Nanoindentation
To test the mechanical properties, nanoindentation experiments were performed on three additional radulae (for detailed protocol see Krings et al., 2022c, 2022d, 2023; Gorb & Krings, 2021). Radulae were arranged on glass object slides and surrounded by a small metallic ring. Afterwarde, each ring was filled with epoxy resin, which covered the radula completely. After polymerization, samples were polished with sandpapers until tooth sections were on display (see Supplementary Figure 1), and smoothened with aluminum oxide polishing powder suspension on a polishing machine. Samples were cleaned in an ultrasonic bath for five minutes. A nanoindenter SA2 (MTS Nano Instruments, Oak Ridge, Tennessee, USA) equipped with a Berkovich indenter tip and a dynamic contact module (DCM) head was employed. Hardness (H) and Young’s modulus (E) were determined from force-distance curves by applying the continuous stiffness mode. All tests were performed under normal room conditions (relative humidity 28–30%, temperature 22–24 °C) and each indent and corresponding curve were both manually controlled. After this, samples were smoothened and polished until the next target localities were on display.
Overall, the inner structure of each tooth was tested at five localities to receive data on mechanical property gradients within each tooth. E and H were determined at penetration depths of 500–1000 nm. For each site indented, we received ~60 values obtained at different indentation depths, which were averaged to receive one H and one E mean value per indent. 413 localities were overall tested:118 on the inner lateral, 59 on the outer lateral A, outer lateral B, outer lateral C, outer lateral D and outer lateral E respectively.