Interference Microscopy Research Articles

A hybrid method of phase shifting interferometry and optical tomographic techniques for determining the optical anisotropy of grafted nylon-6 fibres with PMMA polymer

A method combining the phase shifting interferometry PSI and optical tomographic techniques for detecting the optical properties of grafted nylon-6 fibres is introduced. The method is capable of detecting the optical anisotropy and morphological properties of the sample at same time for the same region of the tested sample. The effect of grafting of nylon-6 with poly(methyl methacrylate), PMMA, polymer on their optical and morphological properties was investigated. The two-beam Pluta polarising interference microscope attached to a fibre rotation device with the suggested method were used to produce both computed tomographic data for morphological characterisation of the grafted fibre and interferometric measurements of birefringence values along the fibre axis. A blank sample of nylon-6 fibre was used to confirm the impact of grafting process with the PMMA polymer on the optical and structural properties of the nylon-6 fibres. Two -beam interference patterns are included for illustration.

Comparison of modulation interference microscopy, DNA spectrometry, DNA cytometry, and flow cytofluorimetry in the assessment of phytohemagglutinin-induced activity of human blood lymphocytes

Rationale: The study of the structural particulars and functional state of immune cells and primarily lymphocytes is of great importance for both fundamental and clinical medicine. It requires the development of simple and reliable analytic methods that would allow for fast and effective real-time assessment of cell activity.Aim: To evaluate the effectiveness of the interference microscopy compared to DNA spectrometry, DNA cytometry, and flow cytometry with an internalized fluorescent label CFSE (carboxyfluorescein succinimidyl ester) in the assessment of PHA-induced proliferation of human blood lymphocytes.Materials and methods: Phytohemagglutinin (PHA)-induced proliferative activity of blood lymphocytes from 10 healthy volunteers was studied with various methodological strategies. Blast transformation of lymphocytes was induced by their incubation for 5 days with PHA 5 μg/mL. The cell proliferative activity was assessed as follows: 1) by DNA spectrometry at 260/280 nm using Tecan Infinite 200 Pro with a specialized NanoQuant Plate™; 2) by cytophotometry followed by cell distribution analysis assessing deoxyribonucleic acid (DNA) content after staining with Felgen's dye with an imaging system based on an Olympus BX41 light microscope with a ProgRes CF camera; 3) by flow cytometry using an internalized fluorescent label CFSE; the analysis was performed with a BD FACS Calibur flow cytometer; 4) by measurement of the lymphocyte interference profile with a modulation interference microscope MIM-340 (Schwabe, Russia). The functional activity of the nucleus (FAN) was determined and used as a criterion for assessment of the lymphocyte functional state.Results: Incubation of lymphocytes with PHA led to an increase in the linear size by 22.2±2.8%, a decrease in phase height by 46.3±4.7% (p=0.019), and an increase in FAN by 75.9±9.4%, vs control (p=0.046). As measured by isolated DNA spectroscopy, PHA stimulation of lymphocytes was associated with an increase in the amount of DNA by 55% vs baseline (409.8±22.3 ng/μL and 264.3±25.0 ng/μL, respectively, p=0.049). Felgen's reaction revealed that the proportion of nuclei containing more than 2n DNA was 2% in the control cells and 14.8% in the PHA-activated lymphocytes, with a difference between the groups of 12.8%. CFSE staining with subsequent incubation and assessment by flow cytofluorimetry demonstrated an increase in the percentage of proliferating cells from 1.68±0.9% in the control to 55.56±5.6% (p=0.00068) in the mitogen-stimulated sample.Conclusion: Modulation interference microscopy does not require the sample preparation and demonstrated comparable and even higher effectiveness compared to conventional methods for assessment of lymphocyte activity. At the same time, it allows for evaluation of the lymphocyte functional state in real time in the process of cultivation. This opens ample opportunities for evaluation immune cells for research and diagnostic purposes.

Study of formation of secondary structures of frictional transfer on surface of steel samples in implementation of metal-plating technology

The state of the surface condition of steel samples (rollers) after tribological tests with modifi cation of working surfaces by applying hard cladding materials on friction surfaces are studied. Metal-plating of the subsystems of the wheel-rail friction mechanical system reduce friction between the wheel ridge and the side surface of the rail head, preventing the wheel from rolling onto the rail head. Surface morphology was investigated using an interference microscope surface analysis system and a scanning (raster) electron microscope with an energy dispersion analyzer prefix.

