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SUMMARY In the processing of cell cultures, grown as a monolayer in tissue culture dishes for electron microscopy, the sectioning of the monolayer is an essential step. The monolayer can be sectioned either parallel or perpendicular to the plane of growth. Several methods for the perpendicular way of sectioning have already been described. We propose a simplified method in which the monolayer is sandwiched between two layers of resin, one of which is a pre-polymerized block, the other being a layer of resin, applied at a second stage. Sectioning of this ‘flat embedded’ specimen yields thin sections perpendicular to the plane of growth of the monolayer without elaborate orientating procedures. The advantage of this procedure is that it can be done using only routine embedding techniques, avoiding special materials or complex manipulations. This sandwich technique provides an excellent mechanical fixation of the monolayer and protects it against external damage.

SUMMARY A quick sampling and preparation method for freezing of cell monolayers is described. The cells are grown on a large Formvar film supported by a frame of polystyrene. A polyvinylpyrrolidone (PVP) solution is applied to one side of the film forming a flat disc when frozen with a pair of pliers precooled in liquid nitrogen. The PVP solution provides the specimen with sufficient strength and may be used as an elemental standard for absolute quantification if salts of known concentrations are added. Manipulation of the cells prior to freezing is thus restricted to a minimum, which eliminates possible harmful treatments like scraping and centrifugation. The procedure is quickly performed, the freezing being completed within 30 s of the cells having been removed from the culture well. The analytical results reveal low and stable Na: K ratios. Our results confirm that cells in vitro are comparable to cells in vivo with respect to elemental composition.

Summary This paper introduces a double-image multiple-beam Fizeau fringes system. The introduced system can dynamically determine the variations of the refractive indices for both parallel and perpendicular polarization simultaneously. This is achieved by the simultaneous capturing of two multiple-beam interference patterns during the mechanical processing of isotactic polypropylene fibre. This parallel determination of the refractive indices of both polarization directions allowed us to determine the full-field distribution of the stress vector, S. To accomplish this, a mathematical model was deduced to calculate the components of the stress vector, S, i.e. parallel stress component, S1, and perpendicular stress component, S2. Double-image Fizeau fringes system and the deduced mathematical model were used to investigate the variation of the refractive index and stress components of the fibre during the stretching process and propagation of necked regions.

A new configuration for Transmission Kikuchi Diffraction (TKD) in a scanning electron microscope is presented; called 'on-axis TKD'. Compared to the usual off-axis configuration, the scintillator is placed perpendicular to the incident beam under the electron-transparent sample, not in vertical position. In this way, the setup benefits from intense forward scattered electrons enabling short acquisition times. At equivalent diffraction pattern quality, the electron dose needed on the sample is estimated to be 20 times lower in comparison to the off-axis configuration. The technique is particularly suited to the characterisation of severe plastic deformation induced ultrafine grained microstructures. The evolution of the microstructure of an Al-Mg alloy deformed by high pressure tube twisting was analysed. It is shown that the grain refinement was in the steady state stage for a shear strain of 24 with a mean grain size of 120 nm.

Behavioural stress has shown to strongly affect neurotransmission within the neocortex. In this study, we analysed the effect of an acute stress model on density and distribution of neurotransmitter-containing vesicles within medial prefrontal cortex. Serial section transmission electron microscopy was employed to compare two groups of male rats: (1) rats subjected to foot-shock stress and (2) rats with sham stress as control group. Two-dimensional (2D) density measures are common in microscopic images and are estimated by following a 2D path in-section. However, this method ignores the slant of the active zone and thickness of the section. In fact, the active zone is a surface in three-dimension (3D) and the 2D measures do not accurately reflect the geometric configuration unless the active zone is perpendicular to the sectioning angle. We investigated synaptic vesicle density as a function of distance from the active zone in 3D. We reconstructed a 3D dataset by estimating the thickness of all sections and by registering all the image sections into a common coordinate system. Finally, we estimated the density as the average number of vesicles per area and volume and modelled the synaptic vesicle distribution by fitting a one-dimensional parametrized distribution that took into account the location uncertainty due to section thickness. Our results showed a clear structural difference in synaptic vesicle density and distribution between stressed and control group with improved separation by 3D measures in comparison to the 2D measures. Our results showed that acute foot-shock stress exposure significantly affected both the spatial distribution and density of the synaptic vesicles within the presynaptic terminal.

A combined scanning electron microscope and focussed ion beam instrument is suitable for micro- and nanopatterning, cross-sectioning and subsequent imaging, of specimens at room temperature as well as under cryo conditions. In order to reveal internal details, samples are conventionally milled with the ion beam positioned perpendicular to the sample surface. Using this approach certain limitations are frequently encountered, e.g. accumulation of redeposited material, shadowing effects, image distortion and a limited imaging area. Here we show an approach in which samples are pre-trimmed using a microtome to obtain a sample block face that is parallel to the ion beam. This new grazing incidence geometry eliminates the need for removal of bulk material with the ion beam and enables immediate fine polishing of a pre-selected area of interest. Many of the limitations previously described are avoided and in addition milling time is reduced, whilst creating larger cross-sectional areas. Another advantage is that electron imaging can be accomplished by tilting the sample surface perpendicular to the electron beam, providing a geometrically undistorted image. The proposed approach is suitable for materials that can be microtomed, both in ambient and cryogenic conditions, and proves to be of particular benefit for biological and food samples.

