bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of high‐pressure freezing on plant microfilament bundles
Authors: Ding Biao , Turgeon Robert , Parthasarathy M. V.
Published: August 2011

Cell structures, particularly cytoskeletal elements, of tobacco (Nicotiana tabacum L. Var. Maryland Mammoth) leaf tissues were studied after high‐pressure freezing and freeze substitution. In general, the freezing quality was superb and all organelles including microtubules, single microfilaments and microtubule‐associated microfilaments were well preserved. However, when compared with those frozen with a propane jet freezer, the microfilament bundles in high‐pressure frozen cells often had a frayed or loosened appearance. We suggest that such an appearance is an artefact, presumably caused by the high pressure developed prior to freezing.
The new stereological tools in metallography: estimation of pore size and number in aluminium
Authors: Karlsson Lars M. , Cruz‐Orive Luis M.
Published: August 2011

A number of simple, unbiased and efficient methods are now available in stereology for estimating the number and size of arbitrary particles or voids in a material, with the only assumption that particles must be identifiable on serial sections or confocal planes through the material. In recent years, these methods have been developed and applied mainly in a biomedical context: this paper reviews and illustrates them with the aid of a metallographic example, namely the pore population of a sand‐cast aluminium alloy. Our goal is to convey the fact that stereology is sampling in three dimensions, and therefore its principles remain valid and applicable in no matter what context. The disector, the selector, and an indirect method to estimate the distance between two parallel planes of polish are thereby illustrated. It is also shown how to split the error variance of the estimator of the pore volume fraction (‘porosity’) into the three components due to blocks, sections within blocks and systematic point counting on sections.
The efficient and unbiased estimation of nuclear size variability using the ‘selector’
Authors: McMillan Anna‐Marie , Sørensen Flemming Brandt
Published: August 2011

The selector was used to make an unbiased estimation of nuclear size variability in one benign naevocellular skin tumour and one cutaneous malignant melanoma. The results showed that the estimates obtained using the selector were comparable to those obtained using the more time consuming Cavalieri‐disector approach. Employing ‘optical sections’, the selector was found to be between five and ten times more efficient than the Cavalieri‐disector method when using physical sections.
Diamonds are a cryosectioner's best friend
Authors: Michel M. , Gnägi H. , Müller M.
Published: August 2011

High‐pressure frozen Golden Delicious apple leaves were cryosectioned at low temperature with diamond knives. Good cryosections were obtained by optimizing the cutting parameters, i.e. sectioning temperature, mechanical stability of the sample, and sectioning velocity. Cutting artefacts were minimized by reducing the electrostatic interactions between the knife surface and the cryosection. This was accomplished by sectioning the sample in the presence of an ionization electrode. The ionization device, with a primary voltage of 7–8 kV, produces positively and negatively charged nitrogen ions which neutralize the surface charges of the knife and the section. This minimizes the friction on the knife surface and results in ultrathin sections without crevasses or knife marks. Compression of the sections could be minimized, but not avoided, by reducing the knife angle to 30°. Improved contrast of the frozen‐hydrated sections was obtained with the Zeiss EM 902 energy‐filter microscope operated in the zero‐loss mode.
Imaging and measurement of cytosolic free calcium in plant and fungal cells
Authors: Read N. D. , Allan W. T. G. , Knight H. , Knight M. R. , Malhó R. , Russell A. , Shacklock P. S. , Trewavas A. J.
Published: August 2011

Calcium plays a central role as a second messenger in plant and fungal cells and as such is involved in controlling numerous biological processes. Direct demonstration of signal‐response coupling via Ca2+requires the measurement and localization of changes in cytosolic free Ca2+, [Ca2+]i, during these processes in living cells. In recent years this has become possible with the introduction of a range of fluorescent dyes (e.g. Indo‐1 Fura‐2 and Fluo‐3) which have a high affinity and selectivity for free Ca2+. When used with recently developed microscope technologies (e.g. fluorescence ratio imaging or confocal scanning laser microscopy), subcellular localization and precise quantification of [Ca2+]idynamics in single cells can be achieved. This review describes the principles of [Ca2+]iimaging and measurement using fluorescent dyes, the equipment required to do it, the problems with botanical material and how they are being overcome, future developments for this approach in plant cell biology, and an entirely different strategy for the imaging and measurement of [Ca2+]iinvolving genetic transformation with the aequorin gene.
Confocal laser microscopy and three‐dimensional reconstruction of nucleus‐associated microtubules in the division plane of vacuolated plant cells
Authors: Lloyd Clive W. , Venverloo Catharina J. , Goodbody Kim C. , Shaw Peter J.
Published: August 2011

