Particle development in apple juice determined by light scattering and electron microscopy

Light scattering was used to detect the kinetic development of particles in apple juices produced with and without oxidation and in procyanidin extracts prepared from these two juices. Particle size developed exponentially inboth oxidized and unoxidized juices, suggesting an enzymatic origin for the particle forming reactions. The procyanidin extract from oxidized juices produced particles which grew linearly, suggesting diffusion-controlled aggregation or coalescence of particles. Procyanidins from unoxidized juice showed no particle development over at least 60 days of storage. Electron microscopy showed particles similar to those seen previously and some newly described morphologies. The mechanism of haze particle development is discussed in the light of the light scattering and electron microscopic results.     

SPARK,a kernel of software programs for spatial reconstruction in electron microscopy

SPARK, an acronym for ‘SPAtial Reconstruction Kernel’, is the nucleus of a software library being developed for the three-dimensional (3-D) reconstruction of objects observed by the electron microscope. A unifying concept is used: the Fourier transform, known in several central sections, is resampled to obtain a 3-D Cartesian grid, which is inverted by a fast Fourier transform. This technique is used for both single-axis tilting (of 2-D periodic layers or of isolated objects) and for the random conical-tilt technique. The principles that make it possible to recover Cartesian grids in the two different geometries are illustrated and some preliminary results are reported. SPARK resamples the Cartesian grids with the use of a fast and efficient algorithm of Shannon interpolation developed by the authors. Compared to back-projection techniques the method shows a considerable improvement in execution time with no sacrifice in accuracy; it therefore allows the effects of a variety of parameters in a given reconstruction to be scrutinized in a reasonable time. Some new possibilities and future extensions of the library are briefly outlined.     

The preparation of cross-section specimens for transmission electron microscopy

The structure and chemistry of thin solid films are best studied by transmission electron microscopy (TEM) when they are viewed in cross-section—that is, when the surface normal of the film is made perpendicular to the electron beam. In this orientation, the substrate, the thin film layers, and the interfaces between them can be imaged either simultaneously or individually. Further, information from each of these regions remains distinct from that obtained from the others, eliminating the problems of superimposition that are a consequence of viewing a layered structure in the conventional manner (i.e., parallel to the surface normal). A technique for fabricating TEM specimens that can be viewed in cross-section is described here. Although the majority of our work is with silicon-based materials, the technique can be readily adapted to the study of other systems.     

Chemical polishing of palladium for transmission electron microscopy

Chemical polishing methods for thinning palladium for TEM are reviewed. The quality of the foils produced by the different solutions is compared and the possible influence of hydrogen absorption on the microstructure is discussed.     

Preparation of lithium specimens for transmission electron microscopy

A method of preparing undeformed thin lithium specimens for TEM is described. A solution of dehydrated methanol and toluene is used both for initial dishing of the foil by chemical polishing and also for final thinning. Under electron beam irradiation in the TEM, new pure Li crystals can grow out of the existing Li specimen. These in situ crystals can be used for the study of the microstructure and electronic structure of Li using TEM and electron energy loss spectrometry.     

A scanning electron microscopy specimen holder for viewing different angles of a single specimen

The specimen holder for scanning electron microscopy described herein allows a single specimen to be examined in any possible view and significantly improves object illumination. The specimen is glued to a fine pin and flexibly mounted on a double‐sided adhesive conductive pad on a rotatable pivot. A milled pot placed beneath the specimen acts as an electron trap. This provides a homogeneous black image background by minimizing noisy signals from the specimen's surroundings. Microsc. Res. Tech. 73:1073–1076, 2010. © 2010 Wiley‐Liss, Inc.     

Scanning electron microscopy of piliated Neisseria gonorrhoeae processed with hexamethyldisilazane

Piliated Neisseria gonorrhoeae are virulent and attach readily to some human mucosal cells. The study of interactions between piliated Neisseria gonorrhoeae and surface structures of eukaryotic cells in tissue culture requires consistent high resolution imaging in scanning electron microscopy (SEM). The combination of the fixatives glutaraldehyde, osmium, tannic acid, and uranyl acetate improves preservation of pili and other delicate structures. Following the critical point drying (CPD) process, pili bundles remained intact, but charging produced image distortion in most of the specimens. The use of hexamethyldisilazane (HMDS) with air drying substantially reduced charging and image distortion. Less contrast and greater resolution of pili bundles and surface structures of bacteria or tissue culture cells were obtained at magnifications of 10,000 or higher. As an alternative to CPD, HMDS processing of cell culture monolayers was simple and was more efficient when a large number of samples was processed.     

