Journal
NATURE PROTOCOLS
Volume 5, Issue 4, Pages 744-757Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2010.2
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Funding
- Joint Science and Technology Office for Chemical and Biological Defense (JSTO-CBD)
- Defense Threat Reduction Agency (DTRA)
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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 (similar to 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 similar to 1 week.
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