Understanding the crystallographic and nanomechanical properties of bryozoans.

This study examines how microscale differences in skeletal ultrastructure affect the crystallographic and nanomechanical properties of two related bryozoan species: (i) Hornera currieae, which is found at relatively quiescent depths of c. 1000 m, and (ii) Hornera robusta, which lives at depths of 50-400 m where it is exposed to currents and storm waves. Microstructural and Electron Backscatter Diffraction (EBSD) observations show that in both species the secondary walls are composed of low-Mg calcite crystallites that grow with their c-axes perpendicular to the wall. Branches in H. currieae develop a strong preferred orientation of the calcite c-axes, while in H. robusta the c-axes are more scattered. Microstructural observations suggest that the degree of scattering is controlled by the underlying morphology of the skeletons: in H. currieae the laminated branch walls are smooth and relatively uninterrupted, whereas the wall architecture of H. robusta is modified by numerous deflections, forming pustules and ridges associated with microscopic tubules. Modelling of the Young's modulus and measurements of nanoindentation hardness indicate that the observed scattering of the crystallite c-axes affects the elastic modulus and nanohardness of the branches, and therefore controls the mechanical properties of the skeletal walls. At relatively high pressure in deep waters, the anisotropic skeletal architecture of H. currieae is aimed at concentrating elasticity normal to the skeleton wall. In comparison, in the relatively shallow and active hydrographic regime of the continental shelf, the elastically isotropic skeleton of H. robusta is designed to increase protection from external predators and stronger omni-directional currents.

Making EBSD on water ice routine.

Electron backscatter diffraction (EBSD) on ice is a decade old. We have built upon previous work to select and develop methods of sample preparation and analysis that give >90% success rate in obtaining high-quality EBSD maps, for the whole surface area (potentially) of low porosity (<15%) water ice samples, including very fine-grained (<10 µm) and very large (up to 70 mm by 30 mm) samples. We present and explain two new methods of removing frost and providing a damage-free surface for EBSD: pressure cycle sublimation and 'ironing'. In general, the pressure cycle sublimation method is preferred as it is easier, faster and does not generate significant artefacts. We measure the thermal effects of sample preparation, transfer and storage procedures and model the likelihood of these modifying sample microstructures. We show results from laboratory ice samples, with a wide range of microstructures, to illustrate effectiveness and limitations of EBSD on ice and its potential applications. The methods we present can be implemented, with a modest investment, on any scanning electron microscope system with EBSD, a cryostage and a variable pressure capability.

Microstructural analysis of Co-Cr dental alloy at the metal-porcelain interface: a pilot study.

OBJECTIVE: The purpose of the study was to observe whether conventional porcelain firings had an effect on the underlying microstructure of cobalt-chromium alloys used in porcelain-fused-to-metal systems. METHODS: One as cast (non-veneered) and two porcelain veneered Co-Cr specimens layered with and without tungsten(W)-metal conditioner were manufactured and analysed. Electron backscatter diffraction was used to determine the crystal structures and grain size across the porcelain-fused-to-metal interface. RESULTS: No difference was found in the microstructure of the alloy in both with and without W-metal conditioner. For the porcelain fired specimens, disparately sized granular structures were observed adjacent to the metal-porcelain interfaces compared to the bulk of the metal. Ellipsoid shaped grains at the alloy surface ranged between 1-11 µm in diameter and averaged 2.70 µm (SD: 2.17 µm) for the specimen layered with W-metal conditioner and 2.86 µm (SD: 1.85 µm) for the specimen layered without W-metal conditioner. Grains located in the bulk were > 200 µm with dendritic-like features. The depth of the fine grain structure adjacent to the surface had an average depth of 15 µm. The crystal structure of the surface layer was found to be predominantly hexagonal close-packed whereas the underlying bulk was a mixture of both face-centered cubic and hexagonal close-packed phases. For the as cast specimen, similar large grains of over 200 µm was observed but exhibited no dendritic like features. In addition, no fine grains were observed at the surface region of the as cast alloy. CONCLUSION: Conventional porcelain firings altered the interfacial and bulk microstructure of the alloy while the presence of the W-metal conditioner had no influence on the underlying alloy microstructure.

