Confocal Measurements Research Articles

Proton Transfer Can Occur at High Rates through Single-Layer Graphene in Nafion | Graphene | Nafion Sandwich Structures

Hydrogen pump cells fabricated from Nafion membranes and Pt-on-carbon electrodes were used to measure rates of proton transfer across single-layer graphene embedded in Nafion | graphene | Nafion sandwich structures. Single-layer graphene prepared by chemical vapor deposition onto copper foil was sandwiched between Nafion membranes by a series of hot-pressing steps coupled with chemical etching to remove the copper. Proton current densities in excess of 1 A / cm2 were observed for proton transfer across the graphene at cell bias voltages less than 200 mV. Confocal Raman spectroscopy measurements of the Nafion / graphene sandwich structures are consistent with single-layer graphene having very few structural defects embedded between Nafion membranes. Ion-transfer rates for lithium, sodium, potassium, and other cations were also measured and found to be much lower than rates for proton transfer, in most cases by many orders of magnitude. Two-layer and few-layer (3-5 layer) graphene sandwich structures were also studied and found to give proton currents much lower than single-layer graphene. Proton currents are described in terms of an area-normalized resistance following correction for all other contributions to series resistance in the cells. Resistances are then analyzed in terms of a rate constant for proton self-exchange across graphene. Figure 1

Etching Shapes the Topography of Silicon Wafers: Lattice-Strain Enhanced Chemical Reactivity of Silicon for Efficient Solar Cells

Multiwire sawing of silicon (Si) bricks is the state-of-the-art technology to produce multicrystalline Si solar wafers. The massive indentation of the abrasive Si carbide or diamond particles used leads to a heavily mechanically damaged layer on the wafer surface. Etching the surface layer using typical HF–HNO3–H2SiF6 acid mixtures reveals an unevenly distributed etch attack with etch rates several times higher than known for bulk Si etching. The present study follows the hypothesis that lattice strain, introduced by the sawing process, leads to an increase of the etch rate and determines the topography of the etched wafer, the so-called texture. Scratches were introduced into single crystalline Si surfaces in model experiments, and the magnitude and local distribution of lattice strain were extracted from confocal Raman microscopy measurements. The essential parameter used to describe the local reactivity of Si is the local etch rate, which was derived by confocal microscopy from the local height before ...

The Power of Shielding: Low Toxicity and High Transfection Performance of Cationic Graft Copolymers Containing Poly(2-oxazoline) Side Chains.

We show the potential of oligo(2-ethyl-2-oxazoline) (Oxn)-shielded graft copolymers of (2-aminoethyl)-methacrylate and N-methyl-(2-aminoethyl)-methacrylate for pDNA delivery in HEK cells. For the effect of grafting density and side chain length concerning improved transfection properties through the concept of shielding to be investigated, copolymers were synthesized via the macromonomer method using a combination of cationic ring opening polymerization and reversible addition-fragmentation chain transfer polymerization to vary the degree of grafting (DG = 10 and 30%) as well as the side chain degree of polymerization (DP = 5 and 20). Investigations of the polyplex formation, in vitro flow cytometry, and confocal laser scanning microscopy measurements on the copolymer library revealed classical shielding properties of the Ox side chains, including highly reduced cytotoxicity and a partial decrease in transfection efficiency, as also reported for polyethylene glycol shielding. In terms of the transfection efficiency, the best performing copolymers (A- g-Ox5(10) and M- g-Ox5(10)) revealed equal or better performances compared to those of the corresponding homopolymers. In particular, the graft copolymers with low DG and side chain DP transfected well with over 10-fold higher IC50 values. In contrast, a DG of 30% resulted in a loss of transfection efficiency due to missing ability for endosomal release, and a side chain DP of 20 hampered the cellular uptake.

Multi-Scale Contact Modeling of Coated Steels for Sheet Metal Forming Applications

Friction in sheet metal forming is a local phenomenon which depends on continuously evolving contact conditions during the forming process. This is mainly influenced by local contact pressure, surface textures of the sheet metal as well as the forming tool surface profile and material behavior. The first step for an accurate prediction of friction is to reliably estimate real area of contact at various normal loads. In this study, a multi-scale contact model for the normal load is presented to predict asperity deformation in coated steels and thus to estimate the real area of contact. Surface profiles of the zinc layer and steel substrate are modelled explicitly obtained from confocal measurements. Different mechanical properties are assigned to the zinc coating and the steel substrate. The model was calibrated and validated relative to lab-scale normal load tests using different samples of zinc coated steel with distinct surface textures. The results show that the model is able to predict the real area of contact in zinc-coated steels for various contact pressures and different surface textures. Current multi-scale model can be used to determine the local friction coefficient in sheet metal forming processes more accurately.

