Inhibition of melanin synthesis pathway by tricyclazole increases susceptibility of Fonsecaea pedrosoi against mouse macrophages

Fonsecaea pedrosoi produces melanin, a pigment related to virulence in pathogenic fungi. To understand the involvement of melanin in the protection of fungi, the authors used tricyclazole to inhibit the melanin pathway in F. pedrosoi. Experiments of pigmentation suggested that F. pedrosoi uniquely produces dihydroxynaphthalene-melanin. Pigments produced on cultures modified or not with tricyclazole were extracted by an alkali-acid method and submitted to infrared and ion exchange chromatography analysis; also cytochemistry analysis for cationized ferritin of whole cells was carried out. This group of experiments showed that the tricyclazole treatment on F. pedrosoi produced a melanin-like pigment, but less negatively charged and with less affinity for iron ions than that without the tricyclazole treatment, and this in turn lead to a less negatively charge cell wall surface. Scanning electron microscopy of such pigments showed that the melanin from control cultures maintained their hyphae-like structures, which have been described as "melanin-ghosts," whereas the tricyclazole pigment showed an amorphous surface. Interaction of conidia from cultures of F. pedrosoi, modified by tricyclazole or not, with peritoneal activated macrophages suggested that tricyclazole causes higher association of fungus with macrophages, weakens the fungus capacity to destroy the macrophages, and diminishes the resistance to dry fracture procedures on samples prepared for high resolution scanning electron microscopy.     

Methods for characterizing plant fibers

The effectiveness of different microscopy techniques for measuring the dimensions of ultimate fibers from harakeke (Phormium tenax, New Zealand flax) was investigated using a factorial experimental design. Constant variables were geographical location, location of specimens along the leaf, season (winter), individual plant, a fourth leaf from a north-facing fan, age of plant, and cultivars (two). Experimental variables were microscopy techniques and measurement axis. Measurements of width and length of harakeke ultimate fibers depended on the microscopic preparation/technique used as well as the cultivar examined. The best methods were (i) transverse sections of leaf specimens 4 microm thick, embedded in Paraplast and observed using light microscopy, and (ii) nonfixed ultimate fibers observed using scanning electron microscopy.     

Characterization of the cutting edge of glass knives for ultramicrotomy by scanning force microscopy using cantilevers with a defined tip geometry

The geometry of glass knife edges for ultramicrotomy was studied with nanoscale resolution using scanning force microscopy (SFM) in the contact mode. The local shape of the cutting edge was estimated from single line profiles of the SFM topographic images by taking into account the exact radius of the ultrasharp silicon tip. The tip radius was estimated from secondary electron micrographs recorded at low voltage by field emission scanning electron microscopy (FESEM). The radius of the investigated cutting edges was found to be in range 5–20 nm. The results obtained illustrate that the combination of SFM and high resolution FESEM provides a unique means to determine precisely the radius of glass knives.     

Irregular snow crystals: structural features as revealed by low temperature scanning electron microscopy

For nearly 50 years, investigators using light microscopy have vaguely alluded to a unique type of snow crystal that has become known as an irregular snow crystal. However, the limited resolution and depth-of-field of the light microscope has prevented investigators from characterizing these crystals. In this study, a field-emission scanning electron microscope, equipped with a cold stage, was used to document the structural features, physical associations, and atmospheric metamorphosis of irregular snow crystals. The crystals appear as irregular hexagons, measuring 60 to 90 mm across, when viewed from the a-axis. Their length (c-axis) rarely exceeds the diameter. The irregular crystals are occasionally found as secondary particles on other larger forms of snow crystals; however, they most frequently occur in aggregates consisting of more than 100 irregular crystals. In the aggregates, the irregular crystals have their axes oriented parallel to one another and, collectively, tend to form columnar structures. Occasionally, these columnar structures exhibit rounded faces along one side, suggesting atmospheric metamorphoses during formation and descent. In extreme cases of metamorphoses, the aggregates would be difficult to distinguish from graupel. Frost, consisting of irregular crystals, has also been encountered, suggesting that atmospheric conditions that favor their growth can also occur terrestrially.     

