Laser Scanning Microscopy Measurements Research Articles

Improving Whole-Cell Biocatalysis by Addition of Deep Eutectic Solvents and Natural Deep Eutectic Solvents

Being considered greener alternatives to ionic liquids (IL), deep eutectic solvents (DES) and natural deep eutectic solvents (NADES) have currently attracted broad interests from academics and industry. In this study, the transformation of isoeugenol to vanillin catalyzed by Lysinibacillus fusiformis CGMCC1347 cells was taken as the model reaction to examine the impacts of 24 DESs and 21 NADESs as cosolvents on whole-cell biocatalysis. Both types of cosolvents showed the ability of improving the production yields up to 142% and 132% of the ones obtained in (NA)DES-free aqueous systems, respectively. The data obtained by confocal laser scanning microscopy and flow cytometry tests and measurements of OD260 and OD280 agreed well with the bioconversion data, suggesting that addition of these cosolvents may be beneficial to whole-cell biocatalysis in enhancing the cellular membrane permeability. Interaction of DES with bacterial cell wall was discussed. The cells immobilized in PVA-alginate beads granted an augmented production yield in the presence of DES and NADES, up to 181% of the one obtained in a pure water system, and their catalytic activity was well maintained after being used for at least 13 cycles.

Effects of Cross-Link Density on Structures and Properties of Dual-Sensitive Semi-Interpenetrating Polymer Networks Hydrogel Microspheres

Semi-interpenetrating polymer network (semi-IPN) strategy is employed to fabricate uniform microspheres with temperature and pH dual-responsive behavior, which is composed of poly(N-isopropylacrylamide) and poly(acrylic acid) in the presence of N,N-methylenebisacrylamide (MBA) as the cross-linker. The influences of MBA amount (M-m) on the structures and properties of the microspheres are investigated in terms of particle size, surface morphology, pH sensitivity, and thermo-sensitivity with low critical solution temperature in the range M-m = 2.5-15%. Additionally, functional group distributions in the microspheres are probed by titration and employing the Henderson-Hasselbalch equation. The results show that all the properties strongly change depending on M-m. Based on transmission electron microscopy and confocal laser scanning microscopy measurements, and these properties, the M-m-induced changes in the structures of the semi-IPN microspheres are discussed.

Development and Technological Characterization of Multi-functional Aeronautical Coating From Lab-Scale to the Relevant Environment

Ice adhesion on critical aircraft surfaces is a serious potential hazard that runs the risk of causing accidents. Frozen contaminants cause rough and uneven surfaces which will disturb smooth air flow and greatly degrade the ability of the wing to generate lift and increasing drag. Amongst icing mitigation systems, passive anti-icing coatings represent a challenge to reduce the ice nucleation and growth, reducing the power consumption of the active de-icing systems and consequently the fuel consumption. In this work the advanced properties and effectiveness of the new multifunctional coating with ice-phobic and aesthetical properties are described. In particular advanced morphological characterizations based on Atomic Force Microscopy and Laser Scanning Microscopy measurements as well as subsequent Power Spectral Density analysis were performed to evaluate the surface roughness. Contact angle measurements were executed in order to determine the wettability and surface free energy as well as work of adhesion in flight conditions. In addition, dynamic analysis of the impact of single water droplets on the new multifunctional coating and the classical livery coating were performed in order to demonstrate the different physical behavior during the impingement. It was also demonstrated that the new multifunctional coating overcome the environmental test similarly to the commercial livery coating in accordance with the aeronautical specification. Finally, two NACA symmetric airfoils were design and developed using 3D printing technology. The surfaces were coated with a commercial coating in one case and with the new multifunctional coating in the other case. Both airfoils were tested in the Icing Wind Tunnel at different conditions in order to evaluate the effectiveness, in terms of reduction of accreted ice, of the new multifunctional coating respect to the commercial one. Tests demonstrated the reduction of accreted ice of 50% using the new multifunctional coating.

