bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of primary cell cultures and intact isolated glandular epithelia for X‐ray microanalysis
Authors: HONGPAISAN J. , ZHANG A.‐L. , MÖRK A.‐C. , ROOMANS G. M.
Published: November 2003

Changes in the elemental composition of cells during isolation of glandular epithelia were studied by electron probe X‐ray microanalysis. Fine chopping of rat submandibular gland followed by enzymatic treatment for 15 min caused marked increases in Na and Cl and a decrease in K concentrations in acinar cells. After enzymatic treatment for 50 min, Na, Cl and K concentrations returned to close to the control level. Mechanical disaggregation of the acinar clumps following enzymatic treatment resulted again in minor increases in Na and Cl and a marked decrease in K concentration. Exposure of isolated acini to cholinergic stimulation in vitro resulted in secretion of Cl and K from the acinar cells. Dissection of the sweat gland from human skin caused a decrease in the K/Na ratio. Incubation of the gland for 30–45 min with collagenase gave rise to a gradual decrease in the K/Na ratio. After mechanical separation of the gland into the secretory coil and reabsorptive duct, a further reduction of the K/Na ratio was seen. However, the duct cells had a much lower K/Na ratio and higher Ca concentration than the coil cells. In primary cultures, the K/Na ratios of the coil and duct cells returned to the in situ level. The elemental composition of sweat gland cells incubated in collagenase‐containing medium was no different from that in cells incubated in collagenase‐free medium. In the intact collagenase‐isolated tissue, Cl− secretion in the coil was elicited by carbachol but not by cAMP, whereas in the duct cells the reverse was the case. In primary cell cultures, Cl− efflux in both coil and duct cells could be elicited by both carbachol and cAMP. In conclusion, although changes in elemental composition of gland cells during the isolation procedure occur, physiological responses can be detected. When primary cell cultures are used, it should be borne in mind that cultured cells may have physiological properties different from those of the intact tissue.
Visualization of volume data in confocal microscopy: comparison and improvements of volume rendering methods
Authors: LUCAS L. , GILBERT N. , PLOTON D. , BONNET N.
Published: November 2003

Confocal microscopes routinely produce three‐dimensional data sets. The visualization of these digital volumes is currently performed by one surface rendering or volume rendering approach. In this paper, we describe improvements developed in the field of volume rendering. We focused on three methods: parallelepiped face mapping: the rotation‐projection method (with or without stereoscopy, with different matters and transparencies); the voxel ray‐tracing method. We compared the possibilities of these different algorithms, in terms of quality of rendering, of computation load and as an essential aid to study the 3D organization of biological specimens.
Thermodynamic limitations on the resolution obtainable with metal replicas
Authors: WOODWARD IV J. T. , ZASADZINSKI J. A.
Published: November 2003

The major factor limiting resolution of metal‐shadowed surfaces for electron and scanning tunnelling microscopy is the granularity of the metal film. This granularity had been believed to result from a recrystallization of the evaporated film, and hence could be limited by use of higher melting point materials for replication, or inhibited by adding carbon or other impurities to the film. However, evaporated and sputtered films of amorphous metal alloys that do not crystallize also show a granularity that decreases with increasing alloy melting point. A simple thermodynamic analysis shows that the granularity results from a dewetting of the typically low surface energy sample by the high surface energy metal film, similar to the beading up of drops of spilled mercury. The metal granularity and the resulting resolution of the metal‐coated surface is proportional to the mobility of the metal on the surface after evaporation, which is related to the difference in temperature between the melting point of the metal and the sample surface temperature.
Probing nuclear ultrastructure by electron spectroscopic imaging
Authors: HENDZEL M. J. , BAZETT‐JONES D. P.
Published: November 2003

Mammalian nuclei are complex organelles containing many functionally distinct nucleoprotein and protein particles in the size range 20–30 nm. This complexity hinders the study of structure‐function relationships within the mammalian nucleus. Element‐specific mapping using the energy‐filtered transmission electron microscope can provide novel information on protein and nucleic acid density within structures, facilitating the identification of biochemical heterogeneity within morphologically similar structures. We demonstrate that imaging phosphorus, nitrogen and carbon can be useful in the characterization of protein and nucleoprotein structures within the nucleus. Additionally, electron spectroscopic imaging (ESI) may be used to map the distribution of stains relative to unstained material when biochemical‐specific staining protocols, such as EDTA‐regressive staining of RNA with uranyl acetate, are used. Relative mass may also be determined from ESI images and can be combined with elemental information further to distinguish biological constituents. Using this approach, heterochromatin was found to be variable in nucleic acid content although the morphology appeared relatively homogeneous. ESI shows substantial promise for the investigation of structure–function relationships in biological specimens.
On the detection of MX‐precipitates in microalloyed steels using energy‐filtering TEM
Authors: Hofer F. , Warbichler P. , Buchmayr B. , Kleber S.
Published: November 2003

The properties of microalloyed steels and HSLA steels depend predominantly on the size and composition of nanometre‐sized MX precipitates consisting of Nb, Ti, V, C and N. In this work we present a rapid and powerful method for visualizing these small precipitates directly in the steel matrix.

