Nanoscale Imaging of Mineral Crystals inside Biological Composite Materials Using X-Ray Diffraction Microscopy

We for the first time applied x-ray diffraction microscopy to the imaging of mineral crystals inside biological composite materials—intramuscular fish bone—at the nanometer scale resolution. We identified mineral crystals in collagen fibrils at different stages of mineralization. Based on the experimental results and biomineralization analyses, we suggested a dynamic model to account for the nucleation and growth of mineral crystals in the collagen matrix. The results obtained from this study not only further our understanding of the complex structure of bone, but also demonstrate that x-ray diffraction microscopy will become an important tool to study biological materials.

Figure 1

(a) Schematic diagram of the x-ray diffraction microscope. (b) Diffraction pattern of a highly mineralized bone particle. (c) Reconstructed image from (b). (d) Power spectral density of the diffraction pattern, indicating a resolution of 24 nm (i.e., a pixel size of 12 nm).

Figure 2

(a) Reconstructed image of unmineralized bone. (b) The zoom-in image indicates that the period of the small island dark spots along the fibril direction (indicated by the arrows) is $\sim 67\mathrm{nm}$. (c) Staggered arrangement of collagen molecules. Collagen molecules with $\sim 300\mathrm{nm}$ long and 1.5 nm in diameter are staggered to each other by D ($\sim 67\mathrm{nm}$), which includes an overlap region O ($\sim 27\mathrm{nm}$) and a hole region H ($\sim 40\mathrm{nm}$).

Figure 3

Reconstructed images of a low mineralized bone particle. (a) The image at 0° reveals the mineralized collagen fibrils are highly aligned. (b) Zoom-in view of the rectangular area (left) and lineout across the individual collagen fibrils (right). The period of dark spots (arrows) is $\sim 67\mathrm{nm}$. (c), (d) Reconstructed images of the same low mineralized bone particle at $-45°$ and $+45°$, respectively.

Figure 4

(a) Reconstructed image of a highly mineralized bone particle. (b) Zoom-in view of the white square region shows a band structure (indicated by the arrows) with a period of $\sim 67\mathrm{nm}$. (c) Reconstructed image of another highly mineralized fish bone particle. (d) Zoom-in view of the rectangular region and the lineout along the fibril direction indicate the high-density hole regions has a periodicity of $\sim 67\mathrm{nm}$.

Figure 5

Schematic illustration of the suggested dynamic mechanism for the nucleation, growth, and orientation of mineral crystals in the collagen matrix at different stages of mineralization.