Effect of La 0.8 Sr 0.2 MnO 3 powder addition in the precursor solution on the properties of cathode films deposited by spray pyrolysis

Films of lanthanum strontium manganite, LSM (La0.8Sr0.2MnO3) were deposited on yttria stabilized zirconia (YSZ) substrates by different methods aiming to establish the most suitable route to prepare cathodes for solid oxide fuel cells (SOFC). Samples were obtained by using a solution of lanthanum, strontium and manganese nitrates or a dispersion of the LSM powder in this solution. Both commercial and synthetized LSM powders were used, the last one obtained by amorphous citrate method. The films were deposited by spray pyrolysis on YSZ substrates prepared by uniaxial and isostatic pressing. Samples were characterized by scanning electron microscopy, confocal laser scanning microscopy, X-ray diffraction and two-probe conductivity measurements. The area specific resistance and relaxation to cathodic activation were measured by electrochemical impedance spectroscopy. The substrate obtained by uniaxial pressing and the commercial LSM produced films with the highest amount of surface cracks. The film obtained from the suspension showed area specific resistance and activation energy lower than the other produced from the solution. For both samples, the cathodic activation process resulted in an initial reduction of the total resistance of around 20%, the sample produced from the suspension being more resistant to relaxation. Therefore, the LSM suspension is more suitable than the salts solution for preparing films by spray pyrolysis on YSZ substrates to obtain efficient cathodes for SOFC.

Stabilization of starch-based microgel-lysozyme complexes using a layer-by-layer assembly technique

The layer-by-layer assembly of polyelectrolyte multilayers of chitosan (CS) and carboxymethyl starch (CMS) on soft and porous pH- and ionic strength-response microgels was determined by confocal laser scanning microscopy (CLSM) and zeta potential measurements. In vitro release of lysozyme from the stabilized microgels under simulated gastric and intestinal fluids was also investigated. The distribution of CS in the microgels was identified by CLSM, and the optimal molecular weight of CS was 100kDa, which could only be absorbed on the microgel surface. The CS was used as the first layer, while the CMS was used as the second layer, and the zeta potential revealed that the optimal weight ratios of CS and CMS to microgels in the complexes were 0.1 and 0.06, respectively. The in vitro release experiments suggested that the stabilized double-layer microgel complexes could potentially be applied as a carrier system to prevent early release in the stomach to target intestinal delivery.

Complexation of lysozyme with sodium caseinate and micellar casein in aqueous buffered solutions

We present an extended structural and morphological study of the complexation of lysozyme (Lys) with sodium caseinate (SC) and micellar casein (MC) by means of turbidity measurements, phase analysis, dynamic, static and electrophoretic light scattering, bright-field and confocal laser scanning (CLSM) microscopy, fluorescence anisotropy and circular dichroism measurements. The solution behavior, structure, effective charge and morphology of the formed complexes as well as the protein structure within the complexes are dependent on the state of the casein molecules (SC versus MC), pH, ionic strength, and the [Cat+]/[An−] charge ratio (ChR). Absorption measurements indicate complexation of Lys with caseins at a pH as high as 11.29 (I = 0.01). At ChR>1, i.e. in excess of lysozyme, CLSM clearly showed formation of complex Lys/SC particles with a neutral core and an exterior part consisting exclusively of hydrophilic Lys macromolecules, whereas in the case of Lys/MC particles a uniform distribution of both proteins was observed. Binding of Lys with SC or MC leads to disruption of the secondary structure of Lys. Binding isotherms from fluorescence anisotropy are well described by an independent binding site model.

