Stepwise Rotation of Nanometric Building Blocks in the Aragonite

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Stepwise Rotation of Nanometric Building Blocks in the Aragonite Helix of a Pteropod Shell Monami Suzuki,† Takenori Sasaki,‡ Yuya Oaki,† and Hiroaki Imai*,† †

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan ‡ The University Museum, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ABSTRACT: Aragonite helical fibers in the shell of a pteropod Cavolinia globulosa are shown to consist of single-crystalline rod-like building blocks ∼1 μm long and ∼100−200 nm wide. Although the building blocks of the orthorhombic crystal share similar c-axis orientations along the surface normal, their a and b directions rotate in a stepwise fashion. The crystallographic rotation of the unit rods and the twisted stacking of the fibers along the helical axis form the specific hierarchical architecture of the pteropod shell.

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INTRODUCTION Calcium carbonate (CaCO3) is a main component of various biominerals. Skeletons and shells of various nonvertebrate organisms are comprised of thermodynamically stable calcite and metastable aragonite.1,2 Unique macroscopic shapes and specific microstructures of CaCO3 crystals possess excellent features of biominerals, such as high mechanical strength3−6 and specific optical properties.7,8 For instance, the nacres and cross-lamellae comprised of aragonite have received attention as an excellent model for lightweight and tough structural materials because of their improved mechanical properties originating from the sophisticated microstructures.6 In recent years, granular textures consisting of oriented nanocrystals less than 100 nm in size have been observed in various calcitic and aragonitic biominerals.9−16 Biogenic CaCO3 crystals are comprised of nanograins that are arranged in the same crystallographic direction. Several biominerals were suggested to be comprised of continuous crystalline bodies, not isolated units such as those in a mesocrystal.17,18 In any case, the biogenic CaCO3 crystals possess mesoscopic particular textures. Characterization of the mesostructures of the biogenic crystals is generally important for understanding the relationship between the specific architecture and its fascinating properties. Very recently, 1 mm aragonitic shells of planktonic pteropods, Cavolinia uncinata,19 Clio pyramidata,20 and Cuvierina columnella and urceolaris21 have been reported to have a unique helical microstructure consisting of interlocking nanofibers. The clade of pteropods, which are small marine planktonic gastropods, has thin, transparent aragonitic shells. A global fiber-like crystallographic texture was observed with local in-plane rotations. The interlocking fibrous building blocks © XXXX American Chemical Society

were further reported to retard crack propagation at the nanometer scale. The c-axis of the tetragonal crystal in the entire helical architecture is perpendicular to the surface of the shell, while the a- and b-axes were suggested to vary in each layer. However, detailed characterization of the crystallographic structure has been still controvertible to clarify the helical architectures of pteropod species. Here, we revealed small-angle rotation of crystalline units in the helical structure and a twisted stacking mode of the curving fibers in the architecture. Our findings are useful for understanding diverse elaborate architectures of biominerals. Helical morphologies have been observed occasionally on solid crystals, which are usually faceted with straight edges.22−33 The crystal growth of helical morphologies is a particularly interesting phenomenon of rigid crystals. The formation of helical crystals has been frequently regarded as the result of intrinsic features such as dislocation stress or the distortion of crystal structures.33,34 Some of the helical crystals produced in the gel matrix reportedly were comprised of single-crystalline blocks that rotated stepwise along the growth direction. The rotation was deduced to be ascribed to periodic changes in the growth direction under a diffusion-limited condition.35 Here, we found a similar helical structure consisting of a twisted assembly of building blocks in the biogenic product of a pteropod. In the present study, the crystallographic structure of the helical fibers in the shell of a pteropod Cavolinia globulosa (C. globulosa) was investigated to clarify the unique architecture of Received: September 25, 2016 Revised: November 24, 2016

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DOI: 10.1021/acs.cgd.6b01417 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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the biomineral. The aragonite helical fibers ∼100−300 nm wide are adequately assembled in a shell ∼50 μm thick. As reported in previous works,19−21 the c axis of the orthorhombic crystal in aragonitic helices is perpendicular to the surface of the shell. On the other hand, aragonitic fibers were found to consist of rodlike building blocks whose orthorhombic crystal has a- and baxes that are rotated in the helical architecture of the shell. Our findings are useful for understanding the unique biogenic architectures.