This can be achieved by energy‐filtering TEM in a 200‐kV microscope. We have recorded elemental maps (three‐window method) and jump‐ratio images (two‐window method) using the inner‐shell ionization edges; e.g. Fe‐M23, Fe‐L23, V‐M23, V‐L23, Nb‐N23, Nb‐M45, C‐K and N‐K edges. We have found that precipitates in a steel matrix can be best visualized by recording the jump‐ratio image of the matrix element. If the specimens are thinner than 30 nm and high resolution in the images and low detection limits are essential, low energy‐loss edges should be used preferentially; in the case of steels the Fe‐M23 edge should be used instead of the Fe‐L23 edge. If the jump‐ratio images are recorded with the Fe‐M23 edge, precipitates about 2 nm in diameter may be detected. With thicker samples and if high resolution is not as essential, higher energy‐loss edges are preferable, because diffraction effects are less possible.

The chemical composition of the larger precipitates (10 nm) could be determined by recording EDX spectra using an STEM probe. We found Nb and V with an atomic ratio of about 3, but we could not detect carbon or nitrogen in the spectra. The Nb and V distribution could be best visualized with a jump‐ratio image including both the Nb‐N23 and the V‐M23 edges. However, it was not possible to image the carbon or nitrogen content of the precipitates unequivocally due to overlapping of the C‐K and N‐K edges with the Nb‐M45 edge.
Microwave‐enhanced fixation of rabbit articular cartilage
Authors: RICHARDS R. G. , KÄÄB M. J.
Published: November 2003

Cartilage, being highly aqueous, is difficult to preserve for electron microscopy without artefacts. Microwave‐enhanced fixation is suggested as a standard method for block samples of this material, with dimensions of up to 12 × 7 × 3 mm. Cartilage samples from the tibial plateau of adult rabbits were fixed by conventional, cryo‐ or microwave‐enhanced fixation. Constant or cyclical microwave irradiation of samples, immersed in fixatives, was carried out to varying final solution temperatures. Microwave‐enhanced fixation and staining is shown to be both rapid and reproducible, giving fine structural preservation. Below 323 K microwave fixation always gave excellent preservation of the fine structure within seconds. At higher temperatures thermal artefacts were introduced. In this study the microwave‐enhanced fixation is equal in quality to the best conventional immersion fixation and is nearly as fast as cryo‐preservation. It provides a standardized, reproducible fixation for morphological studies on cartilage with good process control.
Simple modification of a commercial scanning laser microscope to incorporate dark‐field imaging
Authors: TÖRÖK P. , LACZIK Z. , SKEPPER J. N.
Published: November 2003

Dark‐field imaging modes have attracted less interest in biological confocal microscopy than the extensive applications of immunofluorescence imaging. There are, however, certain biological and materials science applications where it is necessary to use a confocal imaging mode that is capable of partial or full rejection of light specularly reflected from coverslips, microscope slides or specimen surfaces. In this paper we present a simple modification of a commercial confocal microscope to incorporate dark‐field imaging. We discuss theoretical aspects of the resulting dark‐field imaging mode and also give experimental examples.
ESI contrast analysis: a new approach for element analysis with energy‐filtering transmission electron microscopy (EFTEM)
Authors: Körtje K.‐H.
Published: November 2003

Element microanalysis with energy‐filtering transmission electron microscopy can be performed in different ways. Electron energy‐loss spectroscopy (EELS) records the intensity as a function of the energy loss from selected regions. Elemental mapping with electron spectroscopic imaging (ESI) uses energy‐filtered images at an element‐specific energy loss from which a background image has to be subtracted. A combination of these two approaches is Image‐EELS, which records a series of energy‐filtered images, each at a different energy loss and measures the intensity of selected regions as a function of the energy loss. As an additional procedure ESI contrast analysis is introduced; with this we can investigate the image contrast as a function of the energy loss. The contrast can be measured for the total image or for selected regions as the standard deviation of the grey levels divided by the mean grey level. This procedure is added as a new feature to the existing Image‐EELS program, so that EELS intensity spectra and the contrast can be directly compared. Alternatively, the contrast can be calculated step by step from individually recorded electron spectroscopic images, so that only simple equipment for image analysis is sufficient to realize this new method.

Inner shell ionizations produce characteristic, but often weak element edges in energy‐loss spectra which can be detected more sensitively and reliably as a rapid increase in contrast. Regional analysis of the contrast as a function of the energy loss, combination of data from different regions and the possibility to increase the intensity during recording of the images expand the application range of this new analytical method which bridges the gap between ESI and EELS analysis. With ESI contrast analysis it is possible to discriminate between faint element signals and artefacts in elemental mapping.

