Interference Contrast Research Articles

Giardia spp. and Cryptosporidium spp. removal efficiency of a combined fixed-film system treating domestic wastewater receiving hospital effluent.

Giardia and Cryptosporidium have caused numerous outbreaks of diarrhea as a result of the ingestion of water contaminated with sewage. In Brazil, the efficiency of Giardia and Cryptosporidium removal by combined fixed-film systems has rarely been studied. The aims of the present study were therefore to verify the removal efficiency of Giardia and Cryptosporidium by a combined system (anaerobic/anoxic filter and aerated submerged biofilter) and to perform the genetic characterization of these parasites. The (oo)cysts were detected by centrifuge concentration and membrane filtration from raw sewage, effluents, adhered biomass, and sludge samples. Immunofluorescence assay and differential interference contrast microscopy were used for the visualization of the (oo)cysts. Nested PCR was applied to confirm Giardia and Cryptosporidium. Giardia and Cryptosporidium were detected in 27% and 5.5% of the 144 analyzed samples of raw sewage and effluents, respectively. A total of 33,000cysts/L were recovered in the adhered biomass samples (n= 25) from different points of the aerated submerged biofilter, while 6000oocysts/L were registered in a single point. An average of 11,800cysts/L were found in the sludge samples (n= 5). The combined system exhibited a removal efficiency of Giardia cysts of 1.8±1.0 log removal. The C and BIV assemblages of Giardia were identified in the raw sewage while AII was found in the treated effluent sample. It was not possible to calculate the removal efficiency of Cryptosporidium oocysts by the combined system. The combined system exhibited some potential as a suitable treatment for the removal of parasites from sewage.

Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy.

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using visible-range light. The purpose of this protocol is to detail a proven method for preparing plasmonic nanoparticle samples and performing single particle spectroscopy on them with DIC microscopy. Several important steps must be followed carefully in order to perform repeatable spectroscopy experiments. First, landmarks can be etched into the sample substrate, which aids in locating the sample surface and in tracking the region of interest during experiments. Next, the substrate must be properly cleaned of debris and contaminants that can otherwise hinder or obscure examination of the sample. Once a sample is properly prepared, the optical path of the microscope must be aligned, using Kohler Illumination. With a standard Nomarski style DIC microscope, rotation of the sample may be necessary, particularly when the plasmonic nanoparticles exhibit orientation-dependent optical properties. Because DIC microscopy has two inherent orthogonal polarization fields, the wavelength-dependent DIC contrast pattern reveals the orientation of rod-shaped plasmonic nanoparticles. Finally, data acquisition and data analyses must be carefully performed. It is common to represent DIC-based spectroscopy data as a contrast value, but it is also possible to present it as intensity data. In this demonstration of DIC for single particle spectroscopy, the focus is on spherical and rod-shaped gold nanoparticles.

Near Infrared Electrochemiluminescence of Rod-Shape 25-Atom AuAg Nanoclusters That Is Hundreds-Fold Stronger Than That of Ru(bpy)3 Standard.

Near infrared (near-IR) electrogenerated chemiluminescence (ECL) from rod-shape bimetallic Au12Ag13 nanoclusters is reported. With ECL standard tris(bipyridine)ruthenium(II) complex (Ru(bpy)3) as reference, the self-annihilation ECL of the Au12Ag13 nanoclusters is about 10 times higher. The coreactant ECL of Au12Ag13 is about 400 times stronger than that of Ru(bpy)3 with 1 mM tripropylamine as coreactants. Voltammetric analysis reveals both oxidative and reductive ECLs under scanning electrode potentials. Transient ECL signals (tens of milliseconds) and decay profiles are captured by potential step experiments. An extremely strong and transient self-annihilation ECL is detected by activating LUMO and HOMO states sequentially via electrode reactions. The ECL generation pathways and mechanism are proposed based on the key anodic and cathodic activities arising from the energetics of this unique atomic-precision bimetallic nanocluster. Successes in the generation of the unprecedented strong near-IR ECL strongly support our prediction and choice of this nanocluster based on its record-high 40% quantum efficiency of near-IR photoluminescence. Correlation of the properties to the atomic/electronic structures has been a long-pursued goal particularly in the fast growing atomic-precision nanoclusters field. The mechanistic insights provided in this fundamental study could guide the design and syntheses of other nanoclusters or materials in general to achieve improved properties and further affirm the structure-function correlations. The high ECL signal in the less interfered near-infrared spectrum window offers combined merits of high-signal-low-noise/interference or high contrast for broad analytical sensing and immunoassays and other relevant applications.