Anisotropic effects in hexagonal aluminium nitride have been studied by electron energy-loss spectroscopy (EELS) in the N-K energy loss near edge structure (ELNES). Experimental data acquired with different collection angles and with a nearly parallel incident electron beam aligned along the c-axis have been compared to simulations based on ab initio calculations. The extraction of intrinsic parallel I|| (with momentum transfer q || c axis) and perpendicular I perpendicular (with momentum transfer q perpendicular c axis) components has been performed directly from the experimental spectra. This has been done according to their description as linear combinations of I|| and I perpendicular, with adequate weights deduced from a geometrical model of anisotropic behaviour.

In an interferometer-based fluorescence microscope, a beam splitter is often used to combine two emission wavefronts interferometrically. There are two perpendicular paths along which the interference fringes can propagate and normally only one is used for imaging. However, the other path also contains useful information. Here we introduced a second camera to our interferometer-based three-dimensional structured-illumination microscope (I(5)S) to capture the fringes along the normally unused path, which are out of phase by π relative to the fringes along the other path. Based on this complementary phase relationship and the well-defined phase interrelationships among the I(5)S data components, we can deduce and then computationally eliminate the path length errors within the interferometer loop using the simultaneously recorded fringes along the two imaging paths. This self-correction capability can greatly relax the requirement for eliminating the path length differences before and maintaining that status during each imaging session, which are practically challenging tasks. Experimental data is shown to support the theory.

Attention is paid to the stereological estimator of the length density of lineal features in three-dimensional space. In Gokhale (J. Microsc. (1990) 159, 133-141), the estimator based on measurements performed on a projection of the content of vertical slices with a given thickness Delta was derived. The aim of this paper is to show that using five vertical slices with systematically chosen orientations yields a reliable result, i.e. the bias of the estimator is smaller than 5%. In the case of a choice of the vertical axis such that most lineal features are not perpendicular or nearly perpendicular to the vertical axis, a reliable result can be obtained using only three systematic orientations of vertical slices.

Summary Lattice preferred orientations of serpentines induce a strong anisotropy of various properties in serpentine bearing-rocks. Lattice preferred orientations had so far been obtained only by X-ray diffraction techniques. We have applied electron back-scattering diffraction to the measurement of the lattice preferred orientations of antigorite in a naturally deformed high-pressure serpentinite. This technique is very sensitive to sample preparation that can lead to surface amorphization in the case of serpentine. A polishing procedure is described that avoids amorphization and allows accurate electron back-scattering diffraction measurements with optimized experimental conditions in a variable pressure scanning electron microscope. Results indicate that deformation leads to lattice preferred orientations characterized by extremely strong c-axis clustering perpendicular to the foliation, as expected for a layered silicate. In the foliation plane, a significant clustering of the a-axis is observed and tentatively attributed to intracrystalline deformation mechanisms. These data suggest that antigorite deforms mostly by gliding along the basal plane of the layered phyllosilicate structure, but that gliding may occur along directions favouring a-axis alignment. Electron back-scattering diffraction appears to be a reliable method for determining phyllosilicate lattice preferred orientations in deformed rocks, with potential applications for determining anisotropy of properties like seismic velocities or thermal and electrical conductivities.

In a Pd thin film grown on a 6H-SiC (0001) substrate, several twin interfaces have been observed, classified as (1) {111}Sigma = 3 twin interfaces and (2) {221}Sigma = 3 twin interfaces. The first ones are coherent twin interfaces and in some cases exhibit steps of one or more atomic layers in height. The second ones are perpendicular to the first ones. They usually appear with a rigid-body displacement, but in some cases no displacement was observed. Models for the interfaces are proposed.

The performance of a new directional solidification device dedicated to the characterization of solid-liquid interface morphology by means of optical methods is presented in this paper. In contradiction to usual solidification studies on transparent materials carried out on thin films, which eliminates the complex coupling between solidification and convection, this device enables in situ and real time studies on bulk transparent materials. The alloy is contained in a cylindrical crucible and observation is performed in two perpendicular directions: the growth one and the transverse one. In addition to direct observation by light transmission in those directions, an interferometer is also set up in the growth direction to provide information on the shape and the motion of the interface through an analysis of the interferometer fringes. The combined determination of solidification front characteristics by these three observation modes has already given critical information on interface dynamics: front recoil measurements during initial transient, formation of microstructure patterns, and influence of convection on the triggering of instabilities.

Summary Orientational behaviours under the action of linearly polarized light and circularly polarized light of a side-chain azobenzene containing polymer were studied by conoscopic polarized microscopy. The results suggest that the linearly polarized light (473 nm, 20 mW cm−2) results in an in-plane orientation of the azobenzene groups. The irradiation with circularly polarized light (473 nm, 20 mW cm−2) leads to a tilt orientation (out-of-plane) of the azobenzene groups with the long axis of mesogens aligned along the propagation direction of the actinic light. Characteristic features of the in-plane and out-of-plane orientated films were obtained from their interference figures. The in-plane orientated film shows an interference cross consisting of a broad fuzzy bar, and the cross-centre lies in the centre of view field. In-plane orientated film also yields a flash figure upon a less than 10° rotation of the sample under polarized microscopy. The interference figures yielded from the out-of-plane orientated films consist of narrow bar cross. The locations of those interference figures depend on the oblique angle of the irradiation light. A method for distinguishing the in-plane orientation of the mesogens from the vertically out-of-plane orientation is demonstrated, which is based on comparing the bar width of their interference figures, and by whether they can produce a flash figure upon a small angle rotation of the film. The liquid crystalline film is identified as positive and uniaxial anisotropy after annealing of the perpendicularly irradiated film.