This new approach for element detection is especially advantageous for biological objects which usually contain very small amounts of the interesting elements in heterogeneous and complex objects. As an example, nervous tissue of a fish was analysed after cytochemical precipitation of endogenous calcium.
Finite sized coherent and incoherent detectors in confocal microscopy
Authors: WILSON T. , TAN J. B.
Published: November 2003

We consider the effects of finite sized coherent and incoherent detectors on the axial resolution of confocal microscopes. We adopt a high‐angle vector approach which takes the polarization property of the object into account. We further consider polarization imaging and show that coherent detection has an advantage over incoherent detection in terms of the value of the extinction coefficient. The desirability of aberration correction is briefly discussed.
TEM cross‐section preparation with minimal ion milling time
Authors: SCOTT C. P. , CRAVEN A. J. , HATTO P. , DAVIES C.
Published: November 2003

The production of high quality thin film TEM cross‐sections suitable for microanalysis is often a difficult and time‐consuming task. This is particularly so in cases where there exists a large difference between the sputtering rate of the film and that of the substrate. The problem is further exacerbated when the levels of internal stress in the film are high enough to cause the substrate to distort during the thinning process. This paper describes some modifications to existing techniques which allow a greater degree of mechanical thinning prior to the ion etching stage. Consequently, ion milling times are drastically reduced, typically by a factor of at least 5 and by as much as 25 in some cases.
Two‐photon scanning microphotolysis for three‐dimensional data storage and biological transport measurements
Authors: KUBITSCHECK U. , TSCHÖDRICH‐ROTTER M. , WEDEKIND P. , PETERS R.
Published: November 2003

Scanning microphotolysis is a method that permits the user to select, within the scanning field of a confocal microscope, areas of arbitrary geometry for photobleaching or photoactivation. Two‐photon absorption, by contrast, confers on laser scanning microscopy a true spatial selectivity by restricting excitation to very small focal volumes. In the present study the two methods were combined by complementing a laser scanning microscope with both a fast programmable optical switch and a titan sapphire laser. The efficiency and accuracy of fluorescence photobleaching induced by two‐photon absorption were determined using fluorescein‐containing polyacrylamide gels. At optimal conditions a single scan was sufficient to reduce the gel fluorescence by ≈40%. Under these conditions the spatial accuracy of photobleaching was 0.5±0.1 μm in the lateral (xy) and 3.5±0.5 μm in the axial (z) direction, without deconvolution accounting for the optical resolution. Deconvolution improved the accuracy values by ≈30%. The method was applied to write complex three‐dimensional patterns into thick gels by successively scanning many closely spaced layers, each according to an individual image mask. Membrane transport was studied in a model tissue consisting of human erythrocyte ghosts carrying large transmembrane pores and packed into three‐dimensional arrays. Upon equilibration with a fluorescent transport substrate single ghosts could be selectively photobleached and the influx of fresh transport substrate be monitored. The results suggest that two‐photon scanning microphotolysis provides new possibilities for the optical analysis and manipulation of both technical and biological microsystems.
Quantitative electron spectroscopic imaging in bio‐medicine: evaluation and application
Authors: BECKERS A. L. D. , GELSEMA E. S. , DE BRUIJN W. C. , CLETON‐SOETEMAN M. I. , VAN EIJK H. G.
Published: November 2003

Electron spectroscopic imaging (ESI) with the energy‐filtering transmission electron microscope enables the investigation of chemical elements in ultrathin biological sections. An analysis technique has been developed to calculate elemental maps and quantitative distributions from ESI sequences. Extensive experience has been obtained with a practical implementation of this technique. A procedure for more robust element detection has been investigated and optimized. With the use of Fe‐loaded Chelex beads, the measurement system has been evaluated with respect to the linearity of the element concentration scale, the reproducibility of the measurements and the visual usage of image results. In liver specimens of a patient with an iron storage disease the detectability of iron was tested and we tried to characterize iron‐containing components.  The concentration measurement scale is approximately linear up to a relative section thickness of ≈ 0.5. Monitoring of this parameter is therefore considered to be important. The reproducibility was measured in an experiment with Fe‐Chelex. The iron concentration differed by 6.4% between two serial measurements. Element distributions are in many applications interpreted visually. For this purpose the frequently used net‐intensity distributions are regarded as unsuitable. For the quantification and visual interpretation of concentration differences mass thickness correction has to be performed. By contrast, for the detection of elements the signal‐to‐noise ratio is the appropriate criterion.  Application of ESI analysis demonstrated the quantitative chemical capabilities of this technique in the investigation of iron storage diseases. Based on an assumed ferritin iron loading in vivo, different iron components can be discerned in liver parenchymal cells of an iron‐overloaded patient.
Extended energy‐loss fine structure spectroscopy of structural modifications in Nd2CuO4−δ at the oxygen K edge
Authors: VAN AKEN P. A.
Published: November 2003

The discovery of the superconducting electron‐doped compound Nd185Ce015CuO4−δ has stimulated great interest in its micro‐ and crystal structure, since the superconducting properties depend on parameters such as nonstoichiometry, phase composition, heat treatment and microstructure. The work presented herein is focused on the determination of the oxygen environment in the undoped parent compound Nd2CuO4 and in the structural modification Nd2CuO35

