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Characteristics of biogenic calcite in the prismatic layer of a pearl oyster, Pinctada fucata
Micron 41 (2010) 821–826
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Characteristics of biogenic calcite in the prismatic layer of a pearl oyster, Pinctada fucata Taiga Okumura a,∗ , Michio Suzuki a , Hiromichi Nagasawa b , Toshihiro Kogure a a b
Department of Earth and Planetary Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657, Japan
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Article history: Received 22 April 2010 Received in revised form 15 May 2010 Accepted 16 May 2010 Keywords: Biomineral Calcite Prismatic layer Mollusk shell EBSD TEM-EELS
a b s t r a c t The fine structure of the calcite prism in the outer layer of a pearl oyster, Pinctada fucata, has been investigated using various electron beam techniques, in order to understand its characteristics and growth mechanism including the role of intracrystalline organic substances. As the calcite prismatic layer grows thicker, sinuous boundaries develop to divide the prism into a number of domains. The crystal misorientation between the adjacent domains is several to more than ten degrees. The component of the misorientation is mainly the rotation about the c-axis. There is no continuous organic membrane at the boundaries. Furthermore, the crystal orientation inside the domains changes gradually, as indicated by the electron back-scattered diffraction (EBSD) in a scanning electron microscope (SEM). Transmission electron microscopy (TEM) examination revealed that the domain consists of sub-grains of a few hundred nanometers divided by small-angle grain boundaries, which are probably the origin of the gradual change of the crystal orientation inside the domains. Spherular Fresnel contrasts were often observed at the small-angle grain boundaries, in defocused TEM images. Electron energy-loss spectroscopy (EELS) indicated the spherules are organic macromolecules, suggesting that incorporation of organic macromolecules during the crystal growth forms the sub-grain structure of the calcite prism. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Biominerals are not pure inorganic crystals because they contain a certain amount of organic molecules, which may influence polymorph selection, crystal orientation, morphology, and mechanical properties of the biominerals. Mollusk shells are hard tissue with elaborated and well-regulated structures which are mainly composed of CaCO3 crystals. Beside taxonomical studies, mollusk shells have been paid much attention as typical biominerals. Many researches have been conducted to understand recipes to form the shell structures and roles of the intracrystalline organic substances in the forming processes (Gotliv et al., 2003; Levi-Kalisman et al., 2001; Watabe, 1981; Watabe and Wilbur, 1960; Weiner and Addadi, 1991). A columnar calcite prismatic layer is one of the microstructures in shells, observed as an outmost layer in several bivalves (Lutts et al., 1960; Mann, 2001). Each calcite prism is separated by a thick organic compartment wall (Grégoire, 1961; Kennedy et al., 1969; Wada, 1961) and arranged with its crystallographic c-axis perpendicular to the shell surface (Checa et al., 2005; MacDonald et al., 2010; Taylor et al., 1969). Accord-
∗ Corresponding author. Tel.: +81 3 5841 4019; fax: +81 3 5841 4019. E-mail address: [email protected] (T. Okumura). 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.05.004
ing to Kennedy et al. (1969) and Taylor et al. (1969), the outer layers of Pterioida and Mytiloida are composed of calcite “simple prisms”, which means that a prism inside the organic wall is just a single crystal of calcite. Similar conclusions were also reported in Dauphin (2003) and Dauphin et al. (2003a). However, some studies indicated that each prism is not a single crystal but made up of subunits in some species (Watabe and Wada, 1956; Wada, 1961; Dauphin, 2002, 2003; Dauphin et al., 2003a). Watabe and Wada (1956) observed thin sections of the prismatic layer in Pinctada martensii with polarized light and reported that each prism extinguishes inhomogeneously with several smaller domains. Dauphin (2003) also reported a composite structure of the prisms in Pinctada margaritifera owing to similar extinction feature. On the other hand, Baronnet et al. (2008) observed monocrystalline extinction in the prisms of the juvenile shell. Individual prisms of Pinna nobilis exhibit homogeneous extinction (Dauphin, 2003). Besides, the presence of sinuous boundaries on the etched surfaces of the prisms was reported in P. margaritifera by Dauphin (2003), and in Isognomon legumen by Esteban-Delgado et al. (2008). Dauphin et al. (2003a,b) suggested that the sinuous boundaries are organic membranes which divide the calcite prism into domains. Baronnet et al. (2008) explored the prisms of P. margaritifera in more detail and showed that the prisms are made of round-shaped fine domains of ∼0.1 m size surrounded by a cortex. Moreover, Esteban-Delgado et al. (2008) examined the pris-
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matic layers of diverse pteriomorph bivalves and illustrated that the prisms in Pinnidae, Pteriidae and Isognomonidae are built up with calcite nanocrystals whereas those in Pectinidae, Propeamussiidae, Anomiidae and Ostreidae are constituted by micron-sized laths, from scanning electron microscopy (SEM) images. Like these, various features of calcite prismatic layers have been reported or proposed to date, but it seems that complete understanding of the prismatic layers has not been obtained. A reason is that many previous studies are based on morphological observations and investigations for crystallographic aspects of prismatic layers are limited (Checa et al., 2009; MacDonald et al., 2010). Although several studies reported that the calcite prisms contain organic substances (Dauphin, 2002, 2003; Dauphin et al., 2003a,b, 2008, 2010; Nudelman et al., 2007), it is not clear where they locate in the prisms and how they influence the microstructures of the prisms. In this study, we analyzed microscopically and crystallographically the calcite prisms using electron back-scattered diffraction (EBSD) combined with SEM, as well as transmission electron microscopy (TEM) with the focused ion beam (FIB) sample preparation technique. We also performed nanoscopic analysis using the electron energy-loss spectroscopy (EELS) technique to determine the location of organic substances in the crystals.