Data fusion for accurate microscopic rough surface metrology

Data fusion for rough surface measurement and evaluation was analyzed on simulated datasets, one with higher density (HD) but lower accuracy and the other with lower density (LD) but higher accuracy. Experimental verifications were then performed on laser scanning microscopy (LSM) and atomic force microscopy (AFM) characterizations of surface areal roughness artifacts. The results demonstrated that the fusion based on Gaussian process models is effective and robust under different measurement biases and noise strengths. All the amplitude, height distribution, and spatial characteristics of the original sample structure can be precisely recovered, with better metrological performance than any individual measurements. As for the influencing factors, the HD noise has a relatively weaker effect as compared with the LD noise. Furthermore, to enable an accurate fusion, the ratio of LD sampling interval to surface autocorrelation length should be smaller than a critical threshold. In general, data fusion is capable of enhancing the nanometrology of rough surfaces by combining efficient LSM measurement and down-sampled fast AFM scan. The accuracy, resolution, spatial coverage and efficiency can all be significantly improved. It is thus expected to have potential applications in development of hybrid microscopy and in surface metrology.

Surface composition effect of nitriding Ni-free stainless steel as bipolar plate of polymer electrolyte fuel cell

In order to increase the corrosion resistance of low cost Ni-free SUS445 stainless steel as the bipolar plate of a polymer electrolyte fuel cell, a nitriding surface treatment experiment was carried out in a nitrogen atmosphere under vacuum conditions, while an Ar atmosphere was used for comparison. The electrochemical performance, microstructure, surface chemical composition and morphology of the sample before and after the electrochemical measurements were investigated using linear sweep voltammetry (LSV), X-ray diffraction (XRD), glow discharge optical emission spectroscopy (GDS) and laser scanning microscopy (LSM) measurements. The results confirmed that the nitriding heat treatment not only increased the corrosion resistance, but also improved the surface conductivity of the Ni-free SUS445 stainless steel. In contrast, the corrosion resistance of the SUS445 stainless steel decreased after heat treatment in an Ar atmosphere. These results could be explained by the different surface compositions between these samples.

UV Treatment of the Stabilizing Shell for Improving the Photostability of Silver Nanoparticles

Silver nanoparticles or nanoclusters are quite sensitive to light exposure. In particular, irradiation in the localized surface plasmon resonance (LSPR) region brings about a drastic modification of their optical properties due to growth and reshaping of the nanoparticles. In order to obtain luminescent colloids, small silver colloidal nanoparticles were prepared in chloroform using vinylpyrrolidone oligomers as capping agent and their luminescence properties were used to control their stability upon prolonged exposure to visible light. The polymeric shell around the metal clusters was hardened through photo-cross-linking by UV light. This process did not alter the morphology and the optical properties of the nanoparticles but greatly improved the particle photostability as confirmed also by confocal laser scanning microscopy measurements. The data clearly show that UV curing of the stabilizing layer could be a simple postsynthetic procedure to obtain materials with stable properties.

Bioreducible Micelles Self-Assembled from Poly(ethylene glycol)-Cholesteryl Conjugate As a Drug Delivery Platform

The ability of polymeric micelles to self-assemble into nanosized particles has created interest in their application as potential anticancer drug delivery systems. A poly(ethylene glycol)-cholesteryl conjugate (Chol-ss-PEG-ss-Chol) connected by cleavable disulfide linkages was synthesized and used as a nanocarrier for in vitro release of doxorubicin (DOX). Owing to its amphiphilic structure, Chol-ss-PEG-ss-Chol was able to self-assemble into micelles with an average diameter 18.6 nm in aqueous solution. The micelles formed large aggregates due to the shedding of the PEG shell through cleavage of disulfide bonds in a reductive environment. The in vitro release studies revealed that Chol-ss-PEG-ss-Chol micelles released 80% and approximately 9% of the encapsulated DOX within 6 h under reductive and non-reductive conditions, respectively. The glutathione (GSH)-mediated intracellular drug delivery was investigated in a KB cell line. The cytotoxicity of DOX-loaded micelles indicated a higher cellular anti-proliferative effect against GSH-pretreated than untreated KB cells. Furthermore, confocal laser scanning microscopy (CLSM) measurement demonstrated that Chol-ss-PEG-ss-Chol micelles exhibited faster drug release in GSH-pretreated KB cells than untreated KB cells. These results suggest the potential usefulness of disulfide-based polymeric micelles as controlled drug delivery carriers.

