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.

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.

The integration of experimental in-situ EBSD observations and numerical simulations: a novel technique of microstructural process analysis.

The combination of subgrain- and grain-scale microstructural data collected during in-situ heating experiments and numerical simulations of equivalent microstructural development offers an innovative and powerful tool in the advancement of the understanding of microstructural processes. We present a system that fully integrates subgrain- to grain-scale crystallographic data obtained during in-situ observations during heating experiments in a scanning electron microscope and the two-dimensional hybrid numerical modelling system Elle. Such a system offers the unique opportunity to test and verify theories for microstructural development, as predictions made by numerical simulations can be directly coupled to appropriate physical experiments and, conversely, theoretical explanations of experimental observations should be testable with numerical simulations. Discrepancies between data obtained with both techniques suggest the need for an in-depth investigation and thus open up new avenues of theory development, modification and verification. In addition, because in numerical models it is possible to select the processes modelled, the effect of individual processes on the microstructural development of a specific material can be quantified. To illustrate the potential and methodology of the so-called EBSD2Elle system, two in-situ experiments and their equivalent numerical experiments are presented. These are static heating experiments of (a) an annealed Ni-foil coupled with a front tracking model for grain growth and (b) a cold deformed rock salt with kinetic Monte Carlo simulations for subgrain growth.

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.

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.

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.

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.

The molluscan neuropeptide, SCPB, increases the responsiveness of the feeding motor program of Limax maximus.

Small cardioactive peptide B (SCPB) has an excitatory effect on both buccal neurons and musculature in numerous molluscan species. The present study reports the effects of SCPB on the activity of specified buccal neurons and the expression of the feeding motor program of the terrestrial slug, Limax maximus. Superfusion of an isolated CNS preparation with 10(-6)M SCPB results in a 3-4-fold increase in the burst frequency of the fast salivary burster neuron (FSB), while having no effect on the activity of another endogenous burster, the bilateral salivary neuron (BSN). The response of the FSB to SCPB is dose dependent, with a threshold concentration of 2 X 10(-8)M. The response of the FSB to SCPB showed no indication of desensitization, even after long-term exposure (20 min). The feeding motor program (FMP) in Limax is a discrete pattern of cyclical motor activity that can be initiated by lip nerve stimulation. In the presence of SCPB a previously subthreshold stimulus can initiate the full FMP. The pattern of the FMP, once initiated, appears unaffected by SCPB. Thus it is the responsiveness of the initiation process that is enhanced by SCPB. Histochemical studies revealed a number of buccal neuron somata and fibers that stain for SCPB-like immunoreactive material (SLIM).

The pneumostome rhythm in slugs: a response to dehydration controlled by hemolymph osmolality and peptide hormones.

1. One response of the terrestrial slug, Limax maximus to dehydration is the initiation and modulation of the pneumostome rhythm. When a slug has lost 15-20% of its initial body weight by evaporation, the frequency of pheumostome closures, which is less than 0.5 closures/min in fully hydrated slugs, begins to increase. 2. The frequency increases with further dehydration, but the average duration of each closure remains constant. Thus, the proportion of time during which the pneumostome is closed increases. Simultaneously, the area of the pneumostome opening decreases. 3. This behavior appears to be controlled in part by both the osmolality of the slug's hemolymph and by a peptide closely related to arginine vasotocin (AVT) and arginine vasopressin (AVP). Injecting intact slugs with mannitol, which increases the osmolality of the hemolymph, or with AVT or AVP, can initiate the pneumostome rhythm. 4. Mannitol injections, however, do not provoke the decrease in the area of the pneumostome opening which is induced by natural dehydration or by AVT or AVP injection. This suggests that at least two systems may be involved in the overall control of the pneumostome.

Modification of cardiac activity in response to dehydration in the terrestrial slug, Limax maximus.

The level of body hydration in the terrestrial slug Limax maximus modifies several aspects of behavior such as pneumostome activity, feeding responsiveness, huddling, and contact-rehydration. The relationship between water balance and pneumostome activity and respiratory function suggested that cardiac activity might also be affected. To pursue this possibility, intact slugs and isolated heart-central nervous system (CNS) preparations were used to investigate cardiac responses to the increase in hemolymph osmolality which occurs during dehydration. In intact animals, heart rate increased in response to progressive air-dehydration and to increases in hemolymph osmolality resulting from injections of hyperosmotic solutions of mannitol or NaCl. In isolated preparations, the heart or CNS were separately exposed to hyperosmotic saline. Exposure of the heart alone to hyperosmotic saline resulted in decreased heart rate while exposure of only the CNS resulted in an increase in heart rate. These observations suggest that the increase in heart rate that is observed in intact air-dehydrated slugs is primarily mediated by the CNS.

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.

Paracellular water uptake and molecular sieving by the foot epithelium of terrestrial slugs.

A paracellular pathway in the foot epithelium of Lehmannia valentiana can be opened by dehydrating the slug. Movement of water from a wet pad through the opened pathway into the haemolymph of this terrestrial slug is rapid. The sieving properties of this paracellular pathway have been determined using the reference isotope 3HOH and various 14C-labelled solutes. Paracellular uptake of 14C-insulin (Fig. 1) and 3HOH (Fig. 2) is initial rate for at least 3 min. If the wet pad contains 1,000 cpm of 14C per ml of 3HOH, slugs absorb only about 400 cpm of 14C with each ml of 3HOH absorbed representing a sieving ratio of 0.4 for insulin. The sieving ratio of 14C-inulin does not change when the concentration is increased from 0.1 to 2.5 mmol/l. Moreover, the sieving ratio of 14C-inulin was not affected significantly by the nature of the labelling, i.e., 14C-carboxyl vs 14C-methoxy. Sieving ratios for 14C-mannitol (182 Da), 14C-polyethylene glycol (4,000 Da), and 14C-inulin (5,250 Da) were 0.92, 0.63, and 0.39, respectively (Table 1), indicating that sieving is dependent on molecular size. 14C-Dextran (70,000 Da) and blue dextran (200,000 Da) were excluded from the paracellular pathway (Fig. 4). The effective pore size of the paracellular pathway was estimated using the relationships between sieving ratio and molecular weight of 3HOH and the various solutes that can pass through the pathway. The extrapolated pore size is equivalent to that of a sieve having a molecular weight cutoff of about 10,000 Da (Fig 3).

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.