2. Material and methods 2.1. Material The shell of Pinctada fucata, a pteriomorph bivalve and widely used in pearl farming in Japan, was examined in the present study. The shell is made up of an outer prismatic layer made of calcite and an inner nacreous layer (Kennedy et al., 1969; Taylor et al., 1969; Wada, 1961). The shell samples were cultured for two years and collected in May 2008 by the Fisheries Research Institute, Mie Prefecture, Japan. 2.2. Methods 2.2.1. SEM observations Both inner surfaces and polished cross-sections of the prismatic layer were observed by SEM. The samples were etched with 5 mM EDTA for 5 min to reveal microstructures and coated with Pt–Pd. SEM observation was carried out using a S-4500 SEM (Hitachi) with a cold field-emission gun at an acceleration voltage of 2–15 kV. 2.2.2. EBSD analysis Inner surfaces of the prisms were crystallographically analyzed by EBSD. The samples were etched with EDTA in the same way as
Fig. 1. SEM images of the prismatic layer of P. fucata etched by EDTA. (A) Internal surface of the prismatic layer with the initial prisms. (B) Internal surface of the layer with the thick, well-grown prisms, showing intraprismatic sinuous grooves. (C) Enlarged view of the grooves. (D) Longitudinal cross-section of the initial prisms. (E) Longitudinal cross-section of the prisms at the boundary with the nacreous layer, showing the grooves nearly along the growth direction. Some grooves start around the middle of the prism. White arrows indicate the growth direction.
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SEM samples and thinly coated with carbon. The measurements were carried out using a JSM-7000F SEM (JEOL) with a HKL Channel 5 EBSD system (Oxford Instruments). The HKL data processing system was used to obtain crystallographic orientation and pole figures. 2.2.3. TEM-EELS analysis The shells were also observed by TEM to investigate the prismatic layer in more detail. Electron-transparent thin specimens for TEM examination were prepared using a FB-2100 FIB (Hitachi) with the micro-sampling system. The samples were locally coated with the deposition of tungsten and trimmed using Ga ion beam of 30 kV, and then thinned down to a final thickness of 100–200 nm with a low energy beam of 10 kV as the final process. A JEM-2010UHR TEM (JEOL) operated at 200 kV was used to image fine structures in the shells. The bonding states of carbon in the shells were analyzed by a Tridiem EELS imaging filter system (Gatan) equipped to a HF-3300 TEM (Hitachi) with a cold field-emission gun operated at 300 kV. 3. Results 3.1. SEM observations It is known that the prismatic layer of pterioids starts growing on the periostracum and increases its thickness, and then stops growing when the surface is covered with an organic sheet and the inner nacreous layer (Checa, 2000). Therefore, if we observe the inner surface of the prismatic layer, the surface near the shell edge corresponds to the initial stage of the prism growth and the surface near the boundary with the nacreous layer to the final stage of the growth. Fig. 1A–C shows the inner surfaces etched with EDTA. The initial prism surface (Fig. 1A) shows no organization or structure inside the prisms. On the other hand, the prism surface near the boundary with the nacreous layer shows intraprismatic sinuous grooves (Fig. 1B and C), as reported by Dauphin (2003), Dauphin et al. (2003a) and Esteban-Delgado et al. (2008). The density of the grooves in the prisms increases as the prismatic layer grows thicker. The polished and etched longitudinal cross-sections of the prisms were also observed (Fig. 1D and E). The initial part of the prism does not have the sinuous grooves, which is consistent with the surface observation (Fig. 1D). In contrast, the prism near the boundary with the nacreous layer shows the sinuous grooves in the same manner as prism surfaces (Fig. 1E). In the figure, the grooves run nearly along the prism. In addition, some grooves start around the middle of the prism and run towards the growth direction, but others disappear, especially at the thick organic wall. 3.2. EBSD analysis Next, pole figures of crystallographic axes were acquired by EBSD, from the etched surface of a single prism on which sinuous grooves were distinct (Fig. 2A). The EBSD patterns were taken at 1 m step. Six domains around the center of the prism divided by the grooves were denoted by (a)–(f) (Fig. 2A). The directions of [0 0 1], [1 0 0] and [0 1 0] of calcite inside each domain were plotted (Fig. 2B). The crystal orientations of individual domains are similar but not the same, indicating the difference of the crystal orientation across the grooves. Generally the directions of the ai -axis ([1 0 0] and [0 1 0]) are more varied than those of the c-axis ([001]), suggesting that the component of the misorientation is mainly the rotation around the c-axis. Furthermore, the crystal orientation inside the domains is not exactly uniform. As shown in Fig. 2B, the poles of the axes spread out by 8–9◦ in average within the domains (around 12◦ in (b) and (f) whereas 4◦ in (a)). Note that accuracy of the crystal orientation measured by EBSD is generally 0.5–1◦ (Humphreys, 2001;
Fig. 2. (A) SEM image of the etched surface of the prism analyzed by EBSD. Six domains divided by the sinuous grooves are denoted by (a)–(f). (B) Pole figures (upper hemisphere) for each domain of (a)–(f) indicated in (A). The directions of [0 0 1], [1 0 0] and [0 1 0] of calcite are plotted. (C) Misorientation profile along the line NS in (A). Arrow and arrowhead indicate the positions 17 and 28 m away from point N, respectively.