Evaluation of chemical treatments to tensile properties of cellulosic bamboo fibers

Acidified sodium chlorite (NaClO2) and sodium hydroxide (NaOH) solutions were successfully used to eliminate lignin and hemicellulose, respectively, from isolated bamboo fibers to investigate their microstructures, composition and micro-tensile properties in individual cellulose fibers. The results demonstrated that isolated fibers and chemically treated fibers clearly displayed different wrinkles, pores and microfibril aggregations on the cell wall surface. Field emission environmental scanning electron microscope images together with confocal scanning laser microscope measurements revealed that chemical treatments have a reduction effect on micro-tensile strength and modulus. Two methods of freeze-drying and air-drying produced varying interfaces and pores in microfibril aggregations and resulted in different tensile behavior. Notably, the reductions of Young’s modulus and tensile strength in freeze-drying (52.99 and 13.32%) fibers are larger than that (18.21 and 10.93%) in air-drying samples. These results suggest that most fractures occur in the weak interspaces or pores that are caused by the loss of matrix materials during chemical treatments.

Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements.

Single-molecule fluorescence resonance energy transfer (smFRET) remains a widely utilized and powerful tool for quantifying heterogeneous interactions and conformational dynamics of biomolecules. However, traditional smFRET experiments either are limited to short observation times (typically less than 1 ms) in the case of "burst" confocal measurements or require surface immobilization which usually has a temporal resolution limited by the camera framing rate. We developed a smFRET 3D tracking microscope that is capable of observing single particles for extended periods of time with high temporal resolution. The confocal tracking microscope utilizes closed-loop feedback to follow the particle in solution by recentering it within two overlapping tetrahedral detection elements, corresponding to donor and acceptor channels. We demonstrated the microscope's multicolor tracking capability via random walk simulations and experimental tracking of 200 nm fluorescent beads in water with a range of apparent smFRET efficiency values, 0.45-0.69. We also demonstrated the microscope's capability to track and quantify double-stranded DNA undergoing intramolecular smFRET in a viscous glycerol solution. In future experiments, the smFRET 3D tracking system will be used to study protein conformational dynamics while diffusing in solution and native biological environments with high temporal resolution.

High‐Efficiency Broadband Mid‐Infrared Flat Lens

AbstractIntegrated photonic circuits and infrared imaging systems demand compact optical components. Dielectric diffractive optics enables miniaturization of the curved refractive optics into planar structure by encoding phase over a 2D plane. However, in the mid‐infrared wavelength range, such engineered dielectric surfaces are not efficient, because optically transparent dielectric scatterers with high index contrast in the mid‐infrared spectral range suffer from low bandwidth and high thermal noise in long wavelengths. Here, a planar optical lens based on ultrathin gold plasmonic nanostructure operating in the mid‐infrared spectral range is demonstrated. The design enables subwavelength focusing beyond the Abbe‐Rayleigh diffraction limit while maintaining high transmission efficiency (≈60%) with excellent agreement between electromagnetic simulations and confocal measurements. Single and bilayer flat lenses designed for subwavelength polarization‐dependent line and polarization‐independent point focusing, respectively, are demonstrated. Such geometry‐defined tunable optical response overcomes the challenges associated with the unavailability of mid‐infrared transparent materials for low footprint planar integration with thermal imaging systems.

Silica nanoparticles with Tb(III)-centered luminescence decorated by Ag0 as efficient cellular contrast agent with anticancer effect

The present work introduces composite luminescent nanoparticles (Ag0-Tb3+-SNs), where ultra-small nanosilver (4 ± 2 nm) is deposited onto amino-modified silica nanoparticles (35±6 nm) doped by green luminescent Tb(III) complexes. Ag0-Tb3+-SNs are able to image cancer (Hep-2) cells in confocal microscopy measurements due to efficient cell internalization, which is confirmed by TEM images of the Hep-2 cells exposed by Ag0-Tb3+-SNs. Comparative analysis of the cytotoxicity of normal fibroblasts (DK-4) and cancer cells (Hep-2) incubated with various concentrations of Ag0-Tb3+-SNs revealed the concentration range where the toxic effect on the cancer cells is significant, while it is insignificant towards the nonmalignant fibroblasts cells. The obtained results reveal Ag0-Tb3+-SNs as good cellular contrast agent able to induce the cancer cells death, which makes them promising theranostic in cancer diagnostics and therapy.

