Optical Microscopy Experiments Research Articles

An asymmetric orifice-based active micromixer in the microfluidic chip with 3D microelectrode

The fluid mixing in microscale is central to the development of flow patterns in microfluidic chips for particles separation and biological analysis. Yet, as the size of the fluid decreases, the mixing becomes challenging to change this state of flow. In this work, a novel active micromixer for enhancing the mixing performance in the micromixer with asymmetric orifices is presented. To induce the electric-driven micromixing, an AC electric field is employed to the inserted 3D microelectrodes to produce the local electric field through the asymmetric orifices located on the opposite channel walls, which is perpendicular to the main microchannel. Then the mixing experiments were conducted in a microfluidic chip comprising microelectrode with asymmetric orifices, specifically exploiting the mixing efficiency varying with the asymmetric orifice pairs, applied electric potential and frequency of the AC electric field, and flow rate. The bending degree of the mixing interface was calculated and found increasing as a function of applied electric voltage at a given AC frequency, thus enabling effective mixing crossing the microelectrode's area. The predictions are confirmed by optical microscopy experiments and the micromixing can be significantly improved as they experience longer-exposed electrical forces. The integration of multiple asymmetric orifices leads a larger zone to increase mixing efficiency and makes it a promising method for rapid and stable fluid mixing in microscale.

Observation of liquid glass in molecular dynamics simulations.

Molecular anisotropy plays an important role in the glass transition of a liquid. Recently, a novel bulk glass state has been discovered by optical microscopy experiments on suspensions of ellipsoidal colloids. "Liquid glass" is a disordered analog of a nematic liquid crystal, in which rotation motion is hindered but particles diffuse freely. Global nematic order is suppressed as clusters of aligned particles intertwine. We perform Brownian dynamics simulations to test the structure and dynamics of a dense system of soft ellipsoidal particles. As seen in the experiments and in accordance with predictions from the mode coupling theory, on the time scale of our simulations, rotation motion is frozen but translation motion persists in liquid glass. Analyses of the dynamic structure functions for translation and rotation corroborates the presence of two separate glass transitions for rotation and translation, respectively. Even though the equilibrium state should be nematic, aligned structures remain small and orientational order rapidly decays with increasing size. Long-wavelength fluctuations are remnants of the isotropic-nematic transition.

Single-molecule analysis of purified proteins and nuclear extracts: Insights from 8-oxoguanine glycosylase 1

By observing one molecule at a time, single-molecule studies can offer detailed insights about biomolecular processes including on rates, off rates, and diffusivity of molecules on strands of DNA. A recent technological advance (Single-molecule Analysis of DNA-binding proteins from Nuclear Extracts, SMADNE) has lowered the barrier to entry for single-molecule studies, and single-molecule dynamics can now be determined directly out of nuclear extracts, providing information in an intermediate environment between purified proteins in isolation and the heterogeneity of a nucleus. To compare and contrast the single-molecule DNA binding dynamics in nuclear extracts versus purified proteins, combined optical tweezers and fluorescence microscopy experiments were performed with purified GFP-tagged 8-oxoguanine glycosylase 1 (OGG1), purified GFP-OGG1 spiked into nuclear extracts, and nuclear extracts from human cells overexpressing GFP-OGG1. We observed differences in undamaged DNA binding during DNA damage search in each of the three conditions. Purified GFP-OGG1 engaged undamaged DNA for a weighted average lifetime of 5.7 s and 21% of these events underwent DNA diffusion after binding. However, unlike other glycosylases studied by SMADNE, OGG1 does not bind non-damaged DNA efficiently in nuclear extracts. In contrast, GFP-OGG1 binding dynamics on DNA substrates containing oxidative damage were relatively similar in all three conditions, with the weighted average binding lifetimes varying from 2.2 s in nuclear extracts to 7.8 s with purified GFP-OGG1 in isolation. Finally, we compared the purified protein and nuclear extract approaches for a catalytically dead OGG1 variant (GFP-OGG1-K249Q). This variant greatly increased the binding lifetime for oxidative DNA damage, with the weighted average lifetime for GFP-OGG1–249Q in nuclear extracts at 15.4 s vs 10.7 s for the purified protein. SMADNE will provide a new window of observation into the behavior of nucleic acid binding proteins only accessible by biophysicists trained in protein purification and protein labeling.