Lassen, 1996; Prior, 1999; Wilkinson, 2001). Here again the variance of the ai -axis is larger than that of the c-axis. Fig. 2C shows the misorientation profile along the line NS in Fig. 2A. The crystal orientation changes largely at the points of 17 and 28 m away from point N, which correspond to the positions of the grooves. From point N to the groove at 17 m, or inside domain-(d), the crystal orientation changes gradually (Fig. 2C). 3.3. TEM-EELS analysis The cross-section of the prism containing the boundaries, i.e., sinuous grooves, was observed by TEM (Fig. 3A). High-resolution TEM image indicates that the adjacent calcite crystals are intimately connected and no intercrystalline materials like organic membranes are observed at the boundary (Fig. 3B). This result may be in accordance with the high-resolution SEM image (Fig. 1C) in which the sinuous grooves were only etched deeply by EDTA, whereas the thick interprismatic organic wall is prominent in the figure because such organic substances are not etched by EDTA. On the other hand,
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Fig. 3. (A) Bright-field TEM image of the cross-section at the sinuous boundary of the prismatic layer. Black arrow indicates the internal surface of the prismatic layer. Arrowhead indicates the sinuous boundary. (B) High-resolution TEM image from the rectangle indicated by white arrow in (A). Notice that the lattice fringes in the both sides are in contact, indicating no foreign materials at the boundary.
the inside of each crystal is not simple. If the bright-field images are taken with an under-focused condition, many spherular Fresnel contrasts appear in the crystal (Fig. 4). It looks that the spherules are not uniformly distributed but occasionally aligned as if they partition the crystal into sub-grains of a few hundred nanometers. Furthermore, the diffraction contrast is often terminated at the aligned spherules (the arrows in Fig. 4), suggesting that small misorientation is generated there. These spherular contrasts were also well observed in the specimen prepared by crushing the prismatic layer, indicating that they are not artifact induced by the FIB sample preparation. To identify the substance of the spherular contrasts, TEM-EELS analysis was carried out (Fig. 5A). The integrated EELS spectra in the region (a), in which the spherular Fresnel contrasts are distinct, and region (b) without the contrasts are shown in Fig. 5B and C, respectively. If we focus the fine structure of carbon K-edge in the EELS spectra, the peak around 284 eV is most notable for the difference between the spectra and detected intensively
Fig. 4. Bright-field TEM image of the cross-section of the prism with an underfocused condition, showing the spherular Fresnel contrasts. Arrows indicate the aligned spherular contrasts which terminate the diffraction contrast, suggesting that small misorientation is generated there. The rectangle indicates the area from which EELS spectra were acquired.
at the region (a). This peak corresponds to the electron transition from 1s to * (C C) bonds for carbon and is observed from organic matter, graphite, amorphous carbon, etc., but never from the carbonate ion (Gries et al., 2009; Mansot et al., 2003; Martin et al., 1989, 1990; Varlot et al., 1999). On the other hand, the sharp peak around 289 eV and broad peak around 299 eV in the fine structure of the carbon K-edge are characteristic to carbonate materials (Mansot et al., 2003; Martin et al., 1989). The peaks around 347 and 350 eV are calcium L2,3 edges, corresponding to the electron transitions from 2p1/2 to 3d and from 2p3/2 to 3d, respectively (Mansot et al., 2003; Martin et al., 1990). From this result, it is concluded that the spherular contrasts correspond to organic substances surrounded by the crystal.