Optical fiber liquid level sensor realized by cascading two spherical-shape structures

An optical fiber sensor for liquid level measurement of NaCl soultion based on Mach-Zehnder interferometer (MZI) is proposed and demonstrated. The MZI consists of two spherical-shape structures. The simulations of the light propagation of the spherical-shape structure with different diameters are performed, and it shows that the spherical-shape structure with diameter values of 200 μm can couple 50% energy of the core-mode into the cladding mode, and it is helpful to obtain best interference contrast. The liquid level sensitivity is -1.172 nm/cm for pure water, and -1.401 nm/cm for NaCl solution(n = 1.382). The sensor has advantages of easy fabrication, good robustness, and low hysteresis.

Morphological and kinetic study of oral keratinocytes assembly on reconstituted basement membrane: Effect of TEGDMA

ObjectiveOpen wounds of oral cavity require rapid healing. The cytotoxic monomer, triethylene glycol dimethacrylate (TEGDMA) can leach out from dental restoratives, reach the oral epithelial barrier and trigger an immune response. It is speculated that low and moderate concentrations of TEGDMA (0.5 and 1.5 mmol/L, respectively) influence the assembly kinetics and morphology of the keratinocyte layers overlying the extracellular matrix (ECM) in vivo. A three-dimensional cell system composed of immortalized oral keratinocytes (iMOK) cultured on reconstituted basement membrane (ECM) was used to investigate the development of epithelial layers upon exposure to TEGDMA. MethodsAdherence and opposing movement of adjacent keratinocytes using actin protrusions (lamellipodia and filopodia) to create spheroids, and their fusion capacity to establish subsequent layers were tested at different time points. Fluorescent, confocal, differential interference contrast microscopy and image processing were employed to quantify the morphological modifications over time. ResultsIncreasing concentrations of TEGDMA decreased the number of viable cells that utilized the actin protrusions and led to a delay in the communication/interaction among cells. Consequently, cells assembly was affected and the formation of more than a single layer prevented. Areas of basal-like proliferating cells were replaced with the increasing areas of non-replicating large cell population and extended gaps. ConclusionsThese findings suggest that TEGDMA may prevent rapid sealing of open wounds by keratinocytes and suppress the establishment of a resistant and impermeable barrier against pathogen internalization. The iMOK-ECM-based platform facilitated the validation and quantification of solubilized dental materials impact on the reconstitution of epithelial layer.

Erratum: Video-rate quantitative phase analysis by a DIC microscope using a polarization camera: errata.

[This corrects the article on p. 1273 in vol. 10, PMID: 30891345.].

Generalized models for advancing and receding contact angles of fakir droplets on pillared and pored surfaces

Understanding how surface properties determine the mobility of a fakir droplet on patterned surfaces, which is typically characterized by advancing and receding contact angles, is important in the design of superhydrophobic surfaces for tailored applications. However, most analytical models of the contact angles are limited to a specific motion direction and surface type, e.g., receding on a pillared surface. Although it was suggested that the contact angles should be determined by the local configuration and dynamics of the droplet boundary, their link remains vague due to the lack of detailed visualizations. In this study, the contact line dynamics of a fakir droplet in both advancing and receding motions on not only micropillared but also micropored surfaces are visualized in reflection interference contrast microscopy along with the contact angle measurement. Based on the energy change induced by the displacement of a contact line and the deformation of a liquid-gas interface in between contact lines, theoretical models of the contact angles to encompass the two different motion directions and surface types are formulated. Moreover, further generalized models are established to serve as effective analytical models to predict the contact angles only based on the given structure dimensions and the inherent surface hydrophobicity.

Interplay between Spinodal Decomposition and Gelation and Their Role in Two- and Three-Dimensional Pattern Formation at the Gelatin Gel Surface

Understanding the mechanisms underlying the spontaneous formation of Liesegang patterns based on the prenucleation and postnucleation models is of critical importance for understanding pattern formation in nature. In contrast to the rapid experimental and theoretical advances in understanding within the framework of the prenucleation model, discussion of the postnucleation model is mostly limited to numerical analysis. To construct a standard model for a chemical experiment discussed in terms of the postnucleation model, we have investigated the pattern formation mechanism in a mixed system containing gelatin, starch, and sugar. Fluorescence and differential interference contrast microscopy revealed a process of two-dimensional spinodal decomposition into gelatin-rich and gelatin-poor phases. Since the gelation temperature of the gelatin-rich phase was higher, spatially periodic gelation proceeded selectively in the gelatin-rich phase. As both two-dimensional spinodal decomposition and three-dimensional g...

