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PubMed1999

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↗
PubMed2006

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↗
PubMed2006

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↗
PubMed1994

Preparation of unfixed and undecalcified frozen sections of adult rat periodontal ligament during experimental tooth movement.

The upper first molars of adult male rats were moved for 7 days and unfixed, undecalcified frozen sections of the molar periodontal ligament were prepared and observed. The upper jaws of the rats were immersed rapidly in liquid nitrogen and sectioned with a cryostat using a super hard knife. Five micrometer serial sections were cut, collected, freeze-dried and observed with both light and scanning electron microscopy. Electron probe microanalysis (EPMA) was also performed on the sections. On the tension side of the periodontal ligament, periodontal fibers were stretched and the osteoblasts were aligned on the osteoid, which showed metamasia with the toluidine blue stain. On the pressure side where the periodontal ligament was extremely compressed, tissue degeneration was caused by tooth movement and the osteoclasts were observed on the bone surface adjacent to the degenerating tissues. Scanning electron microscopy revealed a network arrangement of the collagen fiber bundles on the tension side, but not on the pressure side of the periodontal ligament. The spectrum obtained from EPMA of the osteoid demonstrated X-ray (Ka) peaks of Na, P, S, K and Ca.

Animals↗
PubMedDate not supplied

Diffraction of circularly polarized light from near-field optical probes.

Diffracted fields from 100-nm aperture near-field scanning optical microscopy (NSOM) probes and uncoated tapered fibres are measured and analysed. Using a solid angle scanner, the two-dimensional intensity distribution and polarization state of the diffracted light are resolved experimentally. Polarization analyses show that circularly polarized input light does not maintain its polarization state for all diffraction angles, and is completely filtered into linearly polarized light at large polar diffraction angles. This drastic decomposition originates from the vector nature of light diffracted by the sub-wavelength aperture. There is a fundamental difficulty in generating circularly polarized light near the aperture of NSOM probes owing to polarization-dependent diffraction in the near-field regime. This is illustrated by the Bethe-Bouwkamp model using circularly polarized input light.

Journal Article↗
PubMed1990

[Studies on gallium alloys for dental restorations. 2. Electron probe microanalysis for hardened gallium alloys].

Hardened gallium alloys were analyzed by scanning electron microscopy (SEM) and electron probe microanalysis (EPMA). The cores, matrix and white irregular shape phases were visible with SEM. The round or irregular shape cores contained Ag, Pd, Cu and Zn but did not contain Ga and In. The dark irregular shape matrix, which surrounded the core, consisted of Ga-Ag, Ga-Pd, Ga-Cu and Ga-Sn phases. However, the amount of Ga-Sn phase was very low. The white phase in the matrix was Ag-In phase.

Dental Alloys↗
PubMed1998

Two-photon laser-scanning microscopy: tests of objective lenses and Ca2+ probes.

The characteristics of objective lenses and Ca2+-sensitive probes were examined for imaging with a two-photon laser-scanning microscope (TP-LSM). The brightness of the images of beads taken by different objectives greatly varied and depended predominantly on their numerical aperture (NA) and less on transmittance and chirping effects. Lateral and axial resolutions, dx and dz, defined as the half decay length of fluorescence intensity of the image of a spherical bead (0.3 m) were 0.12 and 0.42 microm (objective; 40x/0.75). They are far better than those of confocal microscopes (0.3 and 1.5 microm, respectively) measured similarly (Kuba et al., 1994). dx linearly increased with an increase in 1/NA, while dz linearly increased with an increase in n/(NA)2 (n, refractive index) except for an objective of large NA (1.3). The coverslip compensation of objective lenses greatly affected the shape of the X-Z scanned images of 5.0 microm beads as well as resolutions, indicating a large effect of spherical aberration. Two-photon excitation spectra of Ca2+-sensitive fluorescent probes, indo-1, fura-2 and Oregon Green BAPTA-1, lied in a wavelength range shorter than twice that activated by one-photon absorption, while emission spectra were unchanged. Three-dimensional images of a cultured hippocampal neurone loaded with Oregon Green BAPTA-1 showed fine structures of spines, dendrites and axons, while imaging with FM1-43 localized presynaptic boutons and demonstrated synaptic vesicle turnover. Dyes bleached little during the recording of 100 sectioned images. These characteristics of TP-LSM as well as its ability to image deeper tissues provide excellent means to study dynamic, spatial changes in intracellular substances and structures. To achieve the good performance of a TP-LSM, however, the relevant usage of appropriate objectives and fluorescent probes are required.