4. Discussion The present study revealed that the calcite prisms in the outer layer of the pearl oyster have a structure which is significantly different from simple, inorganic calcite single crystals. Dauphin (2003) and Dauphin et al. (2003a) suggested the existence of organic membranes at the intraprismatic sinuous grooves from SEM observations. However, we did not recognize such membranes in SEM/TEM observations. Such sinuous shapes of the grooves (Fig. 1C) seem difficult to explain with a membrane-separated boundary. In contrast, it is reasonable to speculate that the sinuous boundaries reflect the sub-grain structure found in this study. Although the organic spherules are accumulated (Fig. 4) and small misorientation exists at the sub-grain boundaries, the crystal structures of the adjacent grains are still continuous. Li and Huang (2009) reported similar conclusion on aragonite in nacre that pseudosingle-crystal aragonite is assembled with nanoparticles jointed by dislocation and amorphous layers. Alternatively, the sub-grain structure can be analogous to the mesocrystals, which are superstructures of crystalline nanoparticles on the scale of some hundred nanometers to micrometers, but show single-crystalline behavior because of the high orientational alignment of the subunits (Cölfen and Antonietti, 2005). Although we found the sub-grain structure in the calcite prism using TEM, similar results were reported in previous works using other methods. Esteban-Delgado et al. (2008) and Checa et al. (2009) observed the surface of the prismatic layer of P. martensii by SEM and reported that the prisms are made up of nano-units, the size of which is similar to that of the sub-grains observed in this work. Submicron-domains in the prisms of P. margaritifera were reported by atomic force microscopy (AFM) images in Baronnet
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Fig. 5. (A) Bright-field TEM image of the part of the prismatic layer analyzed by EELS (the rectangle in Fig. 4). (B) Integrated EELS spectrum in the region (a) in (A). (C) Integrated EELS spectrum in the region (b) in (A).
et al. (2008). According to Dauphin (2002) and Nudelman et al. (2007), the calcite prisms of P. nobilis and Atrina rigida also have sub-micron units. Hence such fine structure in the calcite prism may be common over the prismatic layers of many bivalve species. It is likely that the origin of the small-angle misorientation is organic substances, as evidenced by TEM imaging and EELS analysis. Baronnet et al. (2008) showed that the sub-grains are surrounded by a cortex from phase images of AFM, which is also interpreted as organic substances. As shown in Fig. 4, the size of the spherular contrasts is several nanometers. Hence, it is reasonable to speculate that they are organic macromolecules, secreted from the mantle cells and incorporated into the calcite crystal via extrapallial fluid. A question is what these organic macromolecules are and why or how they are not distributed homogenously but localized to form the sub-grain structure. Recently several proteins were identified from the prismatic layer of P. fucata and their amino-acid sequences were determined (Sudo et al., 1997; Suzuki et al., 2004; Tsukamoto et al., 2004). Hence it is possible to examine whether the organic macromolecules are these identified proteins or not by using, for instance, the immunogold labeling technique in TEM. Another idea to seek the answer is to perform an in vitro experiment to synthesize calcite with the presence of the organic substances extracted by dissolving the calcite prisms of the outer layer, and investigate the similarity or dissimilarity between the product and the prism. 5. Conclusions The present study for the calcite prismatic layer of P. fucata revealed that the layer has the following hierarchical structures. The first structure consists of prisms (in ∼20 m range) separated by thick organic wall. The second structure is made up of the domains (in ∼5 m range) inside the prisms, partitioned by sinuous grooves. The crystal orientations change largely across the grooves and gradually inside the domains. The sub-grains (∼200 nm in size) surrounded by small-angle grain boundaries form the third structure which was observed by TEM for the first time, and intracrystalline organic macromolecules are probably responsible for the generation of the boundaries. Possible future works are
finding the correspondence between the organic macromolecules imaged in TEM and those extracted from the crystals and reported in previous works, and/or in vitro experiments to reproduce such an organic–inorganic composite nanostructure. Acknowledgements We are grateful to H. Aoki (Mie Prefecture Fisheries Research Institute) for donating the living pearl oysters and to S. Akera and E. Okamoto (Tasaki Shinju Co., Ltd.) for providing us the pearl oyster shells. We also thank H. Matsumoto (Hitachi High-technologies Co., Ltd.) for measuring EELS spectra and H. Raimbourg (University of Orlean) for the assistance of the EBSD analysis. This work was supported by a Grant-in-Aid for scientific research (No. 17GS0311) from the Ministry Education, Culture, Sports, Science and Technology of Japan. References Baronnet, A., Cuif, J.P., Dauphin, Y., Farre, B., Nouet, J., 2008. Crystallization of biogenic Ca-carbonate within organo-mineral micro-domains. Structure of the calcite prisms of the Pelecypod Pinctada margaritifera (Mollusca) at the submicron to nanometre ranges. Mineral. Mag. 72, 617–626. Checa, A.G., 2000. A new model for periostracum and shell formation in Unionidae (Bivalvia, Mollusca). Tissue Cell 32, 405–416. Checa, A.G., Rodríguez-Navarro, A.B., Esteban-Delgado, F.J., 2005. The nature and formation of calcitic columnar prismatic shell layers in pteriomorphian bivalves. Biomaterials 26, 6404–6414. Checa, A.G., Esteban-Delgado, F.J., Ramírez-Rico, J., Rodríguez-Navarro, A.B., 2009. Crystallographic reorganization of the calcitic prismatic layer of oysters. J. Struct. Biol. 167, 261–270. Cölfen, H., Antonietti, M., 2005. Mesocrystals: inorganic superstructures made by highly parallel crystallization and controlled alignment. Angew. Chem. Int. Ed. 44, 5576–5591. Dauphin, Y., 2002. Comparison of the soluble matrices of the calcitic prismatic layer of Pinna nobilis (Mollusca, Bivalvia, Pteriomorpha). Comp. Biochem. Physiol. Part A 132, 577–590. Dauphin, Y., 2003. Soluble organic matrices of the calcitic prismatic shell layers of two Pteriomorphid bivalves. Pinna nobilis and Pinctada margaritifera. J. Biol. Chem. 278, 15168–15177. Dauphin, Y., Cuif, J.P., Doucet, J., Salomé, M., Susini, J., Williams, C.T., 2003a. In situ chemical speciation of sulfur in calcitic biominerals and the simple prism concept. J. Struct. Biol. 142, 272–280.
