Following the white rabbit: multiscale 2D3D correlative imaging of bone structure

Bridging scales in bone research is challenging due to bone's heterogeneities and anisotropy at different scales. High-resolution imaging is often limited to volumes too small to reveal its complete hierarchical structure, a limitation that is further compounded by site-specific variability. Although bone multiscale structural organization has been extensively studied in humans, extrapolating these findings across species remains difficult. The study of bone pathologies or dynamic processes still relies on in vivo models, as these experiments cannot be performed on humans. In particular, rabbit bone represents a common model for bone studies but remains underexplored, especially at small scales. In this work, we investigate bone hierarchical structure and porosities in the rabbit across scales by combining 3D volumetric global (voxel size ∼ 15 μm) and local (voxel size ∼ 2 μm) micro-computed X-ray tomography with high-resolution 2D scanning electron microscopy (pixel size < 0.5 μm) and transmission electron microscopy (pixel size < 50 nm). 2D images were integrated into the spatial context of the 3D data through image registration, to highlight site-specific differences between endosteal, mid-cortex and periosteal regions. Osteocyte lacunae 2D morphology varied between primary osteons, secondary osteons and periosteal bone. These findings motivate further three-dimensional characterization of osteocyte lacunar morphology across osteon types and in larger cohorts, to clarify the role of osteocytes in bone remodeling. More broadly, our work highlights the potential of sequential multimodal workflows in giving a broader spatial context to sub-micron and nanoscale structural findings, reducing the "blindness" compromise that comes with high-resolution detail.