SUMMARY An efficient sampling procedure is presented for estimation of total line length per unit volume Lv. It involves the following steps: (1) choose a vertical axis in the specimen, and cut the specimen to obtain VUR vertical slices of constant thickness Δ such that parallel planes of the slices contain the vertical direction; (2) observe the projected image of a vertical slice using transmission microscopy such that beam direction is perpendicular to the slice; (3) count the number of intersections of the projected images of the lineal features of interest with cycloid-shaped test lines whose minor axis is perpendicular to the vertical axis. The expected value of the number of intersections per unit length prj is related to Lv as follows: Thus, Lv can be estimated from the measurements performed on the projected images of VUR vertical slices.

We compare theoretical images of the same sample obtained with two different scanning near-field optical microscopes. The sample is a two-dimensional periodic array of magnetic sub-micrometric dots. The magnetization is perpendicular to the sample plane (polar magnetization). The first configuration is a scanning tunnelling optical near-field microscope (STOM) where the tip is used in the detection mode and the sample is illuminated by total internal reflection. The second configuration is an inverted STOM: the tip is used in the emission mode and the diffracted field is far-field detected in one direction. We present the models used to describe the two configurations and then explain the main lines of the formalism used to calculate the diffracted fields by a magneto-optical sample.

A method for estimating the mean linear intercept length of anisotropic microstructures using vertical sections is presented. A test system of cycloids and points is overlaid on the vertical sections, and the mean linear intercept length is estimated from a simple counting procedure — no length measurements are required. The vertical direction is either arbitrarily chosen or chosen perpendicular to most of the surface boundaries of the objects of interest. A design with the latter choice of the vertical direction and three vertical sections — a trisector — will optimize the precision of the estimate from only three vertical sections. The method was applied to two metallic structures, but it may also be used in a biomedical context.

PtxCo(1−x) thin films where x =0.24 have been deposited onto Si3N4 windows and studied using transmission electron microscopy. The films are used in ultrahigh-density recording studies and it was found that the surface of the substrate had a strong influence on the microstructure, crystallography and magnetic properties of the film. An investigation of the early growth of the film was made by studying films of different thickness between 100 and 300 A. It was found that the grains were hexagonal in structure with a strong c-axis orientation perpendicular to the surface.

Atomic force microscopy (AFM), in various versions, has had major impact as a surface structural and spectroscopic tool since its invention in 1986. At its present state of development, however, the interpretation of AFM images is limited by the current state of methodologies for calibration over the wide dynamic range of magnification. Also, the parameters of individual tips, as well as the generic characteristics of different kinds of tips, affect both the quality of the images and their interpretation. Finally, the very nature of the tip-to-surface interaction will generate artefacts, in addition to those associated with tip shape, which need to be fully understood by the practitioners of force microscopy. This project seeks to address and shed light on some of these issues. Polystyrene beads deposited on mica substrates form hexagonal close-packed layers. The unit cell parameters are suitable for calibration of the AFM in the lateral plane, while the perpendicular spacing of the layers is appropriate for calibration along the vertical axis. Using different size fractions, it is straightforward to determine the extents of linearity, orthogonality, thermal and instrumental drifts over distances from 100 nm to tens of micrometres. The present results show that the methodologies for contact mode operation can be adapted to noncontact modes. It is known that an AFM image arises from a convolution of surface topography and tip shape, and is mediated by the interaction. In principle it is possible to carry out a deconvolution, if we have complete knowledge about two of the three elements (i.e. tip, surface and interaction). In practice we rarely have the requisite information. Prominent artefacts will occur when the characteristic parameters of the tip are comparable to those of the surface topography, and/or if there is a variable strength, or extent of localization, of the interaction. The present results demonstrate artefacts due to effects of geometry as well as interaction.

Summary A direct microscopic method for determination of birefringence in polarization-maintaining optoelectronic fibres has been developed. The fibre, approximately 0·15 mm in diameter, is embedded in a suitable resin (epoxy). A thin section (approximately 3 mm in thickness) is then cut perpendicular to the fibre axis, ground and polished. Birefringent patterns (isochromatic fringes) can be seen and photographed using a light-polarizing microscope with appropriate magnification. A method for performing a quantitative analysis of birefringence in real optoelectronic fibres using a macroscopic model is indicated.

SUMMARY Carbon-fibre/epoxy-resin unidirectional laminates were prepared in the laboratory by vacuum-bag processing. Three plates were produced using different ‘dwell-times’, before pressure was applied to consolidate the laminate. Sections from the three plates were cut perpendicular to the fibre direction and were examined with a Quantimet 570 Image Analyser. The fibre positions were recorded. The method of microstructural description is based on the size of the Voronoi cell around each fibre; the Voronoi cell around a fibre is defined as the area nearer to that fibre than any other. Thus the dimensions of the cell are a measurement of half the interfibre distance. The measured distributions of Voronoi distances were compared with distributions calculated using the null hypothesis that the fibres are randomly distributed. Comparison showed that fibres from all three sections were clustered, but that the sample from the medium dwell time was most homogeneous. Examination of long Voronoi distances, not predicted by the null hypothesis, showed anisotropy in orthogonal directions arising from the manufacturing process. This method of microstructural analysis revealed a dependence on the plate processing method, and may now be used to evaluate more general processing problems.

