel-sliding-plate, stress-controlled shear cell, optimized to be mounted horizontally on a commercial microscope. Mechanically, soft materials with moduli ranging from few tens of Pa up to tens of kPa can be subjected to a variety of waveforms, ranging from standard step stress and oscillatory stress to more peculiar signals, such as triangular waves or any other signal of interest. Optically, the shear cell is designed to be compatible with different imaging methods (e.g. bright field or confocal microscopy). Most of the components of the shear cell are commercially available, and those that are not can be reproduced by a standard machine shop, easing the implementation of the rheo-microscopy setup in interested laboratories.

Photonic Spin-Hall Differential Microscopy

The visualization of objects such as transparent living cells and tissues plays an important role in biological research. However, weak scattering and absorption of ``phase objects'' make it challenging to obtain high-contrast images, and phase-contrast techniques are needed. This study proposes differential microscopy based on the photonic spin Hall effect at a simple glass interface. The combination of the photonic spin Hall effect and a bright-field microscope can perform spatial differentiation on the phase distribution, which leads to a low-cost differential interference contrast (DIC) system.

Dynamic dentin: A quantitative microscopic assessment of age and spatial changes to matrix architecture, peritubular dentin, and collagens types I and III

To investigate age and site-related changes to human dentin collagen, sound human teeth collected from donors aged 13–29 (young) and 50–74 (aged) years (n = 9/group) were cut to shallow and deep sites. Dentin collagen orientation and fibril bundling was investigated using the Picrosirius Red (PSR) stain observed under cross-polarized light microscopy (Pol), and collagen distribution was investigated using Confocal Laser Scanning Microscopy (CLSM). Collagen types III to I distribution in peritubular dentin (PTD) was revealed using Herovici stain and brightfield microscopy. Image analysis software and linear mixed modelling quantified outcomes. In situ dentin collagen was observed using Xenon Plasma Focussed Ion Beam Scanning Electron Microscopy (Xe PFIB-SEM). The PSR-Pol analysis revealed less coherently aligned and more bundled collagen fibrils in aged dentin (P = 0.005). Deep inner dentin collagen in both groups were less coherently aligned with reduced bundling. Regardless of age, CLSM showed collagen distribution remained stable; and more collagen type III was detectable in PTD located in inner dentin (Young: P = 0.006; Aged: P = 0.008). Observations following Xe PFIB-SEM cross-sectioning showed apatite-like deposits surrounding large intratubular collagen fibers, and evidence of smaller intertubular dentin collagen fibrils in situ. In conclusion, aging changes collagen network architecture, but not distribution or content.

Absolute Counting Method with Multiplexing Capability for Estimating the Number Concentration of Nanoparticles Using Anisotropically Collapsed Gels.

Number concentration─the number of nanoparticles in a given volume─is an important characteristic of any nanoparticle dispersion. However, its estimation for small nanoparticles (∼30 nm) is generally challenging. We introduce an absolute and widely applicable method for analyzing aqueous dispersions of nanoparticles. An innovative immobilization of nanomaterials in the anisotropically collapsed agarose gel is pioneered, followed by optical microscopy and nanoparticle counting. The number of counted nanoparticles is inherently coupled with sampled volume (517 pL) and translates to the number concentration. Photon-upconversion, fluorescence, bright-field, and dark-field microscopy techniques have been proven applicable and used for imaging lanthanide-doped photon-upconversion nanoparticles, their bioconjugates with antibodies, silica dye-doped fluorescent nanoparticles, quantum dots, and pure silica submicron particles. The precision and linearity were characterized by constructing a dilution series of photon-upconversion nanoparticles. The limit of detection was 2.0 × 106 mL-1, and the working range was from 4.4 × 107 to 2.2 × 1010 mL-1. The quantification of nanoparticle clusters was achieved by a thorough analysis of the micrographs. The accuracy was confirmed using gravimetric analysis and transmission electron microscopy as a reference. Multiplexed detection of two nanoparticle types in a mixed dispersion was feasibly demonstrated. The low thickness of the collapsed gel (<1 μm) supported extremely sensitive imaging. This was proven by imaging Tm3+-doped photon-upconversion nanoparticles (17 nm hydrodynamic diameter) with a nanoparticle emission rate of only ∼900 photons/s at a wavelength of 800 nm (excitation wavelength 976 nm).

