bluuebunny/crossref_metadata_2025 huggingface dataset:
Volume and surface area measurement of viable chondrocytes in situ using geometric modelling of serial confocal sections
Authors: GUILAK F.
Published: August 2011

This study describes a technique for noninvasive determination of the surface area and volume of chondrocytes using the confocal scanning laser microscope, and the fundamental limitations associated with its application. Using geometric modelling principles, an isointensity surface contour was formed from a series of optical sections recorded with the confocal microscope. Using a combined surface‐ and volume‐based algorithm, the surface area, volume and other morphometric descriptions were calculated from a polygonal description of the cell surface. The high image contrast required for repeatable identification of the cell border was achieved through the use of a fluorescent dye, which was excluded from cells by an intact membrane. Calibration results indicated that the theoretical modelling algorithm is relatively precise when applied to simulated convex (ellipsoidal) cells, with overall errors of less than 0·5% in surface area and volume measurements. When applied to low‐noise, high‐contrast volume data recorded on the confocal microscope, typical coefficients of variation of 2–4% were determined for length measurements, 2–5% for volume measurements and 3–6% for surface area measurements either for latex microspheres or for chondrocytes. While the precision of the method is comparable to standard histological techniques, its accuracy is difficult to assess, as systematic errors are unpredictable and may be introduced from several sources.
Accurate alignment of sets of images
Authors: SAXTON W. O.
Published: August 2011

Two modifications to the conventional procedure of cross‐correlation are described, widely used for establishing the relative alignment of the members of a set of images from which a higher resolution or more interpretable restoration is sought. Both achieve a high and sharp peak in circumstances where the conventional peak is too ill defined to be recognizable; neither involves significant additional computation time. The more general method requires rough knowledge of the imaging conditions, but a variant applicable to images with axial resolution has no such requirement. In addition, a least‐squares procedure is presented for achieving an optimum compromise between many pair‐wise displacement measurements, preventing the accumulation of alignment errors across a set of images.
Cryo automated electron tomography: Towards high‐resolution reconstructions of plastic‐embedded structures
Authors: BRAUNFELD M. B. , KOSTER A. J. , SEDAT J. W. , AGARD D. A.
Published: August 2011

The use of fully automated data collection methods for electron tomography allows a substantial reduction in beam dose. The goal has been to develop new protocols for data collection defining optimal approaches for maintaining data self‐consistency and maximizing the useful resolution of the reconstruction. The effects of irradiation and post‐cure microwaving were examined for a variety of embedding media (Epon, Epox, Lowicryl) in order to quantify beam damage with the goal of identifying the most beam stable embedding medium. Surprisingly, the substantial dose reduction made possible by automated data collection did not result in a significant decrease in specimen shrinkage even for samples stabilized by preirradiation. We believe that the accelerated shrinkage is a direct consequence of the stroboscopic illumination patterns inherent to automated data collection. Further‐more neither the choice of embedding resin nor microwave post‐curing greatly affected shrinkage. Finally, cryogenic data collection was investigated as a means to minimize the effects of secondary radiation damage. Minimal preirradiation coupled with low‐temperature automated data collection greatly reduces shrinkage and should result in high‐quality data for three‐dimensional reconstructions.
A novel method for mean cell volume estimation
Authors: WEBSTER P. , GRIFFITHS G.
Published: August 2011

A novel method is described for the estimation of the mean cell volume of cell populations grown in suspension. The cells are filtered onto a nitrocellulose filter to form a cylindrical pellet which is embedded in epoxy resin. Using estimates of pellet height and radius, the number of cells in the pellet and of the volume density of the cells in the pellet, it is possible to produce an unbiased estimate of the mean cell volume. This method is compared, using cell suspensions of the blood parasite Trypanosoma brucei, with mean cell volume estimation using a Coulter channellizer. A Coulter channellizer was also used to compare the mean cell volume of living trypanosomes with that of aldehyde‐fixed populations, and the values obtained were compared with those obtained using the new method. The estimated mean cell volume of a T. brucei clone was used to derive values from volume densities obtained by point and intersection counts for the absolute volumes of the flagellar pocket, the nucleus, and endocytic organelles containing internalized horseradish peroxidase and transferrin‐gold after 30‐min incubations at 310 K. Estimated values for the surface area of the flagellar pocket and the surface area of a cell were also obtained. From known data on the total amount of variant surface glycoprotein molecules per cell and the known packing density of membrane proteins, it was estimated that approximately 80% of the molecules must reside in intracellular compartments. It was estimated that the equivalent of 5% of the surface membrane may be internalized per minute, an amount which is almost the size of the entire flagellar pocket membrane.
Astigmatism of a thick cylindrical object in reflective mode CSLM
Authors: WANG CHANG‐GUI , RÄIKKÖNEN H. , LUUKKALA M.
Published: August 2011

The axial response of a basic confocal microscope is determined when the sample is a thick cylindrical or tubular structure. The response from the back wall of the cylindrical sample is split into two separate signals due to basic aberration or astigmatism effects.
ESI in situ analyses of extrachromosomal RNP granules
Authors: ABOLHASSANI‐DADRAS S. , VÁZQUEZ‐NIN G. H. , ECHEVERRÍA O. M. , ROUELLE‐ROSSIER V. BOUTINARD , FAKAN S.
Published: August 2011