The way in which transvacuolar strands radiating from the cell nucleus reorganize to form the phragmosome, within which division occurs, has been thoroughly studied in epidermal explants of Nautilocalyx lynchii. In recent years it has been established that the movement of the nucleus into the centre of large vacuolated cells such as these, in preparation for division, involves actin filaments. In the present study, the appearance and gradual reorganization of nucleus‐associated microtubules (NAMTs) over the premitotic period is described. Epidermal explants fluorescently labelled with anti‐tubulin were optically sectioned by confocal scanning laser microscopy, the sections reconstructed by an image processing computer and projected as rotating stereo pairs. This revealed that the NAMTs are a major component of the phragmosome, and that they change from a radiating to a planar distribution concomitantly with the ‘bunching’ of cortical MTs to form the pre‐prophase band. The continuity of the two sets of MTs indicates that the band contains newly polymerized microtubules. Other recent studies on the division of vacuolated cells are reviewed and factors affecting the alignment of the division plane are discussed.
Complexity in the spatial localization and length distribution of plant cell‐wall matrix polysaccharides
Authors: McCann Maureen C. , Wells Brian , Roberts Keith
Published: August 2011

Distinct polysaccharide fractions can be obtained from onion cell walls by sequential extraction methods. Pectins and xyloglucan molecules from these fractions have been imaged by electron microscopy and their lengths measured. Unexpectedly, instead of a simple distribution of lengths, both classes of matrix polysaccharide showed clear evidence of length periodicity, suggesting block polymer construction using subunits about 30 nm long, each containing a minimum of about sixty backbone sugar residues. These results are discussed in the light of our observation that the average hemicellulose cross‐link between cellulose microfibrils in the wall is also about 30 nm long, and that pectin forms a co‐extensive network with the cellulose/xyloglucan network. Using monoclonal antibodies to pectin we have further shown that even within a thin primary cell wall, containing only a few lamellae, distinct zones of pectin distribution are detectable, thus reinforcing our notion of the importance of domains in wall architecture. The chemical heterogeneity detected amongst pectins indicates that in a primary cell wall each lamella of cellulose microfibrils may be in a unique matrix environment.
Self‐incompatibility: insights through microscopy
Authors: McFadden G. I. , Anderson M. A. , Bönig I. , Gray J. E. , Clarke A. E.
Published: August 2011

The contributions of microscopy to our understanding of gametophytic self‐incompatibility in the ornamental tobacco Nicotiana alata are reviewed. Self‐incompatibility is a genetically based system which prevents self‐fertilization and thereby promotes outbreeding in plants. Pollen bearing an S‐allele identical to either of the S‐alleles in the female sporophytic tissues is rejected. Several S‐allele‐specific style glycoproteins have been isolated from Solanaceous species. All have ribonuclease activity and are termed S‐RNases. The cDNA clones encoding N. alata S‐RNases have been used in in situ hybridization experiments which show that the genes are expressed in the transmitting tract of the style and in the epidermis of the placenta. Immunocyto‐chemistry using an antibody to S2‐RNase demonstrates extracellular accumulation of the enzyme in the same tissues. These tissues are the pathway through which pollen tubes must grow to reach the ovules. The microscopy data are congruent with a model in which self‐pollenation is susceptible to an allele‐specific cytotoxin produced by the sporophyte.
Ultrastructural localization of titanium in chemically modified chrysotile asbestos
Authors: Berghmans Patrick , Jacob Wim , Adams Freddy
Published: August 2011

The ultrastructural localization of Ti in chemically modified chrysotile fibres has been studied. TiCl3‐treated fibres were analysed by electron energy‐loss spectroscopy and electron spectroscopic imaging (ESI) along directions parallel and perpendicular to the fibre axis. ESI of ultrathin sections of conventionally embedded chrysotile fibres was made possible with the use of a cryo‐stage. The ESI images observed in fibre cross‐sections revealed that the Ti is located not only at the surface of the fibres but also inside the fibril tubes. This combination of microanalysis and imaging in the analytical electron microscope is the most suitable technique for relating the microchemistry and microstructure on a submicrometre scale.
The application of electron spectroscopic imaging for quantification of the area fractions of calcium‐containing precipitates in nervous tissue
Authors: Körtje Karl‐Heinz , Körtje Doris
Published: August 2011

Energy‐filtering transmission electron microscopy has been applied to the quantification of area fractions of calcium‐containing cytochemical reaction products in central nervous tissue and the retina of fish. The method of electron spectroscopic imaging using electrons with an energy loss of 250 eV produces images with a very high, structure‐sensitive contrast. This is a suitable imaging condition for the reliable detection of reaction products and structural details in unstained ultrathin sections. The images were recorded with a sensitive TV camera and evaluated with the integrated digital image‐analysis system of the Zeiss CEM 902 energy‐filtering electron microscope. An empirical procedure was developed which objectively detects reaction products and calculates characteristic values, taking into account different staining intensities. This new and sensitive method enabled an assessment to be made of the influence of temperature and light adaptation on cytochemically detectable calcium in nervous tissue of fish. Higher amounts of calcium‐containing reaction product were detected in synaptic clefts of the optic tectum in warm‐adapted fish than in cold‐adapted fish. In synaptic vesicles of photoreceptor cells in the fish retina, higher amounts of reaction product were found in dark‐adapted fish than in light‐adapted fish.
Numerical aspects of the deconvolution of angle‐integrated electron energy‐loss spectra
Authors: Su D. S. , Schattschneider P.
Published: August 2011

