Exploring contrast-enhancing staining agents for studying adipose tissue through contrast-enhanced computed tomography

Contrast-enhanced computed tomography (CECT) offers a non-destructive approach to studying adipose tissue in 3D. Several contrast-enhancing staining agents (CESAs) have been explored, whereof osmium tetroxide (OsO4) is the most popular nowadays. However, due to the toxicity and volatility of the conventional OsO4, alternative CESAs with similar staining properties were desired. Hf-WD 1:2 POM and Hexabrix have proven effective for structural analysis of adipocytes using CECT, but fail to provide chemical information. This study introduces isotonic Lugol’s iodine (IL) as an alternative CESA for adipose tissue analysis, comparing its staining potential with Hf-WD 1:2 POM and Hexabrix in murine caudal vertebrae (MCV) and bovine muscle tissue (BMT) strips. Single and sequential staining protocols were compared to assess the maximization of information extraction from each sample. The study investigated interactions, distribution, and reactivity of iodine species towards biomolecules using simplified model systems and assesses the potential of the CESA to provide chemical information. (Bio)chemical analyses on whole tissues revealed that differences in adipocyte grey values post-IL staining were associated with chemical distinctions between BMT and MCV. More specific, a difference in degree of unsaturation of fatty acids was identified as a likely contributor, though not the sole determinant of grey value differences. This research sheds light on the potential of IL as a CESA, offering both structural and chemical insights into adipose tissue composition.