The analysis of the oxygen K (O 1s) edge extended electron energy‐loss fine structure (EXELFS) of the tetragonal parent compound Nd2CuO4 and of the orthorhombic modification Nd2CuO35 is reported by using electron energy‐loss spectroscopy in combination with transmission electron microscopy. Nd2CuO35 is produced by in situ heating and reduction of Nd2CuO4 in the transmission electron microscope. The EXELFS of the O 1s electron energy‐loss edges is analysed with the classical extended X‐ray absorption fine structure (EXAFS) treatment and compared with ab initio multiple scattering EXAFS calculations for both structural modifications. Highly accurate information on the local atomic environment of the oxygen atoms in Nd2CuO35 is obtained from EXELFS analysis using Nd2CuO4 as a standard. The results are in accordance with the structural data gained from X‐ray diffraction analysis. This applies especially to the more complicated structure of Nd2CuO35 determined recently.
Electrically conducting polymers: preparation and investigation of oxidized poly(acetylene) by EFTEM
Authors: LIESER G. , SCHMID S. C. , WEGNER G.
Published: November 2003

Energy‐filtering transmission electron microscopy (EFTEM) was used to demonstrate that the oxidation of poly(acetylene), necessary to produce a highly conducting phase, proceeds homogeneously on a scale of 100 nm to several micrometres. The distribution of the anions BF4− and PF6− serves as indicator for the penetration of poly(acetylene) by the oxidizing agent. The sample preparation necessitates prevention of sample degradation by air and moisture. It was possible to synthesize, orientate, oxidize, embed and section poly(acetylene) samples under inert conditions. Only mounting of the grids into the sample holder of the electron microscope was performed under ambient conditions. EFTEM was possible for phosphorus and boron, but not for fluorine owing to irradiation damage leading to loss of fluorine from the sample. Polyvinylbutyral was chosen as the embedding medium because it allows embedding of the sample prior to oxidation. Embedding of a previously oxidized poly(acetylene) sample leads to migration of the anions from the interior of the sample into the embedding medium.
Zero‐loss energy filtering under low‐dose conditions using a post‐column energy filter
Authors: GRIMM R. , KOSTER A. J. , ZIESE U. , TYPKE D. , BAUMEISTER W.
Published: November 2003

Electron cryomicroscopy combined with energy filtering can be performed under low‐dose conditions using a post‐column energy filter. The microscope combined with the filter is set up such that it can be used with similar ease as a conventional microscope, the main difference being that all filter and microscope control is performed through a central computer and images are recorded with a cooled slow‐scan CCD camera. The microscope can also still be used for regular imaging on film as without the filter. Owing to the 18 times post‐magnification of the filter, the microscope normally has to be operated at a small magnification, e.g. 3000×, and the beam has to be contracted to a small spot, e.g. 5 mm, in the plane of the microscope viewing screen. Computer control allows one to perform a variety of tasks automatically, such as autofocusing, thickness measurements, most‐probable‐loss imaging, CCD spot‐scanning and tomography. The gain in contrast due to zero‐loss energy filtering is analysed using visual inspection, power spectra and Fourier ring correlation. The thickness range for ice‐embedded specimens in which a filter at 120 kV is most useful appears to be between 100 and 300 nm.
A study of Si‐L and O‐K ELNES in plant material: SiO2, Ca‐ and Zn‐silicate in Minuartia
Authors: LICHTENBERGER O. , NEUMANN D.
Published: November 2003

The formation of vacuolar precipitates containing silicon and calcium, as well as the accumulation of metal‐silicates in cell walls of Minuartia has been investigated by electron microscopy, energy‐dispersive X‐ray analysis and electron spectroscopic imaging. The bond state of the elements concerned is examined by analysing energy‐loss near‐edge structures (ELNES), particularly the Si‐L2,3 and the O‐K ELNES. Experimental ELNES results are compared with local densities of unoccupied states calculated by molecular orbital methods and results of XANES calculations.
Data‐driven, simultaneous blur and image restoration in 3‐D fluorescence microscopy
Authors: AVINASH G. B.
Published: November 2003

A novel algorithm for simultaneous blur and image restoration (SBIR)* in three‐dimensional (3‐D) fluorescence microscopy is presented. All the internal parameters including the point spread function essential for the restoration are estimated from the data. Validation of the SBIR algorithm using simulated signals/images and known real world specimens is provided. Both lateral and axial resolution of images are improved by the application of the algorithm. Finally, the results of the application of the algorithm to unknown specimens are shown, demonstrating the potential of the algorithm in practical applications. Furthermore, evidence is provided to show that this algorithm can provide a turn‐key system to deblur images in 3‐D fluorescence microscopy.
Automated recognition and mapping of immunolabelled neurons in the developing brain
Authors: HIBBARD L. S. , McCASLAND J. S. , BRUNSTROM J. E. , PEARLMAN A. L.
Published: November 2003