Synergistic Stabilization of Emulsions and Emulsion Gels with Water-Soluble Polymers and Cellulose Nanocrystals

The effect of water-soluble polymers on the properties of Pickering emulsions stabilized by cellulose nanocrystals (CNCs) was investigated. Pretreatment of CNCs with excess adsorbing polymer, hydroxyethyl cellulose (HEC) or methyl cellulose (MC), gave smaller and more stable dodecane-in-water emulsion droplets compared to either polymer or CNCs alone, i.e., synergistic stabilization. By contrast, dextran, which does not adsorb on CNCs, gave unstable emulsions, with or without CNCs. CNCs with HEC or MC produced emulsions that showed no significant creaming or phase separation over several months. Interfacial tension, quartz crystal microbalance and confocal laser scanning microscopy measurements indicate that both HEC and MC are surface active and adsorb onto CNCs. 75% of the oil–water interface is covered by CNC particles coated with HEC or MC and the remaining interface is stabilized by HEC or MC chains not bound to cellulose. Viscoelastic emulsion gels were also produced by adding excess MC to the CNC-H...

Polymer–surfactant complexes for microencapsulation of vitamin E and its release

Microencapsulation of vitamin E directly from oil-in-water (o/w) emulsions was carried out by means of a novel practically relevant approach. For the first time, a preformed polyelectrolyte–surfactant complex (sodium polystyrene sulfonate/dodecyl trimethyl ammonium bromide) was simultaneously used as an electrosteric emulsion stabilizer and as a charged precursor for the following build up of microcapsules. Subsequently, a layer-by-layer technique was applied to emulsions leading to the formation of core–shell microcapsules with oily cores and polyelectrolyte shells. The effect of the complexes on the process of emulsion formation and on the stability and characteristics of the resulting emulsions was investigated by measurements of dynamic and equilibrium interfacial tension, size distribution (DLS) and interfacial charge (zeta-potential). The resulting microcapsules were characterized by confocal laser scanning microscopy (CLSM), Cryo-SEM, size distribution and zeta-potential measurements on each stage of the shell assembly. The release kinetics of vitamin E was monitored during the consecutive steps of the encapsulation procedure using UV–vis spectroscopy and showed the progressive enhancement of sustainability.The developed approach may be promising for the practical use in the cosmetic and food industry.

Antibacterial cellulose membrane via one-step covalent immobilization of ammonium/amine groups

This work describes a direct covalent linking of quaternary ammonium salts (QAS) and aminoalkyl groups onto regenerated cellulose (RC) membrane surface to prepare contact active antibacterial membrane materials. The membranes were prepared via alkoxysilane polycondensation reaction with three silane coupling agents, including trimethoxysilylpropyl trimethyl ammonium chloride (QASC0), trimethoxysilylpropyl octadecyldimethyl ammonium chloride (QASC18) and 3-aminopropyltrimethoxysilane (APS). FTIR, XPS, FESEM and AFM were used to characterize the chemical composition and surface morphology of the resulted RC-QASC0, RC-QASC18 and RC-NH2 membranes. Membrane surface charge properties were measured by the streaming potential method. Static water contact angle (WCA) and permeation flux were measured to investigate surface wettability and membrane permeability. Escherichia coli and Staphylococcus aureus were used as model bacteria to evaluate the antibacterial properties of membranes. The bacterial cell viability on membrane surfaces was evaluated by the dynamic shake flask test and the bacterial anti-adhesion properties were evaluated by confocal laser scanning microscopy (CLSM) measurements following the immersion test. RC-QASC18 and RC-NH2 membranes show strong antibacterial and bacteria anti-adhesion properties. Both membranes have a bacteria killing ratio over 99.5%. The stability study shows that the antibacterial property of modified membranes is robust in a wide range of pH and temperature conditions.