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Micron journal homepage: www.elsevier.com/locate/micron
Characteristics of biogenic calcite in the prismatic layer of a pearl oyster, Pinctada fucata Taiga Okumura a,∗ , Michio Suzuki a , Hiromichi Nagasawa b , Toshihiro Kogure a a b
Department of Earth and Planetary Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657, Japan
a r t i c l e
i n f o
Article history: Received 22 April 2010 Received in revised form 15 May 2010 Accepted 16 May 2010 Keywords: Biomineral Calcite Prismatic layer Mollusk shell EBSD TEM-EELS
a b s t r a c t The fine structure of the calcite prism in the outer layer of a pearl oyster, Pinctada fucata, has been investigated using various electron beam techniques, in order to understand its characteristics and growth mechanism including the role of intracrystalline organic substances. As the calcite prismatic layer grows thicker, sinuous boundaries develop to divide the prism into a number of domains. The crystal misorientation between the adjacent domains is several to more than ten degrees. The component of the misorientation is mainly the rotation about the c-axis. There is no continuous organic membrane at the boundaries. Furthermore, the crystal orientation inside the domains changes gradually, as indicated by the electron back-scattered diffraction (EBSD) in a scanning electron microscope (SEM). Transmission electron microscopy (TEM) examination revealed that the domain consists of sub-grains of a few hundred nanometers divided by small-angle grain boundaries, which are probably the origin of the gradual change of the crystal orientation inside the domains. Spherular Fresnel contrasts were often observed at the small-angle grain boundaries, in defocused TEM images. Electron energy-loss spectroscopy (EELS) indicated the spherules are organic macromolecules, suggesting that incorporation of organic macromolecules during the crystal growth forms the sub-grain structure of the calcite prism. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Biominerals are not pure inorganic crystals because they contain a certain amount of organic molecules, which may influence polymorph selection, crystal orientation, morphology, and mechanical properties of the biominerals. Mollusk shells are hard tissue with elaborated and well-regulated structures which are mainly composed of CaCO3 crystals. Beside taxonomical studies, mollusk shells have been paid much attention as typical biominerals. Many researches have been conducted to understand recipes to form the shell structures and roles of the intracrystalline organic substances in the forming processes (Gotliv et al., 2003; Levi-Kalisman et al., 2001; Watabe, 1981; Watabe and Wilbur, 1960; Weiner and Addadi, 1991). A columnar calcite prismatic layer is one of the microstructures in shells, observed as an outmost layer in several bivalves (Lutts et al., 1960; Mann, 2001). Each calcite prism is separated by a thick organic compartment wall (Grégoire, 1961; Kennedy et al., 1969; Wada, 1961) and arranged with its crystallographic c-axis perpendicular to the shell surface (Checa et al., 2005; MacDonald et al., 2010; Taylor et al., 1969). Accord-
∗ Corresponding author. Tel.: +81 3 5841 4019; fax: +81 3 5841 4019. E-mail address: [email protected] (T. Okumura). 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.05.004
