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

Abstract

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.

Quantitative immunocytochemistry at the ultrastructural level: a stereology-based approach to molecular nanomorphomics

“…Reconstruction in 3D by physical slicing can be avoided by electron tomography (Vanhecke et al 2007) and STEM tomography (Baudoin et al 2013). The techniques can be combined with pre-embedding immunolabelling.…”

Morphomics: An integral part of systems biology of the human placenta

“…More recent developments include focussed ion beam/SEM (FIB/SEM) tomography [93e96]. Scanning and transmission EM (STEM) tomography [97,98] can be used also to obtain a tilt series of images through an entire cell.…”

Combined Tilt- and Focal-Series Tomography for HAADF-STEM

“…The method was evaluated on a whole-mount macrophage cell with gold nanoparticles coated with native low-density lipoprotein (LDL) taken up into vesicles [15][16][17][18][19][20][21][22] (Figure 2). The cell had a total thickness of about 1 µm.…”