SUMMARY Length density of lineal features, Lv, is an important stereological parameter. The efficient stereological procedure for the estimation of Lv from the counting measurement performed on the projected images of the vertical slices (foils) is modified and improved: Lv can be now estimated from vertical slices of unknown thickness, and the slices need not be of the same thickness. The required assumption-free stereological relationship is is the average number of intersections of straight test lines parallel to the vertical axis with the projected images of the lineal features in the vertical slices, per unit test line length. is the average number of intersections of the lineal features with the parallel planes of the vertical slices, per unit area. Note that there are two parallel planes in a slice, and therefore their total area is twice the area of the observed projected image frame. is the average number of intersections of orientated cycloid-shaped test lines (minor axis perpendicular to vertical axis) with the projected images of the lineal features in the vertical slices, per unit length. For practical applications of this result, it is necessary uniquely to identify the points of intersections of the lineal features with the parallel planes of the slices in the projected images, so that can be estimated unambiguously. However, in practice, this is not a problem in biological, as well as materials microstructures.

A new type of specimen stage that permits more than 180 degrees of tilting about the axis of a side-entry rod has been developed for a high-voltage electron microscope (HVEM). Roughly cylindrical specimens, with radial dimensions of less than a few micrometers, that can be mounted on the tip of a microneedle or micropipette are applicable. For glass micropipettes, the energy of the 1-MeV beam of the HVEM is sufficient to image specimens through both walls. The stage employs a spindle mechanism that holds these needles or micropipettes coaxial with the tilt axis, allowing the specimen to be rotated without restriction. This arrangement, along with the cylindrical form of the specimen, is an important development for single-axis tomography, because it permits a complete 180 degrees set of projections to be recorded. The angular accuracy of the stage was demonstrated to be within +/- 0.20 degrees, with a cumulative error of less than 1.0 degrees over a 180 degrees span. The new stage was tested using puffball spores mounted on a micropipette. A 180 degrees tilt series was recorded and processed to yield a tomographic three-dimensional reconstruction which was displayed both as a cross-sectional view perpendicular to the tilt axis, and as a shaded surface viewed from different directions. The same computations were repeated using subsets of the tilt series to assess the effect of various amounts of missing information. Visual inspection of a selected cross-section from these reconstructions indicated that limiting the angular range to 160 degrees produced results nearly as good as the full data set. Limiting the range to 140 degrees, however, produced a noticeable geometric distortion, which became increasingly severe with ranges of 120 degrees and 100 degrees.

SUMMARY Under suitable conditions, the region of the aqueous phase immediately adjacent to a glass-water interface can be selectively illuminated using the evanescent wave created when total internal reflection occurs at the interface. Objects in the aqueous phase away from the glass become effectively invisible, since the intensity of the evanescent wave decays exponentially with distance from the interface. Previous methods of generating evanescent waves for light microscopy have employed accessory light sources and optical components that directed the illumination on the specimen from one side. The asymmetric illumination creates a surprising orientation dependence of visibility for some specimens. Objects such as microtubules are totally invisible unless they are orientated nearly perpendicular to the direction of illumination. An explanation of this phenomenon is provided in terms of the geometry of diffraction of light by long thin objects. A simple method of achieving evanescent-wave illumination is described and shown to be useful in practice for biological specimens. In contrast to previously described methods, the present arrangement has the advantage of producing circularly symmetric illumination, and of utilizing only standard optical microscope components. The system has been used for imaging specimens both by light scattering and by fluorescence. It has proved useful for following the fragmentation of flagella into isolated microtubules, observing microtubules gliding over dynein adsorbed to a surface, and also for determining the arrangement of vinculin and alpha-actinin in cell-substratum attachment sites and termini of growing myofibrils in cardiac cells.

SUMMARY Electron microscopy of vitrified ultrathin sections allows cell ultrastructure to be studied in the hydrated state. Sectioning of the frozen material is, however, a limiting step, since the cutting forces cause severe mechanical deformation. In order to address this problem, we have investigated the surface of cryosections. It is shown that cryosections have two fundamentally different surfaces. One surface is rough, deformed by cutting-induced deformation lines which are orientated perpendicular to the cutting direction. The other surface, in comparison, is not affected by those deformation lines. Except for knife marks it is smooth. In order to explain the observations, the following model is proposed. The rough relief corresponds to the former block face. Its roughness originates from material that is squeezed out of the section plane when the section is compressed in the cutting direction and bent away from the specimen block. The smooth section surface is the surface in contact with the knife during the sectioning. This contact keeps the surface smooth while imprinting the knife marks.