Kinesin motor density and dynamics in gliding microtubule motility

Kinesin motors and their associated filaments, microtubules, are essential to many biological processes. The motor and filament system can be reconstituted in vitro with the surface-adhered motors transporting the filaments along the surface. In this format, the system has been used to study active self-assembly and to power microdevices or perform analyte detection. However, fundamental properties of the system, such as the spacing of the kinesin motors bound to the microtubule and the dynamics of binding, remain poorly understood. We show that Fluorescence Interference Contrast (FLIC) microscopy can illuminate the exact height of the microtubule, which for a sufficiently low surface density of kinesin, reveals the locations of the bound motors. We examine the spacing of the kinesin motors on the microtubules at various kinesin surface densities and compare the results with theory. FLIC reveals that the system is highly dynamic, with kinesin binding and unbinding along the length of the microtubule as it is transported along the surface.

Investigation of the 6-Folded Pattern in the Facetted Region of 4° Off-Axis 4H-SiC

We demonstrate 4o off-axis 4H-SiC bulk crystal growth using physical vapor transport method (PVT). A radial 6-folded pattern is discovered in the facetted region of the growth surface by differential interference contrast microscopy. SICA88 Wafer Inspection/Review System was used to further inspect such 6-folded pattern with photoluminescence channel for wafers cut from that 4H-SiC crystal. It was found that the 6-folded pattern exists in all the wafers from the bottom to the top of that crystal. Several radial 6-folded patterns are found in the bottom wafer near the seed, but there is only one in the other two wafers. Defects distribution of these wafers after etched were also researched to find out the relationship between the radial 6-folded pattern and the defects. Based on the results, the formation and evolution mechanism of the radial 6-folded pattern on 4o off-axis 4H-SiC crystal is discussed.

Morphological changes of poly(tetrafluoroethylene) surface due to low current density proton irradiation

The changes of the surface morphology of poly(tetrafluoroethylene) due to proton irradiation was investigated. 1 MeV proton beam was used for the irradiations with relatively low current densities, in the range of 10–100,000 pA/mm2. The applied ion fluences ranged from 0.1 to 2500 nC/mm2 (6.24 * 1010–1.56 * 1015 ion/cm2). The surface was characterized by interference contrast microscopy, surface profilometry and scanning electron microscopy. The results show that at each current density the surface can be machined, and the etching depth is increasing with increasing ion fluence, up to a certain point. However, in each case there is a threshold value, between 50 and 100 nC/mm2, where the process turns back. First, the etching depth starts to decrease. Later, by further increasing the delivered ion fluence, an opposite process begins and the surface becomes protruding.

The life cycles of two species of Myxomycetes in Physarales, Physarum rigidum and Didymium squamulosum

Myxomycetes are eukaryotic microorganisms containing characteristics akin to both fungi and amoebae. They can complete their whole life cycles while being cultured on agar media, and under-laboratory conditions, which favors taxonomic, phylogenetic, and cytological researches. Here, we describe the life cycles of two such species: Didymium squamulosum collected from the field and Physarum rigidum cultured from moist chamber both belonging to the Order Physarales. Three percent oat-agar media (OAM) was used to culture the plasmodia until they aggregated and were almost starved. Natural light was then applied to the plasmodia to induce fructification. Their life cycles share the same common stages, namely: spore, myxamoebae, swarm cell, plasmodia, and sporulation. In this study, we describe the morphogenesis from spore to spore of two species by differential interference contrast (DIC) and stereoscopic microscopies, as well as discuss the differences between the development of both species and interspecies. We found that the spore germination method of both species was the same. However, there were differences noted in time taken and fruiting body formation. Unlike P. rigidum, the species D. squamulosum did not require natural light stimulation. Moreover, the maturation process of both species had similar color transitions but exhibited distinct morphology in each developmental stage except during the swarm cell stage.

