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Enhancement of Raman signals with silver-coated tips.

Silver coated SiN and SiO(2) tips have been fabricated for use with a bottom-illumination tip-enhanced Raman spectroscopy (TERS) setup with a 488 nm laser excitation. SiN tips with 50-60 nm of deposited Ag give the best TERS enhancements for brilliant cresyl blue test analyte spin-coated on a glass slide. Ag nanoparticles on SiN or SiO(2) rather than Si tips are better for TERS because of the proximity of the wavelengths of their surface plasmon resonance to 488 nm. Adjustments of tilt angle of the metallized tip with respect to the surface plane is shown to considerably raise the intensities of the TERS signals, even from tips that initially appear to be rather non-enhancing. This work helps to enable the more frequent use of the 488 nm laser for nanoscale chemical analysis with both TERS and fluorescence imaging in the same setup.

Microscopy, Scanning Probe↗

Carbon nanotubes and nanowires for biological sensing.

This chapter reviews the recent development in biological sensing using nanotechnologies based on carbon nanotubes and various nanowires. These 1D materials have shown unique properties that are efficient in interacting with biomolecules of similar dimensions, i.e., on a nanometer scale. Various aspects including synthesis, materials properties, device fabrication, biofunctionalization, and biological sensing applications of such materials are reviewed. The potential of such integrated nanobiosensors in providing ultrahigh sensitivity, fast response, and high-degree multiplex detection, yet with minimum sample requirements is demonstrated. This chapter is intended to provide comprehensive updated information for people from a variety of backgrounds but with common interests in the fast-moving interdisciplinary field of nanobiotechnology.

Biosensing Techniques↗

Extended, relaxed, and condensed conformations of hyaluronan observed by atomic force microscopy.

The conformation of the polysaccharide hyaluronan (HA) has been investigated by tapping mode atomic force microscopy in air. HA deposited on a prehydrated mica surface favored an extended conformation, attributed to molecular combing and inhibition of subsequent chain recoil by adhesion to the structured water layer covering the surface. HA deposited on freshly cleaved mica served as a defect in a partially structured water layer, and favored relaxed, weakly helical, coiled conformations. Intramolecularly condensed forms of HA were also observed, ranging from pearl necklace forms to thick rods. The condensation is attributed to weak adhesion to the mica surface, counterion-mediated attractive electrostatic interactions between polyelectrolytes, and hydration effects. Intermolecular association of both extended and condensed forms of HA was observed to result in the formation of networks and twisted fibers, in which the chain direction is not necessarily parallel to the fiber direction. Whereas the relaxed coil and partially condensed conformations of HA are relevant to the native structure of liquid connective tissues, fully condensed rods may be more relevant for HA tethered to a cell surface or intracellular HA, and fibrous forms may be relevant for HA subjected to shear flow in tight intercellular spaces or in protein-HA complexes.

Aluminum Silicates↗

Suggested guidelines for the topographic evaluation of implant surfaces.

The bone anchorage components of commercially available oral implant systems differ in surface roughness by at least sixfold. Correct reporting of the surface roughness of implant systems is important, since one cannot exclude the possibility that surface roughness will influence clinical results. However, many confusing statements are found in the literature when the surface topography of implants is described. Different measuring instruments and techniques strongly influence the outcome of a topographic characterization. Furthermore, a screw-type design introduces problems for most measuring instruments. Without a standard procedure, it is generally impossible to compare values from one study with another. The aim of the present study was to suggest standards for topographic evaluation of oral implants in terms of measuring equipment, filtering process, and selection of parameters. It is suggested that the measuring instrument be able to measure all parts of a threaded implant if the investigation relates to such a design. Preferably, 3-dimensional measurements should be performed. On screw-type implants, tops, valleys, and flanks should be evaluated. At least 3 samples in a batch should be evaluated, filter size must be specified, and at least one of each height, spatial, and hybrid parameter should be presented.

Biocompatible Materials↗

Spatial distribution of the beta2 integrin (CD11b/CD18) and L-selectin (CD62L) adherence receptors on human neutrophils by Conventional Optical Scanning Microscopy (COSM).

Receptors such as CD62L and CD11b/CD18, are transmembrane glycoproteins which regulate leukocyte adhesive phenotype. Flow cytometry (FCM) makes it possible to assess a characterization of the cell activation level by receptor quantifying, but that technique does not integrate other factors of adherence regulation, such as spatial distribution and molecular conformation. Our study consisted in exploring the main adherence receptors on Polymorphonuclear Neutrophils (PMN) that were simultaneously analyzed by FCM and Conventional Optical Scanning Microscopy (COSM). FCM analysis showed that TNFalpha induce a decrease in CD62L expression and an increase in beta2 integrins. COSM analysis distinguished three stages of cellular distribution of CD11b/CD18 within resting PMN: most of them (about 80%) had homogeneous distribution (heterogeneous spots distributed over the entire cell surface), for 10-15% of the cells, there was a crown distribution around the widest cell diameter and in less that 10% of them receptor distribution was polarized. CD62L was in the form of heterogeneous spots distributed in a circle on the surface on non-stimulated PMN. PMN stimulation by TNFalpha was associated to a randomized clustering involving both selectin and beta2 integrin. Three-dimensional analysis elicited data not shown by quantitative cytometry. For a single averaged value of the density determined by FMC, various spatial distributions of adherence receptors are found on the surface of non-stimulated PMN. The characterization of the leukocyte adhesive phenotype has to integrate adherence receptors density as well as their spatial distribution.