Mapping the spatiotemporal evolution of solute transport in articular cartilage explants reveals how cartilage recovers fluid within the contact area during sliding

The interstitial fluid within articular cartilage shields the matrix from mechanical stresses, reduces friction and wear, enables biochemical processes, and transports solutes into and out of the avascular extracellular matrix. The balanced competition between fluid exudation and recovery under load is thus critical to the mechanical and biological functions of the tissue. We recently discovered that sliding alone can induce rapid solute transport into buried cartilage contact areas via a phenomenon termed tribological rehydration. In this study, we use in situ confocal microscopy measurements to track the spatiotemporal propagation of a small neutral solute into the buried contact area to clarify the fluid mechanics underlying the tribological rehydration phenomenon. Sliding experiments were interrupted by periodic static loading to enable scanning of the entire contact area. Spatiotemporal patterns of solute transport combined with tribological data suggested pressure driven flow through the extracellular matrix from the contact periphery rather than into the surface via a fluid film. Interestingly, these testing interruptions also revealed dynamic, repeatable and history-independent fluid loss and recovery processes consistent with those observed in vivo. Unlike the migrating contact area, which preserves hydration by moving faster than interstitial fluid can flow, our results demonstrate that the stationary contact area can maintain and actively recover hydration through a dynamic competition between load-induced exudation and sliding-induced recovery. The results demonstrate that sliding contributes to the recovery of fluid and solutes by cartilage within the contact area while clarifying the means by which it occurs.

The specific effect of gallic acid on Escherichia coli biofilm formation by regulating pgaABCD genes expression.

Escherichia coli (E. coli) is associated with an array of health-threatening contaminations, some of which are related to biofilm states. The pgaABCD-encoded poly-beta-1,6-N-acetyl-D-glucosamine (PGA) polymer plays an important role in biofilm formation. This study was conducted to determine the inhibitory effect of gallic acid (GA) against E. coli biofilm formation. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of GA against planktonic E. coli were 0.5 and 4mg/mL, and minimal biofilm inhibitory concentration and minimal biofilm eradication concentration values of GA against E. coli in biofilms were 2 and 8mg/mL, respectively. Quantitative crystal violet staining of biofilms and ESEM images clearly indicate that GA effectively, dose-dependently inhibited biofilm formation. CFU counting and confocal laser scanning microscopy measurements showed that GA significantly reduced viable bacteria in the biofilm. The contents of polysaccharide slime, protein, and DNA in the E. coli biofilm also decreased. qRT-PCR data showed that at the sub-MIC level of GA (0.25mg/mL) and expression of pgaABC genes was downregulated, while pgaD gene expression was upregulated. The sub-MBC level of GA (2mg/mL) significantly suppressed the pgaABCD genes. Our results altogether demonstrate that GA inhibited viable bacteria and E. coli biofilm formation, marking a novel approach to the prevention and treatment of biofilm-related infections in the food industry.

In Situ Measurements of Contact Dynamics in Speed-dependent Hydrogel Friction

The friction behavior of soft, aqueous, biotribological contacts depends on contact geometry, speed, and pressure. Previous efforts to measure the surface profile of soft sliding contacts have been stymied by the matching index of refraction of aqueous materials submerged in water. Here, hydrogel surface deformations were imaged using confocal microscopy to experimentally investigate the contact geometry as a function of sliding speed. In situ fluorescence confocal microscopy measurements of the contact deformation during unidirectional friction experiments revealed the existence of a front/back asymmetry that increased with increasing sliding speed. A polyacrylamide hydrogel disk (96.5% water) was polymerized with fluorescent dyes and used as a rotating countersample below a polished glass hemispherical pin (1mm radius of curvature). All experiments were performed submerged in a dilute (0.7wt%) suspension of 1μm red fluorescent microspheres in ultrapure water. Imaging of the contact was performed using a confocal microscope in situ with unidirectional sliding at 0.1, 1, 10, and 100mm/s. The friction coefficient increased monotonically with increasing speed from μ~0.04 at 0.1mm/s to μ~0.20 at 100mm/s. All imaging was performed relative to the stationary glass probe under steady-state conditions. The contact line measured by connecting the leading and exiting contact points was nearly perpendicular to the loading direction at 0.1mm/s sliding speed, but distorted as sliding speed increased. Consistent with the theories of viscoelastic contributions to polymer friction, the tangent of the contact-line angle correlated with friction.