Unravelling mechanisms of protein and lipid oxidation in mayonnaise at multiple length scales

In mayonnaise, lipid and protein oxidation are closely related and the interplay between them is critical for understanding the chemical shelf-life stability of mayonnaise. This is in particular the case for comprehending the role of low-density lipoprotein (LDL) particles acting as a main emulsifier. Here, we monitored oxidation and the concomitant aggregation of LDLs by bright-field light microscopy and cryogenic transmission electron microscopy. We further probed the formation of protein radicals and protein oxidation by imaging the accumulation of a water-soluble fluorescent spin trap and protein autofluorescence. The effect of variation of pH and addition of EDTA on the accumulation of the spin trap validated that protein radicals were induced by lipid radicals. Our data suggests two main pathways of oxidative protein radical formation in LDL particles: (1) at the droplet interface, induced by lipid free radicals formed in oil droplets, and (2) in the continuous phase induced by an independent LDL-specific mechanism.

Fast Noninvasive Morphometric Characterization of Free Human Sperms Using Deep Learning.

The selection of high-quality sperms is critical to intracytoplasmic sperm injection, which accounts for 70–80% of in vitro fertilization (IVF) treatments. So far, sperm screening is usually performed manually by clinicians. However, the performance of manual screening is limited in its objectivity, consistency, and efficiency. To overcome these limitations, we have developed a fast and noninvasive three-stage method to characterize morphology of freely swimming human sperms in bright-field microscopy images using deep learning models. Specifically, we use an object detection model to identify sperm heads, a classification model to select in-focus images, and a segmentation model to extract geometry of sperm heads and vacuoles. The models achieve an F1-score of 0.951 in sperm head detection, a z-position estimation error within ±1.5 μm in in-focus image selection, and a Dice score of 0.948 in sperm head segmentation, respectively. Customized lightweight architectures are used for the models to achieve real-time analysis of 200 frames per second. Comprehensive morphological parameters are calculated from sperm head geometry extracted by image segmentation. Overall, our method provides a reliable and efficient tool to assist clinicians in selecting high-quality sperms for successful IVF. It also demonstrates the effectiveness of deep learning in real-time analysis of live bright-field microscopy images.

Spatio-temporal segmentation of image sequences for non-invasive analysis of cardiovascular structure and function in Whitefish embryos

Label-free quantitative analysis of the cardiovascular structure and function in wild fish is highly important and still unsolved problem. We developed a robust algorithm for bright field microscopy data processing, which allows reliable detection and quantification of circulatory system across the whole animal. We verified this technique by in vivo experiments on broad whitefish (Coregonus nasus) embryos. Calculated morphofunctional features of heart and vessels are quite specific due to low temperature embryonic development of this fish, and correspond well to the reference data obtained by ex vivo techniques. This research highlights the feasibility of non-invasive cardiovascular imaging and measurements valuable for behavioral, environmental, toxicological, drug discovery and many other studies.

Hybrid brightfield and darkfield transport of intensity approach for high-throughput quantitative phase microscopy

Transport of intensity equation (TIE) is a well-established non-interferometric phase retrieval approach that enables quantitative phase imaging (QPI) by simply measuring intensity images at multiple axially displaced planes. The advantage of a TIE-based QPI system is its compatibility with partially coherent illumination, which provides speckle-free imaging with resolution beyond the coherent diffraction limit. However, TIE is generally implemented with a brightfield (BF) configuration, and the maximum achievable imaging resolution is still limited to the incoherent diffraction limit (twice the coherent diffraction limit). It is desirable that TIE-related approaches can surpass this limit and achieve high-throughput [high-resolution and wide field of view (FOV)] QPI. We propose a hybrid BF and darkfield transport of intensity (HBDTI) approach for high-throughput quantitative phase microscopy. Two through-focus intensity stacks corresponding to BF and darkfield illuminations are acquired through a low-numerical-aperture (NA) objective lens. The high-resolution and large-FOV complex amplitude (both quantitative absorption and phase distributions) can then be synthesized based on an iterative phase retrieval algorithm taking the coherence model decomposition into account. The effectiveness of the proposed method is experimentally verified by the retrieval of the USAF resolution target and different types of biological cells. The experimental results demonstrate that the half-width imaging resolution can be improved from 1230 nm to 488 nm with 2.5 × expansion across a 4 × FOV of 7.19 mm2, corresponding to a 6.25 × increase in space-bandwidth product from ∼5 to ∼30.2 megapixels. In contrast to conventional TIE-based QPI methods where only BF illumination is used, the synthetic aperture process of HBDTI further incorporates darkfield illuminations to expand the accessible object frequency, thereby significantly extending the maximum available resolution from 2NA to ∼5NA with a ∼5 × promotion of the coherent diffraction limit. Given its capability for high-throughput QPI, the proposed HBDTI approach is expected to be adopted in biomedical fields, such as personalized genomics and cancer diagnostics.

