Preservation artifacts in zebrafish X-ray fluorescence imaging

Preservation and embedding procedures can substantially influence elemental distributions measured by X-ray fluorescence imaging (XFI), particularly for mobile endogenous elements. While effects of formalin fixation have been widely studied, comparatively little is known regarding methacrylate embedding. Here, larval zebrafish (Danio rerio) exposed to methylmercury-l-cysteinate (MeHg-l-Cys) were preserved either by JB-4 methacrylate embedding or using cryosectioning/air-drying, called herein cryopreservation, then were sectioned and analyzed using quantitative XFI. Mercury distributions were largely unaffected by preservation method, whereas endogenous elements including potassium, calcium, copper, sulfur, selenium, and zinc showed varying degrees of depletion or redistribution following methacrylate embedding, with potassium showing particularly strong depletion. Despite these changes, methacrylate embedding provided improved anatomical preservation relative to cryopreservation. These results demonstrate that methacrylate embedding is suitable for studies focused primarily on mercury localization, while cryopreservation remains highly preferable when accurate preservation of endogenous elemental distributions is required.