Summary The purpose of the present investigation was to establish the value and suitability of the scanning electron microscope for research on the mechanism of fracture and cohesive failure of the structure of wood. Wood samples, mainly consisting of various pines, were stressed to the point of failure in tension and shear parallel or perpendicular to the main fibre direction. The rate of loading and the temperature were varied between wide limits. The fractured surfaces were studied and gave the following results: (1) In tension parallel to the grain, fissures developed between the microfibrils in the cell walls causing the wood to rupture perpendicular to the longitudinal axis of the fibres. At both high and low temperatures the matrix, in which the microfibrils are embedded, and to a much lesser extent the middle lamella between the fibres failed by plastic flow when the external load was applied very slowly. Under such conditions strands or bundles of microfibrils were pulled out and fractured in an irregular pattern. Cohesive failure in tension caused by sudden impact was characterized by limited slippage of microfibrils and a near brittle failure of the embedding matrix. A remarkable clean cleavage approaching a true brittle failure was observed when the wood was stressed rapidly perpendicular to the grain. (2) Cohesive failure when the wood was exposed to shear along the grain and tension across the grain, showed clearly that the weakest part of the ultrastructure of wood under such conditions was the bonds between parallel strands of microfibrils which were pulled out with little resistance from the matrix. However, the adhesion between the middle lamella and the cell wall or the middle lamella itself failed when the wood was exposed to cleavage parallel to the grain. If the process of cleaving was carried out extremely fast, as in the machining of wood, the rheological properties of the middle lamella did not allow for plastic deformation with subsequent failure, and the bulk structure of the wood tissue behaved almost as a homogeneous material. The use of the scanning electron microscope for the study of the structure of wood under stress and the mechanism of its cohesive failure can supply information which seems to be unobtainable by any other method.

SUMMARY The microstructure of a composite material is commonly defined by the volume fraction occupied by the fibres. An attempt is made to gain a more quantitative definition of the microstructure of unidirectional glass fibre reinforced polyester resin using data derived from a Quantimet 720 Image Analysing Computer. Sections to be examined under the Quantimet were cut perpendicular to the fibre direction and polished and etched such that the fibres appeared black. Values of the area covered by the fibres inside a small test cell, moved around the cross-section in a contiguous grid, were determined; values for larger cells, made up of several adjacent test cells, were calculated from these. Two methods of analysis are presented. The first method involves the determination of the variance of the values of fractional area covered for different cell sizes. Information regarding the nature of the fibre distribution is sought both from comparing the variance values with those predicted for the null hypothesis, a random distribution, and from examining the shape of a surface defined by a variance matrix. The second method of analysis is based on the concept that a structure may be defined by its relationship to a structuring element. In the present case, structuring elements are squares and rectangles of empty space; the probability of finding such empty space is estimated. Information regarding the nature of the fibre distribution is sought from the probability matrices.

This report deals with the reconstruction of the distribution of membrane thickness T from that of orthogonal length Lo, measured in random section planes. In such planes the membrane appears as a band and the linear distance from one of its boundaries perpendicular to the opposite one is the length of the orthogonal intercept. Using a membrane model, an integral equation relating the probability density functions of orthogonal intercept length f(lo) and membrane thickness g(tau) is derived. Relations between moments are derived and the analytic solution to the problem of reconstructing g(tau) from f(lo) is given. The parametric approach by which it assumed that g(tau) has some known analytic form with unknown parameters is considered, and the use of a suggested analytic form for describing the thickness distribution of the human glomerular basement membrane is discussed.

SUMMARY High-voltage electron microscopy (HVEM) of semi-thick sections was evaluated as a technique for studying thylakoid membrane arrangements in cyanobacterial cells. Semi-thick sections (0·25 μm) provided important information that was relatively difficult or impractical to obtain by viewing either randomly or serially cut thin sections. Specifically, the semi-thick sections were better suited for visualizing (i) overall thylakoid arrangements and (ii) interconnections between the thylakoids and the cytoplasmic membrane. By comparison, randomly cut thin sections frequently yielded deceptively incomplete or inconsistent data in regard to these specific features. Tilting of thick sections about two perpendicular axes served to improve the clarity of complex membranous intersections and other cell features.

SUMMARY Since sections intersect membranes at random angles, the mean distances measured between two such membranes are greater than the perpendicular distances between them. Errors of 10–20% will be usual. Equations are derived relating the true lengths with the various observed ones, and the accuracy of the various methods are indicated. These differ for block-stained and section-stained material. In the Appendix it is shown that in section-stained material, while it is the stain near the surface which is of overwhelming importance for the production of contrast, much of this stain lies in the section itself, rather than actually on the surface. This stained depth is shown to be ∼4 nm deep, but this depth may include surface aggregates (∼ 1 nm) as well.

SUMMARY A procedure is proposed for serial sectioning of impregnated porous media, for photographing the sections under the microscope, and for digitizing the image using an IBM PC (XT) with a digitizing board. The result is a three-dimensional matrix containing the enhanced digitized image in a binary form of O's and I's which represent solid and pore spaces respectively. The procedure was tested on three different porous materials: (1) a pack of glass beads, 170–350 μm; (2) Berca sandstone; and (3) Elora clay loam soil. Each of these was impregnated, forty-six to eighty cross-sections were prepared by close parallel polishing of 10–50 μm per section, and then the sections were photographed and digitized. Examples of the real and digitized cross-sectioned samples are given as well as an example of reconstructed images in a plane perpendicular to the plane of sampling. It is proposed that the method of serial sectioning and automated digitization of porous media provides a powerful tool for further three-dimensional geometrical and topological investigation of pore space to be used in models of fluid transport phenomena.

SUMMARY A new method for measuring refractive-index gradients in rotation-symmetric objects is described. Interference micrographs of intact objects, with the symmetry axis perpendicular to the optical axis of the microscope, are used to obtain a phase-shift profile, which is converted into a refractive-index profile by a computer program. The conversion calculations are based on iterative approximations of the gradient slope in a number of presumed shells in the object. The method is fast, convenient and yields highly accurate results even when only a small number of phase shift values can be obtained. The new method is especially advantageous for analysing many invertebrate eyes but it can also be used for measuring manufactured graded-index optical fibres.

