Differential Interference Contrast Microscopy Images Research Articles

A deep learning-based pipeline for analyzing the influences of interfacial mechanochemical microenvironments on spheroid invasion using differential interference contrast microscopic images

Metastasis is the leading cause of cancer-related deaths. During this process, cancer cells are likely to navigate discrete tissue-tissue interfaces, enabling them to infiltrate and spread throughout the body. Three-dimensional (3D) spheroid modeling is receiving more attention due to its strengths in studying the invasive behavior of metastatic cancer cells. While microscopy is a conventional approach for investigating 3D invasion, post-invasion image analysis, which is a time-consuming process, remains a significant challenge for researchers. In this study, we presented an image processing pipeline that utilized a deep learning (DL) solution, with an encoder-decoder architecture, to assess and characterize the invasion dynamics of tumor spheroids. The developed models, equipped with feature extraction and measurement capabilities, could be successfully utilized for the automated segmentation of the invasive protrusions as well as the core region of spheroids situated within interfacial microenvironments with distinct mechanochemical factors. Our findings suggest that a combination of the spheroid culture and DL-based image analysis enable identification of time-lapse migratory patterns for tumor spheroids above matrix-substrate interfaces, thus paving the foundation for delineating the mechanism of local invasion during cancer metastasis.

Statistical image processing quantifies the changes in cytoplasmic texture associated with aging in Caenorhabditis elegans oocytes

BackgroundOocyte quality decreases with aging, thereby increasing errors in fertilization, chromosome segregation, and embryonic cleavage. Oocyte appearance also changes with aging, suggesting a functional relationship between oocyte quality and appearance. However, no methods are available to objectively quantify age-associated changes in oocyte appearance.ResultsWe show that statistical image processing of Nomarski differential interference contrast microscopy images can be used to quantify age-associated changes in oocyte appearance in the nematode Caenorhabditis elegans. Max–min value (mean difference between the maximum and minimum intensities within each moving window) quantitatively characterized the difference in oocyte cytoplasmic texture between 1- and 3-day-old adults (Day 1 and Day 3 oocytes, respectively). With an appropriate parameter set, the gray level co-occurrence matrix (GLCM)-based texture feature Correlation (COR) more sensitively characterized this difference than the Max–min Value. Manipulating the smoothness of and/or adding irregular structures to the cytoplasmic texture of Day 1 oocyte images reproduced the difference in Max–min Value but not in COR between Day 1 and Day 3 oocytes. Increasing the size of granules in synthetic images recapitulated the age-associated changes in COR. Manual measurements validated that the cytoplasmic granules in oocytes become larger with aging.ConclusionsThe Max–min value and COR objectively quantify age-related changes in C. elegans oocyte in Nomarski DIC microscopy images. Our methods provide new opportunities for understanding the mechanism underlying oocyte aging.

Transcriptomic and Morphological Analysis of Cells Derived from Porcine Buccal Mucosa-Studies on an In Vitro Model.

Simple SummaryDomestic pigs express high phylogenetic similarity to humans and are often used as a compatible model in biomedical research. Porcine tissues are used as an accessible biomaterial in human skin transplants and tissue architecture reconstruction. We used transcriptional analysis to investigate the dynamics of complex biological system of the mucosa. Additionally, we performed computer analysis of microscopic images of cultured cells in vitro. Computer analysis of images identified epithelial cells and connective tissue cells in in vitro culture.Transcriptional analysis and live-cell imaging are a powerful tool to investigate the dynamics of complex biological systems. In vitro expanded porcine oral mucosal cells, consisting of populations of epithelial and connective lineages, are interesting and complex systems for study via microarray transcriptomic assays to analyze gene expression profile. The transcriptomic analysis included 56 ontological groups with particular focus on 7 gene ontology groups that are related to the processes of differentiation and development. Most analyzed genes were upregulated after 7 days and downregulated after 15 and 30 days of in vitro culture. The performed transcriptomic analysis was then extended to include automated analysis of differential interference contrast microscopy (DIC) images obtained during in vitro culture. The analysis of DIC imaging allowed to identify the different populations of keratinocytes and fibroblasts during seven days of in vitro culture, and it was possible to evaluate the proportion of these two populations of cells. Porcine mucosa may be a suitable model for reference research on human tissues. In addition, it can provide a reference point for research on the use of cells, scaffolds, or tissues derived from transgenic animals for applications in human tissues reconstruction.