The cerebral cortex is distinguished by layers of neurons of different morphologies and densities. The layers are formed by the migration of newly generated neurons from the ventricular zone to the cortical plate near the outer (pial) boundary of the cortex, along radial paths approximately perpendicular to the cortical surface. Immunochemical labelling makes these cells’ patterns visible in brightfield microscopy so that layer formation can be studied. We developed a suite of programs that automatically digitize the entire cortex, identify the labelled cells and compute cell densities along local radial paths. Cell identification used supervised classification on all the significantly stained objects corresponding to maxima in lowpass filtered versions of the digital micrographs. Classification of all the stained objects as cells or noncell objects was made by a decision rule based on morphometric and grey‐level texture features, including features based on Gabor functions. Detection sensitivity and classification accuracy were jointly maximized on training data consisting of about 3000 expert‐identified neurons in micrographs. Total program performance was tested on a separate (test) set of labelled neurons the same size as the training data set. The program detected 85% of the cells in the test set with a total error of 019. The identified cells’ locations were used to compute population densities along normals to the cortical layers, and these densities served as a measure of neuronal migration. Transcortical density profiles obtained by computation and by manual cell counting were very similar. The cell identification program was built on well‐established methods in statistical pattern recognition and image analysis and should generalize readily to other histological preparations.
Visualization and electron diffraction on cryosections of stratum corneum: a utopia?
Authors: WIJDEVELD M. M. G. , KOERTEN H. K. , ONDERWATER J. J. M. , PARROTT D. T. , BOUWSTRA J. A.
Published: November 2003

The skin acts as an effective barrier to protect the body against penetration of substances from the environment and against desiccation. The main barrier function resides in the stratum corneum, and more specifically in the intercellular lipid domains. Several techniques have been used to elucidate the local lipid crystal arrangements in these domains, but they either needed an extensive pretreatment of the skin with the risk of damaging the native structure, or were not suited to obtain local structure information as bulk quantities of stratum corneum were required. In this paper a method of performing local structure analysis (electron diffraction) on cryo‐fixed specimens is described. Therefore a cold chain procedure was used to obtain cryosections of stratum corneum. On these sections visualization and electron diffraction at low temperature were carried out.

Using a so‐called tape sandwich method, cryosections were prepared in which corneocytes and lipid matrix could easily be distinguished. Moreover, detailed cellular components such as desmosomes and intracellular lipid domains were observed. However, probably due to the limited amount of intercellular lipids in the stratum corneum, electron diffraction on cryosections did not result in diffraction patterns that were undoubtedly originating from the intercellular lipids. In the electron diffraction patterns of a skin lipid model system reflections were present that were indicative of hexagonal and orthorhombic sublattices. The d‐spacings of these reflections were similar to the spacings of the high‐intensity reflections of the X‐ray diffraction pattern of the same mixture. This showed agreement between a bulk and a local technique, X‐ray and electron diffraction.
Methanol as a rapid fixative for the investigation of plant surfaces by SEM
Authors: Neinhuis C. , Edelmann H. G.
Published: November 2003

A rapid fixation/dehydration method of plant specimens for scanning electron microscopy is presented. Prior to critical‐point drying (CPD) the specimens are immersed in pure methanol. Methanol incubation instantly fixes the elastically extended cell walls. Owing to this instant fixation, shrinking of the specimens is prevented, resulting in an improved preservation of cell dimensions comparable to in vivo conditions. The method is most suitable for plant epidermal surfaces. It avoids the time‐consuming fixation/dehydration in routine investigation of plant surfaces prior to CPD, especially for delicate specimens.
Methods for X‐ray microanalysis of epidermis: the effect of local anaesthesia
Authors: GRÄNGSJÖ A. , LINDBERG M. , ROOMANS G. M.
Published: November 2003

The effect of local anaesthesia on the elemental content of cells in human epidermis was studied by electron probe X‐ray microanalysis. Local anaesthesia with lidocaine was given by intracutaneous injection within 1 min prior to taking a skin biopsy. Biopsies taken without local anaesthesia were used as controls. Lidocaine with or without adrenaline caused a significant increase in the concentrations of Na and Cl, and a decrease in the concentration of K in the cells of the stratum basale and the stratum spinosum, compared with the control samples. The presence of adrenalin in the anaesthetic did not change the effect of lidocaine. The effects of local anaesthesia have to be considered in planning and interpretation of clinical applications of X‐ray microanalysis.
A new method for the model‐independent assessment of thickness in three‐dimensional images
Authors: Hildebrand T. , Rüegsegger P.
Published: November 2003

Three‐dimensional (3‐D) structural parameters derived from lower‐dimensional measurements using indirect morphometric methods may be strongly biased if the measured objects deviate from the assumed structure model. With the introduction of 3‐D microscopic measuring techniques it is possible to obtain a complete depiction of complex spatial structures. As a consequence, new 3‐D methods have recently been developed for the estimation of morphometric parameters such as volume, surface area and connectivity by direct processing of the 3‐D images. Structure thickness is an important morphometric parameter which is usually defined for specific structure models only. In this paper we propose a general thickness definition for arbitrary structures allowing us to calculate the mean structure thickness and the thickness distribution of 3‐D objects in a direct way and independently of an assumed structure model. Additionally, an efficient implementation for the practical usage of the method is described using distance transformation. The new method is applied to trabecular bone structures measured with a 3‐D micro‐computed tomography system.
The observation of ferroelastic/ferroelectric domains in synthetic Mn3B7O13X single‐crystal boracites by light and electron microscopy
Authors: CASTELLANOS‐GUZMAN A. G. , CAMPA‐MOLINA J. , REYES‐GOMEZ J.
Published: November 2003