On the optimum off-cut angle for the growth on InP(111)B substrates by molecular beam epitaxy

InGaAs and InAlAs epilayers were grown on InP(111)B substrates by molecular beam epitaxy. Rather than focusing on a specific off-cut angle, the growths were done on rounded wafer edges, which expose a broad spectrum of vicinal surfaces with varying off-cut angle and off-cut azimuth. The epilayers were grown at several different growth conditions by varying the growth temperature, growth rate, and arsenic (As) overpressure. The epitaxial layers were characterized at the center and the edge of the wafers using Nomarski differential interference contrast microscopy and atomic force microscopy. It was shown that a minimum misorientation angle of ∼0.4° should be used in order to avoid pyramidal hillocks. At higher misorientations, 1.7°–3°, step bunching can lead to surface roughening.

Lung cancer diagnosis with quantitative DIC microscopy and a deep convolutional neural network.

We present a study on lung squamous cell carcinoma diagnosis using quantitative TI-DIC microscopy and a deep convolutional neural network (DCNN). The 2-D phase map of unstained tissue sections is first retrieved from through-focus differential interference contrast (DIC) images based on the transport of intensity equation (TIE). The spatially resolved optical properties are then computed from the 2-D phase map via the scattering-phase theorem. The scattering coefficient ( ) and the reduced scattering coefficient ( ) are found to increase whereas the anisotropy factor (g) is found to decrease with cancer. A DCNN classifier is developed afterwards to classify the tissue using either the DIC images or 2-D optical property maps of , and g. The DCNN classifier with the optical property maps exhibits high accuracy, significantly outperforming the same DCNN classifier on the DIC images. The label-free quantitative phase microscopy together with deep learning may emerge as a promising approach for in situ rapid cancer diagnosis.

Elastography of multicellular spheroids using 3D light microscopy.

We have demonstrated a new method of 3D elastography based on 3D light microscopy and micro-scale manipulation. We used custom-built micromanipulators to apply a mechanical force onto multicellular tumor spheroids (200-300 µm in size) and recorded the induced compression with a differential interference contrast (DIC)/confocal microscope to obtain a 4D (x, y, z, and indentation steps) image sequence. Deformation analysis made through 3D pattern tracking without using fluorescence revealed 3D structural and spatial heterogeneity in tumor spheroids. We observed a 20-30 µm-sized spot of locally-induced large deformation within a tumor spheroid. We also found solid fibroblast cores formed in a tumor-fibroblast co-culture spheroid to be stiffer than surrounding cancer cells, which would not have been discovered using only conventional fluorescence. Our new method of 3D elastography may be used to better understand structural composition in multicellular spheroids through analysis of mechanical heterogeneity.

Label-free color staining of quantitative phase images of biological cells by simulated Rheinberg illumination.

Modern microscopes are designed with functionalities that are tailored to enhance image contrast. Dark-field imaging, phase contrast, differential interference contrast, and other optical techniques enable biological cells and other phase-only objects to be visualized. Quantitative phase imaging refers to an emerging set of techniques that allow for the complex transmission function of the sample to be measured. With this quantitative phase image available, any optical technique can then be simulated; it is trivial to generate a phase contrast image or a differential interference contrast image. Rheinberg illumination, proposed almost a century ago, is an optical technique that applies color contrast to images of phase-only objects by introducing a type of optical staining via an amplitude filter placed in the illumination path that consists of two or more colors. In this paper, the complete theory of Rheinberg illumination is derived, from which an algorithm is proposed that can digitally simulate the technique. Results are shown for a number of quantitative phase images of diatom cells obtained via digital holographic microscopy. The results clearly demonstrate the potential of the technique for label-free color staining of subcellular features.

Assessing fractional hair cell survival in archival human temporal bones.

Histopathological analysis of hair cell survival in human temporal bone sections has historically been binarized such that each hair cell row is rated as either present or absent, thereby greatly underestimating the amount of hair cell loss. Here, we describe and validate a technique to reliably assess fractional hair cell survival in archival sections stained with hematoxylin and eosin (H&E) using high-resolution light microscopy and optical sectioning. Technique validation. Hair cell counts in archival temporal bone slide sets were performed by several observers using either differential interference contrast (DIC) or confocal microscopy of the endogenous eosin fluorescence in hair cells. As a further cross-check, additional decelloidinized sections were immunostained with hair cell markers myosin VI and VIIa. Cuticular plates and stereocilia bundles are routinely resolvable in DIC imaging of archival H&E-stained human material using standard research-grade microscopes, allowing highly accurate counts of fractional hair cell survival that are reproducible across observer and can be verified by confocal microscopy. Reanalysis of cases from the classic temporal bone literature on presbycusis suggests that, contrary to prior reports, differences in audiometric patterns may be well explained by the patterns of hair cell loss. NA Laryngoscope, 130:487-495, 2020.

