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
- Andreas S Schneider†1,
- Birgit Heiland1,
- Nicolas J Peter1,
- Christina Guth1,
- Eduard Arzt1, 2 and
- Ingrid M Weiss†1, 3Email author
© Schneider et al.; licensee BioMed Central Ltd. 2012
Received: 2 April 2012
Accepted: 3 September 2012
Published: 12 September 2012
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.
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.
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.
In the need for optimizing the materials performance, hierarchical construction principles became genetically manifest in the growth processes of skeleton forming animals  over millions of years of evolution [2–6]. The mollusk shell is one of the most ancient examples of such a hierarchical material [7–11]. Shells often consist of different crystalline polymorphs of calcium carbonate minerals such as calcite and aragonite, which are embedded in organic components [12–14]. Commonly known structures are nacre, and prismatic and crossed-lamellar shell types [15, 16]. Many aspects regarding the optical and mechanical properties of mollusk shells are well understood [12, 17]. The most studied shell type is nacre [18–21].
Since it is difficult to precisely classify the shell structures according to crystal sizes , it is more appropriate to characterize natural composites according to volume fractions of organic and inorganic phases. In the case of sea shells, the mineral part makes up more than 95 wt% of the natural composite [2, 23]. The relationships between structural and mechanical properties of nacre were identified on different length scales [11, 22, 24]. This shell type consists of mineral tablets of about 0.5–1 μm in thickness and about 5–10 μm in diameter. Its fracture toughness is relatively high (~7 MPa√m) as compared to the inorganic compound (~0.2 MPa√m), whereas the hardness is in a similar range, ~200-300 Hv, for both nacre and aragonite . In this respect, nacre differs from other biological minerals such as corals (~0.1 MPa√m, 5–10 Hv) and calcitic egg shells .
However, the growth mechanisms of the different shell structures remain speculative due to the lack of high-resolution light optical techniques, which are required to study shell formation in vivo, and the semi-transparency of adult shells. A seminal SEM study was performed by Wise . He reported the sheet-like structure of the organic matrix at the nacreous growth front of the gastropod Cittarium pica and discussed the formation of crystal stacks in the light of earlier suggestions for their columnar growth mode in Haliotis (and refs. therein). Wise pointed out that this is in clear contrast to the horizontal layer-by-layer growth modes for pelecypod (bivalve) nacre, e.g. of Pinctada radiata. The morphological study of Wise  could not answer the question regarding whether or not the organic matrix acts as a template for crystal nucleation by epitaxy. Weiner and Traub were using X-ray diffraction and demonstrated the perpendicular orientation of some organic molecules such as chitin fibers  with respect to the c-axes of the aragonite crystals for gastropods, bivalves and cephalopods. In the case of Nautilus the degree of misorientation of the organic matrix was in the same order of magnitude as for the a and b plane of the aragonite crystals . When these authors used electron diffraction in order to gradually reduce the sampling areas down to approximately the size range of a single biological cell, they identified locally ordered aragonite patterns but, on larger scales and depending on the biological species, a mosaic organization of the organic matrix with various degrees of order . This suggests a gradual, kinetic selection of faster growth rates and a small probability of nucleating a new orientation. Recent X-PEEM synchrotron experiments demonstrated a high degree of order within stacks of abalone nacre vs. a considerable degree of disorder towards the neighbouring stacks . The stacks of crystalline aragonite tablets in nacre are misoriented in their c-axis with respect to each other and orientational ordering of tablet stacking occurs not abruptly but gradually over a distance of 50 μm [1, 30]. Initially, the orientation of nucleation sites could be completely random, as shown e.g. in Figure 8 of . It can be assumed that the orientation of the first platelet existing next to the prismatic layer is determined by the roughness of the substrate (e.g. calcite prism, spherulitic aragonite, ACC precursor granules). It is well-known that nucleation sites are located within a gel-like organic matrix [10, 31] and, that certain aragonite-inducing and chitin-binding proteins may form an oriented 3-dimensional nucleation site [27, 32]. Although the distribution and function of such nucleation sites have not yet been identified on the molecular level and many aspects are still under debate , a detailed texture analysis of calcite shells using EBSD suggests that similar mechanisms are valid for molluscs in general and also for members of other phyla such as brachiopods [34, 35].