Self-compensating quantitative differential interference contrast microscopy imaging system using liquid crystal shearing assembly

A quantitative differential interference contrast microscopy (DICM) using a liquid crystal shearing assembly (LCSA) is proposed and demonstrated experimentally. Differential interference contrast (DIC) imaging with arbitrary shearing amount and different shearing direction can be implemented by changing the driving voltage applied to the liquid crystal without mechanical devices. Moreover, by combining a quarter-wave plate and a mirror, not only the shearing amount is enlarged, but also the aberration and the additional phase shift induced by the refractive index difference between ordinary and extraordinary light can be compensated. Using the proposed LCSA/DICM system, we implement DIC imaging of a polystyrene microsphere crown and the quantitative differential phase measurement of an oral epithelial cell.

Two New Species of Sucking Lice (Phthiraptera: Anoplura: Hoplopleuridae and Polyplacidae) from Grant's Rock Mouse, Micaelamys granti, in South Africa.

Two new species of sucking lice (Phthiraptera: Anoplura), Hoplopleura granti n. sp. (Hoplopleuridae) and Polyplax megacephalus n. sp. (Polyplacidae), are described from Grant's rock mouse, Micaelamys granti (Wroughton), from Northern Cape Province, South Africa. Adults of both sexes are illustrated via line drawings and differential interference contrast microscopy images, and are compared with previously described related species that parasitize related hosts: Hoplopleura patersoni Johnson from Aethomys chrysophilus (de Winton) (red veld rat), Aethomys kaiseri (Noack) (Kaiser's aethomys), and Micaelamys namaquensis (A. Smith) (Namaqua rock mouse); Hoplopleura aethomydis Kleynhans from M. namaquensis; Polyplax praomydis Bedford from A. chrysophilus and M. namaquensis; and Polyplax solivaga Johnson from A. chrysophilus. It is not known if these new species of lice are vectors of any pathogens.

Near-infrared scanning cavity ringdown for optical loss characterization of supermirrors.

A cavity ringdown system for probing the spatial variation of optical loss across high-reflectivity mirrors is described. This system is employed to examine substrate-transferred crystalline supermirrors and to quantify the effect of manufacturing process imperfections. Excellent agreement is observed between the ringdown-generated spatial measurements and differential interference contrast microscopy images. A 2-mm diameter ringdown scan in the center of a crystalline supermirror reveals highly uniform coating properties with excess loss variations below 1 ppm.

Cell Membrane Tracking in Living Brain Tissue Using Differential Interference Contrast Microscopy.

Differential interference contrast (DIC) microscopy is widely used for observing unstained biological samples that are otherwise optically transparent. Combining this optical technique with machine vision could enable the automation of many life science experiments; however, identifying relevant features under DIC is challenging. In particular, precise tracking of cell boundaries in a thick ( ) slice of tissue has not previously been accomplished. We present a novel deconvolution algorithm that achieves the state-of-the-art performance at identifying and tracking these membrane locations. Our proposed algorithm is formulated as a regularized least squares optimization that incorporates a filtering mechanism to handle organic tissue interference and a robust edge-sparsity regularizer that integrates dynamic edge tracking capabilities. As a secondary contribution, this paper also describes new community infrastructure in the form of a MATLAB toolbox for accurately simulating DIC microscopy images of in vitro brain slices. Building on existing DIC optics modeling, our simulation framework additionally contributes an accurate representation of interference from organic tissue, neuronal cell-shapes, and tissue motion due to the action of the pipette. This simulator allows us to better understand the image statistics (to improve algorithms), as well as quantitatively test cell segmentation and tracking algorithms in scenarios, where ground truth data is fully known.