Preparation of high-quality ultrathin transmission electron microscopy specimens of a nanocrystalline metallic powder

This article explores the achievable transmission electron microscopy specimen thickness and quality by using three different preparation methods in the case of a high-strength nanocrystalline Cu-Nb powder alloy. Low specimen thickness is essential for spatially resolved analyses of the grains in nanocrystalline materials. We have found that single-sided as well as double-sided low-angle Ar ion milling of the Cu-Nb powders embedded into epoxy resin produced wedge-shaped particles of very low thickness (<10 nm) near the edge. By means of a modified focused ion beam lift-out technique generating holes in the lamella interior large micrometer-sized electron-transparent regions were obtained. However, this lamella displayed a higher thickness at the rim of ≥30 nm. Limiting factors for the observed thicknesses are discussed including ion damage depths, backscattering, and surface roughness, which depend on ion type, energy, current density, and specimen motion. Finally, sections cut by ultramicrotomy at low stroke rate and low set thickness offered vast, several tens of square micrometers uniformly thin regions of ～10-nm minimum thickness. As major drawbacks, we have detected a thin coating on the sections consisting of epoxy deployed as the embedding material and considerable nanoscale thickness variations.     

Sensitivity limits of strain mapping procedures using high-resolution electron microscopy

Strain detection limits achieved by several approaches of strain mapping differing in the procedure of determining positions of intensity maxima have been compared. Algorithms which simultaneously take into account the two-dimensional intensity distribution around maximum positions yield similar detection limits and are superior to those performing successive one-dimensional cuts. Investigations of the effect of image recording imply that the use of photographic plates is superior to direct image recording using a slow-scan CCD camera because of larger sampling rates and a larger field of view. It is finally shown that the choice of specimen preparation technique substantially affects the strain detection limit with cleaved samples yielding results which are a factor of about three better than ion-milled samples.     

Measurement of specimen charging in scanning electron microscopy with a kelvin probe

A vibrating Kelvin probe in form of a platinum wire loop is used to measure the surface potential U_s on electron-irradiated free-floating metal and insulator specimens as a function of electron energy E. This allows an accurate measurement of the critical electron energy E₂ for no charging. At energies below E₂, the positive charging increases with decreasing energy to U_s=2–5 eV at E=0.5 keV and switching off the collector bias of the Everhart-Thornley detector. A two-to threefold increase of U_s is observed when the bias is switched on. For E > E₂, the strong increase of a negative surface potential can be measured. Insulating films free-supported on a conductive substrate show a steep decrease to small positive and negative U_s when the film thickness becomes lower than the electron range at a critical energy E₃ > E₂. At insulating specimen the temporal decrease of charging can be measured when the electron beam is switched off.     

Polylysine as an adhesive for the attachment of nanoplankton to substrates for electron microscopy

Nanoplankton can be readily attached to polylysine-coated substrates for examination by transmission and scanning electron microscopy. This simple technique gives high yields of organisms which are consistently better preserved and less obscured by detritus than samples dried directly on to substrates.     

Improved specimen holders for the scanning electron microscopy of insects and other small objects

Two specimen holders for use in scanning electron microscopy (SEM) of insect and other specimens glued to triangular cardboard points are described. They have important advantages over standard metal stub mounts. Diverse, precisely orientated, viewing angles are possible using single specimens, which can afterwards be re-pinned for return to the collection.     

Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.     

A balanced technique for preparation of specimens from pathogenicity studies for scanning electron microscopy

This paper reports our experiences with preparing delicate biological specimens for scanning electron microscopy. Three different washing methods were evaluated: One method allowed the analysis of the location of the bacterium Mycoplasma mobile on piscine gill epithelium and the optimal evaluation of histopathologic changes caused by this microbe. These results were achieved when specimens were washed three times in a cacodylic acid buffer after completion of the in vitro infection experiment in gill explant cultures. We also found that of three different concentrations of glutaraldehyde, a fixation with a 1.5% solution was sufficient to achieve excellent structural preservation, even without using post fixation in osmium tetroxide. Furthermore, this study showed that the use of acetone-carbon dioxide in the critical point drying procedure resulted in well-preserved piscine gill epithelium and mycoplasmas. Finally, long-term storage of tissue specimens in 0.1 M cacodylic acid buffer is possible if the buffer is changed on a monthly basis to avoid growth of unwanted microorganisms, such as fungi.     