The present study was motivated by the challenge to improve quantitative light-optical investigations of mollusk shells, which would be of particular interest because they bear the potential for in vivo experiments. We performed quantitative polarization analysis using liquid crystal compensator technology (LC-PolScope AbrioTM Imaging System), introduced in 1995 by Oldenbourg [36, 37]. The quality and reproducibility of LC-PolScope data depends not only on sample thickness but also on structural features , which are in the range of the wavelength of light and below [39, 40]. Hence, the strategy used here involved sample preparation according to standard metallographical methods for producing thin sections of defined thickness and orientation . In parallel to LC-PolScope analysis, a detailed light-microscopic investigation of composite interfaces using materials polarization microscopy was performed. We also characterized the mechanical properties of a polished section of the nacre/prismatic (N/P) shell transition region using nanoindentation. Due to the high lateral resolution of nano-indentation, we were able to correlate the observed structural features to mechanical function.
Incident light microscopy
Shells were embedded in two different orientations: For orientation A, the polished section was horizontal to the coronal plane of the shell (Figure 1, left), which results in an inclination angle between the normal of each nacre platelet and the normal of the polished surface. This angle is related to the natural curvature of the Haliotis specimen and also depends on how much of the shell material was removed by the grinding and polishing procedure. For orientation B, the polished surface is a transverse section (Figure 1, right). In this case, the normal of each nacre platelet is perpendicular to the normal of the polished surface.
Besides the well-known regular nacre layers, we observed undefined colored reflexes with mosaic arrangement and dimensions covering several platelets in a stacked configuration in the incident light optical image, which cannot be corelated with surface topography. In the following, we refer to this additional contrast, which covers multiple platelets, as super-structure. (Figure 2c, Additional file 2: Figure S2).
where d is the distance between reflecting planes, θ is the diffraction angle, n is the refractive index and λ is the wavelength of radiation. In case of nacre, where the aragonite layers are separated by organic interfaces in the size range of 10–100 nm, d is approximately the thickness of one aragonite layer. The diffraction angle θ is defined as the angle between the incident light and the reflecting nacre planes and thus depends on the orientation of the nacre platelets with respect to the polished surface.
We do not expect that the orientation as well as the thickness of the nacre platelets vary on a length scale of the super-structure. Therefore, according to Equation (1) the local color contrast can only be assigned to local changes in the refractive index, which is related to the orientation-sensitive dielectric constant of the material. The color contrast does not appear in orientation B (Figure 2b), since the reflective planes (aragonite layers) are oriented parallel to the beam and thus the Bragg condition is not fulfilled.
At this stage of the experiments it remained unclear whether the mineral crystals or the organic interfaces contribute significantly to this effect.
In order to investigate the nature of the super-structure, we chose a comparative approach using different light and electron microscopic techniques for the specimen with orientation A.
Transmitted light microscopy
Further, the previously observed lamellar structure of the nacre (Figure 2c, Additional file 2: Figure S2) is not visible in Figure 3 (N, nacreous shell part). One would expect a strong crystalline birefringence signal for aragonite for this area. However, we could not observe any structure in the sense that well-defined boundaries could be identified. This suggests that the number of interfaces in the sampled volume is too large . In our case, we roughly estimate that the investigated specimen consists of ~ 60 nacre platelets interspaced by organic sheets across the transmitted section. Another explanation would be that the signals originating from crystallographically misaligned aragonite platelets are superimposed, hence causing the blurred polarization.