Differential interference contrast microscopy imaging of single gold nanospheres beyond the quasi-static limit

Herein, we employed single particle dark-field (DF) and differential interference contrast (DIC) microscopy to investigate the particle size dependency for large gold nanospheres (AuNSs) with average diameters of 100nm, 300nm, and 400nm. Higher order plasmon resonances were observed in the single particle scattering spectra of large AuNSs (300nm and 400nm). Furthermore, the DF and DIC intensities were linearly increased with increasing the particle size from 100nm to 400nm. Therefore, the results provide useful experimental information of how the scattering and interference properties of single AuNSs are influenced by the particle size.

Surface morphology improvement of homoepitaxial GaN grown on free-standing GaN substrate by metalorganic chemical vapor deposition

Free-standing GaN is generally regarded as an ideal substrate for GaN-based devices due to its advantage of low threading dislocation density (TDD) and good thermal conductivity. However, new surface features such as hillocks and ridges appear on the GaN homoepitaxy films. In this paper, the influences of the intermediate GaN (IM-GaN) layer on the surface defects and crystal quality of GaN homoepitaxy films grown on c-plane GaN substrates by metalorganic chemical vapor deposition are investigated. It is found that hexagonal hillocks and ridges on the surface can be avoided by inserting an IM-GaN layer grown at an intermediate temperature (650850℃), prior to the growth of GaN at 1050℃. The results based on X-ray diffraction (XRD) measurements and differential interference contrast microscopy images demonstrate that the growth temperature of the IM-GaN layer has a significant influence on GaN homoepitaxy layer, which is one of the most critical parameters determining the surface morphology and crystal quality. As the IM-GaN growth temperature decreases from 1050℃ to 650℃, thed densities of hillocks and ridges on the surface reduce gradually. While, the XRD full width at half maximum (FWHM) values of (002) and (102) peaks for the homoepitaxy films are increased rapidly, indicating the adding of the TDD in the films. The atomic force microscopy (AFM) images show that the quasi-step growth mode change into layer-layer growth mode with the growth temperature decreasing from 1050℃ to 650℃ during the IM-GaN layer growing. It is speculated that the growth mode is determined by the diffusion length of adatom on the growing surface, which is proportional to the growth temperature. In the case of IM-GaN grown at low temperature, the formation of hillocks can be suppressed by reducing the adatom diffusion length. Finally, High crystal quality GaN homoepitaxy films (2 m) without hillocks is achieved by optimizing the growth parameters of IM-GaN layer, which is about 150 nm in thickness and grown at 850℃. The crystal quality of GaN homoepitaxy film is assessed by XRD rocking curve measured with double-crystal optics. The FWHMs of the (002) and (102) peaks are 125arcsec and 85arcsec respectively, indicating that rather low TDD is formed in the film. And well defined steps are observed on the image of AFM test, the root-mean square roughness value of the which is only about 0.23 nm for 5 m5 m scan area.

Detection of retinitis pigmentosa by differential interference contrast microscopy.

Differential interference contrast microscopy is designed to image unstained and transparent specimens by enhancing the contrast resulting from the Nomarski prism-effected optical path difference. Retinitis pigmentosa, one of the most common inherited retinal diseases, is characterized by progressive loss of photoreceptors. In this study, Differential interference contrast microscopy was evaluated as a new and simple application for observation of the retinal photoreceptor layer and retinitis pigmentosa diagnostics and monitoring. Retinal tissues of Royal College of Surgeons rats and retinal-degeneration mice, both well-established animal models for the disease, were prepared as flatmounts and histological sections representing different elapsed times since the occurrence of the disease. Under the microscopy, the retinal flatmounts showed that the mosaic pattern of the photoreceptor layer was irregular and partly collapsed at the early stage of retinitis pigmentosa, and, by the advanced stage, amorphous. The histological sections, similarly, showed thinning of the photoreceptor layer at the early stage and loss of the outer nuclear layer by the advanced stage. To count and compare the number of photoreceptors in the normal and early-retinitis pigmentosa-stage tissues, an automated cell-counting program designed with MATLAB, a numerical computing language, using a morphological reconstruction method, was applied to the differential interference contrast microscopic images. The number of cells significantly decreased, on average, from 282 to 143 cells for the Royal College of Surgeons rats and from 255 to 170 for the retinal-degeneration mouse. We successfully demonstrated the potential of the differential interference contrast microscopy technique’s application to the diagnosis and monitoring of RP.