The development of the modified Hurst orientation transform for the characterization of surface topography of wear particles

A modified Hurst orientation transform (HOT) method for characterization of wear particle surfaces is proposed and described in this paper. The method involves the calculation of self-affine Hurst coefficients in all directions and displays the calculated coefficient values in a form of rose plot. The calculation of individual Hurst coefficients, H, is based on the rescale range (r/s) analysis (r(d)/s∼ d ^H ). The rose plot is then used to obtain three texture surface parameters, i.e.: texture aspect ratio, texture minor axis and texture direction. The effectiveness of this modified HOT and resulting surface texture parameters was evaluated. The method was first applied to computer-generated images of isotropic and anisotropic particle fractal surfaces and then to field emission scanning electron microscope images of wear particles found in synovial joints. The ability of the surface parameters to reveal surface isotropy or anisotropy, measure roughness and determine the dominant direction of surface texture was assessed. The effects of measurement conditions such as noise, gain variations and focusing on the surface parameters were also investigated. The results demonstrate that the HOT and surface texture parameters developed can successfully be used in the characterization of wear particle surface topography. This revised version was published online in August 2006 with corrections to the Cover Date.     

Application of ion-beam etching techniques to the fine structure of biological specimens as examined with a field emission SEM at low voltage

The term “etching,” in electron microscopy, refers to the removal of specimen surface layers and includes chemical, electrolytic, and ion-beam methods. The ion-beam etching process is used to remove layers of a target material by bombarding it with ionized gas molecules. Recently, the method has been applied to the field of biological specimens; however, the practical procedures for such organic materials have not been developed. In the present study, we used an apparatus in which a beam of argon ions is collimated and focused by electrostatic lenses onto an appropriate target. We demonstrated the optimum conditions to observe biological specimens that were treated with osmium tetroxide and tannic acid. The specimens were examined uncoated at low accelerating voltage using a field emission scanning electron microscope. According to our experiments, when a biological specimen was observed under high-resolution conditions at over 50,000x magnification, the optimum condition of ion-beam etching consisted of an accelerating voltage of E = 1 keV and an ion-beam dose of It = 360 ～ 400 μA. min, depending on parts of the specimens. In order to decrease overetching, we had to choose factors such as E = 1 ～ 2 keV and It = 500 μA. min.     

The tetrahedral tip as a probe for scanning near-field optical microscopy at 30 nm resolution

The tetrahedral tip is introduced as a new type of a probe for scanning near-field optical microscopy (SNOM). Probe fabrication, its integration into a scheme of an inverted photon scanning tunnelling microscope and imaging at 30 nm resolution are shown. A purely optical signal is used for feedback control of the distance of the scanning tip to the sample, thus avoiding a convolution of the SNOM image with other simultaneous imaging modes such as force microscopy. The advantages of this probe seem to be a very high efficiency and its potential for SNOM at high lateral resolution below 30 nm.     

Limitations in tandem scanning confocal microscopy as a diagnostic tool for microbial keratitis

One potential application of tandem scanning confocal microscopy is the detection of in vivo pathogens. Our study of an experimental model of Acanthamoeba keratitis demonstrates that while this technology can successfully detect certain organisms, there are currently limitations. These limitations relate to instrument configuration, movement of either the tissue or the microscope, difficulty in reproducibly returning to the area of interest for serial examination, the lack of a distinctive morphology of some pathogens, and limited resolution of the microscope.     

Surface roughness measurements as a method for tribological characterisation of ball bearings

A series of experiments using steel or hybrid balls in an SNFA VEX25 type bearing was conducted, at a rotational speed of 50,000 rpm. The race material was 100Cr6 steel, whereas for the balls various steels, coatings, and ceramics were used. The different materials used for the balls as well as the method of lubrication (air/oil or grease) strongly influenced the surface degradation of the ball bearings. Hybrid bearings with ceramic balls showed very little wear of either the balls or the steel races, and so offer potential for high‐speed applications. The degradation of the bearings was examined using scanning electron microscopy, atomic force microscopy, and laser scanning profilometry. The disadvantages and advantages of these methods are given, along with the results of surface roughness measurements.     