Quadriwave gradient light inteference microscopy for lable-free thick sample imaging

Due to the multiple scattering effect, quantitative phase imaging of thick specimens is challenging in biomedicine research. Phase gradient methods with partially coherent illumination in the reflection geometry have significant potential. However, to reconstruct two-dimensional (2D) phase information robustly, these methods cannot avoid changing the shear direction orthogonally. Here we propose a rotation-free method named quadriwave gradient light interference microscopy (qGLIM). qGLIM excludes conventional shear components and uses an amplitude-type spatial light modulator (SLM) to perform lateral shearing and phase shifting. By displaying checkerboard gratings on the SLM, we can reconstruct phase gradient information in orthogonal shear directions simultaneously. We extract the phase quantitatively by applying the phase-shifting technique and deconvolution algorithm. We demonstrate this approach by standard samples, thin samples, and thick multiple scattering samples.

Azimuthal Beam Scanning Microscope Design and Implementation for Axial Localization with Scanning Angle Interference Microscopy.

Azimuthal beam scanning, also referred to as circle scanning, is an effective way of eliminating coherence artifacts with laser illumination in widefield microscopy. With a static excitation spot, dirt on the optics and internal reflections can produce an uneven excitation field due to interference fringes. These artifacts become more pronounced in TIRF microscopy, where the excitation is confined to an evanescent field that extends a few hundred nanometers above the coverslip. Unwanted intensity patterns that arise from these imperfections vary with path of the excitation beam through the microscope optical train, so by rapidly rotating the beam through its azimuth the uneven illumination is eliminated by averaging over the camera exposure time. In addition to being useful from TIRF microscopy, it is also critical for scanning angle interference microscopy (SAIM), an axial localization technique with nanometer-scale precision that requires similar instrumentation to TIRF microscopy. For robust SAIM localization, laser excitation with a homogeneous profile over a range of polar angles is required. We have applied the circle scanning principle to SAIM, constructing an optimized instrument configuration and open-source hardware, enabling high-precision localization and significantly higher temporal resolution than previous implementations. In this chapter, we detail the design and construction of the SAIM instrument, including the optical configuration, required peripheral devices, and system calibration.

Particle erosion under multiphase bubble flow in horizontal-vertical-upward elbows

The erosion of particles is one of the main reasons of damage to the multiphase flow pipelines in black water treatment systems. In this study, the particle erosion of liquid–solid–gas multiphase bubble flow in a horizontal-vertical-upward elbow was analysed. The optical microscope, scanning electron microscope (SEM) and white light interference microscope were used for detection of failed pipe samples. An erosion simulation method based on the volume of fluid (VOF) model and discrete phase model (DPM) was adopted to study the influencing factors on the erosion. The results showed that the particles and bubbles lead to lots of pits and furrows on the pipe surfaces, and the erosion at the outer elbow is relatively severe due to the effect of multiphase bubble flow. The most severe damage occurred at θ = 25° of the elbow, and erosion rate is positively correlated with the particle diameter, volume fraction and inlet velocity.

Single-Shot Quantitative Polarization Imaging of Complex Birefringent Structure Dynamics.

Polarization light microscopes are powerful tools for probing molecular order and orientation in birefringent materials. While a number of polarization microscopy techniques are available to access steady-state properties of birefringent samples, quantitative measurements of the molecular orientation dynamics on the millisecond time scale have remained a challenge. We propose polarized shearing interference microscopy (PSIM), a single-shot quantitative polarization imaging method, for extracting the retardance and orientation angle of the laser beam transmitting through optically anisotropic specimens with complex structures. The measurement accuracy and imaging performance of PSIM are validated by imaging a birefringent resolution target and a bovine tendon specimen. We demonstrate that PSIM can quantify the dynamics of a flowing lyotropic chromonic liquid crystal in a microfluidic channel at an imaging speed of 506 frames per second (only limited by the camera frame rate), with a field-of-view of up to 350 × 350 μm2 and a diffraction-limit spatial resolution of ~2 μm. We envision that PSIM will find a broad range of applications in quantitative material characterization under dynamical conditions.

Q-factor modification of LN based WGM resonator

We demonstrated a monolithic optical whispering-gallery-mode (WGM) resonator made of lithium niobate (LN) single crystal. The effect of WGM resonator side roughness on the quality factor (Q-factor) of resonator was simulated by finite-difference time-domain (FDTD) method. WGM resonators were annealed at high temperature(1050 °C) to improve the Q-factor, and measured the side roughness of the WGM resonator before and after annealing with a white light interference microscope. It found that Q-factor of WGM resonator can be increased by more than 2 orders of magnitude by thermal treatment at 1050 °C.