Defocused differential interference contrast microscopy imaging of single plasmonic anisotropic nanoparticles.

We present the defocused differential interference contrast (DIC) imaging of gold nanorods. We found that the scattered light and the defocus aberration play an important role in the formation of orientation-dependent DIC image patterns of a gold nanorod. Interestingly, the scattered light from a gold nanorod aligned closer to the polarization directions enables us to directly resolve its spatial orientation under a defocused DIC microscope.

Bacterial cell identification in differential interference contrast microscopy images.

BackgroundMicroscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding.ResultsWe have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides.ConclusionsResults demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins.

WDDD: Worm Developmental Dynamics Database

During animal development, cells undergo dynamic changes in position and gene expression. A collection of quantitative information about morphological dynamics under a wide variety of gene perturbations would provide a rich resource for understanding the molecular mechanisms of development. Here, we created a database, the Worm Developmental Dynamics Database (http://so.qbic.riken.jp/wddd/), which stores a collection of quantitative information about cell division dynamics in early Caenorhabditis elegans embryos with single genes silenced by RNA-mediated interference. The information contains the three-dimensional coordinate values of the outlines of nuclear regions and the dynamics of the outlines over time. The database provides free access to 50 sets of quantitative data for wild-type embryos and 136 sets of quantitative data for RNA-mediated interference embryos corresponding to 72 of the 97 essential embryonic genes on chromosome III. The database also provides sets of four-dimensional differential interference contrast microscopy images on which the quantitative data were based. The database will provide a novel opportunity for the development of computational methods to obtain fresh insights into the mechanisms of development. The quantitative information and microscopy images can be synchronously viewed through a web browser, which is designed for easy access by experimental biologists.

A low-dimensional deformation model for cancer cells in flow.

A low-dimensional parametric deformation model of a cancer cell under shear flow is developed. The model is built around an experiment in which MDA-MB-231 adherent cells are subjected to flow with increasing shear. The cell surface deformation is imaged using differential interference contrast microscopy imaging techniques until the cell releases into the flow. We post-process the time sequence of images using an active shape model from which we obtain the principal components of deformation. These principal components are then used to obtain the parameters in an empirical constitutive equation determining the cell deformations as a function of the fluid normal and shear forces imparted. The cell surface is modeled as a 2D Gaussian interface which can be deformed with three active parameters: H (height), σ(x) (x-width), and σ(y) (y-width). Fluid forces are calculated on the cell surface by discretizing the surface with regularized Stokeslets, and the flow is driven by a stochastically fluctuating pressure gradient. The Stokeslet strengths are obtained so that viscous boundary conditions are enforced on the surface of the cell and the surrounding plate. We show that the low-dimensional model is able to capture the principal deformations of the cell reasonably well and argue that active shape models can be exploited further as a useful tool to bridge the gap between experiments, models, and numerical simulations in this biological setting.

NMR q-space analysis of canonical shapes of human erythrocytes: stomatocytes, discocytes, spherocytes and echinocytes

