Hierarchical super-structure identified by polarized light microscopy, electron microscopy and nanoindentation: Implications for the limits of biological control over the growth mode of abalone sea shells

Abstract

Background: Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 μm for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed.

Results: Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size.

Conclusions: In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM) [Gilbert et al., Journal of the American Chemical Society 2008, 130:17519-17527]. Polarized optical microscopy revealed unprecedented super-structures in the calcitic shell part. This bears, in principle, the potential for in vivo studies, which might be useful for investigating the growth modes of nacre and other shell types.

Figure 1

Sample preparation. Metallographic preparation of Haliotis pulcherina sea shell. Left, orientation A; right, orientation B .

Figure 2

Incident light microscopy of differently oriented shell cross-sections. Incident light microscopy. Top, orientation A; bottom, orientation B. Hierarchical structures of nacreous and prismatic shell parts are visualized best in orientation A and B, respectively. Inserts in the images on the left side are shown at higher magnification on the right. Note that in addition to the nacre lamellae there is another level of hierarchy in the range of ~20 μm, defined here as the super-structure, which is observed in orientation A at higher magnification. P, prismatic layer; N, nacre.

Figure 3

Transmitted light microscopy of Haliotis shell thin section. Polarized light microscopy in transmission of a 60 μm thin section in orientation A. The transition region nacre/prismatic is centred on the microscopy stage. The left and the right image were obtained at the same position by rotating the stage 90°. The structure of the prismatic layer (P) is clearly visible. The tapering nacre structure gradually changes as a function of distance from the prismatic layer. Note that neither the lamellar structure nor the super-structure are visible. P, prismatic layer; N, nacre.

Figure 4

LC-PolScope analysis of nacre/prismatic transition. LC-PolScope ("Abrio™-Imaging") microscopy in transmission of a 60 μm section in orientation A. The birefringent retardance values (left, 0–273 nm from dark blue to dark red) and the orientation of the slow axis vector (right, red 0° / 180°, light blue 90° / 270°) are calculated for each pixel and color coded in the images. The two arrows in the left image indicate two differently oriented calcite prisms. Note that the marked light blue prism with a retardance value of ~109 nm appears orange in the orientation image (right), which corresponds to an azimutal angle of 17°. The marked red prism with a retardance value of ~267 nm appears purple in the orientation image (right), which corresponds to an azimutal angle of 129°. This analysis shows that the prisms consist of different domains, which can be distinguished according to their homogeneous crystallographic orientation. Not all of them are homogeneous. The tapering nacre layer gradually changes as a function of distance from the prismatic layer. Due to the blurred polarization signal, no structure is visible. Arrows in the left image indicate pixels with medium (ligh blue, 109 nm/17°) and high (dark red, 267 nm/129°) birefringent retardance contrast. P, prismatic layer; N, nacre.

Figure 5

Scanning electron microscopy image of the nacre super-structure. Scanning electron microscopy of a 60 μm section in orientation A. Top, overview image showing the transition between prismatic and nacreous layers. Center, super-structure in the size range of 20 μm in the nacreous layer. Domains of homogeneous grey level are formed by stacks of adjacent nacre platelets. Bottom, boundary between two domains which form the super-structure.

Figure 6

Nanoindentation of Haliotis shell sectioned in orientation B. Nanoindentation experiments performed on a polished section in orientation B. Hardness (A) and Young's modulus (B) are shown as a function of indentation position. C, Scanning electron microscopy image of the cross section after the experiments. The positions of some indents with respect to the 20 μm super-structure are exposed. Indents were 20 μm apart from each other and do not exactly match with the periodicity of the super-structure. Note that the super-structure in this image was visualized at a tilt angle of 20°.

Figure 7

Scanning electron microscopy image of nacre super-structures. Scanning electron microscope image of a Mytilus galloprovincialis sea shell. Domains of homogeneous grey level are formed by stacks of adjacent nacre platelets.