Electrocatalysis at Individual Colloidal Nanoparticles: A Quantitative Survey of Four Geometries via Electrochemical Cell Microscopy

Colloidal nanoparticles are inherently heterogeneous, exhibiting variations in size, shape, or composition that will impact their catalytic behavior. Understanding these particle-to-particle variations in catalytic behavior will be critical to realizing more stable, selective, and efficient catalyst systems, but it remains difficult to generate this understanding using conventional characterization techniques. Here, we demonstrate how targeted electrochemical cell microscopy (TECCM) can be utilized to rigorously evaluate the electrocatalytic behavior of hundreds of individual metal nanoparticle catalysts, enabling statistically meaningful insights into these systems to be generated. The electrocatalytic oxidation of hydrazine was studied in a series of Au nanoparticle systems (nanorods, nanospheres, triangular nanoprisms, and nanocubes), directly revealing particle-to-particle variations in key kinetic parameters and catalyst stability. On average, “sharper” nanoparticle geometries were found to exhibit higher initial activities but quickly degraded upon potential cycling. Interestingly, our single particle studies also reveal that while the smoother nanorod and nanosphere geometries exhibit stable behavior in an ensemble sense, this is in fact due to a complicated balance of populations which exhibit increasing or decreasing catalytic behavior over typical experimental time scales. Results from correlated optical spectroscopy and electron microscopy experiments suggest that these observed changes in catalytic behavior are not associated with significant changes in particle structure. Together, these results demonstrate the extensive heterogeneity present in common colloidal nanoparticle systems and the utility of single particle analytical techniques for studying these systems.

Experimental Demonstration of Light-Trapping Transparent Electrode Geometries

The intrinsic trade-off between conductivity and transparency has long impeded the development of truly transparent electrodes. Recently, a transparent electrode design was introduced that promised remarkably high electrical conductivity while allowing near-complete transmission of incident light into the underlying optical device. The approach is based on the use of metallic wires with inclined surfaces, embedded in a transparent dielectric film. Light reflected by metallic regions of the sample can be recovered by total internal reflection, resulting in efficient trapping of light incident on conductive regions. Prototype electrode geometries were fabricated by multiphoton lithography and selective chemical deposition. Light trapping is observed by optical microscopy and laser scanning experiments for surface tilt angles exceeding 25°. Approximately 60% of light reflected by the metallic electrode surface was recovered through total internal reflection. The presented approach could lead to optoelectronic devices with significantly improved performance across a wide spectral bandwidth.

The Influence of Ethynyl In-Chain Crosslinkers on the Properties of 6FDA-Based Polyimides

Two 4,4'-(hexafluoroisopropylidene)diphthalic anhydride-based thermosetting polyimide formulations with varied amounts of crosslinking sites were compared to understand the influence of crosslinking density on fracture toughness, glass transition temperature and thermal oxidative stability. The thermal and mechanical properties of both materials were investigated through a series of single-edge notched beams, differential scanning calorimetry, dilatometry, weight loss, light optical microscopy and nanoindentation experiments. It was found out that the reduced crosslinking resulted in slightly increased fracture toughness but decreased the Tg of the material. No significant difference could be observed in the thermal oxidative stability with the experimental techniques considered.

Sorting of heterogeneous colloids by AC-dielectrophoretic forces in a microfluidic chip with asymmetric orifices

HypothesisThe synthesis of compositionally heterogeneous particles is central to the development of complex colloidal units for self-assembly and self-propulsion. Yet, as the complexity of particles grows, synthesis becomes more prone to “errors”. We hypothesize that alternating-current dielectrophoretic forces can efficiently sort Janus particles, as a function of patch size and material, and colloidal dumbbells by size. ExperimentsWe prepared Janus particles with different patch size and material by physical vapor deposition and colloidal dumbbells via capillarity-assisted particle assembly. We then performed sorting experiments in a microfluidic chip comprising electrodes with asymmetric orifices, specifically exploiting the dielectric contrast between different portions of the particles or their size difference to steer them towards different outlets. FindingsWe calculated that the DEP force for Janus particles may switch from positive to negative as a function of composition at a critical AC frequency, thus enabling sorting different particles crossing the electrodes’ region. The predictions are confirmed by optical microscopy experiments. We also show that intact and “broken” dumbbells can be simply separated as they experience different DEP forces. The integration of multiple asymmetric orifices leads a larger zone with high field gradient to increase separation efficiency and makes it a promising tool to select precise particle populations, isolating fractions with narrowly distributed characteristics.