Effects of carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC) as fat replacers on the microstructure and sensory characteristics of fried beef patties

Microcrystalline cellulose (MCC) or carboxymethyl cellulose (CMC) can be used as potential fat replacers; both are non-digestible fibers. The objective of this study was, therefore, to assess the impact of added CMC/MCC on the microstructural/functional characteristics of beef patties. Therefore, low-fat beef patties were formulated with CMC/MCC at concentrations of 0.5–3.0 wt%. For this formulation, 10 wt% of the ground beef was replaced using a dispersion of CMC or MCC in water. The influence of the addition of MCC and CMC on the sensory characteristics, such as the texture, color, taste, flavor, and juiciness, of fried beef patties was evaluated by using a hedonic scale from 0 to 10 (5 = optimal). The confocal laser scanning microscopy (CLSM) measurements and sensory evaluation all indicated that addition of CMC (>1 wt%) led to destabilization of the microstructure, sensory quality and texture of fried beef patties, which, upon heating, could no longer be converted into a protein network with coarse meat and fat particles, a fact that was also revealed in the CLSM images. By contrast, the CLSM images prove that MCC was highly compatible with the matrix and improved the texture in the sensory evaluation with increasing concentration compared to the control. The best sensory evaluation achieved was with the beef patties containing 2 wt% MCC. Generally, hot samples containing MCC had more juiciness than the control and had a fat-like mouthfeel. Contrarily, CMC is not suitable as a fat replacer in concentrations more than 0.5 wt%.

Spectral analysis of irregular roughness artifacts measured by atomic force microscopy and laser scanning microscopy.

Atomic force microscopy (AFM) and laser scanning microscopy (LSM) measurements on a series of specially designed roughness artifacts were performed and the results characterized by spectral analysis. As demonstrated by comparisons, both AFM and LSM can image the complex structures with high resolution and fidelity. When the surface autocorrelation length increases from 200 to 500 nm, the cumulative power spectral density spectra of the design, AFM and LSM data reach a better agreement with each other. The critical wavelength of AFM characterization is smaller than that of LSM, and the gap between the measured and designed critical wavelengths is reduced with an increase in the surface autocorrelation length. Topography measurements of surfaces with a near zero or negatively skewed height distribution were determined to be accurate. However, obvious discrepancies were found for surfaces with a positive skewness owing to more severe dilations of either the solid tip of the AFM or the laser tip of the LSM. Further surface parameter evaluation and template matching analysis verified that the main distortions in AFM measurements are tip dilations while those in LSM are generally larger and more complex.

LY2157299-loaded carriers inhibiting wound healing in hepatocellular carcinoma

post-translational modifications, of many proteins involved in the cytoskeleton organization with respect to controls. In particular, higher levels of phosphorylated cofilin (p-COF)weredetected in the patient. p-COF was reported to be essential for the remodelling of cellular actin filaments. Here we show that higher levels of pCOF in PDfibroblasts coincidewithanhigher amountof stressfibreswhich appeared randomly oriented with consequent dramatic effects on both the cell morphology and the cell elasticity, as revealed by confocal laser scanning microscopy and atomic force microscopy measurements. Taken together, our results point out an important role of the cofilin pathway in the cellular homeostasis impairment in parkin-mutant human primary fibroblasts.

Surface Charge and Carbon Contamination on an Electron-Beam-Irradiated Hydroxyapatite Thin Film Investigated by Photoluminescence and Phase Imaging in Atomic Force Microscopy

The surface properties of hydroxyapatite, including electric charge, can influence the biological response, tissue compatibility, and adhesion of biological cells and biomolecules. Results reported here help in understanding this influence by creating charged domains on hydroxyapatite thin films deposited on silicon using electron beam irradiation and investigating their shape, properties, and carbon contamination for different doses of incident injected charge by two methods. Photoluminescence laser scanning microscopy was used to image electrostatic charge trapped at pre-existing and irradiation-induced defects within these domains, while phase imaging in atomic force microscopy was used to image the carbon contamination. Scanning Auger electron spectroscopy and Kelvin probe force microscopy were used as a reference for the atomic force microscopy phase contrast and photoluminescence laser scanning microscopy measurements. Our experiment shows that by combining the two imaging techniques the effects of trapped charge and carbon contamination can be separated. Such separation yields new possibilities for advancing the current understanding of how surface charge influences mediation of cellular and protein interactions in biomaterials.

