Human bone mesoscale 3D structure revisited by plasma focused ion beam serial sectioning

Visualizing bone mineralization and collagen microfibril organization at intermediate scales between the nanometer and the 100s of microns range, the mesoscale, is still an important challenge. Similarly, visualizing cellular components which locally affect the tissue structure requires a precision of a few tens of nanometers at maximum while spanning several tens of micrometers. To address this issue, we employed a plasma focused ion beam (PFIB) equipped with a scanning electron microscope (SEM) to sequentially section nanometer-scale layers of demineralized and mineralized human femoral lamellar bone over volumes of approximately 46 × 40 × 9 μm, and 29 × 26 × 9 μm, respectively. This large scale view retained high enough resolution to visualize the collagen microfibrils while partly visualizing the lacuno-canalicular network (LCN) in three-dimensions (3D). We showed that serial sectioning can be performed on mineralized sections, and does not require demineralization. Moreover, this method revealed ellipsoidal mineral clusters, noted by others in high resolution studies, as a ubiquitous motif in lamellar bone over tens of microns, suggesting a heterogeneous and yet regular pattern of mineral deposition past the single collagen fibril level. These findings are strong evidence for the need to revisit bone mineralization over multi-length scales.