Multi-modal and multi-scale microscopy across light, electron, and X-ray microscopy can uncover new insights into diverse hierarchical biological materials. X-ray microscopy (µCT/XRM) reveals previously undiscovered internal microarchitectures, as well as generating 3D representations of complex surface structures. Light and electron microscopy provide microstructural, chemical, and crystallographic characterisation. Fusing these techniques together in correlative workflows can extend the fundamental basis of materials science - structure/property relationships - to structure/property/function studies of organisms.
Here, we demonstrate the correlative potential of numerous coupled systems at different length scales: X-ray microscopy (µCT), scanning electron microscopy (SEM), optical light microscopy (OM), focused ion beam microscopy (FIB), and nanoindentation to investigate the chemical composition, microstructural and nanomechanical properties, and crystallographic orientation of a number of biological materials and systems. Studies on mineralised materials include plate joints (ala) in the 'shell' of the barnacle Semibalanus balanoides, and the internalized shell of the cuttlefish Sepia officinalis. Ala are the mechanical interlocking of neighboring plates. µCT reveals complex interactions and relationships between neighboring plates that can only be identified via non-destructive 3D methods. Correlated OM, SEM, EDS, EBSD and nanomechanical mapping indicate specific chemical distributions, crystallographic orientations, and nanomechanical properties at the ala tips, which has implications for natural strengthening mechanisms and potential biologically-mediated precipitation of elements. FIB is used to isolate micro-scale areas of interest from larger macro-scale structures, linking overall properties to microstructural features. We also demonstrate the use of diffusible iodine-based contrast-enhanced (DICE) CT, to image non-mineralised biological structures such as muscles, bridging the hard/soft material interface, crucial for a holistic biomechanical understanding. From this work we have demonstrated how even small-scale and often overlooked organisms demonstrate naturally-occurring bio-engineering. The work demonstrates that correlative methods that span different platforms and techniques enable the extension of the fundamental basics of material science to broaden the application of bio-inspiration in human-made engineering applications.