Learn to segment single cells with deep distance estimator and deep cell detector

Single cell segmentation is a critical and challenging step in cell imaging analysis. Traditional processing methods require time and labor to manually fine-tune parameters and lack parameter transferability between different situations. Recently, deep convolutional neural networks (CNN) treat segmentation as a pixel-wise classification problem and have become a general and efficient method for image segmentation. However, cell imaging data often possesses characteristics that adversely affect segmentation accuracy: absence of established training datasets, few pixels on cell boundaries, and ubiquitous blurry features. We developed a strategy that combines strengths of CNN and traditional watershed algorithm. First, we trained a CNN to learn Euclidean distance transform (EDT) of the mask corresponding to the input images (deep distance estimator). Next, we trained a faster R-CNN (Region with CNN) to detect individual cells in the EDT image (deep cell detector). Then, the watershed algorithm performed the final segmentation using the outputs of previous two steps. Tests on a library of fluorescence, phase contrast and differential interference contrast (DIC) images showed that both the combined method and various forms of the pixel-wise classification algorithm achieved similar pixel-wise accuracy. However, the combined method achieved significantly higher cell count accuracy than the pixel-wise classification algorithm did, with the latter performing poorly when separating connected cells, especially those connected by blurry boundaries. This difference is most obvious when applied to noisy images of densely packed cells. Furthermore, both deep distance estimator and deep cell detector converge fast and are easy to train.

Propentdyopents as Heme Degradation Intermediates Constrict Mouse Cerebral Arterioles and Are Present in the Cerebrospinal Fluid of Patients With Subarachnoid Hemorrhage.

Delayed ischemic neurological deficit is the most common cause of neurological impairment and unfavorable prognosis in patients with subarachnoid hemorrhage (SAH). Despite the existence of neuroimaging modalities that depict the onset of the accompanying cerebral vasospasm, preventive and therapeutic options are limited and fail to improve outcome owing to an insufficient pathomechanistic understanding of the delayed perfusion deficit. Previous studies have suggested that BOXes (bilirubin oxidation end products), originating from released heme surrounding ruptured blood vessels, are involved in arterial vasoconstriction. Recently, isolated intermediates of oxidative bilirubin degradation, known as PDPs (propentdyopents), have been considered as potential additional effectors in the development of arterial vasoconstriction. To investigate whether PDPs and BOXes are present in hemorrhagic cerebrospinal fluid and involved in the vasoconstriction of cerebral arterioles. Via liquid chromatography/mass spectrometry, we measured increased PDP and BOX concentrations in cerebrospinal fluid of SAH patients compared with control subjects. Using differential interference contrast microscopy, we analyzed the vasoactivity of PDP isomers in vitro by monitoring the arteriolar diameter in mouse acute brain slices. We found an arteriolar constriction on application of PDPs in the concentration range that occurs in the cerebrospinal fluid of patients with SAH. By imaging arteriolar diameter changes using 2-photon microscopy in vivo, we demonstrated a short-onset vasoconstriction after intrathecal injection of either PDPs or BOXes. Using magnetic resonance imaging, we observed a long-term PDP-induced delay in cerebral perfusion. For all conditions, the arteriolar narrowing was dependent on functional big conductance potassium channels and was absent in big conductance potassium channels knockout mice. For the first time, we have quantified significantly higher concentrations of PDP and BOX isomers in the cerebrospinal fluid of patients with SAH compared to controls. The vasoconstrictive effect caused by PDPs in vitro and in vivo suggests a hitherto unrecognized pathway contributing to the pathogenesis of delayed ischemic deficit in patients with SAH.

Radial profile detection of multiple spherical particles in contact with interacting surfaces.

Adhesive interactions of soft materials play an important role in nature and technology. Interaction energies can be quantified by determining contact areas of deformable microparticles with the help of reflection interference contrast microscopy (RICM). For high throughput screening of adhesive interactions, a method to automatically evaluate large amounts of interacting microparticles was developed. An image is taken which contains circular interference patterns with visual characteristics that depend on the probe’s shape due to its surface interaction. We propose to automatically detect radial profiles in images, and to measure the contact radius and size of the spherical probe, allowing the determination of particle-surface interaction energy in a simple and fast imaging and image analysis setup. To achieve this, we analyze the image gradient and we perform template matching that utilizes the physical foundations of reflection interference contrast microscopy.