Bone has a complex hierarchical structure, which is essential for its performance. Bone is typically replete with cells called osteocytes that are embedded in the mineralized bone matrix in osteocyte lacunae, which are interconnected by canaliculi only a few hundred nanometer wide to form a vast cellular network. Our understanding of the osteocyte lacuno-canalicular network has been limited because of difficulties to image the cellular network within the opaque bone matrix in 3D. Synchrotron X-ray computed tomography is ideally suited to study the lacuno-canalicular network in bone because it combines the high penetration power of X rays with sub-micron resolution while retaining a fast acquisition time and thus high throughput. We discuss how synchrotron radiation-based tomography techniques have given insights into the osteocyte network in bone both in the form of regular tomography and, for higher resolution studies, in the form of nanotomography such as holotomography. These studies have provided quantitative measures of osteocyte lacunar properties and their relation to location within bones and bone challenges such as immobilization or lactation. Nanotomography revealed new features of the canalicular network that we term canalicular junctions, which are likely to play an important but hitherto hidden role in fluid flow dynamics within the bone cellular network. The examples illustrate how tomography provides information on complex biological materials like bone and we foresee that these capabilities will continue to improve with future/upgraded synchrotron X-ray sources.
Composite materials, both biominerals such as bone or shells but also increasingly synthetic materials, often have a complex hierarchical 3D structure ranging over several length scales. To fully understand the structure of such materials, a method for probing the nanoscale structure in 3D is needed. Materials such as bone are particularly challenging due to their complex composition and hierarchical structure. Recent developments in synchrotron x-ray focusing optics have paved the way for smaller x-ray beams with high brilliance. Herein, we present how we probe the 3D elemental distribution and crystallographic properties of human bone using combined fluorescence and diffraction tomography (F-CT and XRD-CT) with a 50 nm pencil X-ray beam. The 2.6×3.1 µm2 cross section sample was a FIB-cut rod of human iliac crest bone. We recorded 2D diffraction patterns and fluorescence spectra for each point in a 50 nm raster scan grid pattern from 92 projection angles covering 0-182°. This allowed us to reconstruct tomographically both x-ray diffraction patterns and the elemental composition in a ∼5×5×3 µm3 volume encompassing the sample. We show that tomographic reconstruction of x-ray diffraction and fluorescence information is possible at <140 nm spatial resolution estimated from features in reconstructed images. Thereby it is possible to probe crystalline structure and elemental composition in 3D at length scales an order of magnitude smaller than hitherto available. This allows studying a very broad range of materials from biominerals to energy materials in more detail than ever before.