ing to Kennedy et al. (1969) and Taylor et al. (1969), the outer layers of Pterioida and Mytiloida are composed of calcite “simple prisms”, which means that a prism inside the organic wall is just a single crystal of calcite. Similar conclusions were also reported in Dauphin (2003) and Dauphin et al. (2003a). However, some studies indicated that each prism is not a single crystal but made up of subunits in some species (Watabe and Wada, 1956; Wada, 1961; Dauphin, 2002, 2003; Dauphin et al., 2003a). Watabe and Wada (1956) observed thin sections of the prismatic layer in Pinctada martensii with polarized light and reported that each prism extinguishes inhomogeneously with several smaller domains. Dauphin (2003) also reported a composite structure of the prisms in Pinctada margaritifera owing to similar extinction feature. On the other hand, Baronnet et al. (2008) observed monocrystalline extinction in the prisms of the juvenile shell. Individual prisms of Pinna nobilis exhibit homogeneous extinction (Dauphin, 2003). Besides, the presence of sinuous boundaries on the etched surfaces of the prisms was reported in P. margaritifera by Dauphin (2003), and in Isognomon legumen by Esteban-Delgado et al. (2008). Dauphin et al. (2003a,b) suggested that the sinuous boundaries are organic membranes which divide the calcite prism into domains. Baronnet et al. (2008) explored the prisms of P. margaritifera in more detail and showed that the prisms are made of round-shaped fine domains of ∼0.1 m size surrounded by a cortex. Moreover, Esteban-Delgado et al. (2008) examined the pris-
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matic layers of diverse pteriomorph bivalves and illustrated that the prisms in Pinnidae, Pteriidae and Isognomonidae are built up with calcite nanocrystals whereas those in Pectinidae, Propeamussiidae, Anomiidae and Ostreidae are constituted by micron-sized laths, from scanning electron microscopy (SEM) images. Like these, various features of calcite prismatic layers have been reported or proposed to date, but it seems that complete understanding of the prismatic layers has not been obtained. A reason is that many previous studies are based on morphological observations and investigations for crystallographic aspects of prismatic layers are limited (Checa et al., 2009; MacDonald et al., 2010). Although several studies reported that the calcite prisms contain organic substances (Dauphin, 2002, 2003; Dauphin et al., 2003a,b, 2008, 2010; Nudelman et al., 2007), it is not clear where they locate in the prisms and how they influence the microstructures of the prisms. In this study, we analyzed microscopically and crystallographically the calcite prisms using electron back-scattered diffraction (EBSD) combined with SEM, as well as transmission electron microscopy (TEM) with the focused ion beam (FIB) sample preparation technique. We also performed nanoscopic analysis using the electron energy-loss spectroscopy (EELS) technique to determine the location of organic substances in the crystals.
2. Material and methods 2.1. Material The shell of Pinctada fucata, a pteriomorph bivalve and widely used in pearl farming in Japan, was examined in the present study. The shell is made up of an outer prismatic layer made of calcite and an inner nacreous layer (Kennedy et al., 1969; Taylor et al., 1969; Wada, 1961). The shell samples were cultured for two years and collected in May 2008 by the Fisheries Research Institute, Mie Prefecture, Japan. 2.2. Methods 2.2.1. SEM observations Both inner surfaces and polished cross-sections of the prismatic layer were observed by SEM. The samples were etched with 5 mM EDTA for 5 min to reveal microstructures and coated with Pt–Pd. SEM observation was carried out using a S-4500 SEM (Hitachi) with a cold field-emission gun at an acceleration voltage of 2–15 kV. 2.2.2. EBSD analysis Inner surfaces of the prisms were crystallographically analyzed by EBSD. The samples were etched with EDTA in the same way as
Fig. 1. SEM images of the prismatic layer of P. fucata etched by EDTA. (A) Internal surface of the prismatic layer with the initial prisms. (B) Internal surface of the layer with the thick, well-grown prisms, showing intraprismatic sinuous grooves. (C) Enlarged view of the grooves. (D) Longitudinal cross-section of the initial prisms. (E) Longitudinal cross-section of the prisms at the boundary with the nacreous layer, showing the grooves nearly along the growth direction. Some grooves start around the middle of the prism. White arrows indicate the growth direction.