q-Space plots obtained experimentally using pulsed field-gradient stimulated echo (PGSTE) nuclear magnetic resonance (NMR) spectroscopy from water diffusing in red blood cells (RBCs) of different canonical (distinct variant) morphologies have "signature" features. The experimental q-space plots from suspensions of stomatocytes, echinocytes and spherocytes generated chemically had no diffraction features; in contrast a sample of blood from a patient with hereditary spherocytosis showed diffraction minima. To understand the forms of q-space plots, mathematical/geometrical models of discocytes, stomatocytes, echinocytes and spherocytes were used as restricting boundaries in simulations of water diffusion with Monte Carlo random walks. These simulations indicated that diffusion-diffraction minima are expected for each of the cell shapes considered. The absence of diffusion-diffraction minima in stomatocytes generated by dithiothreitol treatment was surmised to be due to non-alignment of the cells with the magnetic field of the NMR spectrometer. Differential interference contrast microscopy images of the chemically generated spherocyte and echinocyte suspensions showed them to be heterogeneous in cell shape. Therefore, we concluded that the shape heterogeneity caused the loss of the diffusion-diffraction features, which were observed in the more homogeneous sample from a patient with hereditary spherocytosis, and in the simulations of homogeneous cell suspensions. This understanding of factors that affect q-space plots from RBC suspensions will assist morphological studies of other cell and tissue types.

Uniformly thinned optical fibers produced via HF etching with spectral and microscopic verification

A method for producing uniformly thinned (etched) optical fibers is described, which can also be employed to etch optical fibers containing a Bragg grating (FBG) uniformly for evanescent-field-based sensing and other applications. Through a simple modification of this method, the fabrication of phase-shifted FBGs based on uneven etching is also shown. The critical role of how a fiber is secured is shown, and the success of the method is illustrated, by differential interference contrast microscopy images of uniformly etched FBGs. An etched FBG sensor for the monitoring of the refractive index of different glycerin solutions is demonstrated.

Microsystems for controlled genetic perturbation of live Drosophila embryos: RNA interference, development robustness and drug screening

A combination of genetic regulation and microenvironmental perturbation might prove especially useful for identifying the fundamental mechanisms responsible for development biology and critical bio-chemical networks. This could transform our understanding of genomics, proteomics, and drug discovery and lead to personalized drugs and molecular diagnostics for improved healthcare. With the complete sequencing of the Drosophila melanogaster, there has been a growing interest in correlating its genetics to human disease. The Drosophila embryo is an excellent whole-animal model, and is ideal for high throughput analysis. This review highlights research on the recent development of miniature tools to study critical cellular processes in Drosophila embryogenesis. First, we discuss the use of micro-injectors to conduct chip-level RNA interference experiments on self-assembled Drosophila embryos in order to determine how genes contribute to the growth and function of a developing organism. Second, we review recent progress on perturbing the microenvironment as a complementary approach to perturbing the genetic components of the development network. We designed a bio-compatible microfluidic device that automatically positions live embryos within a fluid-filled imaging channel and exposes embryos to constant or biphasic temperature or different drug concentrations. We have demonstrated that a thermal gradient applied across live Drosophila embryos results in asynchronous cellularization. Additionally, preliminary drug screening experiments were carried out on live Drosophila embryos in both 96-well plates and microfluidic channels. Time-lapse differential interference contrast microscopic images were taken to show the effects of colchicine (399.40 Da) and cytochalasin D (507.62 Da) on cellularization.

Partially coherent image formation in differential interference contrast (DIC) microscope

Some different image formation models have been proposed for Nomarski's differential interference contrast (DIC) microscope. However, the nature of coherence of illumination in DIC, of key importance in image formation, remains to be elucidated. We present a partially coherent image formation model for DIC and demonstrate that DIC microscope images the coherent difference of shifted replicas of the specimen; but imaging of the each component is partially coherent. Partially coherent transfer functions are presented for various DIC configurations. Plots of these transfer functions and experimental images provide quantitative comparison among various DIC configurations and elucidate their imaging properties. Approximations for weak or slowly varying specimens are also given. These improved models should be of great value in designing phase retrieval algorithms for DIC.

Automated analysis of differential interference contrast microscopy images of the foveal cone mosaic

An algorithm is presented for processing and analysis of differential interference contrast (DIC) microscopy images of the fovea to study the cone mosaic. The algorithm automatically locates the cones and their boundaries in such images and is assessed by comparison with results from manual analysis. Additional algorithms are presented that analyze the cone positions to extract information on cone neighbor relationships as well as the short-range order and domain structure of the mosaic. The methods are applied to DIC images of the human fovea.

Completing the Candida Loop

Completing the Candida Loop