Article pubs.acs.org/crystal
Ordered Misorientations and Preferential Directions of Growth in Mesocrystalline Red Coral Sclerites N. Floquet* and D. Vielzeuf Aix-Marseille University, CNRS, CINaM UMR7325, 13288, Marseille, France S Supporting Information *
ABSTRACT: The sclerites of the red coral are ideal structures to handle the question of crystallographic registers in biominerals. Microscopy and diffraction techniques are used to reveal the concealed relationships between the elaborate morphology and the crystallographic organization of the sclerites. Sclerites are 50−80 μm large structures, built from concentric layers of submicrometer grains of Mg calcite. Two main types of sclerites are identified: single- and multidomain sclerites. “Single-domain” sclerites are elongated centrosymmetric dumbbells with tubercles in opposite trihedral arrangement. The crystalline grains are similarly oriented with the c axis along the dumbbell length. Each tubercle extends along a ̅ direction, the denser Ca2+CO32− chain of the most stable {104} plane of calcite. Each tubercle develops lobes again along the directions indicating a dendritic like growth process in three-dimensional (3D) space. Crystallite misorientations of about ±15° are correlated to the biogenic curved morphology. Their 3D misorientation patterns are thoroughly identified. They combine the existence of well-identified domains of misorientation in the sclerite with progressive transitions between them. The misorientation patterns are consistent with the absence of misorientation in the central axis of the sclerite, the progressive change of direction and the progressive inversion of direction within a tubercle and between different domains of misorientations. Progressive misorientations are characterized by a rotation along each a axis orthogonal to the direction and to the c axis. “Multi-domain” sclerites present a quadripod shape made of four tubercles, exhibiting two types of four-sector cross shape patterns: the 93° cross pattern and the 64° cross pattern identified by electron backscattered diffraction as penetration {104} twin and {104} symmetric interface, respectively. A model based on calcite rhombohedral structure is proposed and describes accurately all the main crystallographic properties of Corallium rubrum sclerites.
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INTRODUCTION Red coral (Corallium rubrum) has been the subject of various studies by oceanologists, marine biologists and ecologists.1−11 However, until recently little was known regarding the calcitic skeleton or the sclerites of the red coral from textural, structural and crystallographic points of views.7,12 These biomineral structures are now viewed as biomesocrystalline structures. Following previous observations of Grillo et al.,7 Vielzeuf et al.13 presented the main chemical and morphological features of the red coral skeleton. In a subsequent study, Vielzeuf et al.14 described a mesocrystalline organization in the skeleton with hierarchical structural patterns. These authors showed that the red coral skeleton exhibits a multilevel modular mesocrystalline organization, which means that mesocrystals are themselves made of mesocrystalline building units. The “multilevel modular mesocrystal” pattern and the remarkable long distance © 2012 American Chemical Society
arrangement of crystalline units open new questions. For instance, the herringbone arrangement in the skeleton is a side by side ∼45° arrangement of mesocrystalline spindles elongated along their c axes. Are such a radial herringbone arrangement and other characteristic structural patterns related in any way to the crystallographic properties of calcite? Is this arrangement the result of a twinning mechanism as commonly observed in abiotic calcite? Can we extrapolate the twinning laws of abiotic calcite to biotic ones? Should we introduce the mesoscale twinning as a mesoscale organization concept in biomineralization processes? At the present stage of knowledge of natural biomesocrystalline structures, the direct path Received: April 17, 2012 Revised: July 31, 2012 Published: August 30, 2012 4805
dx.doi.org/10.1021/cg300528h | Cryst. Growth Des. 2012, 12, 4805−4820
Crystal Growth & Design
Article
The scanning electron microscopy observation was carried out on a JEOL 6320F coupled with a field emission gun. Separated sclerites were dispersed on the sample holder and coated with carbon prior to analysis. The secondary electron mode for imaging was selected throughout this study; depending on the sample, accelerating voltage and working distances conditions were changed within the range 3−15 kV and 6−15 mm. Samples for electron backscattered dif f raction (EBSD) were prepared by conventional polishing using diamond paste with grit sizes down to 1 or 1/4 μm, followed by a final polish with colloidal silica. EBSD measurements were made on two instruments both equipped with a Oxford/HKL technology “channel 5” EBSD system:20 (1) a LEO 1550VP SEM at Caltech, Pasadena, using an accelerating voltage of 20 kV, a probe current of 2 nA, a working distance of 14 mm and a step size of 2 μm at a speed of ∼0.1 s per step; (2) a CamScan X500FE CrystalProbe at Geosciences Montpellier, using an accelerating voltage of 17 kV, a probe current of 3.5 nA, a working distance of 25 mm and step sizes from 0.5 to 1 μm at a speed of ∼0.1s per step. The mean angular uncertainties for both data sets are less than 1° and the results from the two data sets are consistent. EBSD patterns were indexed using the crystallographic data of Mg calcite (space group 167, hexagonal setting, lattice parameters a = 4.941 Å and c = 16.864 Å). The crystallographic data were analyzed in 3D space with the CaRine