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SEM samples and thinly coated with carbon. The measurements were carried out using a JSM-7000F SEM (JEOL) with a HKL Channel 5 EBSD system (Oxford Instruments). The HKL data processing system was used to obtain crystallographic orientation and pole figures. 2.2.3. TEM-EELS analysis The shells were also observed by TEM to investigate the prismatic layer in more detail. Electron-transparent thin specimens for TEM examination were prepared using a FB-2100 FIB (Hitachi) with the micro-sampling system. The samples were locally coated with the deposition of tungsten and trimmed using Ga ion beam of 30 kV, and then thinned down to a final thickness of 100–200 nm with a low energy beam of 10 kV as the final process. A JEM-2010UHR TEM (JEOL) operated at 200 kV was used to image fine structures in the shells. The bonding states of carbon in the shells were analyzed by a Tridiem EELS imaging filter system (Gatan) equipped to a HF-3300 TEM (Hitachi) with a cold field-emission gun operated at 300 kV. 3. Results 3.1. SEM observations It is known that the prismatic layer of pterioids starts growing on the periostracum and increases its thickness, and then stops growing when the surface is covered with an organic sheet and the inner nacreous layer (Checa, 2000). Therefore, if we observe the inner surface of the prismatic layer, the surface near the shell edge corresponds to the initial stage of the prism growth and the surface near the boundary with the nacreous layer to the final stage of the growth. Fig. 1A–C shows the inner surfaces etched with EDTA. The initial prism surface (Fig. 1A) shows no organization or structure inside the prisms. On the other hand, the prism surface near the boundary with the nacreous layer shows intraprismatic sinuous grooves (Fig. 1B and C), as reported by Dauphin (2003), Dauphin et al. (2003a) and Esteban-Delgado et al. (2008). The density of the grooves in the prisms increases as the prismatic layer grows thicker. The polished and etched longitudinal cross-sections of the prisms were also observed (Fig. 1D and E). The initial part of the prism does not have the sinuous grooves, which is consistent with the surface observation (Fig. 1D). In contrast, the prism near the boundary with the nacreous layer shows the sinuous grooves in the same manner as prism surfaces (Fig. 1E). In the figure, the grooves run nearly along the prism. In addition, some grooves start around the middle of the prism and run towards the growth direction, but others disappear, especially at the thick organic wall. 3.2. EBSD analysis Next, pole figures of crystallographic axes were acquired by EBSD, from the etched surface of a single prism on which sinuous grooves were distinct (Fig. 2A). The EBSD patterns were taken at 1 m step. Six domains around the center of the prism divided by the grooves were denoted by (a)–(f) (Fig. 2A). The directions of [0 0 1], [1 0 0] and [0 1 0] of calcite inside each domain were plotted (Fig. 2B). The crystal orientations of individual domains are similar but not the same, indicating the difference of the crystal orientation across the grooves. Generally the directions of the ai -axis ([1 0 0] and [0 1 0]) are more varied than those of the c-axis ([001]), suggesting that the component of the misorientation is mainly the rotation around the c-axis. Furthermore, the crystal orientation inside the domains is not exactly uniform. As shown in Fig. 2B, the poles of the axes spread out by 8–9◦ in average within the domains (around 12◦ in (b) and (f) whereas 4◦ in (a)). Note that accuracy of the crystal orientation measured by EBSD is generally 0.5–1◦ (Humphreys, 2001;
Fig. 2. (A) SEM image of the etched surface of the prism analyzed by EBSD. Six domains divided by the sinuous grooves are denoted by (a)–(f). (B) Pole figures (upper hemisphere) for each domain of (a)–(f) indicated in (A). The directions of [0 0 1], [1 0 0] and [0 1 0] of calcite are plotted. (C) Misorientation profile along the line NS in (A). Arrow and arrowhead indicate the positions 17 and 28 m away from point N, respectively.
Lassen, 1996; Prior, 1999; Wilkinson, 2001). Here again the variance of the ai -axis is larger than that of the c-axis. Fig. 2C shows the misorientation profile along the line NS in Fig. 2A. The crystal orientation changes largely at the points of 17 and 28 m away from point N, which correspond to the positions of the grooves. From point N to the groove at 17 m, or inside domain-(d), the crystal orientation changes gradually (Fig. 2C). 3.3. TEM-EELS analysis The cross-section of the prism containing the boundaries, i.e., sinuous grooves, was observed by TEM (Fig. 3A). High-resolution TEM image indicates that the adjacent calcite crystals are intimately connected and no intercrystalline materials like organic membranes are observed at the boundary (Fig. 3B). This result may be in accordance with the high-resolution SEM image (Fig. 1C) in which the sinuous grooves were only etched deeply by EDTA, whereas the thick interprismatic organic wall is prominent in the figure because such organic substances are not etched by EDTA. On the other hand,
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Fig. 3. (A) Bright-field TEM image of the cross-section at the sinuous boundary of the prismatic layer. Black arrow indicates the internal surface of the prismatic layer. Arrowhead indicates the sinuous boundary. (B) High-resolution TEM image from the rectangle indicated by white arrow in (A). Notice that the lattice fringes in the both sides are in contact, indicating no foreign materials at the boundary.
the inside of each crystal is not simple. If the bright-field images are taken with an under-focused condition, many spherular Fresnel contrasts appear in the crystal (Fig. 4). It looks that the spherules are not uniformly distributed but occasionally aligned as if they partition the crystal into sub-grains of a few hundred nanometers. Furthermore, the diffraction contrast is often terminated at the aligned spherules (the arrows in Fig. 4), suggesting that small misorientation is generated there. These spherular contrasts were also well observed in the specimen prepared by crushing the prismatic layer, indicating that they are not artifact induced by the FIB sample preparation. To identify the substance of the spherular contrasts, TEM-EELS analysis was carried out (Fig. 5A). The integrated EELS spectra in the region (a), in which the spherular Fresnel contrasts are distinct, and region (b) without the contrasts are shown in Fig. 5B and C, respectively. If we focus the fine structure of carbon K-edge in the EELS spectra, the peak around 284 eV is most notable for the difference between the spectra and detected intensively
Fig. 4. Bright-field TEM image of the cross-section of the prism with an underfocused condition, showing the spherular Fresnel contrasts. Arrows indicate the aligned spherular contrasts which terminate the diffraction contrast, suggesting that small misorientation is generated there. The rectangle indicates the area from which EELS spectra were acquired.