Crystallographic preferred orientation (CPO) of gypsum measured by electron backscatter diffraction (EBSD).

An investigation by electron backscatter diffraction on gypsum shows that this technique can be used to study the microstructures and crystallographic preferred orientation of gypsum. Presented here are the methods, verification tests and data obtained from a naturally deformed sample of gypsum-rich rock. The electron backscatter diffraction data show the sample has a strong crystallographic preferred orientation.

The Weighted Burgers Vector: a new quantity for constraining dislocation densities and types using electron backscatter diffraction on 2D sections through crystalline materials.

The Weighted Burgers Vector (WBV) is defined here as the sum, over all types of dislocations, of [(density of intersections of dislocation lines with a map) x (Burgers vector)]. Here we show that it can be calculated, for any crystal system, solely from orientation gradients in a map view, unlike the full dislocation density tensor, which requires gradients in the third dimension. No assumption is made about gradients in the third dimension and they may be non-zero. The only assumption involved is that elastic strains are small so the lattice distortion is entirely due to dislocations. Orientation gradients can be estimated from gridded orientation measurements obtained by EBSD mapping, so the WBV can be calculated as a vector field on an EBSD map. The magnitude of the WBV gives a lower bound on the magnitude of the dislocation density tensor when that magnitude is defined in a coordinate invariant way. The direction of the WBV can constrain the types of Burgers vectors of geometrically necessary dislocations present in the microstructure, most clearly when it is broken down in terms of lattice vectors. The WBV has three advantages over other measures of local lattice distortion: it is a vector and hence carries more information than a scalar quantity, it has an explicit mathematical link to the individual Burgers vectors of dislocations and, since it is derived via tensor calculus, it is not dependent on the map coordinate system. If a sub-grain wall is included in the WBV calculation, the magnitude of the WBV becomes dependent on the step size but its direction still carries information on the Burgers vectors in the wall. The net Burgers vector content of dislocations intersecting an area of a map can be simply calculated by an integration round the edge of that area, a method which is fast and complements point-by-point WBV calculations.

Cobalt iontophoresis techniques for tracing afferent and efferent connections in the vertebrate CNS.

In recent years several dye and cobalt iontophoresis techniques have been successfully used by invertebrate neurophysiologists for the localization of neuron somata and their processes. The cobalt iontophoresis technique has now been extended for use in the tracing of nerve fiber pathways and the localization of neuron somata in vertebrates. The brain and spinal cord of an animal are removed following perfusion with saline, and placed in a dish of cold saline. A suction electrode, filled with 300 mM cobalt chloride, is then placed over the cut end of the nerve trunk. Cobalt ions are then iontophoresed (by means of a voltage divider) within the nerve fibers, along their course. Following iontophoresis, the brain is bathed in an ammonium sulfide solution to precipitate the cobalt as black cobalt sulfide. The brain is then processed for histological procedures. A wide variety of vertebrates has been used, including amphibians, reptiles, aves and mammals, with uniform success. The cobalt iontophoresis technique presently in use has a wide range of applicability for neuroanatomical studies.

An ultrastructural study of peripheral neurons and associated non-neural structures in the bivalve mollusc, Spisula solidissima.