Observation of unstained ultrathin sections of salivary gland cells of Chironomus thummi and C. tentans, by means of electron spectroscopic imaging (ESI), has revealed that phosphorus is distributed in two types of granular structures in the nucleoplasm of these cells. In addition to a specific type of premessenger ribonucleoprotein (RNP) particle, known as the Balbiani ring (Br) granule, ESI revealed a new type of phosphorus‐rich small granular component. Examination of unstained sections by energy‐filtering transmission electron microscopy (EFTEM) offers the opportunity of obtaining the signal from the specimen alone, thus avoiding the possible contributions of heavy metals present in any staining product.
A high‐sensitivity CCD system for parallel electron energy‐loss spectroscopy (CCD for EELS)
Authors: TANG Z. , HO R. , XU Z. , SHAO Z. , SOMLYO A. P.
Published: August 2011

A cooled frame transfer CCD camera system was developed and tested as a parallel detector in an electron energy‐loss spectrometer mounted on a transmission electron microscope. The use of a shutterless camera with a frame transfer CCD collected virtually 100% of the photon signal with a reasonably fast acquisition time. The system detective quantum efficiency was over 90% under normal experimental conditions. Because of the low channel to channel gain variations in the CCD, the signal‐to‐noise ratio and the detection limit were substantially better than that obtained with a silicon intensified target (SIT) camera, and direct fitting to the standard data was feasible. Quantitation at the phosphorus L edge generated from a phosphoprotein, Phosvitin, showed that, under identical experimental conditions, direct fitting of spectra obtained with this CCD system gave better sensitivity than that given by the SIT camera system. Because of its larger pixel charge well, the CCD system can also operate at a much higher beam current, resulting in a significant reduction in the time required for elemental mapping at a given sensitivity.
Second‐order stereology for pores in translucent alumina studied by confocal scanning laser microscopy
Authors: KARLSSON L. M. , LILJEBORG A.
Published: August 2011

The three‐dimensional (3‐D) arrangement of pores in translucent alumina was investigated with a confocal scanning laser microscope (CSLM). By moving the focal plane of the CSLM down into the material, a stack of serial thin optical sections was obtained to produce a 3‐D image of the pores. Computer‐based image analysis was used to obtain the coordinates of the pore centroids. The distance distribution function G(r) and the second‐order functions K(r), L(r), H(r) and g(r) were used to analyse the spatial point pattern of the pore centroids. Estimates of the preceding functions obtained from eight stacks of sections were compared with the corresponding functions for a 3‐D stationary Poisson point process, which served as a reference model for complete spatial randomness. The analysis suggested that the pore centroids were arranged in an aggregated pattern within a range of about 10 μm.
Modelling of vascular growth processes: A stochastic biophysical approach to embryonic angiogenesis
Authors: SANDAU K. , KURZ H.
Published: August 2011

In a computer simulation, growth of a capillary network is driven by a stochastic process on a planar hexagonal grid. Starting at a point source, the probabilities for the formation of new capillary elements depend on local biophysical knowledge. This knowledge is mainly derived from the flow theorem of Hagen–Poiseuille and the diameter exponent Δ. The hexagonal grid is visualized as being supported by a cylinder or a sphere. An arterial tree results from the adaptive diameter augmentation, and is considered to have limited fractal properties. The dimension of its border, and the time course of growth and of blood pressure are compared with biological data from the chorioallantoic membrane (CAM) of incubated chicken eggs. The model is discussed in view of mechanosensitivity and cell–matrix interactions of endothelial cells, and CAM haemodynamics.
Retention of vacuole contents of plant cells during fixation
Authors: DONG Z. , McCULLY M. E. , CANNY M. J.
Published: August 2011

Changes in the semi‐permeability of tonoplast during fixation were studied using beetroot tissue. Using vacuole betacyanin as the indicator, the permeability of the tonoplast was assessed by the leakage of this pigment as determined by changes in the optical density of the solution bathing the tissue. Cryo‐analytical scanning electron microscopy was used to monitor the changes in ion concentration in the cells during fixation. Fixatives were 3% glutaraldehyde or 4% formaldehyde in 0·025 m phosphate buffer at room temperature or on ice. Results showed that glutaraldehyde, especially at low temperature, takes as long as 30 h to disrupt the semi‐permeability of the tonoplast of beetroot cells, while in formaldehyde on ice, these cells begin to lose their selective permeability in 15 min. This study confirms that the semi‐permeability of the tonoplast may not be lost until long after the cytoplasm has been fixed and suggests that this explains why cold fixation in 3% glutaraldehyde for about 12 h has become the most reliable standard procedure for the successful preservation of vacuolated plant cells.
Central and peripheral nervous structures as seen at the confocal scanning laser microscope
Authors: CASTANO P. , MARCUCCI A. , JR A. MIANI , MORINI M. , VERALDI S. , RUMIO C.
Published: August 2011