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EXPERIMENTAL SECTION

The pteropod shell of C. globulosa was used for the structural analysis of aragonitic helical structures. The samples were collected with a plankton net northwest off Okinawa Island, Japan in the station OT-63 (27°03.55′N, 127°00.25′E to 27°03.89′N, 127°00.24′E) in the cruise KT-08-33 of R/V Tansei-Maru, Atmosphere and Ocean Research Institute; their dry shells were stored in a dry plastic bottle. To remove organic substances, the pteropod shells were immersed in a 5-vol % sodium hypochlorite (NaClO, Kanto Chemical) aqueous solution for 2 days and then washed with purified water. For clarification of the microstructure, the samples were mildly etched in a 0.1% acetic acid (Junsei Chemical) aqueous solution for 4 h. The morphology of the shell’s surfaces and grown crystals was observed using FEI Sirion and JEOL JSM-7600F field-emission scanning microscopes (FESEMs). Crystallographic structures were evaluated using an FEI Tecnai F20 field-emission transmission electron microscope (FETEM) with selected area electron diffraction (SAED). The structures of the helical fibrous building blocks were revealed by TEM imaging of the cross-sectional samples prepared using focused ion beam milling (FIB). The samples for FESEM observations were coated with osmium. The crystalline phase and orientation were also studied by X-ray diffractometry (XRD) using a Bruker D8 Discover. Crystal growth on the biomineral substrates was performed using a supersaturated solution system to evaluate the crystalline orientation of helical aragonite crystals. The formation of CaCO3 was performed in a 10 mmol dm−3 calcium chloride (CaCl2) solution supersaturated by the introduction of carbon dioxide generated by the decomposition of ammonium hydrogen carbonate (NH4 HCO3 , Wako Pure Chemical) at 25 °C for 6 h. We used a mechanically fractured specimen of the C. globulosa shell as a base crystal. The substrate was attached on a glass slide and put in a 120 cm3 polystyrene vessel that contained 100 cm3 of an aqueous solution of CaCl2·2H2O (Junsei Chemical) as the exposed surface was downward. The vessel was then covered with aluminum foil with several pinholes and placed in a closed 600 cm3 polystyrene container with 1.0 g of NH4HCO3.

Figure 1. A photograph of a shell of a pteropod C. globulosa (a) and an XRD pattern (b) with the {002} pole figure (c) of the top part of the pteropod shell. We analyzed the crystallographic direction of the part of the shell indicated by the broken red circle in (a) with a collimator 500 μm in aperture. (ψ: polar angle; φ: azimuthal angle; ND: normal direction; TD: transverse direction: RD: drawing direction).

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RESULTS AND DISCUSSION Crystallographic Structure and Microscopic Morphology of the Shell. The top part of the pteropod shell was characterized as ∼50 μm in thickness as shown in Figure 1a. The crystallographic orientation of a platy piece taken from the shell (a micrograph in Figure 1c) was analyzed using the θ-2θ scanning mode of conventional XRD method (Figure 1b). An intense 002 signal of aragonite indicates the preferred orientation of the crystals in the shell. According to pole figure measurements (Figure 1c), the c-axis of aragonite building blocks is deduced to be aligned perpendicularly to the shell’s surface. This analysis result agrees with that reported in a previous work on other aragonite-based mollusk shells.19−21 Figure 2 shows plan-view and cross-sectional-view SEM images of fractured and mildly etched shells of C. globulosa. We observed the parallel assembly of curving fibers ∼200−300 nm in width on the surface after the removal of organic compounds in the plan-view images (Figure 2a,b). The curvature radius of the fibers was roughly estimated to be ca. 10 μm (Figure 2c).

Figure 2. Plan-view (a−d) and cross-sectional-view (e−j) SEM images of fractured (a−h) and mildly etched (i) shells of a pteropod C. globulosa. A schematic illustration of a helical fiber composing the hierarchical structure (j).

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Figure 3. TEM images (a−c) with SAED patterns of an FIB-cut platy sample (thickness: 100 nm) perpendicular to the shell’s surface of a pteropod C. globulosa. SAED patterns were obtained from yellow circles 1−5 in (a). High-resolution (HR) TEM images (b, c) were obtained from yellow squares b and c in (a). A schematic image of the twisted assembly of the building blocks (d). The actual number of stacking blocks was ∼50−60 for a 180-deg turn in the opposite direction.

