New avenues for characterizing individual mineralized collagen fibrils with transmission electron microscopy

Bone serves as a remarkable example of nature’s architectured material with its unique blend of strength and toughness, all at a lightweight design. Given the hierarchical nature of these materials, it is essential to understand the governing mechanisms and organization of its constituents across length scales for bio-inspired structural design. Despite recent advances in transmission electron miscoscopy (TEM) that have allowed us to witness the fascinating arrangement of bone at micro-down to the nano-scale, we are still missing the details about the structural organization and mechanical properties of the main building blocks of bone – mineralized collagen fibrils (MCFs). Here, we propose a novel approach for extracting individual MCFs from nature’s model material via a dropcasting procedure. By isolating the MCFs onto TEM-compatible substrates, we visualized the arrangement of organic and mineral phases within the individual MCFs at the nanoscale. Using a 4D-STEM approach, the orientation of individual mineral crystals within the MCFs was examined. Furthermore, we conducted first-of-its-kind in situ tensile experiments, revealing exceptional tensile strains of at least 8%, demonstrating the intricate relationship between structural organization and the mechanical behavior of MCFs. The capabilities of TEM allow us to resolve MCF organization and composition down to the nanoscale level. This new knowledge of the ultrastructure of the bone-building blocks and the proposed sample extraction and in situ mechanical testing opens up new avenues for research into nature’s inspired material design.

💡 Research Summary

This paper introduces a comprehensive workflow for isolating, imaging, and mechanically testing individual mineralized collagen fibrils (MCFs), the fundamental building blocks of bone. The authors selected mineralized turkey leg tendon (MTLT) as a source material because its fibrils are highly aligned and can be split into long (>10 µm) fibers using mechanical splitting and ultrasonication. The resulting suspension is deposited onto TEM grids by drop‑casting, preserving the native hydrated state better than conventional dehydration‑embedding protocols. High‑angle annular dark‑field (HAADF) STEM and energy‑dispersive X‑ray (EDX) mapping reveal a clear periodic D‑band with an average spacing of 68.64 ± 0.16 nm and a wide range of calcium‑to‑phosphorus ratios (0.26–1.63). A negative correlation between Ca/P ratio and D‑band spacing suggests that higher mineral content slightly contracts the collagen lattice.

Four‑dimensional STEM (4D‑STEM) is employed to map crystal orientation at the nanoscale. By scanning a convergent electron probe and recording diffraction patterns at each pixel, the authors extract the (002) reflection of hydroxyapatite and generate flow‑line maps. In six of eight scans, the hydroxyapatite c‑axis aligns within 2 ± 12° of the fibril axis, while two scans show a broader distribution, indicating heterogeneity between intra‑fibrillar and extrafibrillar mineral.

The most novel aspect is the in‑situ tensile test performed inside a double‑aberration‑corrected FEI Titan microscope using a custom copper tensile stripe. Individual MCFs are stretched manually while dark‑field TEM images are recorded. Crack initiation occurs in the mineral‑rich gap zone and propagates through the collagen‑rich overlap zone. Tracking the D‑band during deformation shows a relaxation from 69.5 nm (pre‑strained) to 66.6 nm, corresponding to an overall tensile strain of at least 8.2 %. Post‑failure imaging reveals irregular fracture surfaces and evidence of crack deflection at the mineral‑collagen interface.

Overall, the study demonstrates that drop‑casting combined with multimodal TEM (HAADF, EDX, 4D‑STEM) and in‑situ mechanical testing can provide unprecedented insight into the nanoscale structure–property relationships of native MCFs. The methodology avoids the artifacts of traditional sample preparation, enables quantitative measurement of D‑band spacing, mineral chemistry, crystal orientation, and tensile behavior on single fibrils, and can be extended to less mineralized or non‑mineralized collagen systems. These findings bridge a critical gap between synthetic collagen‑hydroxyapatite models and the true architecture of bone, offering valuable guidance for bio‑inspired material design.