Visualizing the 3D Internal Structure of Calcite Single Crystals Grown in Agarose Hydrogels

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Science  27 Nov 2009:
Vol. 326, Issue 5957, pp. 1244-1247
DOI: 10.1126/science.1178583

Crystal Growing Kit

For single crystals to remain intact, there is a limit to the size and number of defects that can be included before the underlying lattice is destroyed. Biological crystals, however, are known to include large macromolecules. H. Li et al. (p. 1244; see the Perspective by Hollingsworth) used electron tomography to study the crystallization of calcium carbonate inside an agarose gel, observing that the crystals physically entrapped the agarose macromolecules. To accommodate the curvature induced by the polymer chains, both high- and low-energy facets formed at the fiber-crystal interfaces. Thus, physical interactions alone may be sufficient for the incorporation of macromolecules in biological crystals and it may be possible to grow unusually shaped single crystals.


Single crystals are usually faceted solids with homogeneous chemical compositions. Biogenic and synthetic calcite single crystals, however, have been found to incorporate macromolecules, spurring investigations of how large molecules are distributed within the crystals without substantially disrupting the crystalline lattice. Here, electron tomography reveals how random, three-dimensional networks of agarose nanofibers are incorporated into single crystals of synthetic calcite by allowing both high- and low-energy fiber/crystal interface facets to satisfy network curvatures. These results suggest that physical entrapment of polymer aggregates is a viable mechanism by which macromolecules can become incorporated inside inorganic single crystals. As such, this work has implications for understanding the structure and formation of biominerals as well as toward the development of new high–surface area, single-crystal composite materials.

Biominerals, such as sea urchin skeletal parts and the calcite prisms in mollusk shells, are examples of organic/inorganic single-crystal composites with improved mechanical properties as compared with pure crystals (17). Sea urchin spines and plates are high-magnesium calcite single crystals, which incorporate not only micrometer-sized cellular tissue networks but also smaller intracrystalline proteins (1, 4). Similarly, prisms from the calcitic layer of mollusk shells (such as Atrina rigida) are single crystals of calcite with an incorporated organic matrix composed of proteins and other biomacromolecules (6, 7). Several techniques have been used to investigate how these macromolecules are distributed inside the single crystals without substantially disrupting the crystalline lattice. On the basis of coherence-length measurements on biogenic calcite crystals, Berman et al. suggested that intracrystalline proteins were concentrated at the mosaic block boundaries (8), whereas transmission electron microscopy (TEM) observation of sea urchin teeth has indicated that organic material may be isolated in nanometer-sized cavities (5). Synchrotron powder diffraction studies of a variety of biogenic calcite crystals indicate that the intra-crystalline organics induce anisotropic lattice distortions, suggesting that individual proteins may be incorporated as impurities in the crystalline lattice (9). In addition to biogenic crystals, synthetic calcite crystals also incorporate polymers when grown in the presence of polymer networks, porous membranes, or colloidal particles (1015). Although it is evident that polymers are occluded in all of these crystals, the mode of distribution of the macromolecules within the crystals (such as individual molecules versus aggregates) and the structure of the polymer/crystal interface are still both poorly characterized and understood.

There are several challenges to the study of the internal structure of inorganic crystals with incorporated organic macromolecules at atomic resolution and in three dimensions by use of traditional EM and x-ray diffraction techniques. These difficulties arise from the small sizes of the incorporated organic molecules and aggregates, the radiation sensitivity (for example, electron damage) of the materials, and the lack of well-developed methods for preparing electron-transparent samples from micron-sized specimens suitable for high-resolution imaging. Aided by advances in focused ion beam (FIB) sample preparation technique, annular dark-field scanning TEM (ADF-STEM), and ADF electron tomography for three-dimensional (3D) imaging, we have visualized the internal structure of calcite crystals (CaCO3; trigonal system, space group REmbedded Imagec) grown in agarose (a linear polysaccharide consisting of alternating 1,3-linked β-d-galactopyranose and 1,4-linked 3,6-anhydro-α-l-galactopyranose) hydrogels.