Alcian Blue Staining to Visualize Intracellular Hyaluronic Acid-Based Nanoparticles.

Investigating at transmission electron microscopy the intracellular trafficking of hyaluronic acid-based nanoparticles remains a challenge due to their intrinsic weak electron density. Here we describe a simple protocol to stain hyaluronic acid that allows visualization of hyaluronic acid-based nanoparticles inside cells at both light and electron microscopy. By applying the critical-electrolyte-concentration Alcian blue method, these nanoparticles were observed as blue dots at bright-field microscopy or filled with fine electron dense precipitates at transmission electron microscopy.

Large (anti)ferrodistortive NaNbO3-based lead-free relaxors: Polar nanoregions embedded in ordered oxygen octahedral tilt matrix

Relaxor ferroelectric perovskites have experienced a revival of interest in recent years accompanying the deeply exploration of the fine local structure of polar nanoregions, which are thought to be the structure root for unique electrical properties. Interestingly, a new type of low-tolerance relaxor ferroelectric, which exhibits both cation displacement and large oxygen octahedral tilt, exhibiting anomalous microdomains were found in NaNbO3-based lead-free perovskites, breaking through the limits of the relaxor ferroelectric models proposed from classical relaxors. Atomic-scale annular bright-field scanning transmission electron microscopy images indicate that the microdomains are formed by long-range ordered oxygen octahedral tilt. At the same time, substructure of randomly embedded nanoregions with inhomogeneous polarization vectors can be mapped, being the main reason for relaxor behavior. Based on this special ferroelectric-improper ferroelastic state, delayed polarization saturation can be obtained under strong electric field, providing an opportunity for generating excellent energy storage properties. The results found in this work not only open up a new kind of relaxor ferroelectrics but also will lead to the reconstruction of relaxor models and theories built for more than half a century.

Ozone at low concentration modulates microglial activity in vitro: A multimodal microscopy and biomolecular study.

Oxygen-ozone (O2 -O3 ) therapy is an adjuvant/complementary treatment based on the activation of antioxidant and cytoprotective pathways driven by the nuclear factor erythroid 2-related factor 2 (Nrf2). Many drugs, including dimethyl fumarate (DMF), that are used to reduce inflammation in oxidative-stress-related neurodegenerative diseases, act through the Nrf2-pathway. The scope of the present investigation was to get a deeper insight into the mechanisms responsible for the beneficial result of O2 -O3 treatment in some neurodegenerative diseases. To do this, we used an integrated approach of multimodal microscopy (bright-field and fluorescence microscopy, transmission and scanning electron microscopy) and biomolecular techniques to investigate the effects of the low O3 concentrations currently used in clinical practice in lipopolysaccharide (LPS)-activated microglial cells human microglial clone 3 (HMC3) and in DMF-treated LPS-activated (LPS + DMF) HMC3 cells. The results at light and electron microscopy showed that LPS-activation induced morphological modifications of HMC3 cells from elongated/branched to larger roundish shape, cytoplasmic accumulation of lipid droplets, decreased electron density of the cytoplasm and mitochondria, decreased amount of Nrf2 and increased migration rate, while biomolecular data demonstrated that Heme oxygenase 1 gene expression and the secretion of the pro-inflammatory cytokines, Interleukin-6, and tumor necrosis factor-α augmented. O3 treatment did not affect cell viability, proliferation, and morphological features of both LPS-activated and LPS + DMF cells, whereas the cell motility and the secretion of pro-inflammatory cytokines were significantly decreased. This evidence suggests that modulation of microglia activity may contribute to the beneficial effects of the O2 -O3 therapy in patients with neurodegenerative disorders characterized by chronic inflammation. HIGHLIGHTS: Low-dose ozone (O3 ) does not damage activated microglial cells in vitro Low-dose O3 decreases cell motility and pro-inflammatory cytokine secretion in activated microglial cells in vitro Low-dose O3 potentiates the effect of an anti-inflammatory drug on activated microglial cells.