Some fundamental aspects of the Fourier and algebra methods for deconvolution of angle‐integrated electron energy‐loss spectra are discussed. A simple algebraic method for resolution improvement is given and tested.
A variable cell culture chamber for ‘open’ and ‘closed’ cultivation, perfusion and high microscopic resolution of living cells
Authors: Pentz S. , Hörler H.
Published: August 2011

A multipurpose chamber is described for growing and testing cultured cells. The chamber can be converted from a perfusion chamber to an ‘Open’ or ‘closed’ culture system. The chamber provides optimum conditions for microscopy using all common objectives and condensers for different microscopic methods.
Fixation for biological ultrastructure. I. A viscometric analysis of the interaction between glutaraldehyde and bovine serum albumin
Authors: Luft John H.
Published: August 2011

The increase in viscosity resulting from mixing concentrated solutions of albumin with dilute glutaraldehyde has been investigated. The viscosity changes are slow at first, very sensitive to small changes in glutaraldehyde concentration, and non‐linear with time. The cross‐linking of the albumin by glutaraldehyde seems to be retarded by the weak mechanical shear forces produced by the viscometer. The significance of these findings to the events in tissue fixation is discussed.
Improved preservation of bacterial exopolymers for scanning electron microscopy
Authors: Vandevivere P. , Baveye P.
Published: August 2011

Two methods of sample preparation, originally developed for transmission electron microscopy and better at preserving bacterial exopolymers than the traditional fixation with glutaraldehyde, were adapted for scanning electron microscopy. The first involves a glutaraldehyde‐lysine mixture as a fixative. The second, based on the use of polycationic ferritin, was modified to include a post‐fixation step with either glutaraldehyde or a glutaraldehyde‐lysine mixture. These techniques were found to improve considerably the preservation of exopolymers secreted by three bacterial strains.
Texture analysis of ceramic films by image processing
Authors: Prod‘Homme Murielle , Chermant Liliane , Coster Michel
Published: August 2011

This paper presents an application of automatic image analysis to the morphometry of ceramic sheets. The sheets can be characterized by granulometrie and stereological texture parameters of the granular and the porous phases. Grey level granulometry, automatic thresholding and filtering have thus been used. The results show that the morphology of green sheets is strongly dependent on the ball‐milling conditions.
Estimation error of Leydig cell numbers in atrophied rat testes due to the assumption of spherical nuclei
Authors: Mendis‐Handagama S. M. L. C.
Published: August 2011

In this study, Leydig cell numbers in control and atrophied testes (induced via subcutaneous implants of testosterone plus 17β estradiol for 16 weeks; TE‐implanted) of rats, estimated via the fractionator method (independent of any assumptions) were compared to those estimated via the disector (unbiased, but dependent on shrinkage) and Floderus (assumes spherical particles, dependent on shrinkage) methods. Estimates of Leydig cell numbers in control rats produced by all three stereological methods were similar. In rats with atrophied testes, both the fractionator and the disector methods produced significantly lower (P < 0.01; 47% and 41 % with fractionator and disector, respectively) Leydig cell number estimates per testis than in the controls. By contrast, the estimates of Leydig cell number in atrophied testes derived via the Floderus equation were not significantly different from those of controls, but larger than those obtained via the fractionator and the disector methods. These results suggested that the assumptions of the Floderus method were violated in the atrophied rat testes.

Why was the Floderus method of estimating Leydig cell number applicable to control rats but not to the TE‐implanted rats? In an attempt to answer this question the diameter measurement together with its correction factor used in the Floderus equation (i.e. D + t − 2H) was also derived from the data collected for the disector method. The values for D + t − 2H used in the Floderus method and also calculated via the disector method were found to be identical in controls, but for the TE‐implanted rats a 32% lower value was obtained with the Floderus equation when compared to the disector. These findings suggested that this estimation error caused an overestimation of Leydig cell numbers in the TE‐implanted rat testes.
Pattern reconstruction in ultrastructural morphology
Authors: Bellon Pier Luigi , Lanzavecchia Salvatore
Published: August 2011

A method of image reconstruction which can be used in ultrastructural morphology when a number of comparable structures are available is described. The method is suitable for quasi‐regular arrays of objects, and an extension of it allows the average of equivalent objects which could be orientated at random to be obtained. When dealing with crystalline arrays it is first necessary to obtain a leakage‐free pattern in which the coarse distortions are suppressed; this pattern is then filtered to obtain a reference. When dealing with several individual objects which must be brought to the same orientation and enlargement, one of the objects is fixed as a reference. In both cases the distortions relative to the reference are eliminated by an iterative process, making a final average possible. In crystalline arrays this method enhances the bandwidth of the pattern in a way that can be estimated quantitatively by figures of merit. Two examples of reconstructions are reported and discussed: a ctenophore macrocilium, regarded as a crystalline structure, and a sectioned flagellum of an insect spermatid.
Combined cathodoluminescence and X‐ray analysis of paint pigments
Authors: Koschek Georg
Published: August 2011

