Nanoscale Whole-Body Expansion Microscopy Revealed the Early Skeletal Developmental Malformation Induced by Silver Nanoparticles

The widely used silver nanoparticles (AgNPs) can cause damage to skeletal development in aquatic organisms, which has a significant impact on their survival and reproduction. We hypothesized that the biological toxicity of AgNPs itself may be the culprit for early skeletal developmental malformation. In this study, we achieved the first nanoscale visualization of the notochord and skeletal muscle anatomy affected by exposure to AgNPs through a novel whole-body expansion microscopy technique (whole-ExM). This technique allows the whole zebrafish to be expanded equally in three dimensions by ∼4.5 times, enabling nanoscale resolution imaging without complex and expensive equipment. Our results suggested that AgNPs caused curvature deformation of the notochord as well as reduced and loosely arranged skeletal muscle. In addition, we demonstrated that the main source of Ag+ was the dissolution of AgNPs in vivo. Compared with that of the released Ag+, the developmental toxicity caused by AgNPs themselves was non-negligible. This work achieved cellular-level visualization of living animals and overcame the limitations of previous macroscopic or microscopic consideration of the toxicity of AgNPs. The study provided a new perspective for assessing the toxicity of nanomaterials by using whole-ExM to achieve ultraclear nanoscale imaging of biological organs.