Three-Dimensional Transfer Functions of Interference Microscopes

Three-dimensional transfer functions (3D TFs) are generally assumed to fully describe the transfer behavior of optical topography measuring instruments such as coherence scanning interferometers in the spatial frequency domain. Therefore, 3D TFs are supposed to be independent of the surface under investigation resulting in a clear separation of surface properties and transfer characteristics. In this paper, we show that the 3D TF of an interference microscope differs depending on whether the object is specularly reflecting or consists of point scatterers. In addition to the 3D TF of a point scatterer, we will derive an analytical expression for the 3D TF corresponding to specular surfaces and demonstrate this as being most relevant in practical applications of coherence scanning interferometry (CSI). We additionally study the effects of temporal coherence and disclose that in conventional CSI temporal coherence effects dominate. However, narrowband light sources are advantageous if high spatial frequency components of weak phase objects are to be resolved, whereas, for low-frequency phase objects of higher amplitude, the temporal coherence is less affecting. Finally, we present an approach that explains the different transfer characteristics of coherence peak and phase detection in CSI signal analysis.

Effects of Contact Load and Torsion Angle on Crack Propagation Behaviors of Inclined Crossed Steel Wires during Tension–Torsion Fretting Fatigue in Acid Solution

The hoisting rope in the kilometer-deep coal mine exhibits the tension–torsion fretting fatigue behaviors of inclined crossed steel wires in acid solution. Distinct contact load and torsion angles of steel wires in the rope cause different crack propagation behaviors, which greatly affect the fatigue lives of steel wires. Therefore, the effects of contact load and torsion angle on the crack propagation behaviors of inclined crossed steel wires during tension–torsion fretting fatigue in acid solution were investigated in the present study. The three-dimensional X-ray tomographic micro-imaging system was used to reveal evolutions of crack profiles and crack propagation depths during the test. The evolution of friction coefficient between steel wires during the test is presented. The three-dimensional white light interference microscope, electrochemical analyzer, and scanning electron microscope were employed to investigate the wear depth profiles, Tafel polarization curves and impedance spectra, and wear scar morphologies, respectively, of steel wires. Effects of contact load and torsion angle on crack propagation behaviors of inclined crossed steel wires during the tests were explored through analyses of friction and wear mechanisms and electrochemical corrosion damage. The results show that as the contact load and torsion angle increase, the crack propagation depth and rate of steel wire both increase and the fatigue life of steel wire decreases. Those are mainly attributed to the increases in the average tangential force between steel wires, wear depth, electrochemical corrosion tendency, and surface damage of steel wire as well as the decrease in corrosion resistance.

Cell composition at the vitreomacular interface in traumatic macular holes

PurposeTo describe characteristics of the vitreomacular interface (VMI) in traumatic macular holes (TMH) compared to idiopathic macular holes (IMH) using immunofluorescence and electron microscopy, and to correlate with clinical data.MethodsFor immunocytochemical and ultrastructural analyses, premacular tissue with internal limiting membrane (ILM) and epiretinal membrane (ERM) was harvested during vitrectomy from 5 eyes with TMH and 5 eyes with IMH. All specimens were processed as flat mounts for phase-contrast microscopy, interference and fluorescence microscopy, and transmission electron microscopy (TEM). Primary antibodies were used against microglial and macroglial cells. Clinical data was retrospectively evaluated.ResultsSurgically excised premacular tissue of eyes with TMH showed a less pronounced positive immunoreactivity for anti-glutamine synthetase, anti-vimentin and anti-IBA1 compared to eyes with IMH. Cell nuclei staining of the flat-mounted specimens as well as TEM presented a lower cell count in eyes with TMH compared to IMH. All detected cells were found on the vitreal side of the ILM. No collagen fibrils were seen in specimens of TMH. According to patients’ age, intraoperative data as well as spectral-domain optical coherence tomography (SD-OCT) analysis revealed an attached posterior vitreous in the majority of TMH cases (60%), whereas all eyes with IMH presented posterior vitreous detachment.ConclusionThe vitreomacular interface in TMH and IMH shows significant differences. In TMH, glial cells are a rare finding on the vitreal side of the ILM.