The ultrastructural morphology of peripheral neurons and associated structures in the bivalve mollusc. Spisula solidissima have been studied in an effort to describe the synaptic topography and to provide anatomical correlates of previous physiological observations. The somata of the peripheral neurons are located within the perineurium at branch points of the siphonal nerves. There are many fiber-fiber synaptic contacts which are characterized by isolated sites of contact with membrane specialization and unilateral accumulation of synaptic vesicles. There are also synaptic contacts involving the somata, both axo-somatic and somato-axonic, the two being distinguishable on the basis of the polarity of vesicle accumulation. All of the observed synaptic profiles were similar in appearance regardless of the neuron loci involved. Much of the non-synaptic soma surface is covered with processes of glial cells. Likewise, in many cases, individual fibers and groups of fibers are encases with glial processes. Unique clusters of membrane bound, pigment containing glial like cells occur throughout the nervous system of Spisula. The heterogenous appearance of the inclusions and the presence of lysosome-like bodies in the cytoplasm of these cells suggest a possible phagocytic role.

Morphological correlate of regional partitioning of integumental water absorption in terrestrial slugs.

An ultrastructural study of the foot surface of the terrestrial mollusc, Limax maximus, has revealed a correlation of epithelial cell type with the functional partitioning of the surface. The lateral absorptive bands of the foot are comprised exclusively of microvillar epithelial cells, while those of the medial locomotor band are all ciliated. Thus, there is a clear partitioning of epithelial cell types between areas of the foot surface with distinct functional roles. Consistent with the proposed role for paracellular absorption, varying states of hydration are shown to affect the extent of the intercellular spaces, but not the intracellular architecture.

High-temperature electron backscatter diffraction and scanning electron microscopy imaging techniques: in-situ investigations of dynamic processes.

In-situ heating experiments have been conducted at temperatures of approximately 1200 K utilising a new design of scanning electron microscope, the CamScan X500. The X500 has been designed to optimise the potential for electron backscatter diffraction (EBSD) analysis with concomitant in-situ heating experimentation. Features of the new design include an inclined field emission gun (FEG) column, which affords the EBSD geometrical requirement of a high (typically 160 degrees) angle between the incoming electron beam and specimen surface, but avoids complications in heating-stage design and operation by maintaining it in a horizontal orientation. Our studies have found that secondary electron and orientation contrast imaging has been possible for a variety of specimen materials up to a temperature of at least 900 degrees C, without significant degradation of imaging quality. Electron backscatter diffraction patterns have been acquired at temperatures of at least 900 degrees C and are of sufficient quality to allow automated data collection. Automated EBSD maps have been produced at temperatures between 200 degrees C and 700 degrees C in aluminium, brass, nickel, steel, quartz, and calcite, and even at temperatures >890 degrees C in pure titanium. The combination of scanning electron microscope imaging techniques and EBSD analysis with high-temperature in-situ experiments is a powerful tool for the observation of dynamic crystallographic and microstructural processes in metals, semiconductor materials, and ceramics.

First combined electron backscatter diffraction and transmission electron microscopy study of grain boundary structure of deformed quartzite.

The structures of boundaries in a deformed and dynamically recovered and recrystallized quartz polycrystal (mylonite) were characterized by transmission electron microscopy, after the misorientation angles across the same grain boundaries had been analysed using electron backscatter diffraction in a scanning electron microscope. In this new approach, a specific sample area is mapped with electron backscatter diffraction, and the mapped area is then attached to a foil, and by the ion beam thinned for transmission electron microscopy analysis. Dislocations in grain boundaries were recognized as periodic and parallel fringes. The fringes associated with dislocations are observed in boundaries with misorientations less than 9 degrees , whereas such fringes cannot be seen in the boundaries with misorientations larger than 17 degrees . Some boundaries with misorientations between 9 degrees and 17 degrees generally have no structures associated with dislocation. One segment of a boundary with a misorientation of 13.5 degrees has structures associated with dislocations. It is likely that the transition from low-angle to high-angle boundaries occurs at misorientations ranging from approximately 9 degrees to 14 degrees . Change in the grain boundary structure presumably influences the mobility of the boundaries. In the studied deformed quartz vein, a relative dearth of boundaries between misorientation angles of theta = 2 degrees and theta = 15 degrees has previously been reported, and high-angle boundaries form cusps where they intersect low-angle boundaries, suggesting substantial mobility of high-angle boundaries.