Central neurons and peripheral nervous structures, e.g. cutaneous free endings, perifollicular nets, Meissners corpuscles and intramuscular fibres, were studied using various impregnation methods. The confocal scanning laser microscopes (CSLMs) used were equipped with different laser sources, in order to evaluate their limitations and advantages with these techniques and to contribute to a better understanding of the general morphology of the nervous system. When staining with silver sections with clouds of tiny silver granules which are beyond the resolution power of the conventional light microscope but which show a high reflectivity with the CSLM are obtained. Golgi‐Cox mercuric impregnation, however, provides specimens which are precipitate‐free, thus ensuring the reliability of information obtained. It does, however, have the disadvantage of being applicable only to the central nervous system. In all cases it is an advantage for the instrument to be fitted with different lasers (e.g. Ar and He–Ne), so as to optimize the images of samples impregnated with different methods. Notwithstanding the possibility that artefacts may distort the geometry of the sample and reduce the resolution, the images presented in this paper show that with careful selection of optical sectioning distances, the use of a suitable stack of sections and, if necessary, the aid of false electronic colours and of partial or complete rotation, it is possible to achieve a more precise interpretation of the morphology and organization of complex structures, such as those of the nervous system.
Rapid estimation of bacterial antibiotic susceptibility with flow cytometry
Authors: MASON D. J. , ALLMAN R. , STARK J. M. , LLOYD D.
Published: August 2011

Bacterial antibiotic susceptibility was rapidly estimated for Escherichia coli and Staphylococcus spp. by flow cytometry. This was achieved by measuring the uptake of a negatively charged membrane potential sensitive dye bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol and observing changes in low‐angle light scatter (excitation light scattered by up to 15°). Estimations of ampicillin, gentamicin and ciprofloxacin susceptibilities were possible within 2–5 h from a plate culture, depending on the species and antibiotic used. This includes the time necessary to establish steady‐state growth in liquid culture.
Rapid assay for pathogenic salmonella organisms by immunofluorescence flow cytometry
Authors: PINDER A. C. , MCCLELLAND R. G.
Published: August 2011

Multi‐parameter flow cytometry was investigated for the rapid detection of specific serotypes of salmonellas (S. typhimurium and S. montevideo) labelled with fluorescent monoclonal antibodies, both in pure culture and in a typical food matrix (full‐fat milk). In all cases, the method was accurate to levels of below 104 target cells per ml for a total assay time of about 30 min. After 6 h non‐selective enrichment in the presence of a 10 000‐fold excess of competing micro‐organisms (Escherichia coli) the corresponding detection limit was about 20 cells ml−1. These results suggest that flow cytometry has significant potential for the detection of pathogenic micro‐organisms in the food industry.
Fractal characterization by frequency analysis: III. Effect of noise
Authors: PANCORBO M. , ANGUIANO E. , AGUILAR M.
Published: August 2011

The effect of noise in the fractal characterization by frequency analysis of surface images obtained by scanning tunnelling microscopy (STM), atomic force microscopy (AFM) or profilometry has been studied. The origin of noise and its relationship to the signal is discussed. A procedure to simulate noisy images is presented. From the study it is concluded that the method usually used to characterize noise in STM is not valid and it is shown that fractal characterization of surfaces when noise is present by traditional frequency analysis methods is not possible. A new method to perform both the noise characterization and the fractal characterization of surfaces when noise is present is proposed.
Vitrification of aqueous suspensions from a controlled environment for electron microscopy: An improved plunge‐cooling device
Authors: BATTERSBY B. J. , SHARP J. C. W. , WEBB R. I. , BARNES G. T.
Published: August 2011

In the process of vitrifying aqueous suspensions for cryotransmission electron microscopy, water is solidified without crystallization. Vitrification can be achieved by rapidly plunging an aqueous thin film into a liquid cryogen. The preparation of aqueous thin films prior to vitrification must be performed in an environmental cabinet at controlled temperature and humidity in order to prevent evaporation and temperature‐induced phase changes in the thin film. The device described here incorporates several important features which make the apparatus simpler and more convenient to use than similar devices described in the literature. One of these features includes the use of a totally enclosed environmental cabinet in which the grid, sample, micropipette and absorbent paper are equilibrated before thin‐film preparation. Other features include a cryogen dewar on a swing arm for easy refilling, a guillotine shutter which is used to trigger the plunger electrically and a semiautomatic system which facilitates rapid transfer of the vitrified specimen from liquid propane to liquid nitrogen for storage and reduces handling of the specimen. To demonstrate the utility of the device, results showing the influence of temperature on the morphology of phospholipid vesicles are presented. A commercial cryotransfer apparatus (which is used for transportation of the vitrified specimen to the electron microscope cold‐stage) has been modified to reduce the possibility of reversion of the vitreous phase to the crystalline ice phases.
The tetrahedral tip as a probe for scanning near‐field optical microscopy at 30 nm resolution
Authors: FISCHER U. C. , KOGLIN J. , FUCHS H.
Published: August 2011