Crystallography21 software. In the EBSD maps of crystallographic orientation, calcite crystallographic planes are color coded using the Euler color key for a hexagonal material: each Euler crystal orientation with the Euler angles (φ1, Φ, φ2) is transformed into a RGB color key using the following formulas: red = 255*φ1/180, green = 255*Φ/180, blue = 255*φ2/120. Pole figures (upper hemisphere stereographic projection) are used to interpret the orientations of the calcite crystallographic {hkl} planes and rows. Each pixel on an EBSD map corresponds to a single analysis, and the corresponding pixel in the stereographic projection is shown with the same color. White pixels represent areas where the crystallographic orientation could not be determined. In this article, emphasis will be put on the characterization of misorientation patterns. For this purpose, a misorientation angle is determined for each indexed pixel of an EBSD map. This misorientation angle corresponds to the angular rotation necessary to bring the crystal lattice associated to each pixel in coincidence with a reference crystal lattice. The reference crystal lattice is deduced from the preferential orientation of the crystallites and determined as the maximum of density distribution in the pole figures. Additional quantitative information was extracted from misorientation angle histograms that relate pixel fractions and misorientation angles within each domain of interest. These histograms are generated from built-in “misorientation angle distribution” subroutine in the HKL Technology Software.20
consisting of a more in-depth study of the skeleton is not the most appropriate way to address these problems. Indeed the three-dimensional (3D) structure of the red coral skeleton is too complex. On the other hand, and because of their sizes and shapes, sclerites which are small grains of calcium carbonate found in the living tissues of the same organism are particularly appropriate for unraveling the complexity of crystallographic organization in octocorals and explore the subtle relationships between morphology and crystallography. The sclerites of C. rubrum have been observed and described by several authors.7,12,15−17 However, no systematic studies of the morphology or crystallography have been carried out so far on a large number of sclerites. In a previous article18 we showed that sclerites can be seen as archetypes of biomesocrystals and that twinning at the mesoscale is an important process of mesocrystalline organization. In this new article, a systematic study of the crystallographic properties of several red coral sclerites is presented through multiscale analyses with optical microscopy, X-ray diffraction, SEM and SEM-electron backscattered diffraction. Our results show the systematic presence of small and progressive crystallographic misorientations between sub-micrometer grains and demonstrate that these misorientations obey systematic patterns. A crystallographic model based on rhombohedral unit blocks is proposed to explain the misorientation pattern. In addition, we demonstrate that the strong periodic bond Ca2+CO32− chains in calcite coincide with important directions of growth for the sclerites. Some data of the previous article18 are reprocessed to emphasize these additional crystallographic features.
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MATERIALS AND METHODS
Colonies of C. rubrum were collected from the rocky coast near Marseille (France) and the Medes islands (Spain). Sclerites were isolated from the soft tissue by immersion in sodium hypochlorite for a few hours and rinsed in distilled water. Some of them were embedded in epoxy resin and polished to allow observation with a reflected light microscope and SEM. Aliquots were analyzed by X-ray powder diffraction (XRPD) and observed by polarized optical microscopy (POM) and by scanning electron microscopy (SEM). Some coral branches were untreated to preserve the sclerites relationships within the soft tissues. Untreated branches were cut in axial or longitudinal sections and the sclerites spread in the soft tissue were observed by POM, SEM, and mapped by electron backscattered diffraction (EBSD). Optical investigation was performed with a Zeiss Scope A1 microscope, equipped in transmission and reflection mode, with a translating and rotating stage, polarizer with or without analyzer. Five magnifications (25, 50, 100, 200, and 500×) are available on the microscope. Images were collected with a Canon digital camera mounted on the microscope and transferred to a computer. Isolated sclerites were examined in reflected and transmitted light. The polished sections comprising the sclerites spread in the soft tissue of untreated coral branches were observed by reflection through polarized light. X-ray analyses were performed on an INEL diffractometer equipped with CPS 120 detectors (4096 channels) placed in horizontal position using Cu Kα radiation produced at 45 keV and 20 mA. The Cu Kα1 Xray line was selected using a bent quartz crystal monochromator. The data were collected from 3° to 90° in 2θ at the angular channel of 0.029°. To avoid textural effect, finely ground air-dried sclerites were put in a quartz capillary tube placed in the rotating sample holder of the diffractometer. The diffractograms were calibrated from NIST standard sample silicium NBS-640a and Iceland spar calcite. The unit cell parameters in the hexagonal setting of the Mg calcite structure were determined by Rietveld refinement of the diffractograms using the Fullprof software.19
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RESULTS Presentation of the Sclerites. Sclerites are organic/ inorganic composites with a low proportion of organic matrix (