Surface ion with Mo, Ti and Al for steel studies

Modification of metals by a surface treatment is one of the first problems of modern mate- rials science. The use of ion-plasma technologies for a modification of initial specimens, in particular, by ion implantation, for this purpose can be considered a promising solution. We study the microstructure, phase and elemental compositions, wetting forces, and electrical properties of steel with implanted ions of titanium, aluminum, and molybdenum. The results obtained by means of XRD, XPS, optical microscopy, and laboratory experiments show sig- nificant differences between the treated and untreated specimens. The modification of metal by ion implantation causes changes in the wettability and electrical characteristics and indicates the prospects of this technology for mechanical engineering, chemical, food, pharmaceutical industries, for the construction of structures and vehicles, including their work under severe atmospheric conditions.

Properties of Steel Modified by Surface Ion Implantation with Mo, Ti, and Al

Modification of metals by a surface treatment is one of the first problems of modern materials science. The use of ion-plasma technologies for a modification of initial specimens, in particular, by ion implantation, for this purpose can be considered a promising solution. We study the microstructure, phase and elemental compositions, wetting forces, and electrical properties of steel with implanted ions of titanium, aluminum, and molybdenum. The results obtained by means of XRD, XPS, optical microscopy, and laboratory experiments show significant differences between the treated and untreated specimens. The modification of metal by ion implantation causes changes in the wettability and electrical characteristics and indicates the prospects of this technology for mechanical engineering, chemical, food, pharmaceutical industries, for the construction of structures and vehicles, including their work under severe atmospheric conditions.

Determination of association equilibrium constant from single molecule fluorescence localization microscopy.

Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants. In this work, we use as a test system two complementary homo-oligonucleotides, one strand marked with Cyanine 3.5 and the other with Alexa Fluor 647. Hybridization is controlled by the amount of each strand, temperature, and the ionic force, and measured in steady state emission. The same samples are examined in Stochastic Optical Reconstruction Microscopy (STORM) experiments with split-field simultaneous two-colour detection. The effect of multiblinking, labelling-detection efficiency, and determination of the critical distance for association are discussed. We consistently determine values in STORM coincident with those of the bulk experiment.

Structural origin of excitations in a colloidal glass-former.

Despite decades of intense research, whether the transformation of supercooled liquids into glass is a kinetic phenomenon or a thermodynamic phase transition remains unknown. Here, we analyzed optical microscopy experiments on 2D binary colloidal glass-forming liquids and investigated the structural links of a prominent kinetic theory of glass transition. We examined a possible structural origin for localized excitations, which are building blocks of the dynamical facilitation theory-a purely kinetic approach for the glass transition. To accomplish this, we utilize machine learning methods to identify a structural order parameter termed "softness" that has been found to be correlated with reorganization events in supercooled liquids. Both excitations and softness qualitatively capture the dynamical slowdown on approaching the glass transition and motivated us to explore spatial and temporal correlations between them. Our results show that excitations predominantly occur in regions with high softness and the appearance of these high softness regions precedes excitations, thus suggesting a causal connection between them. Thus, unifying dynamical and thermodynamical theories into a single structure-based framework may provide a route to understand the glass transition.

Detailed characterisation and evaluation of composite stiffener R-zones based on mono-pulse ultrasonic reflection behavior

R-zones are important part of the skin-stiffener joint in composite panels. Owing to their complex geometric shapes, defect behaviour, and poor accessibility, it is difficult to perform non-destructive testing and evaluation of their defects. A new method based on reflection behaviour generated by mono-pulse ultrasonic (MU) waves in R-zones for characterising and evaluating their defects was developed. Specimens with artificial and actual delaminations were used. The MU reflection behaviours, time-domain signals, and image characteristics originating from the R-zones were studied. Optical microscopy experiments were conducted to verify the MU results. Methods for discriminating the delamination and resin layer as well as determining their accurate depth were established using the MU signals from the R-zones. The experimental results show that delamination, resin layer, and pockets in the R-zones can be characterised and evaluated in detail using the MU method. Its resolution and dead zone reached 1 prepreg ply thickness. The minimum deviation of the layup depth of the detected delamination and resin layer in the R-zone is less than one prepreg ply thickness. Delaminations of ∅ 3 mm at different depths were detected reliably.

WO3 Nanowires Enhance Molecular Alignment and Optical Anisotropy in Electrospun Nanocomposite Fibers: Implications for Hybrid Light-Emitting Systems.