CD18 Antigens↗

Near-field measurement of short-range correlation in optical waves transmitted through random media.

Two-dimensional near-field images of speckle patterns formed by optical waves transmitted through a disordered porous silica glass sample are measured. The corresponding 2D intensity correlation function, C, is extracted. The subwavelength spatial resolution of near-field microscopy allows us to resolve in the spatial distribution of C the expected subwavelength oscillations and to follow their dependence on the excitation wavelength. Finally, we deduce the effective refractive index of the material by fitting the theoretical spatial dependence of C to our experimental results.

Light↗

A multicellular spheroid-based drug sensitivity test by scanning electrochemical microscopy.

The respiratory activity of a multicellular spheroid was non-invasively monitored by scanning electrochemical microscopy (SECM) to investigate the anticancer drug sensitivity. The effects of the three anticancer drugs, cisplatin (CDDP), 5-fluorouracil (5-FU), and paclitaxel (TXL), were continuously evaluated based on respiratory activity for 5 days. The drug sensitivities obtained by SECM were higher compared to those evaluated by the spheroid volume and conformed to those evaluated by a conventional colorimetric assay. Our results show that the SECM-based assay directly correlates with the number of viable cells within the spheroid, whereas the spheroid volume does not necessarily correlate with the number of viable cells. Furthermore, the results obtained by spheroid culture (3-D) were compared to those of cells cultured in a flask (2-D) and within a collagen gel (3-D). The drug sensitivity of cells cultured in 2-D is more pronounced than that of the cells cultured in 3-D. Since the cellular proliferation status in a 3-D culture is similar to that in vivo, the drug sensitivity test performed in the spheroid culture will give meaningful results that can be extended to an in vivo application. A SECM-based assay is perfectly suitable to directly evaluate the drug sensitivity of the spheroid.

Antimetabolites, Antineoplastic↗

Microbiological studies of an anaerobic baffled reactor: microbial community characterisation and deactivation of health-related indicator bacteria.

This WRC funded project has studied the appropriateness of the ABR (anaerobic baffled reactor) for on-site primary sanitation in low-income communities. A 3,000 L pilot reactor was located at the Kingsburgh wastewater treatment plant south of Durban, South Africa. Feed to the reactor was raw domestic wastewater containing a significant proportion of particulate organic matter. The compartments of the ABR were routinely monitored for pH, COD, and gas production, among other physical-chemical determinants. The microbial population in each compartment was analysed by fluorescent in situ hybridisation, using general oligonucleotide probes for eubacteria and archeae and a suite of 10 genera or family specific probes. Scanning electron microscopy was conducted on the sludge fraction of each compartment. Mixed fractions from each compartment were also analysed for health-related indicator bacteria (total coliforms and E. coli). Results indicated that methanogenesis was not occurring to the expected extent in the latter compartments, and that this was probably due to a hydraulic load limitation. This contrasted with earlier studies on industrial effluent, for which the organic load was exclusively in soluble form. Inactivation of health-related indicator bacteria was less than 1 log, indicating the need for an additional post-treatment of the effluent to protect community health.

Bacteria, Anaerobic↗

Preparation of plasmid DNA in transfection complexes for fluorescence and electron spectroscopic imaging.

The aim of this project was to develop procedures necessary to study mechanisms of receptor mediated gene transfer by means of integrated microscopy. Plasmid DNA was incorporated into a transfection complex consisting of poly(L)lysine and transferrin to which the nuclear localization signal was conjugated. This complex was presented to cultured glioma cells. Preparation of the transfected DNA for imaging was pursued by two methods. In the first method tetramethylrhodamine, nanogold, and ferritin were linked through streptavidin to the biotinylated plasmid DNA. Trafficking of the fluorescent derivatives was studied in living cells with fluorescence microscopy. Then, selected cells were rapidly cryo-immobilized. Ultra-structural distribution of the transfected DNA was imaged with energy filtering transmission electron microscopy. In the second method, the unmodified transfected DNA was detected in cryo-immobilized cells by in situ polymerase chain reaction and in situ hybridization. For laser scanning fluorescence microscopy probes were labeled with tetramethylrhodamine. For ultrastructural analysis by electron spectroscopic imaging, probes containing incorporated digoxigenin were labeled with anti-digoxigenin boronated antibodies. Based upon the developed procedures, it has been demonstrated that the presence of the nuclear localization signal in the transfection complex resulted in rapid nuclear import of the transfected DNA.

Cryopreservation↗

Nanowire probes for high resolution combined scanning electrochemical microscopy - atomic force microscopy.