Choosing the optimum accelerating voltage (EO) to visualize submicron precipitates with a field emission scanning electron microscope

With the advent of field emission scanning electron microscopes (FESEM), the observation of small phases in the 5 to 50 nm range seems to be possible at low accelerating voltage using backscattered electron imaging mode. In this context, it is important to understand the contrast of multiphased materials at such low energy. A Monte Carlo program to simulate electron trajectories of multiphased materials (CASINO) was used to compute electron backscattering images. Simulations of images for various compositions of spherical precipitates embedded in a homogeneous matrix as a function of precipitate size and accelerating voltage are presented. These simulations show the concept of an optimum accelerating voltage to maximize the contrast of electron backscattering images. The results presented in this paper show that the contrast of backscattering images of multiphased images in the scanning electron microscope is not only a function of the atomic number difference, but that it is also strongly related to the geometry and the size of the phases.     

Polymer-based materials to be used as the active element in microsensors: a scanning force microscopy study

Polymer-based materials can be incorporated as the active sensing elements in chemiresistor devices. Most of these devices take advantage of the fact that certain polymers will swell when exposed to gaseous analytes. To measure this response, a conducting material such as carbon black is incorporated within the nonconducting polymer matrix. In response to analytes, polymer swelling results in a measurable change in the conductivity of the polymer/carbon composite material. Arrays of these sensors may be used in conjunction with pattern recognition techniques for purposes of analyte recognition and quantification. We have used the technique of scanning force microscopy (SFM) to investigate microstructural changes in carbon-polymer composites formed from the polymers poly (isobutylene) (PIB), poly (vinyl alcohol) (PVA), and poly (ethylene-vinyl acetate) (PEVA) when exposed to the analytes hexane, toluene, water, ethanol, and acetone. Using phase-contrast imaging (PI), changes in the carbon nanoparticle distribution on the surface of the polymer matrix are measured as the polymers are exposed to the analytes in vapor phase. In some but not all cases, the changes were reversible (at the scale of the SFM measurements) upon removal of the analyte vapor. In this paper, we also describe a new type of microsensor based on piezoresistive microcantilever technology. With these new devices, polymeric volume changes accompanying exposure to analyte vapor are measured directly by a piezoresistive microcantilever in direct contact with the polymer. These devices may offer a number of advantages over standard chemiresistor-based sensors.     

A comparative study of thin coatings of Au/Pd, Pt and Cr produced by magnetron sputtering for FE-SEM

Visualization of structural details of specimens in field emission scanning electron microscopy (FE-SEM) requires optimal conductivity. This paper reports on the differences in conductive layers of Au/Pd, Pt and Cr, with a thickness of 1.5–3.0 nm, deposited by planar magnetron sputtering devices. The coating units were used under standard conditions for source–substrate distance, current, HT and argon pressure. Carbon films, deposited by high-vacuum evaporation on small, freshly cleaved pieces of mica, were used as substrate and mounted on copper grids for TEM and SEM inspection. Au/Pd, Pt and, to a lesser extent, Cr coatings varied in particle density, size and shape. Au/Pd coatings have a slightly more granular appearance than Cr and Pt coatings, but this is strongly dependent on the type of sputtering device employed. In FE-SEM images there is almost no difference in contrast and particle size between the Au/Pd layer and the Pt layers of a similar thickness. The nuclei of Au/Pd are rather small with almost no growth to the sides or in height, making Au/Pd coatings a good alternative to chromium and platinum for FE-SEM of biological tissues because of its higher yield of secondary electrons.     

