IN SITU IMAGING OF MINERAL FORMATION BY CALCIFYING MICROALGAE
Coccolithophores, a group of unicellular marine microalgae, are of great ecological significance as they dominate several global elemental cycles. The hallmark of this group is the external calcite-bearing scales called coccoliths. During this biomineralization process, calcium carbonate (CaCO3) is deposited by the alga to form calcite crystals. The calcification process is intracellular and occurs in a specialized organelle called the coccolith vesicle. Inside this vesicle, calcium carbonate is deposited on an organic base plate in a highly regulated process that involves inorganic ions and soluble polysaccharides. When the coccolith is complete, it is exocytosed to form an extracellular shell of tens of interlocked coccoliths. Most of the knowledge of how coccoliths are formed is based on electron microscopy images of chemically fixed and resin embedded calcifying cells. These methods do not preserve the inorganic phases, leaving the mechanism of inorganic mineralization unclear.
In this work, we investigated coccolith formation in Pleurochrysis carterae, a model species for coccolith formation, using cryo-electron tomography (cryo-ET). This methodology enables imaging of vitrified samples in their native hydrated state, at sub nanometer resolution. P. carterae cells were cryo-fixed using plunge freezing, and thinned to a ~ 200 nm thin lamella by cryo Focused Ion Beam (cryoFIB) milling. Then, a 300kV Titan Krios TEM equipped with a Volta phase plate was used to acquire a series of 2D tilt-projections, which were reconstructed and segmented as 3D data sets. The results show the very first stages of crystal nucleation, a crucial stage for the understanding of coccolith formation. The data show dense mineral-loaded particles aggregating on the rim of the base plate, and crystal nuclei, only a few nanometers in size, that appear in the middle of this aggregate. These dense granular deposits develop to minute calcite crystals that are aligned around the rim of the base plate with equal gaps between them. By using this advanced cryo-ET method, the crystallographic habit and orientation of the growing crystals can be determined, as well as the nanoscale environment in which they grow. Such high resolution data will enable us to elucidate this highly controlled crystal nucleation and growth process, thus understanding some of the biological regulation over the intricate morphology of coccoliths.