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.
Scanning force microscopy on live cultured cells: Imaging and force‐versus‐distance investigations
Authors: RICCI D. , GRATTAROLA M.
Published: August 2011

Extensive measurements with the scanning force microscope on living cells in their native liquid environment are described with the purpose of critically assessing the extent of the interaction between the SFM tip and the (soft) cell materials and the effect of such interaction on topographic information. Images are obtained under various force conditions and systematically correlated with force‐versus‐distance curves. As a result, detailed indications about tip indentation are given, thickness estimates deduced and identification of submembranous cytoplasmic structures suggested.
In‐vivo analysis of angiogenesis and revascularization of transplanted pancreatic islets using confocal microscopy
Authors: MERCHANT F. A. , AGGARWAL S. J. , DILLER K. R. , BOVIK A. C.
Published: August 2011

A technique to measure angiogenesis and revascularization in pancreatic islets transplanted at the renal subcapsular site in the rat has been developed. In‐vivo imaging of the microcirculation of transplanted pancreatic islets was conducted using a confocal scanning laser microscope (CSLM) to achieve optical sectioning through the graft in order to perform a computer reconstruction of the three‐dimensional neovascular morphology. Individual islets were harvested by enzymatic digestion of excised pancreas from Fischer 344 rats. Isolated islets were cultured for 24 h, and approximately 300–350 islets were transplanted at the renal subcapsular site of the left kidney in an anaesthetized rat. Six to 14 days post‐transplantation, the animal was anaesthetized and prepared for in‐vivo imaging of the microvasculature on a Zeiss LSM‐10. Optical contrast of the microvasculature was enhanced by the administration of fluorescein‐labelled dextran into the circulating blood. The transplant site was identified and serial sections were obtained through the vascular bed at varying z‐intervals. Complementary fluorescence video images were also obtained via a silicon intensifier tube camera mounted on the CSLM. At completion of the imaging procedure, the kidney was returned into the body cavity, the area was sutured and the animal was allowed to recuperate for subsequent examinations. Image processing algorithms, such as grey‐level thresholding, median filtering, skeletonization and template matching, were applied to compute the vessel density and diameters and extrapolated to measure 3‐D vessel lengths and the tortousity index of the neovasculature.
Simultaneous confocal recording of multiple fluorescent labels with improved channel separation
Authors: CARLSSON K. , ÅSLUND N. , MOSSBERG K. , PHILIP J.
Published: August 2011

Confocal microscopes are often used to study specimens labelled with fluorophores. A commonly used method for simultaneous recording of the distribution of multiple fluorophores is to divide the fluorescent light emitted by the specimen into different wavelength regions using dichroic and bandpass filters. These different wavelength regions are then distributed to multiple detectors. However, the broad and overlapping spectra of commonly used fluorophores often result in considerable crosstalk between channels. A new technique, intensity‐modulated multiple‐beam scanning (IMS) microfluorometry, can be used to reduce this cross‐talk substantially.

The IMS technique is implemented with two laser beams of different wavelengths, intensity‐modulated at different frequencies, which illuminate the specimen simultaneously. The two laser wavelengths predominantly excite one fluorophore each. Fluorescent light from the specimen is divided into two wavelength regions (red and green) which are detected by two photomultiplier tubes. The output signals from the photomultiplier tubes are connected to lock‐in amplifiers. The effect of using modulated laser beams, in combination with lock‐in amplifiers, is strongly to reduce cross‐talk between the channels. The performance of the IMS technique using various types of specimen is compared with the results obtained using the conventional multi‐detector method.
Standard performance criteria for analytical electron microscopy
Authors: ZEMYAN S. M. , WILLIAMS D. B.
Published: August 2011

Users of analytical electron microscopy lack easy‐to‐use standards for assessing the consistency and quality of analytical performance. We propose using a Cr thin film of known thickness to measure three important characteristics related to performance: the Cr Kα peak‐to‐background (P/B) ratio, the X‐ray spectrometer relative efficiency, and the spectrometer energy resolution. We used a Cr specimen to determine the instrumental factors which influence the P/B ratio, finding that the highest P/B ratios are achieved in scanning transmission mode at the highest available accelerating voltage. We present values of the P/B ratio, and the detector relative efficiency and energy resolution which can be used for comparison in other laboratories using the standard film.
Regression methods for automated colour image classification and thresholding
Authors: BREEN E. J.
Published: August 2011

Regression methods are used to perform automated image thresholding and colour pixel classification. This is done by considering threshold levels and pixel classification labels as pattern attributes. A regression equation that performs a mapping from the J dimensional feature‐pattern space to the K dimensional attribute space is derived. The approach is non‐parametric and deterministic, hence no assumptions about the statistical properties of the input patterns or images need be made. Initially a known set of input patterns with associated attributes are used to constitute a training set. A mapping function is then determined from the training patterns and used for estimating attribute values from unknown input patterns, such as images.
Surface ultrastructure of olfactory receptor sense hairs in the silkmoth, Antheraea pernyi
Authors: POPOV V. I. , NIKONOV A. A. , AGAFONOVA N. K. , FESENKO E. E.
Published: August 2011