Facile Fabrication of Redox-Responsive Thiol-Containing Drug Delivery System via RAFT Polymerization

A novel kind of redox-responsive polymeric drug delivery system has been designed and prepared successfully through the coupling of the multithiol branched polymers and thiol-containing drugs. The branched poly((S-(4-vinyl) benzyl S'-propyltrithiocarbonate)-co-(poly(ethylene glycol) methacrylate)) (poly(VBPT-co-PEGMA)) was synthesized by one-pot reaction via reversible addition-fragmentation chain transfer (RAFT) copolymerization. Subsequently, the hydrophobic thiol-containing anticancer drug 6-mercaptopurine (MP) was conjugated to poly(VBPT-co-PEGMA) by thiol-disulfide exchange reaction, resulting in the formation of poly(VBPT-co-PEGMA)-S-S-MP conjugate. Due to its amphiphilicity, poly(VBPT-co-PEGMA)-S-S-MP conjugate self-assembled into amphiphilic micelles in aqueous solution. Under a reductive environment, the disassembly of polymeric micelles resulted in the MP release. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements demonstrated that the poly(VBPT-co-PEGMA)-S-S-MP micelles could be taken up by Raji cells (a Burkitt lymphoma cell line). The viability of the Raji cells incubated with the glutathione (GSH) mediated poly(VBPT-co-PEGMA)-S-S-MP micelles was investigated by Cell Counting Kit-8 (CCK-8) assay. The experimental results showed that the viability of the glutathione monoester (GSH-OEt) pretreated cells was lower than that without pretreatment, while the viability of the buthionine sulfoximine (BSO) pretreated cells was higher than that without pretreatment. The poly(VBPT-co-PEGMA)-S-S-MP micelles could induce the apoptosis of Raji cells, and the apoptosis behavior was dose-dependent. This redox-responsive polymer-drug conjugate provides a promising platform for the delivery of thiol-containing biological molecules.

Metal-enhanced fluorescence using aggregated silver nanoparticles

A simple strategy to prepare aggregated Ag nanoparticle composites in solution by self-assembly is developed. Sodium citrate is used as the reduction agent to produce negatively charged Ag nanoparticles which are then aggregated using cationic polyacrylamide (PAM) to form self-assembled Ag-PAM microcomposites. In these microcomposites, the degree of aggregation of Ag nanoparticles can be controlled by changing the Ag/PAM ratio. This special structural feature can be applied in the study of metal-enhanced fluorescence. Confocal laser scanning microscopy and time-resolved fluorescence measurements are used to examine the fluorescence enhancement phenomenon of the Ag-PAM microcomposites. The microcomposites considerably enhanced the fluorescence intensity of fluorescein isothiocyanate isomer (FITC).

Confocal laser scanning microscopy measurement of the morphology of vanadium pentoxide nanorods grown by electron beam irradiation or thermal oxidation

In order to observe the morphology of nanostructures at the submicroscale, we use a confocal laser scanning (CLS) microscope built in our laboratory. The theoretical resolution of the hand-made CLS microscope is 150 nm and the performance of the microscope is evaluated by observing a USAF target. Vanadium pentoxide nanorods grown by electron beam irradiation and thermal oxidation methods are used as nanostructures and the morphologies of the nanorods observed by confocal laser scanning microscopy (CLSM) are compared with those obtained by scanning electron microscopy. The magnification and resolution of the CLSM were estimated to be approximately 1500 and 800 nm, respectively. From the results, we confirm that the CLSM can be used to measure nanostructures at the sub-micro-scale without a preconditioning process.