Calcite, the most abundant biomineral, has interested scientists because of the exquisite control biological organisms exert over its macro- and microscopic structure and the corresponding advanced mechanical properties of these organic/inorganic composite materials (19). Biogenic minerals, including calcite, are often associated with a fibrous, hydrated, organic matrix. For these reasons, we chose to study the growth of calcite crystals in agarose hydrogels. Similar to biogenic calcite, these gel-grown crystals incorporate the agarose polymers, providing a model system with which to study the internal structure of organic/inorganic composites (10, 11).

Calcite crystals were grown by using the ammonium carbonate method in agarose hydrogels (1 w/v %) (11, 16). The gel-grown crystals are faceted with the characteristic rhombohedral morphology of calcite expressed by six {10Embedded Image4} faces (Fig. 1, A and B) (10, 11). For STEM investigations, electron-transparent sections of the crystals were prepared by using a dual-beam FIB (fig. S1). Because of the radiation sensitivity of the samples, the final surfaces of the sections were finished by means of a 2-keV low-dose ion-polishing step in order to remove the damaged layer left behind by previous high–kilo–electron volt fast milling procedures (16). The resulting ion-thinned area has a wedge shape, giving a usable working thickness ranging from ~20 nm to 1 μm.

Fig. 1

(A) A SEM image of a gel-grown calcite crystal. (B) A model of a calcite crystal expressed by six {10Embedded Image4} faces. (C) A LAADF-STEM image of a thin section cut from a gel-grown calcite crystal by means of FIB. (D) A LAADF-STEM lattice image viewed down the [20Embedded ImageEmbedded Image] zone axis of calcite. (Inset) A SAED pattern of the cut section. The examined area (diameter of 800 nm) contains both crystal and fibers.

After comparing ADF-STEM and TEM images, we selected ADF-STEM for high-resolution imaging and tomography for its more effective suppression of diffraction contrast and the better fulfillment of the projection requirement (the signal is a monotonic function of the projected mass thickness of the sample), which is an essential requirement for directly interpretable and qualitatively reliable tomographic reconstructions. We optimized imaging conditions before the calcite sample was loaded so as to reduce radiation damage (16). Tilt-series images (for tomographic reconstruction) were recorded in areas that were 200 to 500 nm thick, where the features of interest (the incorporated organic fibers) are abundantly sampled and the imaging quality is not substantially degraded by beam divergence and beam spreading (17).

Low-angle ADF-STEM (LAADF-STEM) images of a thin section of gel-grown calcite reveal an interconnected network of darker fibers (average diameter of 13 ± 5 nm) within a brighter matrix (Fig. 1C). These fibrous structures are assigned to be the agarose fibers on the basis of the difference in the elastic scattering cross-section of the two materials. The lower average atomic number (Z) of the organic polymer as compared with that of the calcite results in the observed difference in Z-contrast between the two materials.

In order to examine the 3D structure of the polymer network, tomographic reconstructions were generated from a tilt series of high-angle ADF-STEM (HAADF-STEM) images (16, 18). The result shows that the incorporated polymer aggregates form a 3D random network penetrating throughout the section of the calcite crystal (Fig. 2A, fig. S2, A and B, and movies S1 and S3). The preservation of the fiber network indicates that the agarose fibers are relatively rigid and that calcite grows around the fibers without substantially disrupting the fiber aggregates (19). Although the examined section was lifted out from near the surface of a gel-grown crystal (~10 μm penetration of ~60 μm total height), we believe for two reasons that the images are representative of the interior of the whole crystal. First, the network persists over our largest field of view (~4 μm by 4 μm) (fig. S3). Second, as we have reported previously, gentle etching of fractured crystals revealed that polymer fibers are distributed throughout the crystal (19).

Fig. 2

(A and B) Tomographic reconstructions of (A) an agarose network inside of a section of as-prepared crystal and (B) cavities inside of a section of heated crystal. The fiber/crystal and cavity/crystal interfaces were highlighted (made brighter) by using a 3D Sobel filter for enhanced contrast. A Sobel filter takes the local gradient of the image, removing any slowly varying background (16). The fiber networks are already visible in both the density rendered images (fig. S2) and the raw tilt series (movies S3 and S4). The view is reconstructed from a tilt series of HAADF-STEM images recorded from approximately –70° to 70° [–68° to 60° for (A) and –55° to 61° for (B)] at 2° intervals. Movies of the rotating views showing the 3D structures are provided in the supporting online material (movies S1 and S2). The dimensions of the bounding box of the 3D reconstructions are (A) 1453 nm by 975 nm by 220 nm and (B) 1405 nm by 1196 nm by 554 nm.