The fine structure of silkmoth sense hair surfaces has been investigated by freeze etching with Pt/C rotary shadowing. To do this, the hydrophobic layer in the cuticle was removed using 20–25% methanol or ethanol. Freeze‐etch patterns of sense hair surfaces, as well as the pore structure and pore distribution, are shown. The hair surface has a nonhelical ‘band’ structure, in which every ‘band’ lies at an oblique angle with respect to the axis of the hair. Each ‘band’ is separated from its neighbour by a 30‐nm step. The average density of pores is 11·3 + 2·4 pores μm−2. Freeze‐etch patterns of the single and multiple pore tubules are shown. Evidence for direct contact between the pore tubule and dendrite membrane of an olfactory receptor neuron is presented.
Agar standards for quantitative X‐ray microanalysis of resin‐embedded plant tissues
Authors: FRITZ E. , JENTSCHKE G.
Published: August 2011

Calibration standards for quantitative X‐ray microanalysis of resin‐embedded plant tissue were prepared by adding 6600 mM KC1 to 5% agar. Agar blocks with an edge length of 1–2 mm were rapidly frozen, freeze‐dried and embedded in styrene‐methacrylate. Dry sections 1 μm thick were mounted on adhesive‐coated grids. Apart from fine‐scale inhomogeneities caused by ice crystal formation, the KC1 is evenly distributed in the agar blocks. The peak‐to‐continuum values of K and Cl were highly linearly correlated to the K and Cl contents over the whole concentration range.
Differential imaging in confocal microscopy
Authors: WILSON T. , JUŠKAITIS R. , TAN J. B.
Published: August 2011

A coherent detection system using a two‐mode optical fibre is described which permits confocal, differential amplitude contrast and differential phase contrast images to be obtained simultaneously from a scanning optical microscope. The direction of differentiation may be chosen arbitrarily. The differential imaging modes possess an optical sectioning property.
A new method to correlate acoustic spectroscopic microscopy (30 MHz) and light microscopy
Authors: VAN DER STEEN A. F. W. , THIJSSEN J. M. , VAN DER LAAK J. A. W. M. , EBBEN G. P. J. , DE WILDE P. C. M.
Published: August 2011

A powerful new method is used to investigate the correlation between light microscopic and acoustic properties of biological tissues. Specimens of liver were sectioned into successive slices, 250 μm and 10 μm thick. The thick sections were investigated acoustically, the thin sections by means of light microscopy. Markers that could be detected and located, both optically and acoustically, were used to find and reconstruct corresponding regions in the acoustic and optical sections (2·5 × 2·5 mm).

Parameter images were reconstructed from the sections investigated acoustically. The acoustic parameters were attenuation at 30 MHz, the slope of the attenuation spectrum (between 10 and 50 MHz), backscattering at 30 MHz, the slope of the backscattering spectrum (between 10 and 50 MHz) and the local ultrasound velocity. Acoustic images were obtained in the frequency range from 10 to 50 MHz, yielding a lateral resolution of about 50 μm.

The sections for light microscopy were stained according to the Goldner trichrome staining technique. The histological composition was determined quantitatively, using digital image segmentation techniques. The percentage of collagen‐rich fibrous tissue, luminal structure and interstitial spaces, and the number of nuclei were calculated for regions of 250 × 250 μm. These histological features were correlated with the acoustic parameters obtained from the corresponding regions in adjacent sections. It was thus possible to find the histological components responsible for acoustic parameters.
Three‐dimensional reconstruction of the human renal glomerulus
Authors: PRESTON K. , JOE B. , SIDERITS R. , WELLING J.
Published: August 2011

The capillary bed of the human renal glomerulus is one of the more complex capillary structures in the human body. This paper illustrates three‐dimensional reconstruction of the capillary bed from serial sections. It shows that, although traditional methods of three‐dimensional rendering by computer fail to handle the complexities of the capillary structure, new methods based on filtering using three‐dimensional mathematical morphology are capable of revealing previously unseen details. This is done at the expense of eliminating fine structure (small capillaries). An error analysis allows the degree to which fine details are lost to be estimated.
Computer‐simulated high‐resolution transmission electron microscopy (HRTEM) in tourmaline
Authors: FERROW E. A.
Published: August 2011

The contrast distributions observed in high‐resolution transmission electron microscopy (HRTEM) images of tourmaline depend on the types and magnitudes of the exchange components present and on the degree of atom overlap along the direction of observation. Furthermore, the fractional atomic coordinates in tourmalines are valid only for the specific specimen refined. These properties make the interpretation of experimental HRTEM images of tourmaline using image simulation if not impossible at least extremely difficult. A correct interpretation of experimental HRTEM images of tourmaline is possible provided the structural refinement data on the same crystal are available. Nevertheless, it is possible to interpret the experimental HRTEM images of tourmaline if the composition of the structural model chosen during image simulations approximates the composition of the specimen studied by electron microscopy. A good control of the composition of the specimen studied and an appropriate choice of a structural model for image simulation are therefore as important as properly controlling specimen thickness, specimen tilt, beam tilt and objective lens defocus.
Thick bone section preparation using a silicon‐rubber‐based sealant
Authors: COX H. , WALKER C. , HOWARD C. V.
Published: August 2011

