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

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.

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.

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.

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