LC-PolScope birefringence analysis
The orientation image (Figure 4b) indicates that the prisms cover a large range of crystallographic orientations and that adjacent prisms in a single colony have more or less similar crystallographic orientations. For differently oriented colonies the birefringent retardance values of prisms and colonies were in different ranges (e.g. 109 nm in blue areas, 267 nm in red areas; Figure 4a, arrows). For example, the prism colony with an azimutal angle of 17° (color coded in orange and indicated also by the array overlay of azimutal angles in Figure 4b) has a retardance value of 109 nm, whereas the prism colony with an azimutal angle of 129° (color coded in purple) revealed a retardance of 267 nm. In contrast to synthetic calcite , further comparison of Figures 4a and b shows that areas of homogeneous orientation are not necessarily related to homogeneous birefringent retardance values of the prisms. This could be due to the fact that prisms are not pure single crystals of calcite but consist of complex arrangements of organic and inorganic phases [43, 44]. The interface between adjacent colonies and also adjacent prisms within the colonies appeared with non-uniform colors in both, retardance and orientation mode. This can be attributed to the organic layers surrounding the prisms . Finally, the same colonies which did not change their color after 90° rotation in the conventional polarized light microscopy analysis exhibit low birefringent retardance (dark areas, Figure 4a, dotted circle) and could therefore not be identified with respect to orientation. These areas were identified by Raman imaging spectroscopy to consist of calcite (Additional file 3: Figure S3).
Although the thin section is too thick in order to quantify the birefringence in the bulk of the nacre layer, the tapering overlap region with the calcite layer can be used to obtain information regarding the orientation of individual platelets. On top of the prisms with zero retardance (Figure 4a, dotted circle), individual nacre platelets or fragments of the tapered nacreous layer were identified. Raman imaging spectroscopy confirmed that these platelets consist of aragonite (Additional file 3: Figure S3). These nacre parts appear in the blue range of retardance values. At areas where the nacreous layer is thicker and composed of several platelets stacked on the prisms, the retardance reaches the yellow-red range. This shows that the thickness of the nacreous layer significantly contributes to the observed retardance contrast. In areas where the thin section consists only of the nacreous part, a gradient of retardance values from light blue to red was detected. This phenomenon cannot directly be correlated to the structure of the sea shell and may be caused by the multiple superpositions of differently oriented nacre platelets with internal structures (Additional file 4: Figure S4 shows isolated nacre platelets investigated by LC-PolScope microscopy). Furthermore, the large fraction of interfaces contributes to form birefringence which complicates data analysis.
Scanning electron microscopy
At higher magnification (Figure 5b,c) it can be seen that the domain boundaries are related to the boundaries of individual nacre platelets. From the literature it is known that the c-axis orientation of the aragonite platelets is not necessarily perpendicular with respect to the plane of each individual layer, but varies [1, 33, 45]. Accordingly, the different grey levels of the domains might be explained by an electron channeling contrast caused by the differently oriented areas. It is well known that backscattered electrons (BE) provide information about crystallographic orientation via channeling . Secondary electron (SE) images also contain this channeling contrast, though mostly superimposed by a stronger topography contrast. The LFD detector used for imaging in low vacuum collects a broader spectrum of electrons as compared to the conventional Everhard-Thorneley detector. Therefore, both SE and BE contribute to the contrasting of the image, which might explain the orientation sensitiviy of the SEM experiments with uncoated specimens. Tilting enhances the contrast as it increases the yield of backscattered electrons, reduces multiple scattering and energy loss processes and enhances channeling .
The fact that the super-structure was observed by two different contrasting methods, i.e. incident light microscopy and scanning electron microscopy, indicates that surface topography effects can be ruled out. The super-structure must rather be attributed to the biological growth mode .
In order to investigate the implication of the super-structure on the mechanical properties of the shell, nanoindentation experiments were performed on a polished section in orientation B. This orientation was chosen as in this case the nacre platelets are parallel to the indentation axis, which makes data analysis more straight forward. The indents were performed with a maximum load of 10 mN and a distance between adjacent indents of 20 μm. Furthermore, the indents were positioned in two parallel rows such that they cover a certain part of the calcite and the nacre layer.