A method has been developed, using a silicon‐rubber‐based sealant, which allows 2–3‐mm‐thick specimens to be maintained in a protected fluid environment for a number of months, without risk of dehydration. Following this, the specimen can be retrieved, stained, embedded and sectioned further. For example, 2‐mm‐thick sections of fixed unstained bone are easily examined by means of epi‐illuminated polarized light and fluorescence microscopies using either conventional or confocal optics. The method could easily be extended to other tissues, for example brain tissue.
Application of confocal laser microscopy and three‐dimensional Voronoi diagrams for volume and surface estimates of interphase chromosomes
Authors: EILS R. , BERTIN E. , SARACOGLU K. , RINKE B. , SCHRÖCK E. , PARAZZA F. , USSON Y. , ROBERT‐NICOUD M. , STELZER E. H. K. , CHASSERY J.‐M. , CREMER T. , CREMER C.
Published: August 2011

This study demonstrates the use of Voronoi tessellation procedures to obtain quantitative morphological data for chromosome territories in the cell nucleus. As a model system, chromosomes 7 and X were visualized in human female amniotic fluid cell nuclei by chromosomal in situ suppression hybridization with chromosome‐specific composite probes. Light optical serial sections of 18 nuclei were obtained with a confocal scanning laser fluorescence microscope. A three‐dimensional (3‐D) tessellation of the image volumes defined by the stack of serial sections was then performed. For this purpose a Voronoi diagram, which consists of convex polyhedra structured in a graph environment, was built for each nucleus. The chromosome territories were extracted by applying the Delaunay graph, the dual of the Voronoi diagram, which describes the neighbourhood in the Voronoi diagram. The chromosome territories were then described by three morphological parameters, i.e. volume, surface area and a roundness factor (shape factor). The complete evaluation of a nucleus, including the calculation of the Voronoi diagram, 3‐D visualization of extracted territories using computer graphic methods and parameterization was carried out on a Silicon Graphics workstation and was generally completed within 5 min. The geometric information obtained by this procedure revealed that both X‐ and 7‐chromosome territories were similar in volume. Roundness factors indicated a pronounced variability in interphase shape for both pairs of chromosomes. Surface estimates showed a significant difference between the two X‐territories but not between chromosome 7‐territories.
PHOEBE, a prototype scanning laser‐feedback microscope for imaging biological cells in aqueous media
Authors: WONG T. L. , SABATO S. L. , BEARDEN A.
Published: August 2011

Based on the principle of laser‐feedback interferometry (LFI), a laser‐feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of ∼ 1 nm and a lateral (x, y) resolution of ∼ 200 nm when used with a high‐numerical‐aperture oil‐immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re‐entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back‐reflected light gives information about an object's position and reflectivity. Using a 1‐mW He–Ne (λ = 632·8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 × 256‐pixel images over fields from (10 μm × 10 μm) to (120 μm × 120 μm) in ∼ 30 s. An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back‐reflected light originating from small refractive‐index changes (> 0·02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point‐by‐point by growing or placing a cell suspension on a higher‐reflecting substrate (glass or a silicon wafer). Advantages of the laser‐feedback microscope in comparison to other confocal optical microscopes include: the simplicity of the single‐axis interferometric design; the confocal property of the laser‐feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re‐enters the laser meeting the stringent frequency, spatial (TEM00), and coherence requirements of the laser cavity resonator mode modulate the laser intensity; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.
Near‐field imaging: some attempts to define an apparatus function
Authors: COURJON D.
Published: August 2011

Near‐field microscopy is a promising new tool capable of imaging details smaller than the wavelength. The mechanism of imaging is analysed and an overview of the apparatus functions which could be used to define an image quality criterion is given.
Microstructure and thermo‐mechanical stability of a low‐oxygen Nicalon fibre
Authors: BERGER M. H. , HOCHET N. , BUNSELL A. R.
Published: August 2011

A new Nicalon SiC‐based fibre, characterized by a low oxygen content (0–5% wt) has been studied. The absence in this fibre of a continuous Si–C–O phase, which characterized the previous NLM 202 series of fibres, induces larger mean sizes for the constituents: the fibre is composed of β‐SiC grains 5–20 run in diameter and turbostratic aggregates of carbon 2–5 nm in diameter. The fibre is seen to be suffer at room temperature (E= 300 GPa) and stronger due to a reduction in critical defects thanks to improvements in processing conditions. The Young's modulus remains almost stable up to 1473 K in air and above this temperature the core of the fibre exhibits continuous grain growth up to 1773 K, but without the degradation that occurred in the previous generation of fibres. Fibre strength was seen to be lowered when compared to room temperature values even when exposed in air to temperatures of 1073 K. A comparable fall is not seen with the NLM 202 fibres until 1273 K and this difference is attributed to the oxidation of the carbon‐rich surface of the new fibre. SiC is oxidized at higher temperatures, inducing, above 1473 K, the growth of a silica layer on the surface, with defects at the glass/ceramic interface. The large discrepancies between the good thermo‐mechanical characteristics in inert atmosphere and the behaviour in air may be reduced if a coating resistant to oxidation could be applied to the fibre.
Environmental ageing effects in a silicon carbide fibre‐reinforced glass‐ceramic matrix composite
Authors: PLUCKNETT K. P. , SUTHERLAND S. , DANIEL A. M. , CAIN R. L. , WEST G. , TAPLIN D. M. R. , LEWIS M. H.
Published: August 2011

