Schwarz Tim M, Dumont Maïtena, Garcia-Giner Victoria, Jung Chanwon, Porter Alexandra E, Gault Baptiste
Max-Planck-Institute for Sustainable Materials, Max-Planck-Str. 1, Düsseldorf 40237, Germany.
Max-Planck-Institute for Sustainable Materials, Max-Planck-Str. 1, Düsseldorf 40237, Germany; now at Groupe Physique des Matériaux, Université de Rouen, Saint Etienne du Rouvray, Normandie 76800, France.
Acta Biomater. 2025 May 15;198:319-333. doi: 10.1016/j.actbio.2025.03.051. Epub 2025 Mar 27.
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.
骨结构通常在层次上被组织成有机成分（胶原蛋白、蛋白质等）和无机成分（羟基磷灰石（HAP））。然而，生物矿化过程的许多基本机制，如HAP形成、微量元素的影响、矿物质与胶原蛋白的排列等，目前尚未完全清楚。部分原因在于，在纳米尺度上同时表征生物矿物质的三维（3D）结构和化学成分面临分析挑战，原则上这可以通过原子探针断层扫描（APT）实现。然而，骨的层次结构在样品产量和分析分辨率方面对APT分析构成了关键障碍，特别是对于微量元素而言，并且胶原蛋白中的有机成分在分析过程中似乎会系统性地丢失。在此，我们在用于制备样品的聚焦离子束（FIB）内对APT样品进行原位金属镀膜，并证明样品产量和化学灵敏度得到了极大提高，从而能够分析单个胶原纤维以及诸如镁和钠等微量元素。我们在有无镀膜的情况下，就分析分辨率性能探索了一系列测量参数，并确定了APT分析骨样品的最佳实践参数。为了解析骨样品的复杂质谱，我们获取了纯HAP和胶原蛋白的参考光谱以明确识别信号，从而使我们能够在近原子尺度上分析整个胶原纤维和界面。我们的结果为在近原子水平上理解骨结构的层次结构和化学异质性开辟了新的可能性，并证明了这种新方法在未来为生物矿化过程提供新的、未被探索的见解的潜力。
原子探针断层扫描（APT）总体上是一种用于分析骨骼、牙齿或生物矿物质的相对较新的技术。APT能够在近原子水平上三维地表征材料的微观结构，同时具有高元素灵敏度，低至百万分之一。与金属相比，将APT应用于研究生物矿化现象存在样品产量低和分析性能较差的问题。在此，我们通过对APT样品进行原位金属镀膜克服了这些限制。这可以开启未来的APT分析，以深入了解基本的生物矿化过程，例如胶原蛋白/羟基磷灰石相互作用、微量元素的影响，以及更全面地理解骨疾病或骨生物矿化。
