Enhanced Visualization of Collagen Fibril and Mineral Ellipsoids in Bone Tissue Using FIB-SEM Nanotomography and Advanced Analysis Tools
Abstract
Bone architecture is founded on the arrangement of mineral and organic content. At the nanoscale, collagen fibrils and ellipsoidal-shaped mineral-rich regions create the building blocks of bone and indicate nanoscale bone processes [1–3]. 3D visualization of these features is challenging in mineralized tissue due to the requirements of high-resolution imaging across a large volume[4]. In this work, we used focused ion beam-scanning electron microscopy (FIB-SEM) nanotomography and image analysis to enhance the 3D visualization of collagen fibrils in the context of mineral ellipsoids in human tibial cortical bone tissue. FIB-SEM nanotomography acquisition alongside tools such as deep learning and fast Fourier transform (FFT) and inverse FFT analysis were optimized and implemented for clear visualization of the collagen fibril 67nm D-banding periodicity and mineral ellipsoids in orthogonal planes. FFT analysis highlights the ubiquitous presence of fibrils both in and around mineral ellipsoids and 3D visualization clearly shows fibrils co-aligned with the ellipsoids’ major long axis. 3D analysis also shows fibrils crossing over towards the center of the ellipsoids (Figure 1E- white arrow). Thereby, experiencing twisting at the fibril level. This study is the first to segment collagen fibrils in association with mineralized cortical bone tissue from FIB-SEM and the utilization of inverse FFT analysis to enhance the visualization of collagen fibrils in/around mineral ellipsoids. Thereby, elucidating the interwoven connection between these two distinctive hierarchical features of nanoscale bone architecture.