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Subject: quantitative analysis × WGL Institute: INM ×

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Now showing 1 - 3 of 3
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    Scanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying
    (San Francisco, California, US : PLOS, 2021) Schu, Moritz; Terriac, Emmanuel; Koch, Marcus; Paschke, Stephan; Lautenschläger, Franziska; Flormann, Daniel A.D.
    The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.
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    Enantiomer-selective magnetization of conglomerates for quantitative chiral separation
    (Berlin : Springer Nature, 2019) Ye, X.; Cui, J.; Li, B.; Li, N.; Wang, R.; Yan, Z.; Tan, J.; Zhang, J.; Wan, X.
    Selective crystallization represents one of the most economical and convenient methods to provide large-scale optically pure chiral compounds. Although significant development has been achieved since Pasteur’s separation of sodium ammonium tartrate in 1848, this method is still fundamentally low efficient (low transformation ratio or high labor). Herein, we describe an enantiomer-selective-magnetization strategy for quantitatively separating the crystals of conglomerates by using a kind of magnetic nano-splitters. These nano-splitters would be selectively wrapped into the S-crystals, leading to the formation of the crystals with different physical properties from that of R-crystals. As a result of efficient separation under magnetic field, high purity chiral compounds (99.2 ee% for R-crystals, 95.0 ee% for S-crystals) can be obtained in a simple one-step crystallization process with a high separation yield (95.1%). Moreover, the nano-splitters show expandability and excellent recyclability. We foresee their great potential in developing chiral separation methods used on different scales. © 2019, The Author(s).
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    Whole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography
    (San Francisco, CA : Public Library of Science, 2013) Baudoin, J.-P.; Jerome, W.G.; Kübel, C.; de Jonge, N.
    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.