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Micro-CT analysis is conventionally used to investigate mineralized tissues. However, the potential of phase-contrast and contrast-enhanced micro-CT extends to soft tissue inspection, though optimizing soft tissue contrast alongside bone remains challenging, preventing spatial inspection of bone remodeling including the cellular components. 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 (EDTA) and treated with three different contrast agents: i) iodine in ethanol, ii) phosphotungstic acid (PTA) in water and iii) Lugol’s iodine. Micro-CT scans were performed in the laboratory set-up SkyScan 1172 and at the SYRMEP beamline in ELETTRA. Soft- and hard-tissue contrast-to-noise ratio (CNR) and contrast efficiency after decalcification were measured. In laboratory micro-CT, iodine in ethanol and PTA provided a higher CNR for bone compared to bone marrow, while Lugol’s iodine demonstrated a three-times higher CNR in bone marrow, representing the soft tissue portion. Contrast efficiencies, measured as the percentage increase of gray value compared to its decalcified state prior to contrast enhancement, were consistent with these findings in synchrotron micro-CT. Higher resolutions and the specificity of Lugol’s iodine to cellular structures enabled detailed visualization of bone-forming cells in the epiphyseal plate. The optimized protocol for micro-CT imaging significantly enhances soft tissue visualization alongside bone, facilitating non-invasive anatomical and histological analysis. This approach shows high potential for exploring the cellular intricacies of mineralized tissues in developmental biology and pathology.
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