Anatomical and histochemical structures of Cyperus rotundus L. facilitate success in amphibious environments

The perennial Cyperus rotundus (Cyperaceae) is a noxious weed worldwide that tolerates the extreme environments of the Three Gorges Reservoir, China. Using brightfield and epifluorescence microscopy, we investigated the anatomical and histochemical features that allow this species to thrive in amphibious environments. The apoplastic barriers of C. rotundus consisted of an endodermis, a uniseriate exodermis, and a cuticle in the roots, rhizomes, and culms, respectively, as well as suberized stele and primary xylem poles. These barriers may help C. rotundus to retain oxygen and control water-solute exchanges under anoxic conditions. The mechanical tissues of C. rotundus included lignified sclerenchyma rings and sclerenchyma fibers in the roots, shoots, rhizomes, and tubers. The air spaces had lysed aerenchyma in the roots, leaves, and rhizomes, while schizogenous aerenchyma was observed in the culms. Neither lysed nor schizogenous aerenchyma was found in the tubers. C. rotundus is a typical C4 species with Kranz anatomy; chloroplasts were present in the inner sheaths and parenchymatous sheaths. Thus, our analyses identified several anatomical and histochemical features that may facilitate the adaptive success of C. rotundus in amphibious environments.

Benzimidazole-linked pyrazolo[1,5-a]pyrimidine conjugates: synthesis and detail evaluation as potential anticancer agents.

A library of benzimidazole briged pyrazolo[1,5-a]pyrimidine (6a-q) was designed, synthesized and subjected for evaluation for cytotoxic potential. Antiproliferative activity, ranging from 3.1-51.5μM, was observed against a panel of cancer cell lines which included MCF-7 (breast cancer), A549 (lung cancer), HeLa (cervical cancer) and SiHa (cervical cancer). Among them, 6k, 6l, 6n and 6o have shown significant cytotoxicity and were investigated further to study their probable mechanism of action against MCF-7 cell line. Accumulation of cells at sub-G1 phase was observed in flow cytometric analysis. The detachment of cells from substratum and membrane blebbing seen under bright field microscopy supports the ability of these conjugates to induce apoptosis. Immunostaining and western blot analysis showed EGFR, p-EGFR, STAT3, and p-STAT3 significant downregulation. Western blot analysis demonstrated an elevated level of apoptotic proteins such as p53, p21, Bax, whereas a decrease in the antiapoptotic protein Bcl-2 and procaspase-9, confirming the ability of these conjugates to trigger cell death by apoptosis. EGFR kinase assay confirms the specific activity of conjugates. Molecular docking simulation study disclosed that these molecules fit well in ATP-binding pocket of EGFR. The analysis of docking poses and the atomic interactions of different conjugates rationalize the structural-activity relationship in this series. Benzimidazole-linked pyrazolo[1,5-a]pyrimidine conjugates were synthesized and evaluated for their anticancer potential. All the conjugates have significant anticancer potential. Further mechanistic studies revealed that these conjugates arrest cancer cell growth by EGFR/STAT3 inhibition.

An Attempt to Relate Oleogel Properties to Wax Ester Chemical Structures.