The molecular orientation in polymer fibers is investigated for the purpose of enhancing their optical properties through nanoscale control by nanowires mixed in electrospun solutions. A prototypical system, consisting of a conjugated polymer blended with polyvinylpyrrolidone, mixed with WO3 nanowires, is analyzed. A critical strain rate of the electrospinning jet is determined by theoretical modeling at which point the polymer network undergoes a stretch transition in the fiber direction, resulting in a high molecular orientation that is partially retained after solidification. Nearing a nanowire boundary, local adsorption of the polymer and hydrodynamic drag further enhance the molecular orientation. These theoretical predictions are supported by polarized scanning near-field optical microscopy experiments, where the dichroic ratio of the light transmitted by the fiber provides evidence of increased orientation nearby nanowires. The addition of nanowires to enhance molecular alignment in polymer fibers might consequently enhance properties such as photoluminescence quantum yield, polarized emission, and tailored energy migration, exploitable in light-emitting photonic and optoelectronic devices and for sensing applications.

Characterization of horseshoe-shaped defects in 4H-SiC thick homoepitaxial layers

We report horseshoe-shaped protuberance defects encountered in growing thick homoepitaxial films on 4° off c-axis 4H-silicon carbide substrates. Their morphology, polytype and formation mechanism are investigated by optical microscopy, laser microscopy, micro-Raman spectroscopy, micro-photoluminescence spectroscopy and defect tracking experiments. The defects are observed to protrude from the surrounding area with a facet of (0001) at an angle of about 3.06°. Concomitantly, surface undulations develop and extend about hundreds of microns from the defect perpendicular to the direction of step flow. No polytype inclusions are found in these defects. The defect tracking reveals that the defects are not directly related to the existence of dislocations. We think that the formation of the defects origin from the occurrence of giant step bunching and subsequent faceting.

In-plane reflection phase engineering of graphene plasmons realized by electronic boundary design at the nanoscale

Understanding and controlling the reflection phase picked up by graphene plasmons (GPs) upon scattering at graphene boundaries is a prerequisite for designing the GP propagation and the resonance properties of GPs in nanostructures. However, an efficient method that could continuously change the reflection phase of GPs in a wide range is still lacking. Here, we demonstrate that the reflection phase of GPs can be effectively controlled by electronic boundary design. Specifically, a Fabry–Pérot (F–P) cavity is constructed by two electronic boundaries and then acts as an equivalent reflection boundary. Theoretical results show that the reflection phase of GPs could continuously vary in a wide range, almost 2π, by simply changing the graphene Fermi energy and the width of the F–P cavity. Furthermore, the evolution of GP modes is obtained in the simulated scattering-type scanning near-field optical microscopy experiment, which verifies the feasibility of the reflection phase control by employing our configuration. This work not only paves a way for in-plane plasmon control but also could serve as a valuable reference to various graphene-based plasmonic applications.

A High Sensitivity Transparent Ultrasound Transducer based on PMN-PT for Ultrasound and Photoacoustic Imaging.

We recently introduced piezoelectric lithium niobate (LN) based transparent ultrasound transducers (TUT) as a new platform for developing multimodal optical, ultrasound and photoacoustic imaging systems. However, LN based TUT is limited in its signal-to-noise ratio due to material's low piezoelectricity (d 33). In this paper, we report, for the first time, a 0.2 mm thick transparent lead magnesium niobate-lead titanate (PMN-PT) based TUT (PMN-PT-TUT) for ultrasound and photoacoustic applications and compared its performance with a 0.25 mm thick transparent LN based TUT (LN-TUT). To improve the ultrasound energy transmission efficiency, TUTs were fabricated with a two-matching-layer design. This resulted in a dual frequency response with center frequencies of 7.8 MHz/13.2 MHz and corresponding bandwidths of 28.2%/66.67% for PMN-PT-TUT, and center frequencies of 7.2 MHz/11.8 MHz and bandwidths of 36.1%/62.7% for LN-TUT. The optical transmission rate of PMN-PT-TUTs and LN-TUTs are measured as ~73% and ~91% respectively at 532 nm optical wavelength. The PMN-PT-TUT exhibited higher sensitivity compared to LN-TUT with a nearly three-fold higher pulse echo amplitude and more than two-fold higher photoacoustic amplitude. Furthermore, optical resolution photoacoustic microscopy (ORPAM) experiments on phantom targets demonstrated lateral resolutions of 7 μm and 5.1 μm, and axial resolutions of 285.6 μm and 375.9 μm for PMN-PT-TUT and LN-TUT respectively. These results indicated that PMN-PT is a viable alternative to LN for developing TUT based multimodal ultrasound and photoacoustic imaging systems.