Despite the presence of the organic networks, the crystals maintain their single-crystal nature. Selected-area electron diffraction (SAED) of a large area (diameter of 800 nm), including fibers, gives a single set of diffraction spots (Fig. 1D, inset). In addition, high-resolution LAADF-STEM images directly show the regular 2D lattice image viewed down the [20Embedded ImageEmbedded Image] zone axis of calcite (Fig. 1D).

The incorporation of polymer fibers into single crystals of calcite introduces interfaces between the organic fibers and the inorganic crystals. Indexing the facets that form the internal interfaces shows that the interfaces are a mixture of {10Embedded Image4} low-energy facets and {01Embedded Image2} high-energy (homocharged) facets (Fig. 3 and fig. S6). In addition to these facets, there are other high-energy facets that could not be indexed. If the sample had been imaged at varied orientations (zones), very possibly we would observe additional high-energy facets forming the crystal/polymer interfaces.

Fig. 3

(A and B) LAADF-STEM images of a thin section cut from a gel-grown calcite crystal by means of FIB. In (B), the lattice fringes are continuous across the fiber because there is a considerable volume of crystal above and/or below the fiber. (Inset) Fast Fourier transform (FFT) of (B) [a magnified view of (B) showing lattice fringes is provided as fig. S4]. The vertical streaking in the FFT is due to scan noise (explained in detail in fig. S5). On the basis of the FFT, interfaces between the crystal and fibers are partially indexed. For clarity, faces in the {10Embedded Image4} family are indicated in yellow, whereas faces in the {01Embedded Image2} family are highlighted in white.

At the length scale of hundreds of nanometers (Figs. 1C and 2A), the interfaces are curved and thus are defined by the contours of the flexible polymer fibers. Because the fibers are interconnected as 3D random networks, at large length scales the interfaces are 3D random curved surfaces. At smaller length scales (tens of nanometers), however, the interfaces appear to be faceted and thus are defined by the faceted habit of the crystal (Fig. 3). A combination of high- and low-energy facets is observed surrounding the fibers. Facets in addition to the {10Embedded Image4} facets are required to satisfy the local curvature of the fibers and to minimize the interface area.

There are several possible mechanisms with which to explain the appearance of high-energy facets surrounding the agarose fibers. We will consider two general categories: physical and chemical mechanisms. Chemical (or epitaxial) mechanisms, usually involving molecular recognition between an organic additive and a specific face of calcite, are commonly invoked to explain the expression of non-{10Embedded Image4} surfaces in calcite crystals (2026). For example, {01Embedded Image2} faces are seldom expressed, except in the presence of homocharged (anionic) surfaces, which are believed to template the formation of a layer of calcium ions with the same structure as the {01Embedded Image2} faces (2729). It is possible that the agarose fibers may in a similar way stabilize or template the formation of the {01Embedded Image2} faces. The external morphology of the gel-grown calcite crystals (six {10Embedded Image4} faces), however, suggests that the uncharged polysaccharide, agarose, has a weak affinity for calcite (Fig. 1, A and B). Furthermore, even though both the external and internal surfaces are exposed to the agarose polymers, high-energy facets only appear at the internal surfaces.

As an alternative or supplement to a chemical mechanism, we propose that the coexistence of singular {10Embedded Image4} faces at the external surfaces and the nonsingular faces at the internal surfaces (Fig. 3) possibly originates from the different geometry (convex versus concave) of the growth fronts. According to the theory of crystal growth, the external morphology of crystals is defined by slow-growing (usually low-energy) facets, as depicted by kinetic Wulff diagrams. The external facets, by definition, are convex growth fronts. The picture changes for internal surfaces, which are concave. It is predicted that such concave growth fronts will instead be dominated by fast-growing (usually high-energy) faces (fig. S7) (30). The external surfaces of calcite crystals are convex; therefore, the low-energy (usually slow-growing) {10Embedded Image4} faces dominate the final morphology (Fig. 1, A and B, and fig. S7B). In contrast, the internal growth fronts are concave because the crystals must grow around the fibers to incorporate them, satisfying the curvature of the fibers (fig. S7A). Consequently, we would predict the appearance of multiple, high-energy facets at the internal interfaces, which is consistent with the observation of the high-energy (usually fast-growing) faces, including {01Embedded Image2} faces at the calcite/agarose interfaces (Fig. 3 and fig. S7, A and C). As described earlier, although {01Embedded Image2} faces are exemplified as high-energy (fast-growing) facets there are other high-energy facets present. Although this physical model is consistent with our observations, further work is required to determine the formation mechanism (or mechanisms) of the high-energy facets, including possible contributions from molecular recognition processes, which might favor one set of facets over another.