Animals↗
PubMed1996

Bone-bonding behavior of plasma-sprayed coatings of BioglassR, AW-glass ceramic, and tricalcium phosphate on titanium alloy.

The bone-bonding behavior of three kinds of bioactive ceramics coated on titanium alloy by the plasma-spray technique was investigated. Titanium alloy (Ti-6A1-4V) coated with BioglassR (45S5), apatite-wollastonite containing glass ceramic (AW), or beta-tricalcium phosphate (TCP) was prepared, and rectangular specimens were implanted into the tibial bones of mature male rabbits, which were sacrificed 8 or 24 weeks after implantation. The tibiae containing the implants were dissected out and subjected to detachment tests to measure the failure load. The bone-implant interface was investigated by Giemsa surface staining, contact microradiography, and scanning electron microscopy-electron probe microanalysis (SEM-EPMA). Eight weeks after implantation, the failure loads for implants coated with BioglassR, AW, and TCP were 1.04 +/- 0.94, 2.03 +/- 1.17, and 3.91 +/- 1.51 kg, respectively, and 24 weeks after implantation, the respective failure loads were 2.72 +/- 1.33, 2.39 +/- 1.30, and 4.23 +/- 1.34 kg. Failure loads of AW- and TCP-coated implants did not increase significantly with time. After the detachment test, breakage of the coating layer was observed. Bioactive ceramics can act as stimulants that induce bonding between bone and metal implants. However, failure load of metal implants coated with the bioactive ceramics was lower than that of bulk AW or TCP. It appears impossible to obtain a higher failure load using a bioactive-ceramic coating on titanium alloy. Histologically, the coating layer was found to become detached from the metal implant and the bone tissue bonded to the coating layer. SEM-EPMA observation revealed breakage of the coating layer, although bonding between bone and the coating layer was evident. A Ca-P-rich layer was observed at the interface between bone and the AW coating, and a Ca-P-rich and a Si-rich layer were observed at the interface between bone and the BioglassR coating. For clinical application, it would seem better to use coated metal implants for short-term implantation. However, there is a possibility of breakage of the coating layer because of both dissolution of the bioactive ceramic and mechanical weakness at the interface between the coating layer and the metal implant.

Alloys↗
PubMed1976

Calcification of alpha-elastin coacervates: a bulk property of elastin.

Scanning electron microscopy and electron probe microanalysis studies are reported on thin sections of calcified coacervates of alpha-elastin. It is found that the capacity of elastin coacervates to initiate calcification is a bulk property of the coacervate and not limited to the serum-coacervate interface, that the calcium phosphate deposits act to bind the protein units together and slow the dissolution and spreading of the coacervate as it floats on an airwater interface, and that, within the limits of detectability, there is no involvement of sulfur. As the charged groups of alpha-elastin had been blocked, the initiation of deposition is due to neutral sites in the protein which are tightly bound to the calcium phosphate deposits.

Animals↗
PubMedDate not supplied

Domain formation in thin lipid films probed with near-field scanning optical microscopy.

High-resolution near-field scanning optical microscopy (NSOM) fluorescence and topographic images of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers doped with a fluorescent dye are presented. DPPC monolayers are deposited onto mica substrates from the air-water interface at several surface pressures using the Langmuir-Blodgett technique. Sub-diffraction limit phase domain structures are observed in both fluorescence and topographic NSOM images of the lipid films. The morphology of the resulting monolayers depends strongly on the surface pressure and composition of the subphase used in the film transfer. Mechanisms for lipid domain formation and growth are discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2004

Probing photonic and optoelectronic structures by apertureless scanning near-field optical microscopy.

This report presents the Apertureless Scanning Optical Near-Field Microscope as a powerful tool for the characterization of modern optoelectronic and photonic components with sub-wavelength resolution. We present an overview of the results we obtained in our laboratory over the past few years. By significant examples, it is shown that this specific probe microscopy allows for in situ local quantitative study of semiconductor lasers in operation, integrated optical waveguides produced by ion exchange (single channel or Y junction), and photonic structures.

