Understanding the intricate organisation of biogenic materials across the length scales is essential for a large and diverse range of research areas and applications in biomedicine, environmental science, archaeology and forensics. Many biological materials possess an hierarchical structure, with characteristic patterns of assembly from the nanoscale to the macroscopic level giving the resulting structure superior mechanical properties to those of the individual components. In particular biomineralising systems such as bone, teeth corals or sea shells show such a fascinating multi-level organisation. Biominerals are composites of a soft organic and a hard crystalline phase with both components affecting the formation of each other and hence the structure and properties of the entire product. Recently, increasing evidence has emerged that the structure formation is often the result of self-similarity with characteristic patterns of assembly repeating across the length-scales [1, 2].

To identify such patterns it is important to obtain access to a wide range of length scales to determine a possible linking of the structural patterns. On the subnanometer level electron tomography in transmission electron microscopy (TEM) has shown to be extremely powerful to reveal the three-dimensional structure of biominerals such as cortical bone, nacre in abalone sea shells and egg shells [1, 3, 4]. To avoid Bragg diffraction related contrast and to obtain directly interpretable images scanning TEM (STEM) was found to be most useful for electron tomography. Furthermore, it is extremely important that preparation and imaging have a minimal effect on the studied samples to avoid artefacts. Therefore, ion beams used for sample thinning (e.g. in focused ion beam or precision ion polishing systems) need to be carefully controlled to avoid beam damage and sensible imaging conditions need to be applied to reduce electron irradiation damage. This presentation will discuss the identification of hierarchical patterns of organisation in bone and nacre using electron tomography, focused ion beam in conjunction with scanning electron microscopy (FIBSEM) as well as X-ray absorption and scattering and will provide a critical evaluation of the necessary sample preparation steps and data acquisition procedures.

[1] N. Reznikov et al., Science 360, eaao2189 (2018).

[2] T. Grünewald et al., Sci. Adv. 6, eaba4171 (2020).

[3] K. Gries et al., Acta Biomat. 5,  3038 (2009).

[4] D. Athanasiadou et al., Sci. Adv. 4, eaar3219 (2018).