Microscale analysis reveals nature of hydrophobic biopolymers in aragonite bivalve shells with crossed-lamellar architecture

[Display omitted] •Confocal microRaman and fluorescence microscopy simultaneously showed the nature of hydrophobic biopolymers within bivalve shells.•The methods used involved etching of interfacial biominerals to expose the organic phase.•The methods used for this study do not involve chemical fixation or epoxy resin that influence organic matrix.•Minor chitin with a collagen-like matrix is responsible for the mechanical properties of bivalve shells with crossed-lamellar layers. Bivalve shells with crossed-lamellar architecture consist of random orientation of anisotropic crystallographic directions that adds to the optimization of the mechanical properties of shell biominerals. The source of these anisotropic distortions has been attributed to the organic macromolecules in the inorganic unit. However, very little is known about the organic macromolecules in shells with crossed-lamellar architecture because it is believed that the organics in this shell ultrastructure can hardly be characterized owing to its low content, about 1 wt%. Here, the shells of two bivalve species (Callista kingii and Anadara trapezia) with crossed-lamellar architecture, have been studied by microRaman spectroscopy, FTIR, and histochemical analysis. For the first time, organic macromolecules and inorganic components were simultaneously identified. Using spectroscopic techniques (microRaman and FTIR) and a confocal scanning fluorescence microscopy, the results herein demonstrated minor chitin in the studied samples and document structural protein, collagen-like matrix as the major organic component. The SEM observations revealed a morphological biopolymer meshwork composed of a polygonal network. Based on those combined evidence, this study suggests that the interactions between polysaccharide-based biomolecules and collagen-like matrix are crucial for the mechanical properties of crossed-lamellar layers in bivalve shells. This study highlights the importance of using in-situ approaches for these types of biomaterials and provides another source of biomaterials.