Another observation in Figure 6 is the systematic variation in hardness and Young’s modulus for calcite and aragonite. As the distance between adjacent indents is similar to the characteristic size of the previously observed super-structure, this variation could reflect the different properties of individual domains of the super-structure. This is demonstrated in Figure 6c, which shows an SEM image of residual indents in the polished section. The image was obtained under a defined tilt angle of 20° in order to visualize the super-structure in the SEM even for the sample in orientation B. Note that indents were randomly distributed over areas (platelet stacks) with different grey levels.
The biological control mechanisms are well understood on the level of a single nacre platelet . However, mechanisms explaining the overall structure of shells are still not clear. With respect to the super-structure observed here, we suggest the following scenario: The formation of the very first nacre layer starts at the irregular growth front with the deposition of nucleation seeds , which are most likely specific proteins secreted by the tissue. Hence, the nacre platelets originating from these seeds will be randomly oriented with respect to their aragonite c-axis. Since the growth kinetics is directly related to the crystallographic orientation of the growing crystal, it is more likely that platelets with a prefered orientation grow on the expense of less well oriented platelets and thus occupy more space in a given time. Once another layer is deposited and new seed crystals start to grow, those growing on top of the "well oriented" platelets, which occupy already a much larger space than the less well oriented ones, have a higher probability to grow faster and with the prefered orientation. Although mineral bridges could contribute to transfer crystal orientation on the level of adjacent nacre platelets within one stack of platelets, they can not account for the super-structure, which is composed of differently oriented platelet stacks. On the level of competing stacks of platelets, the orientation, concentration and arrangement of nucleation seeds would be predetermined by the organic matrix.
The same may apply for prisms, which finally appear as prism colonies with aligned orientations of adjacent crystals.
In order to see whether the super-structures affect the mechanical properties, which would be an evolutionary advantage [47, 48], indentation experiments were performed on the size scale of the super-structure.
Overall, hardness and Young's modulus values found for Haliotis pulcherina nacre are very similar to those recently reported [49–52]. Using microindentation, for example Fleischli et al.  obtained a hardness of 3.9 GPa for Haliotis rufescencs. The Young's modulus in their study was only measured by nanoindentation. At low penetration depths, an average value of 72 GPa was detected. However, it was realized that with increasing penetration depth, the Young's modulus droped significantly. For example, at a penetration depth of 160 nm they found a modulus of 65 GPa, which is very similar to the results of the current study. The strong influence of the penetration depth in  was explained by the indentation size effect and an increasing contribution of the soft organic matrix with increasing penetration depth. Note that for geological aragonite without any polymer inclusions the nanohardness is in a range of 6.2 ± 0.3 GPa and Young's modulus is in the order of 102.8 ± 2.4 GPa .
The influence of the organic matrix may also contribute to the different mechanical properties of the calcite and aragonite layer. Both layers consist of mineralized calcium carbonate and a small fraction of biopolymer. However, in the regular brick and mortar structure of the nacre, each calcium carbonate platelet is surrounded by a soft matrix on a length scale, which is sensed by nanoindentation. Therefore, in nacre the organic phase has to carry the same load as the hard particles, which causes the lower hardness and Young's values compared to calcite. The influence of the organic layer can be estimated by simple composite models  or, more realistically, by the "shear lag model" .
The gradual change of the mechanical properties in the nacre layer is likely related to changes of structural features along the cross-section of the sample. In particular, Pokroy et al.  have shown that the thickness of the nacre platelets increases from the interface calcite/aragonite to the inner surface of the shell. Similar changes in structural features were observed by Nudelman et al.  at the growth front of nacre. Therefore, it can be assumed that the contribution of the softer organic matrix to the mechanical properties decreases as the positions of the indents approach the inner surface. This explains the higher hardness and Young’s modulus values for indents performed closer to the inner surface of the shell.