A silicon carbide fibre‐reinforced glass‐ceramic composite, based upon a BaO–MgO–Al2O3–SiO2 (BMAS) matrix, has been used for a study of microstructural stability (specifically interface stability) after environmental exposure at elevated temperature. Characterization of the as‐received material demonstrated the presence of a thin ‘carbon‐rich’ interfacial layer between fibre and matrix, as typically observed in glass‐ceramic/silicon carbide fibre composite systems. Samples have been subjected to heat‐treatments in an oxidizing atmosphere at temperatures between 723 and 1473 K, for up to 500 h. Intermediate‐temperature ageing, between 873 and 1073 K, results in strong fibre/matrix bonding, with consequent degradation of strength and composite ‘ductility’. This is due to oxidative removal of the carbon interfacial layer and subsequent oxidation of the fibre surface, forming a silica bridge. Carbon is retained at higher ageing temperatures due to the formation of a protective surface oxide scale at exposed fibre ends. Attempts to pretreat the BMAS composite at high temperature (1273–1473 K), designed to inhibit intermediate‐temperature degradation via the formation of silica plugs at exposed fibre ends, has given mixed results due to the high residual porosity content in these materials, allowing paths of ‘easy’ oxygen ingress to be retained.
Synthesis of silicon carbide ceramics from a polysilastyrene at low temperatures
Authors: McMILLAN S. M. , BROOK R. J.
Published: August 2011

The conventional route for preparation of silicon carbide ceramics is by the use of pressureless sintering, hot pressing, or hot isostatic pressing of silicon carbide starting powders. High sintering temperatures (2073–2473 K) and the addition of sintering additives are normally used to enhance densification. These sintering additives, however, form second phases at grain boundaries which impair the mechanical properties of the material, particularly at high temperatures. It is therefore desirable that new processing routes are developed that overcome these difficulties. A proposed route is to use a polymeric pressure which can provide a Silicon carbide matrix as binding agent for silicon carbide powders, thus making the requirement for high temperatures and sintering additives unnecessary. This paper reports observations of the direct transformation of a polymeric precursor into amorphous Si–C, and crystalline SiC at low temperatures, and the use of this precursor as a binder for the production of SiC powder/ex‐precursor SiC composites.
A comparison of the microstructure of silicon nitride‐silicon carbide composites made with and without deoxidized starting material
Authors: TURAN S. , KNOWLES K. M.
Published: August 2011

Two different types of silicon carbide (SiC) matrix composites, with either 10 wt% or 20 wt% silicon nitride (Si3N4) reinforcement, were fabricated to investigate the effect of pretreatment on the resulting composite micro‐structure. The first type of composite was prepared from as‐received α‐SiC and α‐Si3N4 powders, while the second type was prepared from powder compacts that had been deoxidized to eliminate surface silica on the powder particles. The composites were hot isostatically pressed in tantalum cans at 2373 K for 1h under a pressure of 200 MPa. Density measurements showed that full theoretical density was achieved for the composites prepared from the as‐received powders, while much lower densities were obtained for the composites prepared from the deoxidized green compacts. Almost all of the α‐SiC transformed into β‐SiC, and almost all the α‐Si3N4 transformed into α‐Si3N4 in the composites made from the as‐received powders, while in the composites made from the deoxidized material the α‐SiC remained untransformed and both α‐Si3N4 and β‐Si3N4 phases were present in significant quantities. High‐resolution transmission electron microscopy and Fresnel fringe imaging were used to identify the grain boundary and interphase boundary structure. Most interfaces were found to be covered with ≅ 1 nm thick amorphous intergranular films in the composites prepared from as‐received powders, whereas most interfaces were found to be free of such amorphous intergranular films in the composites prepared from the deoxidized material. Taken together, the presence of intergranular films at the interfaces and the results from density measurements are consistent with the densification and reverse α → β‐SiC transformation taking place in the composites made from as‐received powders by a liquid‐phase sintering route. An incomplete liquid‐phase sintering mechanism is also able to explain the microstructure observed in the composites made from the deoxidized material.
Microstructural origins of corrosion in a 20% SiCp/2124 aluminium alloy metal matrix composite
Authors: IMESON D. , BARTLETT D. L.
Published: August 2011