Crystals of halogen manganese boracite, the mineral Mg3B7O13Cl, which is currently found in bedded sedimentary deposits of anhydrite, gypsum and halite, have been grown by chemical transport reactions and were examined by polarizing light microscopy and scanning electron microscopy. For both methods the same as‐grown samples were used without having to metallize the crystal faces. Comparative electron microscope images were useful not only for observing the charging mechanism of an insulating sample bombarded by an electron beam but also for revealing the domain structure of these ferroelectric/ferroelastic materials previously observed between the crossed polars of a light microscope. EDS qualitative analysis of the crystal faces was performed for the three compositions under study, i.e. Mn3B7O13Cl, Mn3B7O13Br and Mn3B7O13I.
Stereology of single objects
Authors: Cruz‐Orive L. M.
Published: November 2003

Systematic stacks of Cavalieri type, spatial grids, vertical sections and projections, etc., are recent sampling tools for the stereology of single objects, namely of isolated objects that can be orientated and scanned at will in a prescribed way. The increasing use of modern noninvasive scanning devices is facing potential users with the challenge of encompassing the necessary knowledge to implement ‘good’ stereology. The present paper presents a coherent set of recent stereological methods for single objects in a historical perspective, emphasizing the fact that all relevant techniques — old and new — emanate from a common, relatively small set of basic principles.
Nonlinear deterministic analysis of tissue texture: a stereological study on mastopathic and mammary cancer tissue using chaos theory
Authors: Mattfeldt T.
Published: November 2003

Signals from some dynamical systems look like stochastic processes although their latent mechanism is deterministic. When such systems show sensitive dependence on small changes of initial conditions, they are denoted as deterministic chaos. Motivated by recent advances in statistical inference methods for chaotic systems and by the concept of spatial chaos, we present a deterministic approach to the study of epithelial tissue texture. Methods for estimation of the autocorrelation function, for evaluation of the power spectrum, for attractor reconstruction, for estimation of the Lyapunov exponent and of the correlation dimension, and for the generation of surrogate data sets are outlined. In our biological example, these methods are applied to 20 cases of mastopathy as compared to 20 cases of mammary cancer. The input signals for the analysis were estimates of epithelial fraction measured at low magnification within 5100 equally spaced line segments per case perpendicular to an arbitrarily directed axis. The results suggest the existence of a low‐dimensional deterministic attractor in mastopathic tissue texture, which is replaced by coloured noise in the majority of mammary carcinomas. Biological mechanisms for this finding and scale effects are discussed, and some methodological aspects and possible extensions of our approach are outlined.
Stereological analysis of spatial surface processes
Authors: Krejčíř P. , Beneš V.
Published: November 2003

The aim of the project presented here is to study statistical properties of surface intensity estimators using the model‐based approach to stationary surface structures. The class of estimators investigated enables a clear description of second order quantities in the anisotropic case, where the geometry of the surface structure, sampling design and observation window interact in a complex manner. Although the paper does not deal with real data, it includes theoretical examples and offers the readers a better understanding of stereological formulas up to second moments.
Generalized region growing operator with optimal scanning: application to segmentation of breast cancer images
Authors: BELHOMME P. , ELMOATAZ A. , HERLIN P. , BLOYET D.
Published: November 2003

Segmentation of medical images is a complex problem owing to the large variety of their characteristics. In the automated analysis of breast cancers, two image classes may be distinguished according to whether one considers the quantification of DNA (grey level images of isolated nuclei) or the detection of immunohistochemical staining (colour images of histological sections). The study of these image classes generally involves the use of largely different image processing techniques. We therefore propose a new algorithm derived from the watershed transformation enabling us to solve these two segmentation problems with the same general approach. We then present visual and quantitative results to validate our method.
The structure of SiC‐reinforced Mg casting alloys
Authors: Yang W. , Weatherly G. C. , McComb D. W. , Lloyd D. J.
Published: November 2003

The nature of the interface and interfacial reaction products formed between SiC particles and magnesium has been studied in two SiCp‐reinforced magnesium casting alloys, ZC63 and ZE63. In ZC63, a thin amorphous film was observed at the interface of the SiC particles. Energy‐dispersive X‐ray and electron energy‐loss spectroscopy methods were used to characterize the films. They were found to contain Si, O and Mg, with the Si tetrahedrally coordinated to the O. The alloy ZE63 contains Zr and mischmetal (Ce, La, Nd, Pr), all of which are strong oxide formers. In the as‐cast state, patches or continuous fine‐grained oxide films were found at the interface of the SiC reinforcing phase in this alloy. Electron diffraction was used to identify these oxides as the tetragonal form of ZrO2 and cubic CeO2.
Large‐area, high‐resolution image analysis of composite materials
Authors: DAVIDSON N. C. , CLARKE A. R. , ARCHENHOLD G.
Published: November 2003

