ISM 2022 (Microscopy)


Emanuel Avrahami 1 Lothar Houben 2 Lior Aram 1 Assaf Gal 1
1Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
2Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel

Microscopic organisms are responsible for constructing some of the most intricate, diverse and intriguing crystal structures observed in nature. Unlike inorganic crystals that grow in solution, according to defined thermodynamic paths with defined shapes, the growth environment created by organisms allows far-from-equilibrium shapes to emerge. It was believed that specialized and chiral interactions, between biomacromolecules and the inorganic building blocks, facilitate this ‘sculpting’ ability.

An exemplary for such complex biominerals are coccoliths - plates of calcium carbonate produced by unicellular algae. Coccoliths form intracellularly, inside a specialized vesicle into which calcium and carbonate ions are delivered. Each coccolith is made of individual, repeating, single-crystal subunits with alternating c-axis orientations. Two unit-types exist, which grow and interlock until the final, chiral, structure is complete. Presumably, organic growth modifiers regulate coccolith growth in a ‘fine-tuned’ and complicated manner. However, evidence for this mode of control are limited and primarily stem from works that used 2D images and thin slices to infer the crystallography of coccoliths, which can be misleading when considering the nature of an intricate 3D object. Understanding growth control of coccoliths has thus remained an open challenge, attracting the interest of scientists from various field – from chemists and biologists, to geologists and materials scientists.

In this work (Avrahami et al., Science 2022, in press), we studied the growth process of the large coccoliths of Calcidiscus leptoporus, by investigating their evolving structures in 3D, using high-resolution electron microscopy techniques. We first developed a novel method to extract growing intracellular coccoliths from the cells of C. leptoporus at a spectrum of morphological stages, and then proceeded to characterize their shapes in detail. These different growth stages have therefore served as snapshot-in-time of the dynamic process of coccolith development.

To understand the crystallographic nature of the crystals, we opted for a scanning transmission electron microscope (STEM) analysis, and collected tomograms of different growth stages, best reflecting the major morphological states of the coccoliths. Similar to other works of Materials Science, we decided to utilize the high-angle annular dark field (HAADF) detector for data collection, as it is well suited for crystalline samples. The penetration power and resolvability of HAADF-STEM imaging proved highly beneficial for studying these thick, crystalline samples. The acquired data has allowed, for the first time, to inspect the full volume of coccolith crystals, as well as the interfaces between them, in unprecedented resolution. Segmentations of the volumes permitted rendering of entire coccoliths and of individual crystals, allowing detailed examination of their morphologies in 3D. We additionally implemented tomography-diffraction collection, to corroborate the morphological information obtained from the 3D visualizations.

Detailed crystallographic characterization of individual crystals along their growth process, showed they develop with only a single set of crystallographic facets, belonging to the {104} rhombohedral habit of calcite. This mode of growth is inconsistent with the notion of specific and chiral interactions, as previously hypothesized, given that the symmetry relation between the habit’s facets renders them all chemically equivalent with no clear, inherent, reason to develop anisotropically. Further examination of individual facets and their development revealed that it is through differential growth rate with which the crystals grow anisotropically, and not through complicated materialization of various crystallographic facets.

We propose that within the confined environment of the vesicle, symmetry related facets grow in a differential manner due to ion availability being anisotropic. Hence, the results of the 3D investigation presented here demonstrate that the complex architecture of coccoliths is a consequence of the geometry of their basic units, and can be explained with straightforward principles. At the early stages of coccolith assembly, the rhombohedral habit’s different edges distinguish between the basic units’ orientations, and convey ultrastructural chirality. As the coccolith matures, different atomic step orientations lead to differential growth of symmetry-related facets, in response to an anisotropic environment.

Thus, combined data from different state-of-the-art electron microscopy techniques has unlocked a way to examine these complex structures, in a way essential for revealing their true shape. It has allowed us to uncover a vital mechanistic aspect, through which complex microscopic biogenic crystals are made. We hold that applying these mechanisms on coccoliths assembled with slight alterations of initial conditions, e.g., unit positioning, may yield dramatically different final morphologies. Consequently, it is possible that such principles will aid those interested in fabricating evermore-complex crystalline materials, by allowing them to reconsider the mechanistic intricacy that is required of the system in actuality.