The systematic variation in hardness and Young’s modulus for calcite and aragonite, might partially be explained by topography effects, or locally different contributions of the soft organic matrix. However, also the crystallographic orientation of the indented structural features might contribute to this effect. Our experiments confirm the original observations made by Schmidt , who showed that individual prisms in the calcite layer have different crystallographic orientations. Therefore, the scatter in the Young’s modulus can be explained by the fact, that indents were performed in prisms with a soft or hard crystallographic direction [57, 58].
For the nacre layer, where the size of the structural features is smaller than the indented area, the influence of the crystallographic orientation is less obvious. Normally it would be expected that the influence of the crystallographic orientation of the platelets is averaged out, if several platelets are indented at the same time. However, the super-structure observed in this work and the work by Gilbert et al.  indicate that groups of adjacent platelets have an equal crystallographic orientation. Thus the scatter in the indentation data likely reflects the different orientations of these tablet stacks and is an example of where the observed super-structure influences the mechanical properties of sea shell.
All methods applied in this study enable a fast and easy screening of different types of mollusc shells. This is important to identify different growth modes as a function of cell physiology and biochemistry. Here we successfully demonstrated using combined methods that both, nacre and prismatic shell parts consists of characteristic super-structures at the nacre-prismatic interface. We also demonstrate that the super-structures correlate with distinct mechanical properties. These results allow us to draw conclusions regarding the limits of biological control.
Extensive studies targeted the crystallographic orientation of aragonite platelets in the nacreous layer and calcite prisms in the prismatic layer with respect to the overall shape and function of mollusc shells [12, 17]. However, hierarchical super-structures have to the best of the authors knowledge not yet reported to be obtained with conventional light optical and SEM techniques, only with much more sophisticated methods such as synchrotron X-PEEM and TEM. Here, we identified an additional contrast in the incident light microscopy and interpret this as additional nacre and prismatic super-structures. Both super-structures encompass several subunits of the structure such as nacre platelets and calcite prisms. Our observations have major implications for the growth mode, the limits of biological control, and the potential consequences for shell mechanics in various species and developmental stages. This bears an enormous potential for in vivo studies using conventional optical techniques, which might be useful to shed new light on the growth modes of nacre, the prismatic interface and other shell types in real time.
Haliotis pulcherina shells of 10–20 mm diameter were obtained commercially (U.S. Shell Inc., Los Fresnos, Texas, U.S.A.). We followed established procedures for sample processing [59–61]. In brief, shell pieces were mounted in two different orientations at room temperature in epoxy resin (Epofix, Struers, Willich, Germany) using a vacuum impregnation device (Cast N’Vac-1000, Buehler, Düsseldorf, Germany).
Flat cross-sections were obtained by SiC paper grinding at grain sizes of 46, 26, 15, and 8 μm (Buehler, Düsseldorf, Germany), subsequent polishing with “Dur” cloths and abrasives of 6 μm and 3 μm, and “Mol” cloths and abrasives of 1 μm (Struers, Willich, Germany), respectively. Specimens were endpolished using “NAP” cloths and OP-S suspension (Struers), rinsed extensively with water and ethanol, and air dried. The quality of the cross-sections and the cleanliness of the surfaces were confirmed in reflected bright field mode.
Thin sections were obtained by mounting the polished flat cross-section onto a glass slide using the epoxy resin (Epofix, Struers, Willich, Germany) and abrasing the epoxy resin from the top towards the shell to a final specimen thickness of 60 μm. The final thin section was again endpolished.
Incident light microscopy
Reflected bright field mode microscopy was performed using an inverted research metallurgical microscope PMG3 (Olympus, Hamburg, Germany) equipped with NeoSPlan 10 NIC 0.25 ∞/- f = 180, NeoSPlan 20 NIC 0.4 ∞/0 f = 180 and NeoSPlan 50 NIC 0.7 ∞/0 f = 180 objectives. Images were recorded at 2080 × 1544 pixel resolution with the Olympus Digital Color Camera UC30 and "analySIS start" (Olympus Soft Imaging Solutions GmbH; http://www.olympus-sis.com; Münster, Germany).
