Direct Observation of Nacre Proteins in the Whole Calcite by Super resolution Microscopy Reveals Diverse Occlusion Patterns

Biominerals are directed by macromolecules, especially proteins, and incorporate organic occlusions contributing to their overall mechanical properties. However, their distribution patterns in biominerals at the microscale are unclear. Here, we labeled nacre proteins of the pearl oyster Pinctada fucata with cyanine 5 and then directly observed their distribution inside the whole synthetic calcite by using stochastic optical reconstruction microscopy (STORM). Compared to traditional microscopy, we found three forms of occlusions in the crystals, i.e., chain-like, islet-like, and haze-like clusters. In addition, in the whole calcite, a two-ring pattern occurred. Based on the images, we studied the crystal growth process in the presence of nacre proteins with an overgrowth experiment. This study provides a novel approach to directly observe the distribution of nacre proteins in the whole calcite, which has implications for understanding the structures and mechanisms of formation of biominerals as well as for the development of nacre-like materials.