The use of combined cathodoluminescence and EBSD analysis: a case study investigating grain boundary migration mechanisms in quartz.

Grain boundary migration is an important mechanism of microstructural modification both in rocks and in metals. Combining detailed cathodoluminescence (CL) and electron backscatter diffraction (EBSD) analysis offers the opportunity to relate directly changes in crystallographic orientation to migrating boundaries. We observe the following features in naturally heated quartz grains from the thermal aureole of the Ballachulish Igneous Complex (Scotland, U.K.): (a) propagation of substructures and twin boundaries in swept areas both parallel and at an angle to the growth direction, (b) development of slightly different crystallographic orientations and new twin boundaries at both the growth interfaces and within the swept area and (c) a gradual change in crystallographic orientation in the direction of growth. All these features are compatible with a growth mechanism in which single atoms are attached and detached both at random and at preferential sites, i.e. crystallographically controlled sites or kinks in boundary ledges. Additionally, strain fields caused by defects and/or trace element incorporation may facilitate nucleation sites for new crystallographic orientations at distinct growth interfaces but also at continuously migrating boundaries. This study illustrates the usefulness of combined CL and EBSD in microprocess analysis. Further work in this direction may provide detailed insight into both the mechanism of static grain growth and the energies and mobilities of boundaries in terms of misorientation and grain boundary plane orientation.

Control of Central and Peripheral Targets by a Multifunctional Peptidergic Interneuron.

In the terrestrial slug, Limax maximus, feeding activity and cardiovascular function have been shown to be correlated. For example, in intact animals, both feeding responsiveness and heart activity are suppressed during dehydration (Grega and Prior, 1986). The paired peptidergic buccal ganglion neurons RB1 and LB1 have dramatic modulatory effects on both the feeding motor program (FMP) and the force of heart contraction (Welsford and Prior, 1991). The B1 neurons appear to contain the small cardioactive peptides (SCPs). Observations have a frequency dependent excitation of both the FMP and the heart demonstrated by intracellular stimulation of B1. Thus, interneuron B1 may serve to mediate the coincident modulation of multiple responses to physiological stresses.

Analysis of contact-rehydration in terrestrial gastropods: osmotic control of drinking behaviour.

Contact-rehydration in slugs is mediated by a specific behavioural pattern in which dehydrated slugs move onto a moist surface, assume a flattened posture while water is absorbed through the surface of the foot and move off once they are rehydrated. 'Drinking behaviour' is initiated when slugs have been dehydrated to the threshold level of 60-70% of initial body weight (IBW). Drinking behaviour is terminated once slugs have rehydrated to their individual rehydration set-points. The mean 'rehydration set-point' for Limax is 93.6 +/- 12.2% IBW (+/- S.D.). Slugs can achieve their individual set-point regardless of the extent of initial dehydration. Drinking behaviour can be initiated by injections of hyperosmotic mannitol solution and terminated by injections of dilute saline. This indicates that variation in the osmolality of the haemolymph is involved in the control of the behavioural sequence.

Modulation of heart activity in the terrestrial slug Limax maximus by the feeding motor program, small cardioactive peptides and stimulation of buccal neuron B1.

Activation of the feeding motor program (FMP) increases the force of ventricular contractions in heart/central nervous system (CNS) preparations of the terrestrial slug Limax maximus (Linnaeus). The FMP-induced increase in ventricular activity requires innervation of the heart by abdominal ganglion nerves N9 and N11. Application of the small cardioactive peptides SCPA and SCPB to isolated preparations of the heart causes dose-dependent increases in the force of ventricular contractions. In addition, the SCPs induce rhythmic contractions in quiescent heart preparations. The effects of the SCPs appear to be specific in that the neuropeptide FMRFamide has an inhibitory effect on ventricular activity. SCP-like and FMRFamide-like immunoreactive material is found in the heart, kidney and pericardium and in the nerves that innervate these organs. Unilateral intracellular stimulation of buccal neuron B1, which contains SCP-like and FMRFamide-like immunoreactive material, mimics the FMP- and SCP-induced increases in ventricular activity. The effect of B1 on ventricular activity is frequency dependent and requires innervation of the heart by N11. These results are consistent with the hypothesis that the SCPs are involved in feeding-related alterations in heart activity in Limax and that the control of this effect involves neuron B1.