Algorithms↗
PubMed2006

Characterization of microfabricated probes for combined atomic force and high-resolution scanning electrochemical microscopy.

A combined atomic force and scanning electrochemical microscope probe is presented. The probe is electrically insulated except at the very apex of the tip, which has a radius of curvature in the range of 10-15 nm. Steady-state cyclic voltammetry measurements for the reduction of Ru(NH3)6Cl3 and feedback experiments showed a distinct and reproducible response of the electrode. These experimental results agreed with finite element simulations for the corresponding diffusion process. Sequentially topographical and electrochemical studies of Pt lines deposited onto Si3N4 and spaced 100 nm apart (edge to edge) showed a lateral electrochemical resolution of 10 nm.

Journal Article↗
PubMed2004

Application of integrated SECM ultra-micro-electrode and AFM force probe to biosensor surfaces.

The integration of scanning electrochemical ultra-micro-electrode (UME) with atomic force microscope cantilever probe have been achieved by using a homemade photolithography system. A gold-film-coated AFM cantilever was insulated with photo resist coating and a pointed end of the AFM probe was opened by illuminating with maskless arbitrary optical micro-pattern generator. To realize precise control of probe sample distance constantly, the resulting scanning electrochemical microscopy (SECM)-AFM probe was operated using a dynamic force microscopy (DFM) technique with magnetic field excitation. From a steady-state voltammetric experiment, the effective electrode diameters of the probes thus prepared were estimated to be from 0.050 to 6.2 microm. The capability of this SECM-AFM probe have been tested using gold comb in the presence of Fe(CN)(6)(3-). The simultaneous imaging of the topography and electrochemical activity of the strip electrode was successfully obtained. We also used the SECM-AFM to examine in situ topography and enzymatic activity measurement. Comparison of topography and oxidation current profiles above enzyme-modified electrode showed active parts distribution of biosensor surface.

Biosensing Techniques↗
PubMed2004

Observation of polarization property in near-field optical imaging by a polarization-maintaining fiber probe.

We fabricated an original near-field scanning optical microscopy (NSOM) fiber probe made of polarization-maintaining and attenuation-reducing (PANDA)-type polarization-maintaining optical fiber, and observed the polarization property of propagation light in a polymer optical waveguide. The distribution of the transmission coefficient in polarization angles through this NSOM probe showed that the linear polarization is maintained in the two crossing directions: the fast and slow axes. The polarization degree parallel to the slow axis decreases from 1000:1 to 2:1 by bending the fiber probe and the decrease is independent of the bending direction. Using this PANDA-type NSOM probe, we investigated the polarization property of periodic intensity modulation. It was found that the intensity modulation was observed clearly with the electric vector parallel to the radius direction of the waveguide, but was observed vaguely with the electric vector perpendicular to the radius direction.

Journal Article↗
PubMed1991

Cytoskeleton in microridges of the oral mucosal epithelium in the carp, Cyprinus carpio.

Microridges produce a characteristic fingerprint-like pattern on the surface of fish oral mucosa. The cytoskeleton in these microridges was examined by immunofluorescence microscopy and transmission electron microscopy after detergent extraction and decoration with myosin subfragment 1. The effect of cytochalasin B on microridges was probed with scanning electron microscopy. Immunofluorescence microscopy revealed that actin filaments were present throughout the periphery of the epithelial cells and were especially localized beneath the free surface of the epithelium. In thin sections treated with Triton X-100, the majority of filaments in the microridges and their bases were found to be actin filaments and a plexus of keratin filaments that underlay the network of actin filaments. A part of the plexus of keratin filaments entered the microridges. After extraction with Triton X-100 and decoration with myosin subfragment 1, decorated actin filaments were found in the microridge cores, connected to the keratin filaments. The keratin filaments aggregated in the pattern of microridges and a few of them protruded into the microridges. Treatment with cytochalasin B caused microridges to disappear or to become thinner and lower or to change short or microvillus-like microridges. When most microridges disappeared, the surface of the superficial cells was prominently swollen, but the cell boundaries were fastened, and the microridges in the periphery were preserved. On the basis of these observations, the possible roles of actin and keratin filaments in the maintenance and the formation of microridges are discussed.

Actins↗