Methanol as a rapid fixative for the investigation of plant surfaces by SEM

A rapid fixation/dehydration method of plant specimens for scanning electron microscopy is presented. Prior to critical-point drying (CPD) the specimens are immersed in pure methanol. Methanol incubation instantly fixes the elastically extended cell walls. Owing to this instant fixation, shrinking of the specimens is prevented, resulting in an improved preservation of cell dimensions comparable to in vivo conditions. The method is most suitable for plant epidermal surfaces. It avoids the time-consuming fixation/dehydration in routine investigation of plant surfaces prior to CPD, especially for delicate specimens.     

Intercellular junctions and the application of microscopical techniques: the cardiac gap junction as a case model

Intercellular junctions are fundamental to the interactions between cells. By means of these junctions, the activities of the individual cells that make up tissues are co-ordinated, enabling each tissue system to function as an integrated whole. In this review, the work of the authors on one specific type of junction—the cardiac gap junction—is presented as a case model to illustrate how the application of a range of microscopical methods, as part of a multidisciplinary approach, can help extend our understanding of cell junctions and their functions. In the heart, gap junctions form the low-resistance pathways for rapid impulse conduction and propagation, enabling synchronous stimulation of myocyte contraction. Gap junctions also form pathways for direct intercellular communication, a function of particular importance for morphogenetic signalling during development. The work discussed demonstrates some of the applications of techniques in electron microscopy, immunocytochemistry and confocal scanning laser microscopy to the understanding of the structural basis of the function of gap junctions in the normal adult heart, the developing heart and the diseased heart. Freeze-fracture electron microscopy of heart tissue prepared by rapid freezing techniques, in which excision-related structural damage to the cells is minimized or avoided, makes it possible to deduce the structure of the functioning gap junction in vivo. Gap junctions in hearts that are beating normally in the living animal until the very instant of freezing consist of connexons (transmembrane channels) organized in a quasicrystalline arrangement, not a ‘random’ arrangement as proposed in the original hypothesis on the structural correlates of gap junction function. Alterations in connexon arrangement occur in response to ischaemia and hypoxia, though the relationship of these to gap-junctional permeability is indirect. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera to synthetic peptides matching portions of the sequence of connexin43, the major gap-junctional protein reported in the heart, were raised. The specificity of the antisera was confirmed by dot blotting, Western blotting and by immunogold labelling of isolated gap junctions. One antiserum (that raised to residues 131–142) was found to be particularly effective as a cytochemical probe. An immunofluorescence labelling procedure for use with confocal scanning laser microscopy was developed to enable the three-dimensional precision mapping of gap junctions through thick slices of cardiac tissue. By exploiting the serial optical sectioning ability of the confocal microscope, we have succeeded in (1) elucidating the organization of gap junctions at the intercalated disc, (2) establishing temporal and spatial patterns of gap-junctional protein expression in embryogenesis that correlate with functional differentiation in subsets of cardiac cells, and (3) demonstrating abnormalities of gap-junction distribution and quantity that may contribute to the genesis of arrhythmias in ischaemic heart disease.     

Fabrication of a versatile substrate for finding samples on the nanometer scale

With increasing interest in nanometer scale studies, a common research issue is the need to use different analytical systems with a universal substrate to relocate objects on the nanometer scale. Our paper addresses this need. Using the delicate milling capability of a focused ion beam (FIB) system, a region of interest (ROI) on a sample is labelled via a milled reference grid. FIB technology allows for milling and deposition of material at the sub 20-nm level, in a similar user environment as a standard scanning electron microscope (SEM). Presently commercially available transmission electron microscope (TEM) grids have spacings on the order 100 μm on average; this technique can extend this dimension down to the submicrometre level. With a grid on the order of a few micrometres optical, FIBs, TEMs, scanning electron microscopes (SEMs), and atomic force microscopes (AFM) are able to image the ROI, without special chemical processes or conductive coatings required. To demonstrate, Au nanoparticles of ∼ 25 nm in size were placed on a commercial Formvar^®- and carbon-coated TEM grid and later milled with a grid pattern. Demonstration of this technique is also extended to bulk glass substrates for the purpose of sample location. This process is explained and demonstrated using all of the aforementioned analytical techniques.     