Application of modified cellulose-based antiscalant in reverse osmosis membrane scaling control

<p indent=0mm>Applying membrane scaling is a key problem in reverse osmosis (RO) desalination processes. By dosing antiscalant, we can effectively inhibit inorganic scale and control RO membrane scaling. Accordingly, in this work, an environmentally friendly modified cellulose-based antiscalant is synthesized by the etherification of cellulose and subsequent graft copolymerization using the etherification product carboxymethyl cellulose (CMC), acrylic acid (AA), and sodium allyl sulfonate (SAS): CMC-<italic>g</italic>-P(AA-<italic>co</italic>-SAS). The influence of molecular structure, CMC-<italic>g</italic>-P(AA-<italic>co</italic>-SAS) dose, and coexisting anions with respect to inhibiting the calcium-sulfate scale in the RO system are investigated. The results indicate that dosing CMC-<italic>g</italic>-P(AA-<italic>co</italic>-SAS) can significantly prolong the induction time of calcium-sulfate crystallization, inhibit the formation of calcium-sulfate scale, greatly alleviate both membrane fouling and membrane flux decline caused by membrane-surface scale formation, and effectively improve the membrane filtration efficiency. Static tests, scanning electron microscopy, optical microscopy, X-ray diffraction, conductivity measurements, and dispersion experiments suggest that the scale inhibition mechanism of CMC-<italic>g</italic>-P(AA-<italic>co</italic>-SAS) can be attributed to the chelation and dispersion effects of the introduced anionic groups, and that the carboxyl groups are more efficient in the inhibition of calcium-sulfate scale than the sulfonic groups. In addition, an appropriate number of sulfonic groups on the graft chain of CMC-<italic>g</italic>-P(AA-<italic>co</italic>-SAS) can improve the water solubility of CMC-<italic>g</italic>-P(AA-<italic>co</italic>-SAS), as well as effectively stabilize the scale-formed substances and enhance the scale inhibition efficiency. If the proportion of sulfonic groups is too high, the content of the carboxyl groups decreases accordingly, which reduces the chelation and dispersion effects of CMC-<italic>g</italic>-P(AA-<italic>co</italic>-SAS), which, in turn, reduces the scale inhibition performance.

Blinking statistics and molecular counting in direct stochastic reconstruction microscopy (dSTORM).

Many recent advancements in single-molecule localization microscopy exploit the stochastic photoswitching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging, preventing key insight into the cellular structures and processes under observation. Modelling the photoswitching behaviour of a fluorophore as an unobserved continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photoswitching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a direct stochastic optical reconstruction microscopy experiment involving an arbitrary number of molecules. We demonstrate that when training data are available to estimate photoswitching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations. Finally, we demonstrate the method on experimental data by quantifying the number of adapter protein linker for activation of T cells on the cell surface of the T-cell immunological synapse. Software and data available at https://github.com/lp1611/mol_count_dstorm. Supplementary data are available at Bioinformatics online.

Identification of voids and interlaminar shear strengths of polymer‐matrix composites by optical microscopy experiment and deep learning methodology

AbstractThe internal void defects induced during the manufacturing process of polymer‐matrix composites can significantly degrade the mechanical properties of the composite, particularly the interlaminar shear strength (ILSS). In this study, we developed an innovative integrated methodology based on a deep learning semantic segmentation algorithm, named DeepLabV3+, and a theoretically driven equation to automatically identify voids in optical images and investigate the relationship between the microscopic voids and macroscopic ILSS parameters of the composite laminates. Results suggest that for the best fine‐tuned DeepLabV3+ framework, the corresponding mean pixel accuracy and intersection over union scores on the testing set were 99.84% and 90.82%, respectively, thereby indicating the potential of the generalized trained model. In addition, detailed experiments revealed that the proposed method can successfully obtain the ILSS values of laminates with different void contents. In addition, the ILSS values of the carbon/epoxy laminates decreased by approximately 27% with an increase in the void content from 0.07% to 3.14%.

Determination of Tensile Shear Strength and Corrosion Behavior of Circular Bead Produced by a Laser Beam for Overlapped AISI 304 Steel Sheets

Abstract: A circular bead produced by a laser beam (CLW) is a method of joining superposed sheets by scanning circularly a laser beam. Among the various applications, it could be highlighted the welding of austenitic stainless steel AISI 304 sheets for heat exchangers. This work aims to report the effect of fiber laser CLW in tensile shear strength of superposed sheets as well as the corrosion behavior in relation to the process parameters. It was carried out circular beads using stainless steel AISI 304 sheets varying the laser power and speed. The samples were then submitted to optical and electronic microscopy, tensile-shear tests and electrochemical experiments. Among the experimental conditions, the weld sample with 800 W power and 50 mm/s speed presented an ultimate tensile strength of 159 MPa, 17% higher than the sample with the lowest result. The corrosion rate, corrosion and pitting potentials did not change significantly in the welded joint with different conditions compared to the base metal.