Tracking Photoexcited Carriers in Hybrid Perovskite Semiconductors: Trap-Dominated Spatial Heterogeneity and Diffusion.

We use correlated confocal and wide-field fluorescence microscopy to probe the interplay between local variations in charge carrier recombination and charge carrier transport in methylammonium lead triiodide perovskite thin films. We find that local photoluminescence variations present in confocal imaging are also observed in wide-field imaging, while intensity-dependent confocal measurements show that the heterogeneity in nonradiative losses observed at low excitation powers becomes less pronounced at higher excitation powers. Both confocal and wide-field images show that carriers undergo anisotropic diffusion due to differences in intergrain connectivity. These data are all qualitatively consistent with trap-dominated variations in local photoluminescence intensity and with grain boundaries that exhibit varying degrees of opacity to carrier transport. We use a two-dimensional kinetic model to simulate and compare confocal time-resolved photoluminescence decay traces with experimental data. The simulations further support the assignment of local variations in nonradiative recombination as the primary cause of photoluminescence heterogeneity in the films studied herein. These results point to surface passivation and intergrain connectivity as areas that could yield improvements in perovskite solar cells and optoelectronic device performance.

Confocal spectroscopic imaging measurements of depth dependent hydration dynamics in human skin in-vivo

We present confocal spectroscopic imaging measurements applied to in-vivo studies to determine the depth dependent hydration profiles of human skin. The observed spectroscopic signal covers the spectral range from 810 nm to 2100 nm allowing to probe relevant absorption signals that can be associated with e.g. lipid and water-absorption bands. We employ a spectrally sensitive autofocus mechanism that allows an ultrafast focusing of the measurement spot on the skin and subsequently probes the evolution of the absorption bands as a function of depth. We determine the change of the water concentration in m%. The water concentration follows a sigmoidal behavior with an increase of the water content of about 70% within 5 μm in a depth of about 14 μm. We have applied our technique to study the hydration dynamics of skin before and after treatment with different concentrations of glycerol indicating that an increase of the glycerol concentration leads to an enhanced water concentration in the stratum corneum. Moreover, in contrast to traditional corneometry we have found that the application of Aluminium Chlorohydrate has no impact to the hydration of skin.

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells.

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 μs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

ScAlN etch mask for highly selective silicon etching

This work reports the utilization of a recently developed film, ScAlN, as a silicon etch mask offering significant improvements in high etch selectivity to silicon. Utilization of ScAlN as a fluorine chemistry based deep reactive ion etch mask demonstrated etch selectivity at 23 550:1, four times better than AlN, 11 times better than Al2O3, and 148 times better than silicon dioxide with significantly less resputtering at high bias voltage than either Al2O3 or AlN. Ellipsometry film thickness measurements show less than 0.3 nm/min mask erosion rates for ScAlN. Micromasking of resputtered Al for Al2O3, AlN, and ScAlN etch masks is also reported here, utilizing cross-sectional scanning electron microscope and confocal microscope roughness measurements. With lower etch bias, the reduced etch rate can be optimized to achieve a trench bottom surface roughness that is comparable to SiO2 etch masks. Etch mask selectivity enabled by ScAlN is likely to make significant improvements in microelectromechanical systems, wafer level packaging, and plasma dicing of silicon.

Highly wear-resistant and low-friction Si3N4 composites by addition of graphene nanoplatelets approaching the 2D limit

Graphene nanoplatelets (GNPs) have emerged as one of the most promising filler materials for improving the tribological performance of ceramic composites due to their outstanding solid lubricant properties as well as mechanical and thermal stability. Yet, the addition of GNPs has so far enabled only a very limited improvement in the tribological properties of ceramics, particularly concerning the reduction of their friction coefficient. This is most likely due to the challenges of achieving a continuous lubricating and protecting tribo-film through a high GNP coverage of the exposed surfaces. Here we demonstrate that this can be achieved by efficiently increasing the exfoliation degree of GNPs down to the few-layer (FL) range. By employing FL-GNPs as filler material, the wear resistance of Si3N4 composites can be increased by more than twenty times, the friction coefficient reduced to nearly its half, while the other mechanical properties are also preserved or improved. Confocal Raman spectroscopy measurements revealed that at the origin of the spectacular improvement of the tribological properties is the formation of a continuous FL- GNP tribo-film, already at 5 wt% FL-GNP content.

