Background Lugol's solution is well known for its unique contrasting properties to biological samples in in microcomputed tomography imaging. On the other hand, iron oxide nanoparticles (IONPs), which have much lower attenuation capabilities to X-ray radiation show decent cell penetration and accumulation properties, are increasingly being used as quantitative contrast agents in biology and medicine. In our research, they were used to stain 3D cell structures called spheroids. Aim In this

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... the micro computed tomography (µCT) technique was used to visualize and compare the uptake and accumulation of two contrast agents, Lugol's solution and iron (II, III ) oxid e nanoparticles (IONPs) in the in vitro human spheroid tumour model. Methods The metastatic human melanoma cell line WM266-4 was cultured, first under standard 2D conditions, and after reaching 90% confluence cells was seeded in a low adhesive plate, which allows spheroid formation. On the 7th day of growth, the spheroids were transferred to the tubes and stained with IONPs or Lugol's solution and subjected to µCT imaging. Results Our research allows visualization of the regions of absorption at the level of single cells, with relatively short incubation times - 24h - for Lugol's solution. IONPs proved to be useful only in high concentrations (1 mg/ml) and long incubation times (96h). Conclusions When comparing the reconstructed visualizations of the distribution of these stating agents, it is worth noting that Lugol's solution spreads evenly throughout the spheroids, whereas IONPs (regardless of their size 5 and 30 nm) accumulate only in the outer layer of the spheroid structure.