In order to investigate the stability of the internal porous structure, we removed the organic network by heating the crystals at 400°C under a flowing air atmosphere for one hour (16). The removal of agarose fibers at this temperature was confirmed through thermogravimetric analysis (fig. S8) (19). Detailed ADF-STEM observations show the differences between the internal structure of the crystals before and after heating (Fig. 4). After pyrolysis, cavities are observed instead of continuous fiber-like interfaces, as highlighted by the arrows in Fig. 4A. Tomographic reconstructions show in three dimensions that these cavities often are arranged in lines (Fig. 2B, fig. S2, C and D, and movies S2 and S4). This observation suggests that the original, continuous channels fragmented into discrete cavities after the polymer fibers were removed. This surface evolution could be due to reorganization of the internal surfaces to reduce surface area. ADF-STEM images of individual cavities reveal that the high-energy {01Embedded Image2} facets are still present at the internal surfaces after removal of the organic material (Fig. 4, B to D). SAED and LAADF-STEM lattice images show that even after pyrolysis, the crystals behave as single crystals (Fig. 4, E and F). Therefore, crystalline integrity and high-energy internal facets are both preserved after pyrolysis. The internal surfaces, however, become isolated and disconnected from the external surfaces and are no longer accessible.

Fig. 4

(A to D) ADF-STEM images of a thin section (prepared by means of FIB) cut from a gel-grown calcite crystal after heating. The (B) inset shows an FFT of (B), and the (D)inset shows an FFT of (D). On the basis of the FFTs, the interfaces between the crystal and cavities are partially indexed. For clarity, faces in the {10Embedded Image4} family are indicated in yellow, whereas faces in the {01Embedded Image2} family are highlighted in white. (A) and (B) were taken in LAADF imaging mode in order to give a better signal-to-noise ratio and higher contrast between the calcite and the cavities in thin regions of the section. (C) and (D) were taken in HAADF imaging mode in order to eliminate diffraction contrast, allowing the calcite/cavity interfaces to be imaged sharply in thicker regions of the sample. (E) A SAED pattern of the section. The examined area (diameter of 800 nm) contains both crystals and internal cavities. (F) A LAADF-STEM image showing the lattice of a heated calcite crystal viewed along the [20Embedded ImageEmbedded Image] zone axis.

In summary, we have identified the mode of distribution of agarose fibers inside calcite single crystals and observed the polymer/crystal interfaces at high resolution. In these synthetic single crystals, the incorporated macromolecular aggregates are distributed as a 3D network of nanofibers as opposed to isolated molecules or individual fibers. The internal, curved polymer/crystal interfaces are formed by a combination of high- and low-energy facets. These results support physical entrapment of polymer aggregates (19) as a possible mechanism by which macromolecules can become incorporated inside inorganic single-crystals. In contrast to other mechanisms, which invoke a specific chemical interaction between the organic component and the growing crystal (4, 15), physical entrapment can occur for a wide range of organic material. This work suggests an approach for modifying the internal structures of crystals and synthesizing single-crystal composites with large, potentially accessible, internal surface areas. Potential uses for the gel method include the preparation of materials that require both high crystallinity and high surface areas, such as photovoltaic materials (31).

Supporting Online Material

Materials and Methods

Figs. S1 to S8


Movies S1 to S4

  • * These authors contributed equally to this work.

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. L.A.E. acknowledges partial support from NSF (DMR 0845212), the J. D. Watson Investigator Program (New York State Office of Science, Technology, and Academic Research contract C050017), and the Cornell Center for Materials Research (CCMR), a Materials Research Science and Engineering Center of NSF (DMR 0520404). H.L.X. and D.M. acknowledge the Semiconductor Research Corporation and CCMR. Particular acknowledgement is made of the use of the EM and polymer characterization facilities of CCMR.
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