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

Nature Protocols volume 5, pages 744–757 (2010)Cite this article

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Abstract

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 (∼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 ∼1 week.

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Figure 1: Cellular structures, possible mechanisms of nanoparticle (NP) uptake and some potential NP physicochemical uptake factors.
Figure 2: Issue of nanoparticle (NP) agglomeration and dispersion.
Figure 3: Overview of sample preparation process (a–h).
Figure 4
Figure 5: Sample and equipment used for thin sectioning of resin-embedded cells (a–g).
Figure 6: Overview of the uptake of carbon nanomaterials into neuroblastoma (N2A) cells after 24 h at 100 μg ml−1.

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Acknowledgements

A.M.S. received funding from the National Research Council (NRC) Fellowship program funded by the Joint Science and Technology Office for Chemical and Biological Defense (JSTO-CBD), a program administered by the Defense Threat Reduction Agency (DTRA).

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Authors and Affiliations

  1. AFRL/711 HPW/RHPB, Wright-Patterson Air Force Base, Dayton, Ohio, USA

    Amanda M Schrand, John J Schlager & Saber M Hussain

  2. Department of Chemical Engineering, Case Western Reserve University, Cleveland, Ohio, USA

    Liming Dai

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A.M.S. developed the protocol, carried out experiments, analyzed data and wrote the paper under the close supervision of S.M.H. and J.J.S. The carbon nanomaterials were provided by L.D. All the authors discussed the results and implications and commented on the manuscript.

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Correspondence to Saber M Hussain.

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Schrand, A., Schlager, J., Dai, L. et al. Preparation of cells for assessing ultrastructural localization of nanoparticles with transmission electron microscopy. Nat Protoc 5, 744–757 (2010). https://doi.org/10.1038/nprot.2010.2

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