Wax esters are considered to have a dominant contribution in the gelling properties of wax-based oleogels. To understand their gelling behavior, oleogels of seven different wax esters (total carbon number from 30 to 46; c = 10% [m/m]) in medium-chain triglycerides oil were characterized. Scanning electron microscopy revealed that wax esters crystallize in rhombic platelets with a thickness of 80 to 115 monomolecular layers. Bright field microscopy showed that the regularity and face length of the crystals increased with the total carbon number and molecular symmetry of the respective wax ester. Oscillatory rheology was used to characterize the gel rigidity (). Here, wax ester oleogels with smaller total carbon numbers yielded higher values than those of wax esters with higher total carbon numbers. The gel rigidity () inversely correlated with the crystal face length. Smaller and optically less well-defined platelets promoted higher gel rigidities. In the case of the microstructure of a specific oleogel composition being manipulated by a variation in the cooling rates (0.8; 5; 10 K/min), this relationship persisted. The information compiled in this manuscript further elucidates the crystallization behavior of wax esters in oleogels. This contributes to the understanding of the composition–structure–functionality relationship of wax-based oleogels supporting future food applications.

Atorvastatin attenuates NS1 (Non-structural protein-1) of dengue type-2 serotype-induced expressions of matrix metalloproteinases in HL-60 cells, differentiated to neutrophils: Implications for the immunopathogenesis of dengue viral disease

BackgroundThe dengue is a vector borne viral infection in humans. Bite of mosquito infected with a dengue virus transmits the disease. The neutrophils support more to the innate immune response by switching to infected tissues and triggering immunomodulatory mechanisms including the release of proteases and host defence peptides. MethodsCell viability by MTT and trypan blue dye exclusion assay, bright field microscopy for assessment of cell morphology, cytokines measurements by ELISA, estimation of protein by Bradford assay were done. Assessments of matrix metalloproteinase genes mRNA expressions were done using real-time PCR. ResultsIn the present study, we have for the first time unveiled that, NS1 antigen of dengue type-2 serotype, induce and stimulate the neutrophils cells to express high levels of matrix metalloproteases. NS1 exposure of HL-60 cells differentiated to neutrophils affected cell morphology and in 24 h of exposure. We have demonstrated that, the NS1 antigen has induced MMP-2, MMP-14 and MMP-9 expressions in neutrophils in a 24hrs exposure time. NS1 exposure has also further upregulated MMP-1, MMP-13, and MMP-8 expressions in neutrophils in a 24hrs exposure time. Notably, treatment with atorvastatin concentrations downregulated the expression profile of the all matrix metalloprotease significantly. Importantly, NS1 antigen has significantly increased the IL-6, IL-13 release by the HL,60 cells which was reversed by atorvastatin. On the other hand, NS1 exposure enhanced the mRNA expressions of VEGF-A and VEGF-D which was reversed by atorvastatin. However, we found that, NS1 exposure reduced the mRNA expressions profile of VEGF-C, which was reversed by atorvastatin. ConclusionIn conclusion, we report that, neutrophils associated matrix metalloprotease are involved in the pathogenesis of dengue viral disease. VEGF growth factors may also be released by the neutrophils which may subsequently participate in the endothelial dysfunctions leading to dengue shock syndrome.

A Recent Systematic Review of Cervical Cancer Diagnosis: Detection and Classification

Women around the world are frequently diagnosed with cervical cancer. In the beginning, there were no symptoms for the fourth most common cause of fatality in women. Cells of cervical cancer develop gradually at the cervix. Several studies have mentioned that the initial detection of cervical tumours is essential for cancer to be properly treated and to make sure cancer can be successfully treated while minimizing deaths due to cervical cancer. The diagnosis of such cancer before it spreads fast is currently a pressing issue for healthcare professionals. This also provides an extensive understanding with respect to the physical characteristics of the healthy and unhealthy cervix and aids in early treatment planning by giving detailed information about one another. Utilizing image segmentation, several techniques are employed to find malignancy. The dataset contains four distinct pathological pictures, including normal, malignancy, and high-grade squamous intraepithelial lesions (HSIL). While pap tests are the most popular way to diagnose cervical cancer, their accuracy depends a lot on how well cytotechnicians can use brightfield microscopy to spot abnormal cells on smears.

Origin of spurious intensity in vacuum near sample edge in bright field TEM images

Bright-field transmission electron microscope (BFTEM) images exhibit spurious image intensity in the vacuum near the sample edge. The spurious intensity gradually decreases with increasing distance from the sample edge. By taking into account angular and energy loss distribution of the scattered electrons and lens aberrations, the origin of the spurious intensity of BFTEM images can be explained. The spurious intensity extent and magnitude can be significantly reduced by using either electron energy filtering or a small collection semiangle.