The suitability of cathodoluminescence (CL) measurements for differentiating between different paint pigments is demonstrated with an example of a combined energy‐dispersive X‐ray (EDX) and CL analysis of different zinc oxide (ZnO) pigments in a paint fragment where the EDX spectra are virtually identical, but where the CL spectra show significant differences. Consequently, it is possible to distinguish different pigments on the basis of CL spectra and monochromatic CL micrographs.
An environmental sample chamber for X‐ray microscopy
Authors: Goncz K. K. , Batson P. , Ciarlo D. , Loo B. W. , Rothman S. S.
Published: August 2011

The manufacture, construction and performance of a special specimen holder for biological samples suspended in aqueous solution is described. The holder was designed for use in a scanning transmission X‐ray microscope. Using it, we have successfully obtained high‐resolution images of fresh cellular and subcellular specimens in suspension and have been able to vary the sample environment during viewing in the microscope.
A Monte Carlo code to simulate the effect of fast secondary electrons on κAB factors and spatial resolution in the TEM
Authors: Gauvin Raynald , L'Espérance Gilles
Published: August 2011

Fast secondary electrons (FSE), which result from inelastic scattering of incident electrons, are known to generate a significant number of X‐rays for light elements, and also to degrade the spatial resolution of X‐ray microanalysis in thin foils. A Monte Carlo program simulating the generation and diffusion of FSE in binary systems has been developed to study the effect of composition on κAB factors and spatial resolution. The effect of accelerating voltage and thickness is also presented.
High‐resolution scanning electron microscopy of frozen‐hydrated cells
Authors: Walther Paul , Chen Ya , Pech Louis L. , Pawley James B.
Published: August 2011

Cryo‐fixed yeast Paramecia and sea urchin embryos were investigated with an in‐lens type field‐emission SEM using a cold stage. The goal was to further develop and investigate the processing of frozen samples for the low‐temperature scanning electron microscope (LTSEM).

Uncoated frozen‐hydrated samples were imaged with the low‐voltage backscattered electron signal (BSE). Resolution and contrast were sufficient to visualize cross‐fractured membranes, nuclear pores and small vesicles in the cytoplasm. It is assumed that the resolution of this approach is limited by the extraction depth of the BSE which depends upon the accelerating voltage of the primary beam (V0). In this study, the lowest possible (V0) was 2.6 kV because below this value the sensitivity of the BSE detector is insufficient. It is concluded that the resolution of the uncoated specimen could be improved if equipment were available for high‐resolution BSE imaging at 0.5–2kV.

Higher resolution was obtained with platinum cryo‐coated samples, on which intramembranous particles were easily imaged. These images even show the ring‐like appearance of the hexagonally arranged intramembranous particles known from high‐resolution replica studies. On fully hydrated samples at high magnification, the observation time for a particular area is limited by mass loss caused by electron irradiation. Other potential sources of artefacts are the deposition of water vapour contamination and shrinkage caused by the sublimation of ice.

Imaging of partially dehydrated (partially freeze‐dried) samples, e.g. high‐pressure frozen Paramecium and sea urchin embryos, will probably become the main application in cell biology. In spite of possible shrinkage problems, this approach has a number of advantages compared with any other electron microscopy preparation method: no chemical fixation is necessary, eliminating this source of artefacts; due to partial removal of the water additional structures in the cytoplasm can be investigated; and finally, the mass loss due to electron beam irradiation is greatly reduced compared to fully frozen‐hydrated specimens.
A simple way to encapsulate small samples for processing for TEM
Authors: Mariné M. C. Hernández
Published: August 2011

A variation of the technique of encapsulating samples in agar cubes is presented. The samples are enclosed by bubbles in the agar. The hollow space is occupied by the specimen and a drop of melted agar is used to fill the remainder of the cavity and to cover it. The solidified agar is cut into a small cube containing the bubble, and is processed for TEM in the usual way.
Signal‐to‐noise ratio in confocal microscope systems
Authors: Sheppard C. J. R. , Gu Min , Roy Maitreyee
Published: August 2011

The effects of various system parameters on the imaging performance of confocal microscopy are discussed, including the effects of pinhole size, type of detector and imaging algorithms on signal‐to‐noise ratio. Careful consideration of these parameters allows the microscope user to obtain the best performance for particular applications.
High‐resolution imaging of chromosome‐related structures by atomic force microscopy
Authors: Grooth Bart G. De , Putman Constant A. J.
Published: August 2011

An atomic force microscope (AFM) was combined with a conventional optical microscope. The optical microscope proved to be very convenient for locating objects of interest. In addition, the high‐resolution AFM image can be compared directly with the traditional optical image. The instrument was used to study chromosome structures. High‐resolution chromosome images revealed details of the 30‐nm chromatide structure, confirming earlier electron microscopic observations. Chromosomes treated with trypsin revealed a banding pattern in height which is very similar to the optical image observed after staining with Giemsa. Furthermore, it is shown that the AFM can be used to locate DNA probes on in situ hybridized chromosomes. Images of the synaptonemal complex isolated from rat spermatocytes revealed details that improve the understanding of the three‐dimensional structure of this protein.
Quick‐freezing of cultured cardiac cells in situ with special attention to the mitochondrial ultrastructure
Authors: Dalen Helge , Lieberman Melvyn , LeFurgey Ann , Scheie Paul , Sommer Joachim R.
Published: August 2011

