Not all that glitters is gold - Electron microscopy study on uptake of gold nanoparticles in Daphnia magna and related artefacts

Increasing use of engineered nanoparticles has led to extensive research into their potential hazards to the environment and human health. Cellular uptake from the gut is sparsely investigated and microscopy techniques applied for uptake studies can result in misinterpretations. Various microscopy techniques are used to investigate internalization of 10 nm gold nanoparticles in Daphnia magna gut lumen and gut epithelial cells upon 24h exposure and outline potential artefacts, i.e. high contract precipitates from sample preparation related to these techniques. Light sheet microscopy confirmed accumulation of gold nanoparticles in the gut lumen. Scanning transmission electron microscopy and elemental analysis revealed gold nanoparticles attached to the microvilli of gut cells. Interestingly, the peritrophic membrane appeared to act as a semipermeable barrier between the lumen and the gut epithelium, permitting only single particles through. Structures resembling nanoparticles were also observed inside gut cells. As elemental analysis could not verify these to be gold they were likely artifacts from the preparation, such as osmium and iron. Importantly, gold nanoparticles were in fact found inside holocrine cells with disrupted membranes. Thus, false positive observations of nanoparticle internalization may result from either preparation artefacts or by mistaking disrupted cells for intact. These findings emphasize the importance of cell integrity and combining elemental analysis with the localization of internalized nanoparticles using transmission electron microscopy.