at the region (a). This peak corresponds to the electron transition from 1s to * (C C) bonds for carbon and is observed from organic matter, graphite, amorphous carbon, etc., but never from the carbonate ion (Gries et al., 2009; Mansot et al., 2003; Martin et al., 1989, 1990; Varlot et al., 1999). On the other hand, the sharp peak around 289 eV and broad peak around 299 eV in the fine structure of the carbon K-edge are characteristic to carbonate materials (Mansot et al., 2003; Martin et al., 1989). The peaks around 347 and 350 eV are calcium L2,3 edges, corresponding to the electron transitions from 2p1/2 to 3d and from 2p3/2 to 3d, respectively (Mansot et al., 2003; Martin et al., 1990). From this result, it is concluded that the spherular contrasts correspond to organic substances surrounded by the crystal.
4. Discussion The present study revealed that the calcite prisms in the outer layer of the pearl oyster have a structure which is significantly different from simple, inorganic calcite single crystals. Dauphin (2003) and Dauphin et al. (2003a) suggested the existence of organic membranes at the intraprismatic sinuous grooves from SEM observations. However, we did not recognize such membranes in SEM/TEM observations. Such sinuous shapes of the grooves (Fig. 1C) seem difficult to explain with a membrane-separated boundary. In contrast, it is reasonable to speculate that the sinuous boundaries reflect the sub-grain structure found in this study. Although the organic spherules are accumulated (Fig. 4) and small misorientation exists at the sub-grain boundaries, the crystal structures of the adjacent grains are still continuous. Li and Huang (2009) reported similar conclusion on aragonite in nacre that pseudosingle-crystal aragonite is assembled with nanoparticles jointed by dislocation and amorphous layers. Alternatively, the sub-grain structure can be analogous to the mesocrystals, which are superstructures of crystalline nanoparticles on the scale of some hundred nanometers to micrometers, but show single-crystalline behavior because of the high orientational alignment of the subunits (Cölfen and Antonietti, 2005). Although we found the sub-grain structure in the calcite prism using TEM, similar results were reported in previous works using other methods. Esteban-Delgado et al. (2008) and Checa et al. (2009) observed the surface of the prismatic layer of P. martensii by SEM and reported that the prisms are made up of nano-units, the size of which is similar to that of the sub-grains observed in this work. Submicron-domains in the prisms of P. margaritifera were reported by atomic force microscopy (AFM) images in Baronnet
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Fig. 5. (A) Bright-field TEM image of the part of the prismatic layer analyzed by EELS (the rectangle in Fig. 4). (B) Integrated EELS spectrum in the region (a) in (A). (C) Integrated EELS spectrum in the region (b) in (A).
et al. (2008). According to Dauphin (2002) and Nudelman et al. (2007), the calcite prisms of P. nobilis and Atrina rigida also have sub-micron units. Hence such fine structure in the calcite prism may be common over the prismatic layers of many bivalve species. It is likely that the origin of the small-angle misorientation is organic substances, as evidenced by TEM imaging and EELS analysis. Baronnet et al. (2008) showed that the sub-grains are surrounded by a cortex from phase images of AFM, which is also interpreted as organic substances. As shown in Fig. 4, the size of the spherular contrasts is several nanometers. Hence, it is reasonable to speculate that they are organic macromolecules, secreted from the mantle cells and incorporated into the calcite crystal via extrapallial fluid. A question is what these organic macromolecules are and why or how they are not distributed homogenously but localized to form the sub-grain structure. Recently several proteins were identified from the prismatic layer of P. fucata and their amino-acid sequences were determined (Sudo et al., 1997; Suzuki et al., 2004; Tsukamoto et al., 2004). Hence it is possible to examine whether the organic macromolecules are these identified proteins or not by using, for instance, the immunogold labeling technique in TEM. Another idea to seek the answer is to perform an in vitro experiment to synthesize calcite with the presence of the organic substances extracted by dissolving the calcite prisms of the outer layer, and investigate the similarity or dissimilarity between the product and the prism. 5. Conclusions The present study for the calcite prismatic layer of P. fucata revealed that the layer has the following hierarchical structures. The first structure consists of prisms (in ∼20 m range) separated by thick organic wall. The second structure is made up of the domains (in ∼5 m range) inside the prisms, partitioned by sinuous grooves. The crystal orientations change largely across the grooves and gradually inside the domains. The sub-grains (∼200 nm in size) surrounded by small-angle grain boundaries form the third structure which was observed by TEM for the first time, and intracrystalline organic macromolecules are probably responsible for the generation of the boundaries. Possible future works are
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