Summary High resolution and conventional studies have been made on the inclined twin boundaries occurring near the tip of wedge microtwins in the case of repeated wedge microtwins occurring at twin intersections. These inclined boundaries appear to be continuous and all the atomic planes are also continuous so that we suggest the use of the term ‘inclined coherent boundaries’ instead of ‘incoherent’ which one generally employs, in analogy to the case of precipitates in a matrix. This is different from what occurs in the case of isolated wedge microtwins for the same material and for the same kind of twins where the inclined twin boundary is really not coherent, being made of non-inclined coherent regions separated by steps containing a dislocation. The coherent inclined boundaries of repeated wedge microtwins appear to be rotated about two perpendicular axes of the habit plane (tilt and twist) giving rise to a distribution of microdislocations whose role has been studied. The values of the tilt and twist angles are related to a minimization of the total energy by a mechanism that is likely to appear only in the case of thin foils. A comparative study of the different roles related to the different structures of inclined twin boundaries for isolated and repeated wedge microtwins has been made, and also a comparative study of the mechanisms of stress relaxation in each case. The studies have been made on (313) twins of a monoclinic samarium sesquioxide.

SUMMARY Using a large range scanning tunnelling microscope (STM) we have obtained images of the complete outer cell wall of the archaebacterium Methanospirillum hungatei, deposited on a graphite substrate and over-coated with an Au or Pt evaporated film. The width of the collapsed cylindrical sheath structure was found to be 5000 A ± ***10%, which agrees closely with the value from previously published electron microscope (EM) studies. The double thickness of the collapsed sheath was found to be 160 A ± 10%, which is about 20% smaller than that from the EM results. Higher resolution STM images taken on top of the collapsed sheaths show corrugations running perpendicular to the cylinder axis and having widths which are multiples of ∼ 30 A, the minimum period expected from EM studies. The height of the corrugations have a minimum value of about 4 A. The expected 2-D crystalline structure was not seen in the STM images.

Freeze-fracture electron microscopy was used to study the morphology of proteins in solution. The size of the particles appearing on the fractured surface, replicated with tungstentantalum, were measured in a direction perpendicular to the shadowing angle. The distributions of the measured particle sizes could be correlated with the known shape and dimensions for each protein. It is concluded that freeze-fracture electron microscopy is a useful technique to study the morphology of biological molecules in solution, particularly hydrophobic proteins which may be difficult to study by other microscopic techniques.

Specimens approximately a micron thick are routinely examined in the HVEM as a result of its increased penetrating power. This large specimen thickness is advantageous for the study of three-dimensional structure, and it increases the sampling statistics significantly for observing a particular image detail. However, each image in the HVEM represents a volume of the specimen and cannot be assumed to be a plane as is common practice for the CTEM. Therefore, image details in the HVEM often overlap or are not optimally oriented with respect to the beam. Thus, a specimen stage with sufficient degrees of freedom to position the specimen in any orientation relative to beam is required (Turner and Chang, these proceedings). Our double-tilt stage shown in fig. 1 and built by P. R. Swann (1972) has this capability.

Samples of Late Devonian/Early Mississippian New Albany Shale from the Illinois Basin, having maturities ranging from early mature to postmature, were analysed using micro-Fourier transform infrared (FTIR) spectroscopy, ImageJ processing software and scanning electron microscopic X-ray spectroscopy to explore the distribution, connectivity and chemical composition of organic matter, clay minerals, carbonate minerals and quartz, and to further test the applicability of micro-FTIR mapping to study shale heterogeneity. Each sample was analysed in planes parallel and perpendicular to the bedding to investigate anisotropy in component distribution, with a possible implication for better understanding anisotropy in porosity and permeability in organic-matter-rich shales. Our results show that for low-maturity samples, organic matter is better connected in the plane parallel to the bedding than in the plane perpendicular to the bedding. Organic matter connectivity decreases with increasing maturity as a result of kerogen transformation. Clay minerals are very well connected in both planes, whereas carbonate minerals are not abundant whilst dominantly isolated in most samples, independent of maturity. This study demonstrates that micro-FTIR mapping is a valuable tool for studying shale heterogeneity on a micrometre to millimetre scale that becomes even more powerful in combination with scanning electron microscopy techniques, which extend observations to a nanometre scale. However, to obtain meaningful and comparable results, micro-FTIR mapping requires very careful standardization, precise selection of peak heights/areas and mapping conditions (such as aperture size, scan numbers, resolution, etc.) well suited for the analysed samples.

In this review, I ask the question of what is the relationship between growth and the orientations of microtubules and cellulose microfibrils in plant cells. This should be a relatively simple question to answer considering that text books commonly describe microtubules and cellulose microfibrils as hoops that drive expansion perpendicular to their orientation. However, recent live imaging techniques, which allow microtubules and cellulose synthase dynamics to be imaged simultaneously with cell elongation, show that cells can elongate with nonperpendicular microtubule arrays. In this review, I look at the significance of these different microtubule arrangements for growth and cell wall architecture and how these resultant walls differ from those derived from perpendicular arrays. I also discuss how these divergent arrays in stems may be important for coordinating growth between the different cell layers. This role reveals some general features of microtubule alignment that can be used to predict the growth status of organs. In conclusion, nonperpendicular arrays demonstrate alternative ways of cell elongation that do not require hooped arrays of microtubules and cellulose microfibrils. Such nonperpendicular arrays may be required for optimal growth and strengthening of tissues.