A new method has been developed which allows quick‐freezing in situ of primary, cardiac cell cultures grown to confluence on gas‐permeable membranes (Petriperm dishes). Small pieces of the growth substratum, with rhythmically beating myocardial cells, were slam‐frozen, without cryoprotectants, against the surface of a helium‐cooled copper block at approximately 16 K. The quality of the cellular cryopreservation, as judged by ultrastructural criteria, was studied in freeze‐substituted specimens processed for transmission electron microscopy. The ultrastructure of cryofixed cardiac cells was compared with that of unfrozen, chemically fixed samples. The severity of cryodistortions increased progressively with increasing distance from the point of first impact. Of particular interest were the dramatic alterations of the mitochondrial ultrastructure. The concept that the reticular and the outer mitochondrial membranes are intimately and strongly associated was clearly demonstrated. Optimally frozen material revealed cryopreserved ultrastructure of high quality. The method described appears to offer an ideal model system for correlating the information gained by phase‐contrast microscopy of living cell cultures with the ultrastructure of the same samples fixed in situ by chemical or physical techniques. Cryofixation would be particularly useful for studying dynamic cellular processes associated with physiological and pathophysiological conditions, e.g. metabolic inhibition, anoxia and substrate deprivation.
Real‐time three‐dimensional tracking of fast‐moving microscopic objects
Authors: Teunis P. F. M. , Bretschneider F. , Machemer H.
Published: August 2011

Anaxial illumination has been used so far for either enhancement of visual acuity, or three‐dimensional (3‐D) measurements of static objects. Here we report the use of anaxial illumination for 3‐D tracking of fast‐moving objects, with only slight modifications to the standard microscope. To achieve this, we have investigated highspeed recording of stereoscopic images in two different set‐ups.
The accurate alignment of energy‐dispersive X‐ray detectors for quantitative microanalysis
Authors: Nicholson W. A. P. , Craven A. J.
Published: August 2011

Modern collimator design for energy‐dispersive X‐ray detectors requires very accurate positioning of the crystal/collimator assembly in order to achieve the maximum solid angle of collection for the irradiated volume on the specimen. Thus it is important to have a method of checking the alignment of the detector when mounted on the microscope and under vacuum. This paper describes a number of techniques, principally X‐ray mapping, for performing such an alignment check. These techniques are applicable to windowless detectors as well as to those with integral windows which will support atmospheric pressure. Methods of obtaining the non‐standard modes of microscope operation suitable for this task are described, and some suggestions are made for ways of moving the crystal/collimator assembly and monitoring this movement while it is in progress.
Mathematical morphology: from two dimensions to three dimensions
Authors: Meyer F.
Published: August 2011

After a discussion of the best grid for the digitization of three‐dimensional (3‐D) images, two grids are retained for which the approximations of the elementary disc are respectively the cuboctahedron and the rhombododecahedron. These are used to derive the formulae for the basic Minkowski measures. The extension from 2‐D to 3‐D is demonstrated to be straightforward for most transformations of mathematical morphology, and covers almost all practical situations.
Colour confocal reflection microscopy using red, green and blue lasers
Authors: Cogswell C. J. , Hamilton D. K. , Sheppard C. J. R.
Published: August 2011

To obtain colour reflected confocal images we have incorporated three lasers (HeNe: 633 nm; NdYAG: 532 nm; HeCd: 442 nm) and three photomultiplier detectors into our on‐axis scanning system then adjusted the registration of the simultaneous output signals to produce full‐colour images on a video monitor. Colour confocal images were produced from multi‐stained fixed tissue as well as from natural pigments in fresh plant material. Rayleigh scattering properties of immunogold‐labelled specimens were studied to show how variations in colour response can be utilized to identify subwavelength gold particles. Colour stereo pairs were produced to illustrate the accuracy with which the three‐laser microscope system can record depth information without incurring problems due to chromatic aberration effects.
Confocal‐line microscopy
Authors: Benedetti P. A. , Evangelista V. , Guidarini D. , Vestri S.
Published: August 2011

The confocal‐line (CL) technique combines some of the characteristics of confocal‐scanning microscopy with those of conventional‐imaging methods. It is based on the introduction of line‐shaped illumination and linear image detection, as an alternative to the current confocal‐point (CP) approach. Although confocal only in one dimension, the proposed solution offers performance and features adequate to a variety of biological and non‐biological applications and is also adaptable to an increased number of microscopical observations and measurements. The absence of moving components in the optical path and the use of electronic linear imagers permits flexible and fast operation that appears particularly relevant in many fields of basic and applied research. For instance, transmission, reflection and emission images can simultaneously be collected from the same area of the specimen, with slight adjustments to the optical setup. Useful extensions of CL microscopy to the field of spectral imaging are obtained with the introduction of a slit, a polychromator and an area detector, substituting for the linear imager. Prototype instrumentation has been constructed working from the cited principles and some tests have been performed on selected applications.
Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy
Authors: Jester James V. , Petroll W. Matthew , Garana Randa M. R. , Lemp Michael A. , Cavanagh H. Dwight
Published: August 2011

