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- ItemLiquid-Phase Electron Microscopy for Soft Matter Science and Biology(Weinheim : Wiley-VCH Verlag, 2020) Wu, H.; Friedrich, H.; Patterson, J.P.; Sommerdijk, N.A.J.M.; de Jonge, N.Innovations in liquid-phase electron microscopy (LP-EM) have made it possible to perform experiments at the optimized conditions needed to examine soft matter. The main obstacle is conducting experiments in such a way that electron beam radiation can be used to obtain answers for scientific questions without changing the structure and (bio)chemical processes in the sample due to the influence of the radiation. By overcoming these experimental difficulties at least partially, LP-EM has evolved into a new microscopy method with nanometer spatial resolution and sub-second temporal resolution for analysis of soft matter in materials science and biology. Both experimental design and applications of LP-EM for soft matter materials science and biological research are reviewed, and a perspective of possible future directions is given.
- ItemAnti-correlation of HER2 and focal adhesion complexes in the plasma membrane(San Francisco : Public Library of Science, 2020) Weinberg, F.; Han, M.K.L.; Dahmke, I.N.; Campo, A.D.; de Jonge, N.Excess presence of the human epidermal growth factor receptor 2 (HER2) as well as of the focal adhesion protein complexes are associated with increased proliferation, migratory, and invasive behavior of cancer cells. A cross-regulation between HER2 and integrin signaling pathways has been found, but the exact mechanism remains elusive. Here, we investigated whether HER2 colocalizes with focal adhesion complexes on breast cancer cells overexpressing HER2. For this purpose, vinculin or talin green fluorescent protein (GFP) fusion proteins, both key constituents of focal adhesions, were expressed in breast cancer cells. HER2 was either extracellularly or intracellularly labeled with fluorescent quantum dots nanoparticles (QDs). The cell-substrate interface was analyzed at the location of the focal adhesions by means of total internal reflection fluorescent microscopy or correlative fluorescence- and scanning transmission electron microscopy. Expression of HER2 at the cell-substrate interface was only observed upon intracellular labeling, and was heterogeneous with both HER2-enriched and -low regions. In contrast to an expected enrichment of HER2 at focal adhesions, an anti-correlated expression pattern was observed for talin and HER2. Our findings suggest a spatial anti-correlation between HER2 and focal adhesion complexes for adherent cells.
- ItemWhole-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.
- ItemCorrelative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin(Lausanne : Frontiers Media, 2020) Dahmke, I.N.; Trampert, P.; Weinberg, F.; Mostajeran, Z.; Lautenschläger, F.; de Jonge, N.Epidermal growth factor receptor 2 (ErbB2) is found overexpressed in several cancers, such as gastric, and breast cancer, and is, therefore, an important therapeutic target. ErbB2 plays a central role in cancer cell invasiveness, and is associated with cytoskeletal reorganization. In order to study the spatial correlation of single ErbB2 proteins and actin filaments, we applied correlative fluorescence microscopy (FM), and scanning transmission electron microscopy (STEM) to image specifically labeled SKBR3 breast cancer cells. The breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and labeled with quantum dot (QD) nanoparticles attached to specific anti-ErbB2 Affibodies. FM was performed to identify cellular regions with spatially correlated actin and ErbB2 expression. For STEM of the intact plasma membrane of whole cells, the cells were fixed and covered with graphene. Spatial distribution patterns of ErbB2 in the actin rich ruffled membrane regions were examined, and compared to adjacent actin-low regions of the same cell, revealing an association of putative signaling active ErbB2 homodimers with actin-rich regions. ErbB2 homodimers were found absent from actin-low membrane regions, as well as after treatment of cells with Cytochalasin D, which breaks up larger actin filaments. In both latter data sets, a significant inter-label distance of 36 nm was identified, possibly indicating an indirect attachment to helical actin filaments via the formation of heterodimers of ErbB2 with epidermal growth factor receptor (EGFR). The possible attachment to actin filaments was further explored by identifying linear QD-chains in actin-rich regions, which also showed an inter-label distance of 36 nm.
- ItemElectron microscopy of nanoparticle superlattice formation at a solid-liquid interface in nonpolar liquids(Washington, DC : American Association for the Advancement of Science, 2020) Cepeda-Perez, E.; Doblas, D.; Kraus, T.; de Jonge, N.Nanoparticle superlattice films form at the solid-liquid interface and are important for mesoscale materials, but are notoriously difficult to analyze before they are fully dried. Here, the early stages of nanoparticle assembly were studied at solid-liquid interfaces using liquid-phase electron microscopy. Oleylamine-stabilized gold nanoparticles spontaneously formed thin layers on a silicon nitride (SiN) membrane window of the liquid enclosure. Dense packings of hexagonal symmetry were obtained for the first monolayer independent of the nonpolar solvent type. The second layer, however, exhibited geometries ranging from dense packing in a hexagonal honeycomb structure to quasi-crystalline particle arrangements depending on the dielectric constant of the liquid. The complex structures formed by the weaker interactions in the second particle layer were preserved, while the surface remained immersed in liquid. Fine-tuning the properties of the involved materials can thus be used to control the three-dimensional geometry of a superlattice including quasi-crystals.