This microscope with the same objectives, and with crossed polarizers and Nicol's prism was used for quality control of polished surfaces in DIC (differential interference contrast) mode.
Transmitted light microscopy
Transmitted light microscopy was performed using a wide-field polarization microscope Orthoplan-Pol (Leitz, Wetzlar, Germany) equipped with NPL FLUOTAR 16×/0.45 P and NPL FLUOTAR 25×/0.55 P objectives. Images were recorded with an external 7.5 MPixel Live View Digital SLR Camera (E-330 ZUIKO DIGITAL 14–45 mm 1:3.5–5.6 ; Olympus Imaging Corp., http://www.olympus-europa.com) with a C-mount adaptor. The camera settings for taking the electronic images were optimized according to the best match with manual adjustments for visual analysis.
LC-PolScope birefringence analysis
An LC-PolScope image processing system (CRI, LOT-Oriel, Darmstadt, Germany) was used for quantitative birefringence analysis at 546 nm using the standard Abrio™ filter. The LC-PolScope camera was mounted on a Zeiss inverted microscope (Zeiss Observer Z1, Göttingen, Germany) equipped with A-Plan 10×/0.25Ph1, LD Plan Neofluar 20×/0.4 Korr Ph2, LD Plan Neofluar 40×/0.6Korr Ph2, and Plan Achromat 63×/1.40 Oil DIC objectives. All images were obtained according to the manufacturers instructions. Background images were taken from a selected area of the transparent glas slide without the biological sample. The Abrio™ image processing software (CRI/LOT-Oriel; [62, 63]) was used for quantitative data evaluation per pixel with respect to both, the retardance mode and the orientation of the slow optical axis (azimuth) mode.
Raman imaging microscopy
The mineralogy of the specimens was investigated at room temperature in air using a confocal LabRAM ARAMIS Vis Raman Imaging Spectrometer (Horiba Jobin Yvon, Bensheim, Germany), equipped with 50×/0.75 WD = 0.37 mm air objective Olympus MPlan (Olympus, Germany), and a diode laser 785 nm, 80 mW (Pilot P500, Sacher Lasertechnik Group, Marburg, Germany). Spectra represent the average of 2 measurements with an exposure time of 4 sec.
Scanning electron microscopy (VP-SEM)
Specimens were investigated without any coating in low-vacuum mode at 100 Pa using a FEI Quanta VP-SEM (FEI, Eindhoven, Netherlands), equipped with a secondary electon detector, ETD Everhard-Thornley-Detector and a LFD Large-Field-Detector for low vacuum imaging. Images were obtained at 10 keV and 15 keV (working distance 7 and 10 mm) at room temperature.
Mechanical testing, nanoindentation
Nanoindentation experiments were performed on polished sections of shell specimens using a Hysitron Triboindenter TI 950 equipped with a standard transducer and a Berkovich tip. In total, two rows of 25 indents with peak loads of 10 mN were applied along the cross-section of the shell. The distance between two adjacent indents was 20 μm and tests were run in open-loop mode. For the extraction of hardness and Young’s modulus from the data, the unloading segment of the curves was analyzed by the Oliver and Pharr method .
Isolation of single nacre platelets
Abalone shells (Haliotis pulcherina) were rinsed with tap water and dried with clean tissue. The outer prismatic calcite shell layers were mechanically and chemically removed using 0.6 M HCl. The remaining acid was completely removed with pure water and dried in air. Nacreous shell parts were treated with sodium hypochlorite solution (12% active chlorine) for 24 hours and the platelet fraction was recovered from the turbid supernatant by 2 min. centrifugation at 3000 rpm (Rotanta 460 RS with swing out rotor, Hettich, Germany) at room temperature. Subsequent washing steps of the solid phase were performed 20× with pure water until any chlorine vapour was completely removed.
The authors thank Roland Bennewitz and Marcus Koch for constructive comments on the manuscript, and Caroline Bylda and Sarah Schmidt for technical assistance.
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