Preparation of cells for assessing ultrastructural localization of nanoparticles with transmission electron...

We describe the use of transmission electron microscopy (TEM) for cellular ultrastructural examination of nanoparticle (NP)-exposed biomaterials. Preparation and imaging of electron-transparent thin cell sections with TEM provides excellent spatial resolution (approximately 1 nm), which is required to track these elusive materials. This protocol provides a step-by-step method for the mass-basis dosing of cultured cells with NPs, and the process of fixing, dehydrating, staining, resin embedding, ultramicrotome sectioning and subsequently visualizing NP uptake and translocation to specific intracellular locations with TEM. In order to avoid potential artifacts, some technical challenges are addressed. Based on our results, this procedure can be used to elucidate the intracellular fate of NPs, facilitating the development of biosensors and therapeutics, and provide a critical component for understanding NP toxicity. This protocol takes approximately 1 week.