Whole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography

Baudoin, Jean-Pierre; Jerome, W. Gray; Kübel, Christian; de Jonge, Niels

doi:10.1371/journal.pone.0055022

Whole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography

Journal Article · Wed Jan 30 23:00:00 EST 2013 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0055022· OSTI ID:2471410

Baudoin, Jean-Pierre ^[1]; Jerome, W. Gray ^[1]; Kübel, Christian ^[2]; de Jonge, Niels ^[3]

Vanderbilt University School of Medicine, Nashville, TN (United States)
Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshaffen (Germany)
Vanderbilt University School of Medicine, Nashville, TN (United States); INM – Leibniz Institute for New Materials, Saarbrücken (Germany)

Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 2471410

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 1 Vol. 8; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

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Whole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography

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