Using the tandem scanning microscope, in vivo confocal microscopic images of living eyes were compared to images obtained from ex vivo, freshly enucleated or fixed tissue in the rabbit. In the normal cornea, microscopic details of the superficial epithelium, basal lamina, stromal fibrocyte nuclei, nerves and endothelial cell borders were easily discernible. Removal of the eye from the intact animal resulted in loss of detail with distortion of the normal structural interrelationships within the corneal stroma whilst enhancing details of the corneal epithelium. Formalin fixation further enhanced details of the basal and suprabasal corneal epithelial cell nuclei and the stromal fibrocyte cell borders whilst inducing prominent brightly reflecting folds in the thickened stroma with concomitant enhancement of the edge contrast of the collagen lamellae. These changes appeared to be related, in part, to hydration of the cornea and artefactual pooling of water between structures that may enhance reflectivity by increasing the difference between the refractive index of the cellular and extracellular elements. We conclude that microscopic examination of ex vivo preparations of corneal tissue, although providing increased resolution similar to conventional light microscopic techniques, significantly altered the normal structural relationships and could lead to erroneous measurements of the physiological properties of the tissue as compared to in vivo microscopy of undisturbed, intact tissue.
Measuring the surface area of a cell by the method of the spatial grid with a CSLM—a demonstration
Authors: Howard C. Vyvyan , Sandau Konrad
Published: August 2011

The spatial grid is a method for estimating the surface area of particles. A stack of perfectly registered sections is the essential prerequisite for its use. The confocal scanning light microscope provides such a stack by optical sectioning. The spatial grid method is briefly described and applied to an osteocyte lacuna in dry mineralized human mandible. This type of cell was chosen because of its very complex shape. The variance of the area estimate is studied and compared with the results of a simulation.
Amplification and noise in high‐pressure scanning electron microscopy
Authors: DURKIN R. , SHAH J. S.
Published: August 2011

High‐pressure scanning electron microscopy (HPSEM) is a promising new family of techniques. The present knowledge of these techniques is reviewed and a new set of criteria developed for optimizing signal detection in HPSEM with a view to preserving specimen integrity. For this purpose, amplification of contrast signals generated in HPSEM was examined by computing the effect of ionization over a range of pressures and biasing fields, routinely used for this technique. The influence of secondary ionization due to ion impact was included in the calculations. To check the calculated results, the experiments were performed in the HPSEM apparatus in a nitrogen atmosphere. A divergence between the experimental values and the calculated values was found. This was removed by taking into account the effect of recombination of charge carriers. Inductive currents generated in the HPSEM environment are transient and do not affect the conclusions of this study. The gas pressure, biasing fields and beam current for preserving specimen integrity and obtaining good micrographs were selected from the data. Experimental measurements of noise are reported, and criteria for optimizing the signal‐to‐noise ratio for performing HPSEM are discussed. The application of these criteria has shown that specimen current detection can be more advantageous than other methods and configurations of detection and was capable of obtaining high/medium‐resolution micrographs.
Optimizing microstructure in spray‐formed and squeeze‐cast metal‐matrix composites
Authors: CANTOR B.
Published: August 2011

This paper discusses the methods of optimizing microstructure in the manufacture of spray‐formed and squeeze‐cast A‐ and Ti‐based metal–matrix composites (MMCs) for a range of automotive and aerospace applications. Three specific MMC material/process/product combinations are considered: spray‐formed/extruded Al alloy/SiC particulate‐reinforced MMCs for aerospace structural components; squeeze‐cast Al alloy/SiC particulate‐ and Al2O3 fibre‐reinforced MMCs for automotive components; and spray‐formed/diffusion‐bonded Ti alloy/SiC fibre‐reinforced MMCs for aeroengine components. Important features, which are emphasized in each case, are (i) using specific end applications to define desirable property and therefore microstructural goals; (ii) understanding the underlying process physics to control the resulting mechanisms of microstructural development; and (iii) using numerical modelling and associated experimental validation to allow subsequent implementation of process control systems.
Interface compatibility in Nicalon‐fibre reinforced metal and ceramic composites
Authors: SCOTT V. D. , BLEAY S. M. , CHAPMAN A. R. , LOVE G.
Published: August 2011

Microstructural characteristics of the fibre–matrix interface of two composite systems which utilize Nicalon fibre reinforcement are analysed and discussed.

An Al‐based composite produced by liquid‐metal infiltration was found to contain crystals of aluminium carbide and alumina at the fibre–matrix interface, which produced a strong interfacial bond, restricted fibre pullout, and resulted in an essentially brittle composite.

A ceramic‐matrix composite based upon calcium aluminosilicate and produced by hot pressing exhibited substantial fibre pullout during testing; microstructural analysis of the interface showed the presence of a C‐rich layer. Treatment of the composite in air over a range of temperatures (600–1200°C) progressively oxidized the carbon and formed silica ‘bridges' between fibre and matrix, which resulted in increased brittleness.