Use of a Laser Interference Microscope for Estimating Fluctuations and the Equivalent Elastic Constant of Cell Membranes

Use of a Laser Interference Microscope for Estimating Fluctuations and the Equivalent Elastic Constant of Cell Membranes

The properties of microarc oxidation coatings installed on AlSi10Mg specimens produced by selective laser melting

This paper examines the properties of coatings produced by microarc oxidation (MAO) on AlSi10Mg alloy specimens, which were obtained by selective laser melting. Two different electrolytes were used to install the coatings: silicate and silicate-phosphate. Modes were used that are recommended for the microarc oxidation of high-silicon aluminium casting alloys. As a result, double-layer coatings of different thicknesses were produced in relatively close timeframes of the MAO process. The thickness, roughness and wear spots were analyzed with the help of a laser interference microscope; the hardness was measured on a microhardness tester; the morphology and the elemental composition of the MAO coatings were analyzed by means of scanning electron microscopy (SEM); an X-ray diffractometer was used for phase analysis. The measurements showed that the coatings produced in the silicate-phosphate electrolyte have higher microhardness, corrosion resistance and specific electrical strength. Based on the results of friction tests and having analyzed the Ffr – Dr hysteresis loops obtained for reciprocating wear of the specimens, with the friction force Ffr and the travel D being simultaneously measured, the authors established that the wear resistance of the coatings produced in silicate-phosphate electrolyte is ~1.4 times higher than in the case of silicate electrolyte. The conducted study confirmed the efficiency of using silicate-phosphate electrolyte for producing high-quality protective coatings, which applies to almost all parameters. The paper shows that MAO coatings installed on AlSi10Mg alloy specimens produced by selective laser melting offer high performance. Thus, they can be recommended for pilot production of spacecraft components.This research was carried out as part of an assignment of the Ministry of Education and Science of Russia, Subject No.: FSFF-2020-0014.

Research stand for microplasma surface treatment of materials at atmospheric pressure

A research stand for microplasma treatment of object surfaces with the ability to move the discharge zone along the object using a program-controlled linear stepper motor has been developed. The design of the stand allows the use of different types of plasma generation systems, as well as processing with feeding of various gases such as air, nitrogen, oxygen, etc. into the discharge zone. The research bench is equipped with measuring equipment for monitoring the electrical and physical characteristics of the discharge (digital oscilloscopes, optical emission spectrometer, air ion meter, etc.). A microhardness tester, goniometer, interference microscope, tribometer, tensile testing machine, etc. can be used to further evaluate the quality and characteristics of the treated surfaces. Examples of the electrical characteristics of discharge devices tested as part of the research stand, optical emission spectroscopy of plasma, and results of measurements of the contact angle of treated objects surfaces are given.

Adaptive investigation of the optical properties of polymer fibers from mixing noisy phase shifting microinterferograms using deep learning algorithms.

In this article, an adaptive denoising method is suggested to accurate investigate the optical and structural features of polymeric fibers from noisy phase shifting microinterferograms. The mixed class of noise that may produce in the phase-shifting interferometric techniques is established. To our knowledge, this is an early study considered the mixing noises that may occur in microinterferograms. The suggested method utilized the convolution neural networks to detect the noise class and then denoising, it according to its class. Four convolution neural networks (Googlenet, VGG-19, Alexnet, and Alexnet-SVM) are refined to perform the automatic classification process for the noise class in the established data set. The network with the highest validation and testing accuracy of these networks is considered to apply the suggested method on realistic noisy microinterferograms for polymeric fibers, polypropylene and antimicrobial polyethylene terephthalate)/titanium dioxide, recoded using interference microscope. Also, the suggested method is applied on noisy microinterferograms include crazing and nanocomposite material. The demodulated phase maps and the three-dimensional birefringence profiles are calculated for tested fibers according to the suggested method. The obtained results are compared with the published data for these fibers and found to be in good agreements.

Analysis of morphometric and fractal properties of phase-contrast images of native and apoptotically changed MCF-7 cells

This work was aimed at the determination of the morphometric and fractal characteristics of native and apoptotic MCF-7 cells on the basis of the analysis of phase contrast images obtained using laser interference microscopy. MCF-7 cells were cultured under standard conditions. The cell phase images were obtained using a laser interference microscope (model MIM-340) at Perm Federal Research Center of the Ural Branch of the Russian Academy of Sciences. In the work, 30 native MCF-7 cells and 30 MCF-7 cells apoptotically changed under the action of doxorubicin were studied. The phase images obtained were treated to highlight the cell contour so as to subsequently determine the morphometric parameters and fractal dimension of the cell phase image contour. The maximal and minimal diameters, perimeter and volume of the cell phase images were chosen as geometric characteristics of the images. The fractal dimension of the phase image contour was calculated as a scale-invariant parameter indicating the indented character of the cell contour. The comparative analysis of the morphology of native and apoptotic MCF-7 cells enabled differentiating the cells under study using the two-sample non-parametric Wilcoxon–Mann–Whitney test.