Behavioural and physiological aspects of swimming in cercariae of the digenetic trematode, Proterometra macrostoma.

1. Cyclical swimming behaviour of the cercariae of the digenetic trematode, Proterometra macrostoma, involves a highly regular alternating swim-sink sequence. During periods of swimming the cercariae are propelled upward through the water by alternating lateral contractions of the tail. Following each burst of swimming the cercariae passively drift downward. 2. Suction electrode recordings from the tail during swimming reveal that a single biphasic potential precedes each contraction in a burst. 3. The site of initiation of the rhythmic activity is the transverse band at the junction of the furcae and body of the tail. Sensory feedback does not play a major role in maintenance of the rhythm. 4. A burst of swimming can be initiated by tactile stimulation of the tail. At no time is a cercaria refractory to tactile stimulation. 5. The tail of P. macrostoma cercariae is an autonomous locomotor organ specialized for the brief free-living period between emergence from the snail host and infestation of the primary host.

Body hydration and haemolymph osmolality affect feeding and its neural correlate in the terrestrial gastropod, Limax maximus.

When terrestrial slugs (Limax maximus) are dehydrated to 65-70% of their initial body weight (IBW) their feeding responsiveness is greatly decreased. There is a 90% decrease in feeding responsiveness when slugs are injected with hyperosmotic mannitol solution that raises the haemolymph osmolality to that of slugs dehydrated to 65-70% IBW (i.e. 200 mosmol kg-1 H2O). The duration of the Feeding Motor Programme (FMP) that can be recorded from an isolated CNS-lip preparation is reduced by increasing the osmolality of the saline bathing the preparation. The osmolality of the saline that can modify the FMP corresponds to that of the haemolymph of a slug dehydrated to 65-70% IBW. The pattern of the motor programme is not affected. A gradual increase in saline osmolality which temporally mimics the progressive increase in haemolymph osmolality of a dehydrating slug also causes a decrease in the duration of the FMP. The neural network underlying the FMP appears to adapt to hyperosmotic saline since the duration of FMP bouts gradually returns to normal levels after long-term exposure (6-8 h).

Locomotor activity of the terrestrial slug, Limax maximus: response to progressive dehydration.

The circadian locomotor rhythm of the terrestrial slug, Limax maximus, was measured with activity wheels during exposure to both humid and drying conditions. Slugs kept in wet wheels (100% RH) remained fully hydrated while those in dry wheels (less than 30% RH) experienced progressive dehydration. Transfer of slugs from a wet wheel to a dry wheel resulted in an increase in the intensity and duration of their patterned locomotor activity that persisted for 3 days. Once the slugs were returned to wet wheels, their locomotor activity returned to normal.

Effects of temperature on the endogenous activity and synaptic interactions of the salivary burster neurones in the terrestrial slug Limax maximus.

(1) The activity of the endogenously active salivary burster neurones (SBs) shows temperature acclimation and has characteristic cold and warm blockade temperatures. (2) Temperature acclimation affects the upper and lower limits of the temperature range over which SBs are active. The absolute range, in centigrade degrees, during warming, is unaffected by acclimation. (3) Acclimatization of burster activity is a slow response to the mean ambient temperature. (4) There is increased synchrony of activity between the right and left salivary bursters at low temperature which is correlated with an increased electrical coupling between the SBs and protractor motoneurones (B7s). There is a corresponding increase in the input resistance of B7 at low temperatures.