The microstructure of a metal matrix composite (MMC) consisting of 20 wt% 3 μm SiC particles in a 2124 Al alloy matrix has been examined and the relationship to corrosion pitting processes investigated. Standard bulk corrosion tests show that the MMC forms a higher density of pits than the unreinforced alloy, although the overall performance is similar as the pits are shallower. In a new addition to conventional characterization techniques, transmission electron microscopy samples have been directly subjected to ‘flash’ corrosion treatment and subsequently examined. This technique is shown to be effective in studying the initiation of pits. The SiC particles and the widespread 9 intermetallic precipitates are shown to play little, if any, role. A sparse population of features introduced during powder processing and consolidation procedures, probably linked with strong magnesium segregation, is suggested to be responsible for pit initiation.
Ion microprobe studies of reactions in squeeze‐cast aluminium alloy matrix composites
Authors: SONI K. K. , KANG H. G. , GRANT P. S. , CANTOR B. , ADRIAENS A. G. , GAVRILOV K. L. , MOGILEVSKY R. , LEVI‐SETTI R. , TSENG M. W. , WILLIAMS D. B.
Published: August 2011

An ion microprobe with high lateral resolution has been used to study the chemical reactions at the fibre/matrix interface of metal–matrix composites. During the squeeze‐casting process, the Al–Si–Mg matrix reacts with the preform made of Saffil fibres (96% Al2O3, 4% SiO2). The reaction occurs mainly between the silica binder and Mg from the matrix according to SiO2 + 2Mg = 2MgO + Si. A continuous layer of MgO was formed around the fibres, even on surfaces that were not covered by the silica binder. Possible reasons are discussed for the formation of MgO in areas where binder coating was missing. In such areas, Mg reduces SiO2 that is contained in the fibre. However, the fibres (A12O3) are not attacked by Mg. In the isolated case of fibres that were completely uncoated, no reaction products were observed at the interface. The presence of silica binder seems to be an essential requirement for this reaction to occur. When squeeze‐casting is performed with sufficiently high melt temperature, Al from the matrix also reduces silica.
Preliminary confocal scanning laser microscopy study of fluid inclusions in quartz
Authors: PETFORD N. , MILLER J. A. , RANKIN A. H.
Published: August 2011

The initial results of the first dedicated confocal scanning laser microscopy (CSLM) study of fluid inclusions in quartz are presented. CSLM imaging of a large inclusion shows the quartz crystal to contain numerous small (< 1 μm), highly reflective inclusions arranged along planes in at least two directions that are not readily visible in transmitted light. The technique allows measurements to be made of the angular intersection and orientation of the planes in both two and three dimensions. Results suggest that larger inclusions (> 10 μm) occur where two planes of small inclusions intersect, and that the shape of the large inclusions is controlled by the angular relationship between intersecting planes.
STM imaging of metal‐coated cell plugs of the archaeobacterium Methanospirillum hungatei GP1
Authors: XU W. , BLACKFORD B. L. , MULHERN P. J. , JERICHO M. H. , FIRTEL M. , BEVERIDGE T. J.
Published: August 2011

Scanning tunnelling microscopy (STM) images of Pt/Ir‐ and Pt/Ir/C‐coated cell plugs of Methanospirillum hungatei showed paracrystalline structures with P6 symmetry and an 18‐nm lattice constant, in agreement with electron microscopy studies. The three‐dimensional STM images unambiguously distinguished the two morphologically different proteinaceous plug assemblies and led to an improved understanding of the natural internal organization of whole plugs. Tip convolution effects and the grain size of the metal coating complicated interpretation of finer structures. We discuss possible imaging mechanisms to explain observations in which part of the film was removed but the remaining part of the structure was still imaged reproducibly.
Limits of electron probe formation
Authors: CREWE A. V.
Published: August 2011

The performance of many electron optical instruments is fundamentally limited by the dimensions of the focused probe. This is true of the scanning electron microscope and the scanning transmission electron microscope and, by inference, it may affect the transmission electron microscope. There has been very little improvement over the past few years and it seems reasonable to look for the explanation. It is possible to arrive at some simple expressions for the limiting performances of conventional instruments in a way that is independent of the design details and depends upon practical limits of field strength. Experiment and theory also appear to be in agreement with the fact that the limit for high‐voltage instruments has been reached, although there is still room for improvement for low voltages.
Environmental scanning electron microscopy of marine aggregates
Authors: LAVOIE D. M. , LITTLE B. J. , RAY R. I. , BENNETT R. H. , LAMBERT M. W. , ASPER V. , BAERWALD R. J.
Published: August 2011

Marine aggregates were examined for the first time in the hydrated state using an environmental scanning electron microscope (ESEM). Sample preparation consisted of fixation followed by rinsing with distilled water to remove excess salts and fixative. Aggregates were continuously observed at resolutions comparable to conventional scanning electron microscopy through stages of hydration, from completely immersed to desiccated. Because no metallic coating is required, energy‐dispersive X‐ray spectroscopy (EDXS) can be used to analyse rapidly constituent elements occurring at low concentrations with no spectral interference. Subtle differences in mineral particles were seen in both EDXS spectra and in direct observation of relative hydration, reflecting apparent differences in mineralogy. ESEM enabled examination of effects of desiccation and rehydration on individual particles composed primarily of hydrated polymer and eliminated dehydration artefacts in delicate organisms.