Electron‐probe microanalysis combined with electron microscopy of the Nicalon fibre showed that approximately half the material consisted of microcrystalline β‐SiC and the remainder was free carbon and silicon oxycarbide. Thus the carbon constituent was largely responsible for carbide formation in the Al‐based material, which restricted fibre pullout, whilst free carbon, plus the additional free carbon formed by chemical reaction between silicon carbide in the fibre and the calcium aluminosilicate matrix, provided the interfacial carbon layer which gave enhanced fibre pullout in the ceramic‐based composite; the decreased fibre pullout and increased brittleness of the latter after heat treatment in air could thus be explained by the removal of the carbon layer and the development of silica bridges between fibre and matrix.
Microstructural observations of reaction zones in Ti‐based metal‐matrix composites
Authors: LORETTO M. H. , JOHNSON T. P.
Published: August 2011

The interaction between Ti‐alloy matrices and ceramic particulate and fibres in Ti‐based metal‐matrix composites (MMCs) is reviewed. It is concluded that the stability of surface‐treated SiC fibres is adequate for some applications of MMCs, but no other fibres have yet been developed which are useful additions to Ti alloys. Although there is less interest at this stage in particulate‐reinforced Ti MMCs, there are two ways in which useful particulate MMCs could be developed. First, the approach used in fibre‐reinforced composites, of using special coating techniques to stabilize the particulate against any reaction with the matrix. Second, either C or B additions could be used, both of which give rise to particulate phases which are stable in the presence of a Ti matrix, and which can therefore be manufactured using a wider range of process routes than is possible for other composites. The significance of these conclusions with regard to the ability to shape particulate composites into final products is briefly discussed.
Raman microscopy of fibres and composites
Authors: DAY R. J. , YOUNG R. J.
Published: August 2011

Some of the progress made in using Raman microscopy to examine the structure and micromechanics of polymer and inorganic fibres is reviewed. Examples of how the technique has been used to verify some of the theories of composite reinforcement are then given. Finally, a specific example of how Raman microscopy has been used, in conjuction with other approaches, to understand the micromechanics of the microbond test, which is often used to measure interfacial shear strength in composite systems, is presented.
Scanning ion microprobe analysis of composite materials
Authors: WILLIAMS D. B. , SONI K. K. , TSENG M. W. , CHABALA J. M. , LEVI‐SETTI R.
Published: August 2011

Applications of scanning ion imaging with high lateral resolution in the microchemical investigation of metal – and ceramic‐matrix composites are described. The technique, which combines a scanning ion microprobe with secondary ion mass spectrometry (SIMS), is ideally suited to the study of complex, multicomponent composite structures. Most elements can be detected with good sensitivity, enabling the determination of spatial distributions for major and minor elements. Analytical images obtained with this technique reveal unprecedented chemical information about interfacial segregation and interdiffusion phenomena. As examples, the characterization of both ceramic–matrix (Al borate–SiC) and metal–matrix (Ni alloy–Al2O3) composite materials is described.
The use of microscopy in the evaluation of fibre‐matrix interfacial properties
Authors: PITKETHLY M. J.
Published: August 2011

Microscopy plays an important role in the evaluation of the micromechanical properties of fibre‐matrix interfaces. The methods rely on the use of microscopy to undertake each test successfully. Physical parameters are evaluated quantitatively and the interface morphology is assessed qualitatively. This paper reviews the use of microscopy in evaluating the fibre–matrix interfacial properties of composites.
Characterization of subsurface damage in ceramic‐matrix composites by confocal scanning laser microscopy
Authors: POWELL K. L. , YEOMANS J. A. , SMITH P. A.
Published: August 2011

The techniques of confocal scanning laser microscopy (CSLM) and reflected‐light microscopy (RLM) have been combined in a study of the subsurface damage in continuous‐fibre reinforced ceramic‐matrix composites (CMCs).

The damage was induced by subjecting a composite to Vickers hardness indentation tests and single‐particle impacts. The subsurface damage was viewed conventionally by RLM at specific depths, following material removal by standard metallographic techniques. Mechanical sectioning, however, is time‐consuming and may introduce artefacts in the crack patterns. Consequently CSLM was employed to provide a rapid, non‐destructive analysis of the subsurface damage.

In the classic description of elastic‐plastic indentation of monolithic ceramics, there are two major types of crack: radial cracks, which grow out from the elastic‐plastic boundary perpendicular to the surface, and lateral cracks, which are on planes approximately parallel to the surface. In CMCs radial cracks were found to be restricted to the near‐surface region. Lateral cracks, however, were located a few micrometres from the free surface.

Preliminary analysis of damage about impact sites by mechanical sectioning and CSLM has shown that the crack patterns are similar to those observed in indentation fracture. The topographical‐mapping facility of the CSLM has been used to detail the radius of damage about an impact site, which has been found to increase with increasing impact velocity.
Silicon carbide/SRBSN composites
Authors: RAZZELL A. G. , LEWIS M. H.
Published: August 2011

Ceramic‐matrix composites have been produced using unidirectionally aligned Textron SCS‐6 fibres in a sintered reaction‐bonded matrix. A tape‐casting technique was used to produce a prepreg sheet that could be cut and stacked to form a layup. Al2O3 and Y2O3 were used as sintering aids, final sintering being carried out in a hot press at 1700°C.