We describe a method for the production of nanoelectrodes at the apex of atomic force microscopy (AFM) probes. The nanoelectrodes are formed from single-walled carbon nanotube AFM tips which act as the template for the formation of nanowire tips through sputter coating with metal. Subsequent deposition of a conformal insulating coating, and cutting of the probe end, yields a disk-shaped nanoelectrode at the AFM tip apex whose diameter is defined by the amount of metal deposited. We demonstrate that these probes are capable of high-resolution combined electrochemical and topographical imaging. The flexibility of this approach will allow the fabrication of nanoelectrodes of controllable size and composition, enabling the study of electrochemical activity at the nanoscale.

Journal Article↗

Interactions between hydrophobic and hydrophilic silicon surfaces using a tapered probe in near-field scanning optical microscopy.

We examined the tip-sample interaction of a Si(111) surface processed with HF solution. The interaction length of the probe with its hydrophobic surface is approximately 30, and the frequency responses show an interaction force of elastic type. By contrast, the damping curve on a hydrophilic surface, Si with thin oxide, operates on a longer length of 100. Here, the frequency responses indicate an interaction force of the viscous type.

Journal Article↗

Scanning and transmission electron microscopy, and electron probe analysis of the interface between implants and host bone. Osseo-coalescence versus osseo-integration.

Bioinert materials (e.g., alumina implants) and bioactive ceramics (e.g., calcium phosphate ceramics, glass-ceramics) are now extensively used in dentistry. However, the physico-chemical interactions at the interfaces between the implant and the host bone are poorly understood. The purpose of this study was to define the interactions at these interfaces using a combination of analytical techniques: light microscopy, scanning and transmission electron microscopy, electron probe microanalysis, X-ray microradiography, X-ray diffraction, and infrared spectroscopy. Bioinert (pure titanium) and bioactive materials (hydroxyapatite, beta-tricalcium phosphate and biphasic calcium phosphate) were implanted in dogs, and the implants, recovered after various periods of implantation, were analyzed. The results demonstrated the following: the bioactive materials interact with the biological fluid and the living tissues in a specific manner. This process includes biodissolution/biodegradation, apatite crystal precipitation, and bone formation on the implant surface at the expense of the material. The results are discussed according to the limitations of the analytical techniques used. The medical and chemical word coalescence is suggested to describe the specific interactions of bioactive materials and interaction for the phenomenon of physical contact of the bioinert materials with the host bone.

Animals↗

Fast element mapping of titanium wear around implants of different surface structures.

The effect of unintended titanium release around oral implants remains a biological concern. The current study was undertaken to evaluate a new detection system of element mapping in biological probes. A new scanning electron microscopy-energy dispersive spectroscopy detection method was used to map the features of titanium contamination in peri-implant bone around implants with different surface structures. The amount of titanium wear was highest adjacent to titanium-plasma-sprayed surfaces, followed by sandblastered large grid acid-etched and smooth surfaces. A high sensitivity of titanium detection over large areas of bone tissue was observed. A high spatial resolution of titanium wear particles (20 nm) could be reached and correlated to the ultrastructural morphological features of peri-implant tissue. Cells adjacent to titanium wear revealed no signs of morphological alterations on a nanoscale level at early periods of implant/bone interaction. The new technique may serve as a fast and effective tool to evaluate titanium release effects in biological probes.

Acid Etching, Dental↗

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy.

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g, washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 mM ammonium acetate, 300 mM sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10-11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.

Cell Compartmentation↗

Mechanism of corrosion inhibition of AA2024 by rare-earth compounds.

The mechanism of corrosion protection of the widely used 2024-T3 aluminum alloy by cerium and lanthanum inhibitors in chloride media is described in detail in the present work. The corrosion process was investigated by means of scanning Kelvin probe force microscopy (SKPFM), in situ atomic force microscopy, and scanning electron microscopy coupled with energy dispersive spectroscopy. Employment of the high-resolution and in situ techniques results in a deep understanding of the details of the physical chemistry and mechanisms of the corrosion processes. The applicability of the SKPFM for mechanistic analysis of the effect of different corrosion inhibitors is demonstrated for the first time. The inhibitors under study show sufficient hindering of the localized corrosion processes especially in the case of pitting formation located around the intermetallic S-phase particles. The main role of Ce(3+) and La(3+) in the corrosion protection is formation of hydroxide deposits on S-phase inclusions buffering the local increase of pH, which is responsible for the acceleration of the intermetallics dealloying. The formed hydroxide precipitates can also act as a diffusion barrier hindering the corrosion processes in active zones. Cerium nitrate exhibits higher inhibition efficiency in comparison with lanthanum nitrate. The higher effect in the case of cerium is obtained due to lower solubility of the respective hydroxide. A detailed mechanism of the corrosion process and its inhibition is proposed based on thermodynamic analysis.

Journal Article↗

Waveguide analysis of heat-drawn and chemically etched probe tips for scanning near-field optical microscopy.

We analyze two basic aspects of a scanning near-field optical microscope (SNOM) probe's operation: (i) spot-size evolution of the electric field along the probe with and without a metal layer, and (ii) a modal analysis of the SNOM probe, particularly in close proximity to the aperture. A slab waveguide model is utilized to minimize the analytical complexity, yet provides useful quantitative results--including losses associated with the metal coating--which can then be used as design rules.

Computer-Aided Design↗