Seed morphoanatomy of the genus Passiflora L. (Passifloraceae) by scanning electron microscopy and light microscopy

Morphoanatomical analysis of seeds contributes to knowledge of the development of seedlings and identification of species, as well as supporting conservation studies. The conservation of the species belonging to the Passiflora genus is crucial due to of the threats to the genetic resources of these species. Thus, the objective of this study was to morphoanatomically characterize Passiflora seeds, verify possible injuries to the tissues after cryopreservation and thus contribute to the conservation strategies of the species of this genus. Initially, seeds of Passiflora coccinea, P. edulis, P. gibertii, P. maliformis, P. morifolia, P. setacea, P. suberosa, and P. tenuifila collected from the Passion Fruit Active Germplasm Bank of the Embrapa Cassava and Fruits research unit (Embrapa Mandioca e Fruticultura) were analyzed. Then, their length, width and thickness, shape of the base and tip, and ornamentations present on the body and edge of the seeds were evaluated. The seeds of the species were placed in cryotubes and immersed in liquid nitrogen to assess possible cryoinjuries. The tegument and tissues of the seeds were examined by scanning electron microscopy. The seeds had varied biometric data, with average values of 4.63 mm for length, 3.28 mm for width, and 1.51 mm for thickness. Six ornamentation types were observed: reticulate for the species P. coccinea; finely reticulate for P. edulis; foveolate reticulate for P. gibertii and P. setacea; alveolate reticulate for P. maliformis and P. tenuifila; coarsely reticulate for P. morifolia; and falsifoveolate reticulate for P. suberosa. Some seeds suffered tegument cracks due to the freezing in liquid nitrogen, but without physiological damages to the embryo and endosperm. The cryopreservation of the seeds in the presence of the tegument significantly reduced the cryoinjuries caused to the embryo. Cryopreservation can be promising for long-term conservation of passion fruit seeds.     

Characterization of tribological surfaces by scanning electron acoustic microscopy

Scanning electron acoustic microscopy, a useful new technique for analyzing wear surfaces, can be carried out in a modified scanning electron microscope. With this technique the thermal and elastic properties of surfaces can be imaged and surface and subsurface cracks throughout the specimen can be detected from their effect on the image. The technique is valuable in the study of surface fatigue wear, the identification of cracks likely to lead to spalling and the mapping of alloy phases in the upper 5–10 μm of the wear surface. Some examples illustrate the origin and interpretation of scanning electron acoustic micrographs and the use of the technique in the study of wear.     

Transmission electron microscopy of interfaces in structural ceramic composites

Ceramic composites based either on a particulate, fibre or a lamellar architecture are potentially useful as damage-tolerant high-temperature engineering materials. The ability of the interfaces in such systems to deflect cracks is vital to the damage tolerance of these materials. Transmission electron microscopy techniques enable the chemical and physical characterization of these interfaces, providing information on interlayer thicknesses, chemical species, local bonding and the microstructural features which give rise to the interfacial properties, thereby enabling a full understanding not only of composites after processing, but also after exposure to aggressive environments such as air at high temperature. Examples of the application of transmission electron microscopy to all three composite architectures are described.     

Silica-rubber microstructure visualised in three dimensions by focused ion beam-scanning electron microscopy

A focused ion beam-scanning electron microscope (FIB-SEM) technique for three-dimensional reconstruction and representation of material microstructures was applied to a silica-filled synthetic rubber for the first time. Backscattered electron imaging allowed differentiation between rubber matrix, silica filler and zinc oxide (used as an activator for the sulphur vulcanisation reaction). Subsequent image processing allowed three-dimensional isosurface model generation of the particulate structure within the rubber composite and separation of zinc oxide from the silica filler. The potential for development and application of this technique using finite element analysis modelling is also highlighted.     

On the size of small protein particles determined by electron microscopy of unidirectionally shadowed freeze-etched preparations

The influence exerted by the thickness of the deposited metal layer and the ionic strength of the solution on the apparent size of particles of bovine serum albumin in unidirectionally shadowed freeze-etch preparations of spray-frozen specimens was investigated. It appeared that the size increase due to shadowing is nearly twice the thickness of the deposited metal layer. Apparent particle size was shown to increase linearly with the inverse square root of the ionic strength of the solution. At ionic strength 0·001 the particles appeared about 30% larger than at infinite ionic strength.