Adsorbate deformation as a contrast mechanism in STM images of biopolymers in an aqueous environment: images of the unstained,hydrated DNA double helix

A stable residual aggregate remains on a submerged gold surface after electrophoretic deposition of DNA. We present scanning tunnelling microscope (STM) images of these aggregates which show many objects with the geometry of DNA, clearly displaying the 3·4 nm helix pitch. These images are quite distinctive, and cannot be generated when the deposition technique is used without DNA in the buffer solution. A characteristic of these images is that the tip is observed to dip down over the DNA molecule at the same time as the apparent barrier height drops by a factor of about four. The tip displacement is accounted for by a model in which contrast is dominated by local fluctuations in the deformability of the adsorbate layer, a quantity deduced from measurements of the apparent barrier heights in air, water, over small molecule adsorbates, and over DNA.     

New method for characterizing paper coating structures using argon ion beam milling and field emission scanning electron microscopy

We have developed a new method for characterizing microstructures of paper coating using argon ion beam milling technique and field emission scanning electron microscopy. The combination of these two techniques produces extremely high-quality images with very few artefacts, which are particularly suited for quantitative analyses of coating structures. A new evaluation method has been developed by using marker-controlled watershed segmentation technique of the secondary electron images. The high-quality secondary electron images with well-defined pores makes it possible to use this semi-automatic segmentation method. One advantage of using secondary electron images instead of backscattered electron images is being able to avoid possible overestimation of the porosity because of the signal depth. A comparison was made between the new method and the conventional method using greyscale histogram thresholding of backscattered electron images. The results showed that the conventional method overestimated the pore area by 20% and detected around 5% more pores than the new method. As examples of the application of the new method, we have investigated the distributions of coating binders, and the relationship between local coating porosity and base sheet structures. The technique revealed, for the first time with direct evidence, the long-suspected coating non-uniformity, i.e. binder migration, and the correlation between coating porosity versus base sheet mass density, in a straightforward way.     

Advantages of stereo imaging of metallic surfaces with low voltage backscattered electrons in a field emission scanning electron microscope

High emission current backscattered electron (HC-BSE) stereo imaging at low accelerating voltages (≤ 5 keV) using a field emission scanning electron microscope was used to display surface structure detail. Samples of titanium with high degrees of surface roughness, for potential medical implant applications, were imaged using the HC-BSE technique at two stage tilts of + 3° and − 3° out of the initial position. A digital stereo image was produced and qualitative height, depth and orientation information on the surface structures was observed. HC-BSE and secondary electron (SE) images were collected over a range of accelerating voltages. The low voltage SE and HC-BSE stereo images exhibited enhanced surface detail and contrast in comparison to high voltage (> 10 keV) BSE or SE stereo images. The low voltage HC-BSE stereo images displayed similar surface detail to the low voltage SE images, although they showed more contrast and directional sensitivity on surface structures. At or below 5 keV, only structures a very short distance into the metallic surface were observed. At higher accelerating voltages a greater appearance of depth could be seen but there was less information on the fine surface detail and its angular orientation. The combined technique of HC-BSE imaging and stereo imaging should be useful for detailed studies on material surfaces and for biological samples with greater contrast and directional sensitivity than can be obtained with current SE or BSE detection modes.     

A procedure for increasing the contrast of biological specimens in edge-projection TEM

Rotary shadowing has been used to increase the image contrast of biological specimens during edge-projection imaging in the transmission electron microscope (TEM). In this imaging mode, biological specimens are adsorbed from aqueous solution onto a highly curved substrate and observed in a direction parallel to its surface. High contrast TEM images are obtained at 200 kV when a 1–3 nm layer of tungsten is thermally evaporated onto the substrate at an angle of about 9°. Individual adsorbates are clearly delineated by the smooth, continuous, and fine-grained tungsten layer that surrounds them. TEM images obtained with this technique can provide a unique view of biological adsorbates on metal, insulator or semiconductor substrates.