Na+i/K+i imbalance contributes to gene expression in endothelial cells exposed to elevated NaCl

High-salt consumption contributes to the development of hypertension and is considered an independent risk factor for vascular remodelling, cardiac hypertrophy and stroke incidence. Alterations in NO production, inflammation and endothelial cell stiffening are considered now as plausible mediators of cardiovascular dysfunction. We studied early responses of endothelial cells (HUVEC) caused by a moderate increase in extracellular sodium concentration. Exposure of HUVEC to elevated sodium within the physiological range up to 24 h is accompanied by changes in monovalent cations fluxes and Na,K-ATPase activation, and, in turn, results in a significant decrease in the content of PTGS2, IL6 and IL1LR1 mRNAs. The expression of NOS3 and FOS genes, as well as the abundance of cytosolic and nuclear NFAT5 protein, remained unchanged. We assessed the mechanical properties of endothelial cells by estimating Young's modulus and equivalent elastic constant using atomic force and interference microscopy, respectively. These parameters were unaffected by elevated-salt exposure for 24 h. The data obtained suggest that even small and short-term elevations of extracellular sodium concentration affect the expression of genes involved in the control of endothelial function through the Na+i/K+i-dependent mechanism(s).

Structures and topological defects in pressure-driven lyotropic chromonic liquid crystals

Lyotropic chromonic liquid crystals are water-based materials composed of self-assembled cylindrical aggregates. Their behavior under flow is poorly understood, and quantitatively resolving the optical retardance of the flowing liquid crystal has so far been limited by the imaging speed of current polarization-resolved imaging techniques. Here, we employ a single-shot quantitative polarization imaging method, termed polarized shearing interference microscopy, to quantify the spatial distribution and the dynamics of the structures emerging in nematic disodium cromoglycate solutions in a microfluidic channel. We show that pure-twist disclination loops nucleate in the bulk flow over a range of shear rates. These loops are elongated in the flow direction and exhibit a constant aspect ratio that is governed by the nonnegligible splay-bend anisotropy at the loop boundary. The size of the loops is set by the balance between nucleation forces and annihilation forces acting on the disclination. The fluctuations of the pure-twist disclination loops reflect the tumbling character of nematic disodium cromoglycate. Our study, including experiment, simulation, and scaling analysis, provides a comprehensive understanding of the structure and dynamics of pressure-driven lyotropic chromonic liquid crystals and might open new routes for using these materials to control assembly and flow of biological systems or particles in microfluidic devices.

Investigation of measurement data of low-coherence interferometry at tilted surfaces in the 3D spatial frequency domain

Abstract The 3D transfer characteristics of interference microscopes and their effect on the interference signals occurring at surface slopes are studied. The interference image stacks acquired during a depth scan are 3D Fourier transformed. This allows a comprehensive frequency domain analysis of the interferograms. The double foil model introduced in a previous publication enables the interpretation of the signal spectra and the underlying transfer behavior of the interferometer using the concept of the Ewald sphere, which is limited by the numerical aperture (NA) of the imaging system. Analysis in the 3D spatial frequency domain directly discloses that the lateral dimensions of the transfer function depend on the axial spatial frequency. In this contribution we investigate measuring objects produced by Nanoimprint-Lithography. The corresponding signal spectra bear information that can be utilized to optimize the subsequent signal processing algorithms. These include envelope and phase evaluation procedures of the interference signals. A narrow bandpass filter is used to actively select certain frequency components in order to improve the robustness of the estimation of the envelope position. Although the shape and width of the envelope are affected, this procedure increases the reliability of the evaluation process and improves the accuracy of the measured topography especially at steeper surface slopes.

Monitoring reactivation of latent HIV by label-free gradient light interference microscopy

SummaryHuman immunodeficiency virus (HIV) can infect cells and take a quiescent and nonexpressive state called latency. In this study, we report insights provided by label-free, gradient light interference microscopy (GLIM) about the changes in dry mass, diameter, and dry mass density associated with infected cells that occur upon reactivation. We discovered that the mean cell dry mass and mean diameter of latently infected cells treated with reactivating drug, TNF-α, are higher for latent cells that reactivate than those of the cells that did not reactivate. Cells with mean dry mass and diameter less than approximately 10 pg and 8 μm, respectively, remain exclusively in the latent state. Also, cells with mean dry mass greater than approximately 28-30 pg and mean diameter greater than 11–12 μm have a higher probability of reactivating. This study is significant as it presents a new label-free approach to quantify latent reactivation of a virus in single cells.