Advanced soft tissue visualization in conjunction with bone structures using contrast-enhanced micro-CT

Purpose: Micro-computed tomography (CT) analysis of soft tissues alongside bone remains challenging due to significant differences in X-ray absorption, preventing spatial inspection of bone remodeling including the cellular intricacies of mineralized tissues in developmental biology and pathology. The goal was to develop a protocol for contrast-enhanced micro-CT imaging that effectively visualizes soft tissues and cells in conjunction with bone while minimizing bone attenuation by decalcification.

Approach: Murine femur samples were decalcified in ethylenediaminetetraacetic acid and treated with three different contrast agents: (i) iodine in ethanol, (ii) phosphotungstic acid in water, and (iii) Lugol's iodine. Micro-CT scans were performed in the laboratory setup SkyScan 1172 and at the synchrotron radiation for medical physics beamline in synchrotron radiation facility Elettra. Soft and hard tissue contrast-to-noise ratio (CNR) and contrast efficiency after decalcification were measured.

Results: In laboratory micro-CT, Lugol's iodine demonstrated a threefold higher CNR in the bone marrow, representing the soft tissue portion, compared with the bone. Contrast efficiencies, measured in synchrotron micro-CT, were consistent with these findings. Higher resolutions and the specificity of Lugol's iodine to cellular structures enabled detailed visualization of bone-forming cells in the epiphyseal plate.

Conclusions: The combination of decalcification and the utilization of the contrast agent Lugol's iodine facilitated an enhanced soft tissue visualization in conjunction with bone.