Summary The graphical representation of single-frequency phase-modulation fluorescence lifetime imaging data, referred to as the AB plot, is extended to take into account measurements of the polarized components of the fluorescence. For a hindered rotator model (characterized with a single excited-state lifetime, a single rotational correlation time and limiting initial and final anisotropies) the rotational correlation time and the excited lifetime can be determined from the AB plot of any two of the following emission components: parallel, perpendicular, total emission or combinations thereof. A strategy for resolving the component hindered rotations and lifetimes for mixtures of two hindered rotators from measurements of the total, parallel and perpendicular components of the emission is developed. The analysis does not require prior knowledge of the initial limiting anisotropy or of the steady-state anisotropy or of the excited state lifetime. Plots in polarized AB space derived for heterogeneous systems are constructed to aid interpretation of frequency-domain dynamic depolarization imaging microscopy experiments. These plots can be used to distinguish spatially dependent rotational correlation time heterogeneity from heterogeneity in limiting anisotropies. The effects of noise and aperture depolarization are discussed. It is anticipated that the polarized AB plot will provide a useful adjunct to existing methods for visualizing and analysing dynamic polarization phenomena arising from molecular dynamics and homo-energy transfer in single-frequency microscopy applications.

Using the general approach to image formation in collection near-field optical microscopy, I derive the symmetry relations for the amplitude coupling coefficients in the case of a weakly guiding single-mode fibre terminated with a probe tip possessing axial symmetry. It is shown that, for the symmetrical detection configuration, six elements of the coupling matrix can be expressed by using only three independent coupling coefficients. The obtained relations are further applied to describe near-field mapping of surface plasmon polariton (SPP) fields. I demonstrate that, for the symmetrical detection configuration, the near-field optical image reflects the intensity distribution of the SPP field components parallel to the surface plane, even though the strong perpendicular component is also being detected. This conclusion is supported with numerical simulations that elucidate the influence of symmetry of the fibre probe on the resulting near-field optical image. The near-field optical images simulated for scattering systems typical for SPP microoptics and localization are presented. It is found that the presence of asymmetry in the detection configuration increases the contribution of the perpendicular field component and results in the images approaching the corresponding SPP intensity distributions.

Summary Background: Spatial characterization of vertical organization of neurons in human cerebral cortex, cortical columnarity or minicolumns, and its possible association with various psychiatric and neurological diseases has been investigated for many years. New method: In this study, we obtained 3D coordinates of disector sampled cells from layer III of Brodmann area 4 of the human cerebral cortex using light microscopy and 140-μm-thick glycolmethacrylate sections. A new analytical tool called cylindrical K-function was applied for spatial point pattern analysis of 3D datasets to see whether there is a spatially organized columnar structure. In order to demonstrate the behaviour of the cylindrical K-function, the result from brain tissues was compared with two models: A homogeneous Poisson process exhibiting complete spatial randomness, and a Poisson line cluster point process. The latter is a point process model in 3D space, which exhibits spatial structure of points similar to minicolumns. Results: The data show in three out of four samples nonrandom patterns in the 3D neuronal positions with the direction of minicolumns perpendicular to the pial surface of the brain – without a priori assuming the existence of minicolumns. Comparison with existing methods: Studies on columnarity are difficult and have mainly been based on two-dimensional images analysis of thin sections of the cerebral cortex with the a priori assumption that minicolumns existed. Conclusions: A clear difference from complete spatial randomness in the data could be detected with the new tool, the cylindrical K-function, although classical functional summary statistics are less useful in this connection. Lay description The methods to detect or quantify the minicolumns in human cerebral cortex have long been subject to controversial discussions. Studies on minicolumns are basically based on two-dimensional (2D) images analysis of thin sections. The main drawback of such techniques is that they fail to reflect precisely the 3D structures organized radially in cerebral cortex. In contrast to the 2D image techniques, current available modern quantitative light microscopy enables to register 3D coordinates of cells. It yields data that help to test the hypotheses about existence of minicolumns. We aim to develop a robust 3D method for quantitative analysis of minicolumnar structure of the human cerebral cortex and moreover search for changes.

Summary In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensionalspecimensobservationfromdifferentviews is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be

