Advancing atom probe tomography capabilities to understand bone microstructures at near-atomic scale

Bone structure is generally hierarchically organized into organic (collagen, proteins, ...), inorganic (hydroxyapatite (HAP)) components. However, many fundamental mechanisms of the biomineralization processes such as HAP formation, the influence of trace elements, the mineral-collagen arrangement, etc., are not clearly understood. This is partly due to the analytical challenge of simultaneously characterizing the three-dimensional (3D) structure and chemical composition of biominerals in general at the nanometer scale, which can, in principle be achieved by atom probe tomography (APT). Yet, the hierarchical structures of bone represent a critical hurdle for APT analysis in terms of sample yield and analytical resolution, particularly for trace elements, and organic components from the collagen appear to systematically get lost from the analysis. Here, we applied in-situ metallic coating of APT specimens within the focused ion beam (FIB) used for preparing specimens, and demonstrate that the sample yield and chemical sensitivity are tremendously improved, allowing the analysis of individual collagen fibrils and trace elements such as Mg and Na. We explored a range of measurement parameters with and without coating, in terms of analytical resolution performance and determined the best practice parameters for analyzing bone samples in APT. To decipher the complex mass spectra of the bone specimens, reference spectra from pure HAP and collagen were acquired to unambiguously identify the signals, allowing us to analyze entire collagen fibrils and interfaces at the near-atomic scale. Our results open new possibilities for understanding the hierarchical structure and chemical heterogeneity of bone structures at the near-atomic level and demonstrate the potential of this new method to provide new, unexplored insights into biomineralization processes in the future. STATEMENT OF SIGNIFICANCE: Atom probe tomography (APT) is a relatively new technique for the analysis of bones, teeth or biominerals in general. APT can characterize the microstructure of materials in 3D down to the near-atomic level, combined with a high elemental sensitivity, down to parts per million. APT application to study biomineralization phenomena is plagued by low sample yield and poorer analytical performance compared to metals. Here we have overcome these limitations by in-situ metal coating of APT specimens. This can unlock future APT analysis to gain insights into fundamental biomineralization processes, e.g. collagen/hydroxyapatite interaction, influence of trace elements and a better understanding of bone diseases or bone biomineralization in general.