STEM tomography of a whole macrophage after sequential incubation with large (14 nm diameter), and small (5 nm) LDL gold nanoparticles.

<p>(A) 0° STEM view through the thickness of a whole-mount macrophage. The magnification was 57,000×. The pixel size was 1.35 nm. (B) Tomogram reconstructed from an 80° tilt series with 2° increments containing the 0° view in A. The vertical position of the LDL gold nanoparticles clusters is color-coded. (C) Quantification of the vertical positions of the LDL gold nanoparticles clusters within 0.9±0.1 µm thick perinuclear regions. The mean number of clusters per 100 nm-thick virtual cell region (color bars), and standard deviations (black bars) are represented.</p

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

<div><p>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.</p> </div

STEM tomography of nanoparticle clusters with LDL gold nanoparticles.

<p>(A) Selected region of a horizontal (xy) slice of a tomogram depicting a LDL gold nanoparticles cluster. The tilt series was recorded with a tilt range of 76° and 4° increments. The magnification was 115,000×. The pixel size was 0.67 nm. (B) Side view (xz) projection of the tomogram along the white dashed line in A. The arrow points towards the same nanoparticle depicted in A. (C) Side view (yz) projection of the tomogram along the black dashed line in A. (D) Intensity profile in xy direction of the nanoparticle depicted in (A). Intensity is plotted along the white dashed line in A. (E) Intensity profile in xz direction of the nanoparticle depicted in A. The intensity is plotted along the white dashed line in B.</p

Quantitative characterization of the three main types of clusters formed after sequential incubation with large, and small LDL gold nanoparticles.

<p>(A–C) Representative images of the three main categories of clusters. (D) Population of each cluster type in an average thickness of 0.73±0.29 µm. Scale bars, 100 nm.</p

Transmission electron microscopy (TEM) of negatively stained low-density lipoprotein conjugated to gold nanoparticles (LDL gold nanoparticles).

<p>(A) LDL gold nanoparticles of 14 nm diameter (of the gold nanoparticle). (B) A gold nanoparticle (white arrow) is surrounded by low-density lipoproteins (black arrow). (C) 5 nm-diameter nanoparticle.</p

Scanning TEM (STEM) images recorded of whole-mount macrophage cells.

<p>(A) Nucleus and surrounding cellular materials. Clusters of LDL gold nanoparticles are visible as bright spots. The magnification was 5,000×. (B) Nucleus and section of cell containing many gold nanoparticles. (C) Image from a tilt-series showing gold nanoparticles of different sizes, recorded as a magnification of 115,000×. The location of this image is shown as square with * in A.</p

Lipid Droplet Maturation in Adipocytes.xlsx

The files provide raw data derived from studies on the role of microsomal triglyceride transfer protein in lipid droplet biogenesis in adipocytes

Effect of high fat diet on weight gain in control and knockdown mice.

<p>Male mice from <i>aP2</i> crosses were fed the high-fat diet from weaning (4 weeks) until 28 weeks of age. Mice were weighed bi-weekly. Numbers in parentheses represent the number of mice in each group. Data, mean ± s.e.m.</p

Effects of MTP knockdown on lipid droplet size and distribution.

<p>(A) Lipid droplet size in white fat from control and knockdown mice was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181046#pone.0181046.g005" target="_blank">Fig 5</a>. The decrease in lipid droplet size (%) in MTP knockdown mice compared with control was calculated and plotted versus MTP knockdown (%). (B,C) Size distribution of lipid droplets in brown fat from control (B) and knockdown (C) mice. Raw data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181046#pone.0181046.g005" target="_blank">Fig 5</a> were used in this analysis.</p

Tissue weights from control and knockdown mice fed regular rodent diet or high fat diet^*.

<p>Tissue weights from control and knockdown mice fed regular rodent diet or high fat diet^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181046#t001fn001" target="_blank">*</a>}.</p