Low kv EBSD using AZtecHKL and NordlysNano: Characterisation of a mollusc shell
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1 Introduction Marine shells are of high interest to environmental sciences as they are excellent archives of past environmental change to geosciences as the fundamental material carrying the fossil record of the history of life and to materials sciences as prototypes for high-strength and environmentally friendly materials. A very common mollusc found in European waters is Mytilus edulis (Figure 1), which is also cultured for food. The shell of M. edulis consists of a thick outer layer of fibrous calcite, and a thin inner layer of aragonite nacre. The nanoscale laminated hybrid composite of nacre provides outstanding mechanical properties (Mayer, 2005, Barthelat, 2010). While the micro-structure of many calcitic marine shell materials is accessible by standard 20 kv EBSD (Schmahl et al., 2009, Goetz et al., 2009), the investigation of nacre requires spatial resolution well below 1 micrometre. The resolution of EBSD can be significantly enhanced at low acceleration voltages. This application note presents EBSD results acquired at low acceleration voltages to characterise the two calcite and nacre layers in Mytilus edulis shells as well as the interface between these two layers. Figure 1. Macro images of Mytilus edulis shells. Experimental The Mytilus edulis shell was sectioned longitudinally from its posterior to the anterior shell portion. The section surface was carefully polished with a finally etch-polish followed by a thin layer of carbon coating for examination in the SEM. Some shells were also powdered to determine the crystal structures using XRD. XRD data were determined on a Seifert XRD diffractometer (3003 TT). EBSD data acquisition and post-processing were done with HKLNordlysNano EBSD and X-Max 80 EDS detectors attached to a FEGSEM with AZtec software. EBSD maps were collected at 8 kv for imaging and between 8-10 kv for mapping with a resolution from nm. Figure 2. XRD diffactogram from powdered shells. Results and Discussion Figure 2 shows an XRD diffractogram from the powdered Mytilus edulis shell indicating the presence of two phases: Calcite and Aragonite, the crystal structures are given in Table 1. This data of-course gives no information with regard to the distribution of the phases in the shell, for which SEM imaging and EBSD maps are used. 1
2 Table 1. XRD crystal structure of Mytilus edulis shell Phase Calcite Aragonite Laue group trigonal orthorhombic Space group R-3c Pmcn Lattice parameters a,b,c (nm) α,β,γ (degs) , , , 90.00, 90.00, , , , 90.00, 90.00, Volume fraction % ~60 % ~40 % In Figure 3 is shown a back-scattered electron SEM from a polished section of the shell, showing evidence of two distinct crystallite morphologies: an inner blocky or brick like and an outer scaly layer. The two layers are separated by a distinctive transition layer or zone. Figure 4. a and 8 kv 4nA EBSD patterns raw and indexed with the aragonite phase. Figure 3. SEM back-scattered map showing the two morphologies of grains separated by a transition zone in the polished section of Mytilus Edullis shell. EBSD patterns are possible from both layers and are shown in Figures 4 and 5 respectively. These were acquired at 8 kv acceleration voltage. The pattern from the inner blocky layer indexed with an aragonite phase Figure 4a, while patterns from the scaly layer indexed with a calcite phase Figure 5b. It is well known that in 3D the blocky layers are actually equiaxed disc shaped crystallites, while the calcite are fibrous, as shown in Figure 6. 2 EBSD Analysis
3 An EBSD IPF Z coloured plus band contrast and grain boundary map from the inner nacre aragonite layer is shown in Figures 7. The orientation map clearly shows that some platelets have distinct orientations, while a majority lie in a domains of similar orientations. A set of {100} pole figures from this region are shown in Figure 8, indicating that the a-axis of aragonite is perpendicular to the nacre platelets. In addition, we find three distinct crystallographic orientations of the nacre nanoplatelets, with an almost common a-axis orientation, and the b-axis orientation differing by about 30 degrees, suggesting a regular orientation relationship between adjacent grains. This is the world s first successful EBSD data showing the nacre nanostructure and the corresponding crystallite orientation. Figure 5. and 8 kv 4nA EBSD patterns raw and indexed with the calcite phase. Figure 7. EBSD IPF Z coloured plus band contrast and grain boundary map from the Aragonite layer. Thick black lines represent grain boundaries>then 10 o misorientation, while the thin black lines are >2 o misorientation. Figure 6. SEM image taken from fracture surfaces of the fibrous, calcite and the blocky, nacreous aragonite shell portions of Mytilus edulis respectively. Figure 8. Set of {100}, {010},and {001} pole figures from the data in Figure 6. EBSD Analysis 3