From the fractured portion of the top surface (Figure 2d), the longitudinal direction of the curving fibers was gradually changed as their parallel assembly was stacked in a direction perpendicular to the top surface. According to the cross-sectional images (Figure 2e,f), the shell possesses a layered structure consisting of curving fibers. In the middle layer, rectangular cross sections of the fibers 100−200 nm wide and 200−300 nm high were exposed on the fractured surface (Figure 2g,h). The longitudinal direction of the fibers was rotating along the vertical axis of the shell. The appearance of these fractured surfaces revealed that the microstructure was comprised of densely packed curving fibrous building blocks, similar to those of other pteropod and heteropod shells.15−17 In previous works, the shell was deduced to be constructed of parallel assembly of helical fibers in which the helical axis is normal to the shell’s surface. The upper and lower convex layers ∼20 μm thick and the concaved middle layer ∼10 μm thick are explained by the assembly of fibrous units. After mild etching in a diluted acetic acid, we observed the helical architecture more clearly (Figure 2i). These results suggest that right-handed helical fibers whose radius is about 10 μm compose the specific architecture of the shell (Figure 2j). The longitudinal direction of the fibers gradually changed from the left to the front side by going up steps. FETEM Observations of FIB-Cut Samples. Figure 3 shows cross-sectional TEM images of an FIB-cut platy sample perpendicular to the shell’s surface. In the cross-sectional images, we observed a layered structure consisting of positively sloped fibers in the upper region and negatively sloped fibers in the bottom region (Figure 3a,b).

The upper and bottom regions exhibit an oblique angle with respect to the surface by 15−20°, which is consistent with the helical model, as shown in Figure 2. The middle part was comprised of rectangular cross sections of the fibers (Figure 3a,c). From the crystal lattices in the HRTEM images, the c-axis of aragonite is normal to the shell’s surface. These structures are in agreement with those evaluated using the XRD technique and the SEM images. Moreover, here, we obtained the spot patterns of SAED assignable to aragonite from a small part ∼1 μm diameter in the TEM images to clarify the crystallographic structures. The SAED patterns mean that a few adjacent fibers are arranged in the same crystallographic direction and share similar c-axis orientations along the surface normal. On the other hand, the a- and b-axes of the orthorhombic crystal in the fibers in the middle layer gradually deviate from those of the upper and lower layers, seen as a schematic illustration in Figure 3a,d. We observed a 180-degree turn in the opposite direction along the stacking of building blocks ca. 10 μm in height. Figure 4 shows TEM images with SAED spots of an FIB-cut platy sample that is parallel to the shell’s surface. Rod-like building blocks 1−2 μm in length were found in the curving fibers as a part of the helical architecture (Figure 4a). The longitudinal directions of the blocks deviated slightly and were classified into five groups. In the SAED pattern (Figure 4b) from the red circle in Figure 4a, five groups of SAED spots were recognized for the (221), (222), and (240) planes of the aragonite lattice. These signals indicate that the c axis of the aragonite fibers was perpendicular to the surface, although their a- and b-axes are varied as shown in Figure 4b. Consequently, the curving fibers are deduced to be comprised of several rodC



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arrays were produced along the fibrous structure after additional growth in the supersaturated solution for 6 h (Figure 5). The rods were exactly perpendicular to the basal

Figure 5. SEM images (a−c) of the surface of a shell of a pteropod C. globulosa after the additional growth of aragonite crystals. Schematic illustrations of a hexagonal rod grown on the curving fibers (d). Dashed pink and yellow lines indicate hexagonal facets of aragonite crystals. Blue lines indicate the longitudinal direction of the rod-like basal blocks.

fibers. Moreover, the facets of the hexagonal rods were aligned along the same orientation in a small region ∼2 μm in size. Thus, epitaxial growth occurred on the aragonite surface in the solution. In this case, the c direction of aragonite is vertical to the surface. Because the orientation of the hexagonal facets was slightly changed, the a and b directions of the orthorhombic crystal are suggested to be rotating along the fiber. The deviation of adjacent blocks is estimated to be ca. 10°. This value is almost consistent with that obtained from the SAED pattern of the plan-view TEM image (Figure 4b). Assuming that a pair of (010) and two pairs of {110} are exposed on the hexagonal facets, the b-axis intersects at a ca. 5° angle with the longitudinal direction of the rod-like basal blocks. These results support the presence of the stepwise rotation of the nanometric single-crystalline domains in the fibers. Helical Structure of the Shell. Willinger et al. reported detailed crystallographic textures of a pteropod Cuvierina.21 Crystallographically continuous helical fibers of aragonite were shown to be constructed by rotation of twined crystal units. The curving growth in the desired direction could be permitted through abrupt changes across twins. In the present study, we propose another structural model of helical architectures. Our results indicate that the spiral fibers are comprised of singlecrystalline units ∼1−2 μm in size. The c-axis of the orthorhombic crystal is perpendicular to the surface of the shell. However, the a- and b-axes of the building blocks are rotated intermittently along the fiber. Figure 6 shows a schematic illustration of the fiber consisting of several units. The shell has a pseudofiber-like texture with the aragonite c-axis oriented along the surface normal and with the presence of large in-plane rotations of the a- and b-axes. The c-axes of the aragonitic crystals are generally aligned along the surface normal direction. Both the a- and b-axes are parallel to the shell’s surface with large in-plane rotations. The crystallographic rotation of the unit rods and the twisted stacking of the fibers along the helical axis form the specific hierarchical architecture of the pteropod shell. Previous works have already shown helical structures of pteropods similar to our results.19−21 However, their models of