Matrix, fibre and interfacial microstructure has been characterized using analytical microscopical techniques. X‐ray mapping of the carbon and silicon distribution at the fibre–matrix interface was carried out, and evidence of reaction between the outer carbon‐rich layer of the fibre and the matrix was found.

Micromechanical behaviour of interfaces has been investigated and compared with interfacial microstructure and macromechanical properties.
Structure and composition of interfaces in an aluminium metal‐matrix composite
Authors: GOURBILLEAU F. , RUTERANA P. , NOUET G.
Published: August 2011

The microstructure of a 2024 type aluminium alloy, reinforced with 13 vol.% SiC particles, and made by a spray technique, has been investigated by transmission electron microscopy and X‐ray microanalysis in the as‐extruded and T6 conditions. High resolution electron microscopy studies and energy‐dispersive X‐ray spectrometry microanalysis were performed on the interfaces between the SiC particles and the metal matrix. The interfaces were found to be abrupt: some exhibit an amorphous layer, rich in oxygen; in many others, silver‐rich crystallites have grown coherently on the SiC particles.
The measurement of local plastic deformation in a metal‐matrix composite by electron back‐scatter patterns
Authors: WILKINSON A. J. , GONZALEZ G. , DINGLEY D. J.
Published: August 2011

High dislocation densities in the matrices of metal‐matrix composites (MMCs) contribute to the strengthening of particulate MMCs, whilst heterogeneous nucleation of precipitates at these defects can cause marked differences in the age hardening of many matrix alloys. Electron back‐scatter patterns (EBSPs) and transmission electron microscopy (TEM) have been used to examine the dislocation distribution in continuous‐fibre SiC/Al alloy MMCs. TEM observations showed dislocation densities as high as 3 times 1014m−2, with a marked increase as thinner matrix regions were observed. From EBSP measurements on bulk specimens, a much lower dislocation density of 2 times 1012 m−2 was estimated. To resolve this discrepancy, EBSP measurements were made from thinned TEM specimens and these showed that the dislocation density had increased during specimen thinning. However, both TEM and EBSP measurements from unreinforced matrix material prepared for TEM using the same procedures showed no such increase in dislocation density. Redistribution of internal stresses during thinning of the composite to a transparent foil must therefore be the cause of the observed rise in dislocation density. The EBSP technique was demonstrated to be an excellent means of examining the distribution of plastic deformation in MMCs. It was concluded that TEM measurements of dislocation densities in continuous‐fibre MMCs can be erroneously high.
Microstructural characterization in diffusion‐bonded SiC/Ti–6Al–4V composites
Authors: GUO Z. X. , DERBY B.
Published: August 2011

The microstructural evolution during the diffusion bonding consolidation of a Ti–6Al–4V/SiC fibre composite was investigated by optical, scanning and transmission electron microscopy. The effects of processing parameters, particularly temperature, on the microstructures of the matrix and the fibre and their bonding were considered. Processing at too high a temperature can result in growth of SiC crystals in the fibre in addition to rapid interfacial reaction, while interfacial bonding cannot be established if the temperature is too low. Various defects can be caused by inadequate fabrication practices. These include micro‐pores, matrix‐cracking, cracking, bending and impingement of fibres, and heterogeneous fibre distribution. Methods for avoiding these are discussed. A defect‐free and uniformly distributed fibre composite can only be achieved by optimizing the processing parameters (such as temperature, pressure, time and cooling rate) and adequately combining fibre spacing and matrix thickness with accurate fibre alignment.
Three‐dimensional distribution of damaged cells in cryopreserved pancreatic islets as determined by laser scanning confocal microscopy
Authors: MERCHANT F. A. , AGGARWAL S. J. , DILLER K. R. , BARTELS K. A. , BOVIK A. C.
Published: August 2011

The technique of serial optical sectioning by confocal microscopy, in conjunction with off‐line digital image analysis, was used to quantify the radial distribution of damaged cells in rat pancreatic islets following cryopreservation. The process consists of imaging frozen‐thawed islets of Langerhans using laser scanning confocal microscopy (LSCM). The three‐dimensional (3‐D) distribution and analysis of the two populations of viable and damaged cells was visualized via acridine orange/propidium iodide (AO/PI) fluorescent staining. In preparation for cryopreservation, isolated and cultured rat pancreatic islets were brought to a 2 m concentration of dimethyl sulphoxide (DMSO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at −10°C, and individual specimens were frozen to −70°C at cooling rates of 1, 3, 10 and 30°C/min in a programmable bulk freezer and subsequently stored in liquid nitrogen. After rapid thawing and serial dilution to remove DMSO, individual islets were prepared with AO/PI stains for imaging on the LSCM. Serial sections of the islets, 2–7 μm in thickness, were obtained and processed to obtain high‐contrast images. Analysis algorithms consisted of template masking, grey‐level thresholding, median filtering and 3‐D blob colouring. The radial distribution of damaged cells in the islets was determined by isolating the cell and computing its distance from the centroid of the 3‐D islet volume. An increase in the number of blobs corresponding to single and/or aggregates of damaged cells was observed progressively with distance from the centre towards the periphery of the islet. This pattern of freeze‐induced killing of cells within the islet was found to occur consistently in the numerous individual specimens processed.