4 An EBSD IPF X coloured plus band contrast and grain boundary map from the outer calcite layer is shown in Figure 9, while the corresponding {0001}, {10-10} and {11-20} poles figure is given Figure 10. This layer, surprisingly, consists of a single-crystal-like 3-dimensional orientational correlation of the fibres over the scanned 26x45 micrometer-sized area. Figure 11a shows an FSD image of the interface in the section of the shell, the nacreous aragonite is at the top and the fibrous calcite at the bottom separated by a distinctive interfacial layer. High-resolution EBSD phase and orientation maps from the interface are shown in Figure 11b and c, clearly showing that this interlayer material is composed of submicronsized aragonite grains that do not have the characteristic brick like layered microstructure and their crystallographic orientation correspond to adjacent nacre platelets, Also the change from calcite to aragonite in the shell is not a continuous transition, but a sharp switch. Again this is a first set of EBSD data showing the transition zone in such detail, which has not been possible with the traditional laboratory XRD equipment. c) Figure 9. EBSD IPF X colured plus band contrast and grain boundary map from the Calcite layer. Thick black lines represent grain boundaries>then 10 o misorientation, while the thin black lines are >2 o misorientation. Figure 11. FSD image acros nacre *(top blocky) and calcite interface EBSD phase plus band contrast maps across the interface red=aragonite and blue=calcite and c) EBSD IPF X colour plus band contrast and grain boundary map. Thick black lines represent grain boundaries>then 10 o misorientation, while the thin black lines are >2 o misorientation re phase boundaries. Figure 10. Set of {100}, {010},and {001} pole figures from the data in Figure 8. 4 EBSD Analysis
5 EBSD Conclusion This study has for the first time successfully shown low kv EBSD mapping of aragonite, a regular orientation relationship between adjacent grains in aragonite, and more importantly that the transition zone in Mytilus edulis shell between the Aragonite and calcite phases is composed of submicronsized aragonite grains with crystallographic orientation corresponding to the adjacent nacre platelets, and that the change from calcite to aragonite should not be regarded as a continuous transition, but a sharp switch. While XRD can give information regarding the crystal structure of the constituents in the Mytilus edulis shell, SEM/EBSD has been successfully used to show the distribution, grain structure and local orientation relation with the fibrous nature of these phases in the shell section. High-resolution EBSD combined with SEM is a key method for a detailed understanding of the microstructures of nacre and other mineral components of mollusc shells. References G. Mayer (2005) Rigid Biological Systems as Models for Synthetic Composites, Science 310, F. Barthelat (2010) Nacre from mollusc shells: a model for high-performance structural materials.- Bioinspiration and Biomimetics 5, Schmahl, W.W., Griesshaber, E., Neuser, R.D. Götz, A., Lüter, C. (2009) Electron Back-scatter Diffraction Study of Brachiopod Shell Calcite - Microscale Phase and Texture Ana-lysis of a Polycrystalline Biomaterial.- Particle & Particle Systems Characterization, 25, Goetz, A., Griesshaber, E., Neuser, R.D., Luter,C., Hühner, M., Harper, E., Schmahl, W.W. (2009) Calcite morphology, texture and hardness in the distinct layers of rhynchonelliform brachiopod shells.- Eur. J. Mineral. 21, Acknowledgements We thank Dr Erika Griesshaber (E.G.) and Professor Wolfgang W. Schmahl, for this contribution. E.G. is grateful for a research grant from the Deutsche Forschungsgemeinschaft DFG and would also like to thank Dorrit Jacob, Mainz University, for providing the mollusc shell, Patrick Voss and Dirk Hennemann (BUEHLER GmbH, Düsseldorf) for surface finishing preparation and Mrs. Renate Enders, LMU Munich, for initial polishing of the specimens. visit for more information The materials presented here are summary in nature, subject to change, and intended for general information only. Performances are configuration dependent, and are based on AZtec Release 1.1. Additional details are available. Oxford Instruments NanoAnalysis Quality Management System is certified to meet ISO 9001: AZtec is a Registered Trademark of Oxford Instruments plc, all other trademarks acknowledged. Oxford Instruments plc, All rights reserved. Document reference: OINA/EBSD/AN109/0212 FM
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