Figure 4. TEM images (a, c) and an SAED pattern (b) of an FIB-cut platy sample (thickness: 100 nm) parallel to the shell’s surface of a pteropod C. globulosa. The longitudinal directions of the blocks deviated slightly and were classified into five groups (red, blue, yellow, green, and purple) in (a). Five groups of SAED spots in (b) were recognized for the (221), (222), and (240) planes of the aragonite lattice. Panel (a′): schematic illustration of a curving fiver consisting of rod-like blocks. Panel (a″): an enlarge image of rod-like blocks in (a).

like building blocks whose a- and b-axes of the orthorhombic crystal are intermittently deviated. We observed the lattice images of the rod-like building blocks in HRTEM of the FIB-cut sample (Figure 4c). The lattice images of the fiber in HRTEM images indicate the deviation of the crystallographic direction. From the lattice images, the b direction was estimated to be deviated from the long axis of the blocks. The intersecting angle of the b-axis with respect to the long axis of the rods is ca. 15°. The crystallographic orientation was changed in a stepwise fashion along the fibers. Additional Growth on the Shell. We performed additional growth of aragonite crystal on the surface of the shell in a supersaturated solution containing Ca2+ ion by introducing carbon dioxide. After the organic species were removed by immersion in a NaClO solution, the fibrous structure was exposed on the shell’s surface. Characteristic hexagonal rod D



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Figure 6. A schematic illustration of the building blocks in a helical fiber of the pteropod shell.

Notes

the helical structures are different from the architecture proposed in the present study. Zhang et al. presented a model involving a continuous helical fiber structure.19 Li et al. showed a noncontinuous model with crystallographic discontinuities.20 Willinger et al. proposed the stepwise rotation of building blocks due to crystal twinning in a continuous fiber.21 In the present study, our results indicate that a helical structure is formed through small-angle (∼15°) stepwise rotation of rodlike building blocks without specific twinning. Basically, the nanostructures of different species are probably different because species of the samples are not the same. Detailed reports on a wide variety of helical structures of several kinds of pteropods are potentially important to clarify the diversity of biominerals. Moreover, we revealed a twisted stacking mode of the curving fibers as shown in Figures 3 and 6. Although the helical fibers have been shown in previous works, the stacking mode of them has not been discussed sufficiently. Clarification of the assembly mode in the helical architectures is important to understand the elaborate ultrastructures of biominerals.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by Grant-in-Aid for Challenging Exploratory Research (15K14129) and for Scientific Research (A) (16H02398) from Japan Society for the Promotion of Science.

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CONCLUSION We investigated the crystallographic structure of the shells of pteropods C. globulosa that consist of unique aragonitic helical fibers ∼100−300 nm wide. The helical fibers are comprised of single-crystalline rod-like building blocks ∼1 μm long. The caxis of the aragonitic building blocks is perpendicular to the surface of the shell. On the other hand, whenever their a and b directions are rotated along the helical axis in a stepwise fashion, the stepwise rotation of their a and b directions in the c plane of aragonitic crystalline nanometric building blocks produces the helical fibers. The crystallographic rotation of the unit rods and the twisted stacking of the fibers along the helical axis form the specific hierarchical architecture of the pteropod shell.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +81-45-566-1556. Fax: +81-45-566-1551. E-mail: [email protected]. ORCID

Hiroaki Imai: 0000-0001-6332-9514 E



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Stepwise Rotation of Nanometric Building Blocks in the Aragonite

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