An image analysis system based on a small network of parallel processors hosted within a PC has been developed to measure the orientations of glass and carbon fibres in a polymer matrix from a polished section using optical microscopy. A computer‐controlled XY translation stage is used to scan a large sample area (mm × mm), at a high magnification to minimize measurement errors. The distance that the stage moves between adjacent image frames is accurately measured allowing partial images at the edges of image frames to be reconstructed and so produce a continuous area. By removing a small amount of material from the sample by polishing and rescanning the same sample area, data from a number of serial sections can be obtained. Pattern matching is then used to correlate the fibre images from each section, allowing full three‐dimensional orientation data to be extracted, overcoming the limitations of measurements from a single, two‐dimensional section plane.
Microscopy of interfaces in model oxide composites
Authors: Johnson M. T. , Heffelfinger J. R. , Kotula P. G. , CARTER C. B.
Published: November 2003

Solid‐state reactions are known to occur in composite materials during fabrication, processing or service. In the present study, a model approach for studying such reactions is illustrated. The goal of this approach is to understand the effects of, for example, temperature, time and other driving forces on the earliest stages of such reactions. Three different materials systems were used in order to investigate some of the fundamental processes occurring. Two of the systems involve spinel formation while the other is more complicated, since three different compounds can form between the end‐members. For all of these systems a thin‐film reaction geometry was utilized. High‐quality thin films of the various oxides were deposited on bulk substrates by pulsed‐laser deposition.

NiO was deposited on four orientations of α‐Al2O3 in order to study the effect of crystal orientation (and therefore interfacial structure) on the growth kinetics of the nickel–aluminate spinel. The effect of an external electric field at elevated temperatures on the spinel‐forming reaction between Fe oxide and monocrystalline (001) MgO has been analysed. Thin films of Y2O3 were reacted with monocrystalline substrates of α‐Al2O3 at elevated temperatures to determine the reaction sequence: up to three different yttrium aluminates can form.
Structure of carbon nanotube‐based nanocomposites
Authors: Ajayan P. M. , Redlich P. , Ru¨hle M.
Published: November 2003

The structure of nanocomposites fabricated using carbon nanotube templates using transmission electron microscopy and electron energy‐loss spectroscopy will be described here. Examples for filled, network substituted and uniformly coated nanotubes will be described. The novelty of such nanocomposite structures and their possible role in future technology will be discussed.
Examination of microhardness indentation‐induced subsurface damage in alumina platelet reinforced borosilicate glass using confocal scanning laser microscopy
Authors: Winn A. J. , Boccaccini A. R. , Imam N. , Trusty P. A.
Published: November 2003

Borosilicate glass and borosilicate glass matrix composites reinforced with 10, 15 and 30 vol.% alumina platelets have been indented and the subsurface lateral cracking examined by confocal scanning laser microscopy (CSLM). The suitability of the CSLM technique as a quick and nondestructive method of obtaining three‐dimensional information of subsurface damage in dispersion reinforced brittle matrix composite materials was demonstrated. The addition of alumina platelets to the glass matrix has resulted in a reduction in the extent and depth of subsurface damage due to indentation, and hence may make the material more resistant to erosive wear. This damage development may be a consequence of the presence of residual compressive stresses in the matrix and the strong platelet/matrix interfacial bonding.
Scanning (atomic) force microscopy imaging of earthworm haemoglobin calibrated with spherical colloidal gold particles
Authors: Xu S. , Arnsdorf M. F.
Published: November 2003

Scanning (atomic) force microscopy (SFM) permits high‐resolution imaging of a biological specimen in physiological solutions. Untreated extracellular haemoglobin molecules of the common North American earthworm, Lumbricus terrestris, were imaged in NH4Ac solution using calibrated SFM. Individual molecules and their top and side views were clearly identified and were comparable with the images of the same molecule obtained by scanning transmission electron microscopy (STEM). A central depression, the presumed mouth of the hole, was detected. We analysed 75 individual molecules for their lateral dimensions. Compression varied for different molecules, presumably because of the variation of the interaction between the SFM tip and the protein molecule. Two effective heights which correspond to the heights of the points of the haemoglobin molecules first and last touched by the tip, h1 and h2, respectively, were measured for each protein and ranged between 1.58 and 16.2 nm for h1 and 1.23 and 13.6 nm for h2. The apparent diameter was measured and ranged from 44.9 to 86.6 nm (63.2±10.5 nm, n =75), which is about twice the diameter of the molecule reported by STEM for the top view orientation. The higher the measured effective heights, the worse was the tip convolution effect. In order to determine the tip parameters (semivertical angle, curvature of radius and the cut‐off height) and to calibrate images of earthworm haemoglobin molecules, spherical gold particles were scanned as standards. The tip sectional radii at distances of h1 and h2 above the tip apex were subtracted from the apparent diameter of the protein. The calibrated lateral dimension was 29.1 ±3.85 nm, which is close to the reported scanning transmission electron microscopy data 30.0 ±0.8 nm. The results presented here demonstrate that the calibration approach of imaging gold particles is practical and relatively accurate. Calibrated SFM imaging can be applied to the study of other biomacromolecules.
Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells
Authors: Braet F. , De Zanger R. , Wisse E.
Published: November 2003

