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Understanding biomineralization relies on imaging chemically heterogeneous organic–inorganic interfaces across a hierarchy of spatial scales. Further, organic minority phases are often responsible for emergent inorganic structures from the atomic arrangement of different polymorphs, to nano- and micrometer crystal dimensions, up to meter size mollusk shells. The desired simultaneous chemical and elemental imaging to identify sparse organic moieties across a large field-of-view with nanometer spatial resolution has not yet been achieved. Here, we combine nanoscale secondary ion mass spectroscopy (NanoSIMS) with spectroscopic IRs-SNOM imaging for simultaneous chemical, molecular, and elemental nanoimaging. At the example ofPinctada margaritiferamollusk shells we identify and resolve ~ 50 nm interlamellar protein sheets periodically arranged in regular ~ 600 nm intervals. The striations typically appear ~ 15 µm from the nacre-prism boundary at the interface between disordered neonacre to mature nacre. Using the polymorph distinctive IR-vibrational carbonate resonance, the nacre and prismatic regions are consistently identified as aragonite ($${\overline{\nu }}_{a}=860$$${\overline{\nu }}_a=860$cm⁻¹) and calcite ($${\overline{\nu }}_{c}=880$$${\overline{\nu }}_c=880$cm⁻¹), respectively. We observe previously unreported morphological features including aragonite subdomains encapsulated in extensions of the prism-covering organic membrane and regions of irregular nacre tablet formation coincident with dispersed organics. We also identify a ~ 200 nm region in the incipient nacre region with less well-defined crystal structure and integrated organics. These results show with the identification of the interlamellar protein layer how correlative nano-IR chemical and NanoSIMS elemental imaging can help distinguish different models proposed for shell growth in particular, and how organic function may relate to inorganic structure in other biomineralized systems in general.

 

Abstract Coral reef ecosystems support significant biological activities and harbor huge diversity, but they are facing a severe crisis driven by anthropogenic activities and climate change. An important behavioral trait of the coral holobiont is coral motion, which may play an essential role in feeding, competition, reproduction, and thus survival and fitness. Therefore, characterizing coral behavior through motion analysis will aid our understanding of basic biological and physical coral functions. However, tissue motion in the stony scleractinian corals that contribute most to coral reef construction are subtle and may be imperceptible to both the human eye and commonly used imaging techniques. Here we propose and apply a systematic approach to quantify and visualize subtle coral motion across a series of light and dark cycles in the scleractinian coral Montipora capricornis . We use digital image correlation and optical flow techniques to quantify and characterize minute coral motions under different light conditions. In addition, as a visualization tool, motion magnification algorithm magnifies coral motions in different frequencies, which explicitly displays the distinctive dynamic modes of coral movement. Specifically, our assessment of displacement, strain, optical flow, and mode shape quantify coral motion under different light conditions, and they all show that M. capricornis exhibits more active motions at night compared to day. Our approach provides an unprecedented insight into micro-scale coral movement and behavior through macro-scale digital imaging, thus offering a useful empirical toolset for the coral research community.