Summary 1D Al/Al2O3 nanostructures have been synthesized by chemical vapour deposition (CVD) of the molecular precursor [tBuOAlH2]2. The deposited nanostructures grow chaotically on the substrate forming a layer with a high porosity (80%). Depending on the deposition time, diverse nanostructured surfaces with different distribution densities were achieved. A three-dimensional (3D) reconstruction has been evaluated for every nanostructure density using the Focus Ion Beam (FIB) tomography technique and reconstruction software tools. Several structural parameters such as porosity, Euler number, geometrical tortuosity and aspect ratio have been quantified through the analysis with specified software of the reconstructions. Additionally roughness of the prepared surfaces has been characterized at micro- and nanoscale using profilometry and AFM techniques, respectively. While high aspects ratio around 20–30 indicates a strong anisotropy in the structure, high porosity values (around 80%) is observed as a consequence of highly tangled geometry of such 1D nanostructures. Lay Description 1D nanostructures have tube or wire-like shapes with diameters below 100 nm and lengths of several micrometers. There are various methods to fabricate such ultrafine structures. The growth out of a chemical synthesis is the most elegant and simplest approach for fabrication of these ultra-small wires. Some of these methods are known as wet chemical syntheses since the material fabrication takes place within a solution. The chemical synthesis may also be carried out in the gas phase. Chemical Vapour Deposition (CVD) is such a chemical method, which leads to the fabrication of solid materials through accumulation of the vapour phase chemical species on a solid substrate. In this work, we have used a special CVD process to fabricate the nano-wires. The obtained wires have core-shell geometry. The inner core is made of aluminium and the outer surrounding shell is made of aluminium oxide. These ultrafine and extremely long wires form an assembly, which is similar to a bundle of spaghettis. It is difficult to analyse their shape, assembly, voids and other structural properties easily, just by looking from the top-view using a high-resolution electron microscopy. This microscopic method can give extremely high-resolution images (more than 500,000 times magnification) to analyse any fine detail. On the other hand, the gathered information is limited only to the planar 2D surface of the material. To get 3D information, we cut several slices perpendicular to the surface of the nano-wire assembly by edging and analysed every slice in terms of their morphology (destructing process). Afterwards, all these 2D projection images are brought together in order to get a reconstructed 3D image. This method allows observing every fine detail in the assembly just by rotating 3D image along any axis of interest.

This work provides an electrodeposition-based methodology for synthesizing multicomponent nanowires containing Ag, Co and Ni atoms. Nanowire morphology was obtained by using an anodic alumina membrane with cylindrical pores of similar to 200-nm diameter. Structural, compositional and magnetic characterization revealed that the as-synthesized nanowires adopted a core-shell microstructure. The core (axial region) contained pure Ag phase volumes with a plate-like morphology oriented perpendicular to the nanowire axis. The shell (peripheral region) contained pure Ag nanoparticles along with superparamagnetic Co and Ni rich clusters.

Summary In this paper, we propose a semiautomatic procedure for estimation of particle surface area. It uses automatic segmentation of the boundaries of the particle sections and applies different estimators depending on whether the segmentation was judged by a supervising expert to be satisfactory. If the segmentation is correct the estimate is computed automatically, otherwise the expert performs the necessary measurements manually. In case of convex particles we suggest to base the semiautomatic estimation on the so-called flower estimator, a new local stereological estimator of particle surface area. For convex particles, the estimator is equal to four times the area of the support set (flower set) of the particle transect. We study the statistical properties of the flower estimator and compare its performance to that of two discretizations of the flower estimator, namely the pivotal estimator and the surfactor. For ellipsoidal particles, it is shown that the flower estimator is equal to the pivotal estimator based on support function measurements along four perpendicular rays. This result makes the pivotal estimator a powerful approximation to the flower estimator. In a simulation study of prolate and oblate ellipsoidal particles, the surfactor also performs well for particles which are not extremely elongated. In particular, the surfactor is not very much affected by the singularity in the surfactor formula or by possible inaccuracies in the necessary angle measurements. We also assess the performance of the semiautomatic procedure in a study of somatostatin positive inhibitory interneurons from mice hippocampi. Only 35% of the cells needed to be analysed manually and an important decrease in workload was obtained by using the semiautomatic approach.

Summary This paper made a different attempt of real-time observation of the meiotic spindle movements in living mouse oocyte using a convenient method. This method was based on an experimental phenomenon discovered in our work. In living mouse oocytes, a high concentration of calcium ions (Ca2+) was observed throughout the region occupied by the initial meiotic spindle. After Ca2+ labelling with Fura-2, a weakly fluorescent area (WFA) appeared on each side of the chromosomes. The activities of the WFAs changed during spindle development. By real-time tracking of WFAs, we were able to indirectly observe the meiotic spindle movements. Occasionally, it was observed that the first meiotic spindle rotated from an orientation parallel to the cortex to become perpendicular, instead of migrating from the oocyte centre to the cortex along its axis. Moreover, we analysed this uncommon rotation of the first meiotic spindle and found that the whole rotation process can be divided into two phases: the early slow-speed rotation and the subsequent rapid-speed rotation. We further characterized this rotation with respect to rotational speed and acceleration at all the stages of development. By using a two-photon laser-scanning microscope in combination with Fura-2 dye that is nondamaging to oocytes, we provide a convenient method for indirect visualization and quantitative analysis of spindle movements by real-time tracking of WFAs. This method is easy to operate and master, and economical with time and effort.

The forward scattering of light in a conventional inverted optical microscope by nanoparticles ranging in diameter from 10 to 50nm has been used to automatically and quantitatively identify and track their location in three-dimensions with a temporal resolution of 200ms. The standard deviation of the location of nominally stationary 50-nm-diameter nanoparticles was found to be about 50nm along the light path and about 5nm in the plane perpendicular to the light path. The method is based on oscillating the microscope objective along the light path using a piezo actuator and acquiring images with the condenser aperture closed to a minimum to enhance the effects of diffraction. Data processing in the time and spatial domains allowed the location of particles to be obtained automatically so that the technique has potential applications both in the processing of nanoparticles and in their use in a variety of fields including nanobiotechnology, pharmaceuticals and food processing where a simple optical microscope maybe preferred for a variety of reasons.