Stiffness signatures along early stages of Xylella fastidiosa biofilm formation

The pathogenicity of Xylella fastidiosa is associated with its systematic colonization of the plant xylem, forming bacterial biofilms. Mechanisms of bacterial transport among different xylem vessels, however, are not completely understood yet, but are strongly influenced by the presence of extracellular polymeric substances (EPS), which surrounds the assembly of cells forming the biofilm. In this work, we show quantitative measurements on the elastic properties of the system composed by EPS and bacterial cell. In order to investigate the mechanical properties of this system, force spectroscopy and confocal Raman measurements were carried out during Xylella fastidiosa subsp. pauca initial stages of adhesion and cluster formation. We show that stiffness progressively decreases with increasing culture growth time, from two to five days. For early adhesion samples, stiffness values are quite different at the bacterial polar and body regions. Lower stiffness values at the cell pole suggest a flexible mechanical response at this region, associated with first cell adhesion to a surface. These results correlate very well with our observations of cell motion within microchannels, under conditions simulating xylem flow. Both the oscillatory movement of vertically attached single cells, as well as the transport of cell clusters within the biofilm matrix can be explained by the presence of softer materials at the cell pole and EPS matrix. Our results may thus add to a more detailed understanding of mechanisms used by cells to migrate among vessels in plant xylem.

Demarcating wetting states in textured microchannels under flow conditions by Poiseuille number

In this work, flow friction in microchannels decorated with micropillars was investigated experimentally, with an interest to understand the wetting transition through two simple means: Poiseuille number and scaling laws. Different wetting states were demarcated by qualitatively assessing the behaviour of Poiseuille number (Po = f·Re, where f is friction factor and Re is Reynolds number), which are further corroborated by confocal microscopy-based measurements and numerical simulations. The wetting transition ensued smoothly with an increase in Re, independent of the gas fraction (a ratio of area covered by the liquid–gas interface to the total projected area), for moderate gas fractions, whereas an early breakdown of the Cassie–Baxter state occurred irrespective of Re at high gas fractions. Additionally, the scaling laws were found to correlate well with the underlying state of the flow. Our observations revealed that the liquid–gas interface exhibits a partial slip, contrary to the common notion that it is shear free. It is inferred that an increase in effective flow area leads to a reduction in flow friction in textured microchannels. The present work underlines three important outcomes. The first is the identification of wetting states in flow conditions shown by tracking the Poiseuille number. The second is that the liquid–gas interface is deduced to behave like a partial slip boundary. The third is that a textured microchannel can be worse than an enlarged dimension microchannel.

Ultra-Low Reflectivity Anti-Reflection Coating on a Plastic Cover Slip in Liquid for He-Ne Laser Light

An ultra-low anti-reflection optical coating on both surfaces of a plastic cover slip was studied for use in confocal image measurements. The optical reflectance at a wavelength of 632.8 nm was less than 0.1% when the coated sample was placed in a liquid having a refractive index of 1.34, close to the aqueous solution of the biomaterial. The high- and low-index coating films, Substance-2 (PrTiO3) and silicon dioxide (SiO2), were measured by an ellipsometer to determine their optical refraction indices and extinction coefficients. Theoretically, when the two layer thicknesses are designed using the optical admittance diagram of the cover slip to approach the equivalent index of 1.34, a reflectance of 1.6 × 10−5% in the liquid could be obtained. Experimentally, the reflectance of the sample deposited on the two faces of the cover slip was 4.223 ± 0.145% as measured in the air; and 0.050 ± 0.002% as measured by a He-Ne laser in the liquid.

The influence of the pyrolysis process on mechanical parameters and tightness of the black glasses layers on titanium substrates

Abstract Black glasses are amorphous materials in which structure Si–O bonds coexist with Si–C bonds. The substitution of divalent oxygen ions by tetravalent carbon ions causes the change of properties, especially mechanical ones. The materials were obtained from the sol-gel synthesized ladder-like polysilsesquioxane organosilicon precursors. After deposition on titanium substrates and ceramization, continuous and hermetic layers were obtained. Adhesion, Young's modulus, hardness, roughness and corrosion resistance were examined as the most relevant mechanical properties of the obtained layers. Nanoindentation, surface mapping, electrochemical, SEM and confocal microscope measurements results are discussed in relation to the pyrolysis temperature of the black glasses layers.