Critical point drying (CPD) is a common method of drying biological specimens for scanning electron microscopy (SEM). Drying by evaporation of hexamethyldisilazane (HMDS) has been described as a good alternative. This method, however, is infrequently used. Therefore, we reassessed HMDS drying. Cultured rat hepatic sinusoidal endothelial cells (LEC), possessing fragile fenestrae and sieve plates, were subjected to CPD and HMDS drying and evaluated in the scanning electron microscope, atomic force microscope (AFM) and transmission electron microscope (TEM). We observed no differences between the two methods regarding cellular ultrastructure. In contrast with CPD, HMDS drying takes only a few minutes, less effort, low costs for chemicals and requires no equipment. We conclude that HMDS‐dried specimens have equal quality to CPD ones. Furthermore, the method also proved useful for drying whole‐mount cells for TEM and AFM.
Immunohistochemical processing of tissue which allows for embedding in Araldite®, and orientation and correlation between light and electron microscopic staining
Authors: Oates P. S. , Light A. , Thomas C.
Published: November 2003

This paper describes a simple and efficient method for the immmunohistochemical processing of tissue sections on glass slides and the subsequent transfer of the entire section to an Araldite® medium. These sections present in one plane can then be orientated and when cut provide the user with whole tissue, free from gaps due to folding. This procedure therefore enables the changes in protein concentration and distribution to be assessed and correlated within and between different cells at the light and electron microscopic level. This procedure may be of use in other microscopic techniques studies.
Development of an optimal protocol for the ultrastructural examination of skin by transmission electron microscopy
Authors: Van den Bergh B. A. I. , Swartzendruber D. C. , Bos‐van der Geest A. , Hoogstraate J. J. , Schrijvers A. H. G. J. , Boddé H. E. , Junginger H. E. , Bouwstra J. A.
Published: November 2003

The intercellular lipid bilayers of the stratum corneum provide the permeability barrier of the skin. To perform an electron microscopical examination of the ultrastructure of these bilayers, ruthenium tetroxide fixation is required. In this study an optimal fixation protocol was developed and selected upon comparing seven different fixation procedures, using glutaraldehyde (GA) and the postfixatives ruthenium red, osmium tetroxide (OsO4) and ruthenium tetroxide (RuO4) in combination with potassium ferrocyanide (K4Fe(CN)6) and potassium ferricyanide (K3Fe(CN)6). Instead of fixing skin with either OsO4 or RuO4, these two fixatives were combined in one protocol. In addition, the use of RuRed was introduced and the influence of both K4Fe(CN)6 and K3Fe(CN)6 in combination with RuO4 were examined. Furthermore, we compared two dehydration solvents, methanol and acetone.

The most satisfying results were obtained when the skin was prefixed in GA and postfixed in OsO4 and RuO4 with K3Fe(CN)6, i.e. with Fe in its highest oxidation state (Fe3+). No differences were observed between dehydration in methanol and acetone.
A porous carbon film for the collection of atmospheric aerosol for transmission electron microscopy
Authors: Dye A. L. , Rhead M. M. , Trier C. J.
Published: November 2003

This paper describes a method of direct sampling of fine urban aerosol using porous carbon films, to increase collection efficiency and retain the structural integrity of the aerosol. A nuclepore membrane filter was coated with carbon and washed with chloroform to leave a clean porous carbon film on a 200‐mesh transmission electron microscope copper grid. The grid was attached to a clean nuclepore filter using carbon cement. Sampling was conducted for 1 h at 1.9 L min−1 flow rate. The porous carbon grid with sample was then assayed using transmission electron microscopy, and negatives were suitable for image analysis. The collection of fine airborne particles using this method is appropriate for electron microscopy, with a minimum of interference to the structure of collected particles.
Improved preservation of the ram spermatozoan plasma membrane using betaine in the primary fixative
Authors: SWAN M. A. , ALBOGHOBEISH N.
Published: November 2003

Improved preservation of ram spermatozoa was obtained by filtration onto a Millipore filter followed by fixation in a fixative containing 23 mm betaine, which raised fixative osmolality only slightly. In samples fixed in control preparations with betaine omitted the plasma membrane had a ruffled appearance, while the betaine‐containing fixative gave the plasma membrane a smooth contour which closely followed underlying structures.

Betaine is known to function as an osmoprotectant in other situations, and may protect the cells from osmotic damage during the initial stages of fixation.
An improved model for gaseous amplification in the environmental SEM
Authors: Thiel B. L. , Bache I. C. , Fletcher A. L. , Meredith P. , Donald A. M.
Published: November 2003

We present a new model for the gas amplification effect used in many environmental scanning electron microscopes, wherein molecular complexity is shown to be the critical factor. Monte Carlo simulations, based on experimental electron scattering cross‐sections, are used to deduce a predictive model for the amplification process that is superior to the Townsend gas capacitor model. These predictions are compared with experimentally obtained amplification curves. Significantly, it is shown that the ionization efficiency of the electrons changes dramatically over the gap distance, and a constant value cannot be assumed. Atomic and molecular excitations affect the amplification process in two ways: first, they serve to lower the average kinetic energy of the imaging electrons, thereby keeping a greater fraction near the ionization threshold energy. Second, molecular normal modes determine the effectiveness of positive gas ions in producing additional secondaries upon surface impact. Practical implications such as signal gain and fraction of useful signal as a function of operating conditions are discussed in the light of the new model. Finally, we speculate on potential new contrast mechanisms brought about by the presence of an imaging gas.