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- ItemHigh temporal-resolution scanning transmission electron microscopy using sparse-serpentine scan pathways([London] : Macmillan Publishers Limited, part of Springer Nature, 2021) Ortega, Eduardo; Nicholls, Daniel; Browning, Nigel D.; de Jonge, NielsScanning transmission electron microscopy (STEM) provides structural analysis with sub-angstrom resolution. But the pixel-by-pixel scanning process is a limiting factor in acquiring high-speed data. Different strategies have been implemented to increase scanning speeds while at the same time minimizing beam damage via optimizing the scanning strategy. Here, we achieve the highest possible scanning speed by eliminating the image acquisition dead time induced by the beam flyback time combined with reducing the amount of scanning pixels via sparse imaging. A calibration procedure was developed to compensate for the hysteresis of the magnetic scan coils. A combination of sparse and serpentine scanning routines was tested for a crystalline thin film, gold nanoparticles, and in an in-situ liquid phase STEM experiment. Frame rates of 92, 23 and 5.8 s-1 were achieved for images of a width of 128, 256, and 512 pixels, respectively. The methods described here can be applied to single-particle tracking and analysis of radiation sensitive materials.
- ItemQuantification of EGFR-HER2 Heterodimers in HER2-Overexpressing Breast Cancer Cells Using Liquid-Phase Electron Microscopy(Basel : MDPI, 2021) Peckys, Diana B.; Gaa, Daniel; de Jonge, NielsCurrently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρR, as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.
- ItemSynthesis of 3,4-Dihydro-2H-Pyrroles from Ketones, Aldehydes, and Nitro Alkanes via Hydrogenative Cyclization(Weinheim : Wiley-VCH, 2022) Klausfelder, Barbara; Blach, Patricia; de Jonge, Niels; Kempe, RhettSyntheses of N-heterocyclic compounds that permit a flexible introduction of various substitution patterns by using inexpensive and diversely available starting materials are highly desirable. Easy to handle and reusable catalysts based on earth-abundant metals are especially attractive for these syntheses. We report here on the synthesis of 3,4-dihydro-2H-pyrroles via the hydrogenation and cyclization of nitro ketones. The latter are easily accessible from three components: a ketone, an aldehyde and a nitroalkane. Our reaction has a broad scope and 23 of the 33 products synthesized are compounds which have not yet been reported. The key to the general hydrogenation/cyclization reaction is a highly active, selective and reusable nickel catalyst, which was identified from a library of 24 earth-abundant metal catalysts.
- ItemKey Parameters for the Synthesis of Active and Selective Nanostructured 3d Metal Catalysts Starting from Coordination Compounds – Case Study: Nickel Mediated Reductive Amination(Weinheim : WILEY-VCH Verlag, 2021) Klarner, Mara; Blach, Patricia; Wittkämper, Haiko; de Jonge, Niels; Papp, Christian; Kempe, RhettThe design of nanostructured catalysts based on earth-abundant metals that mediate important reactions efficiently, selectively and with a broad scope is highly desirable. Unfortunately, the synthesis of such catalysts is poorly understood. We report here on highly active Ni catalysts for the reductive amination of ketones by ammonia employing hydrogen as a reducing agent. The key functions of the Ni-salen precursor complex during catalyst synthesis have been identified: (1) Ni-salen complexes sublime during catalyst synthesis, which allows molecular dispersion of the metal precursor on the support material. (2) The salen ligand forms a nitrogen-doped carbon shell by decomposition, which embeds and stabilizes the Ni nanoparticles on the γ-Al2O3 support. (3) Parameters, such as flow rate of the pyrolysis gas, determine the carbon supply for the embedding process of Ni nanoparticles.
- ItemStrategy for optimizing experimental settings for studying low atomic number colloidal assemblies using liquid phase scanning transmission electron microscopy(Amsterdam : Elsevier Science, 2022) Kunnas, Peter; Moradi, Mohammad-Amin; Sommerdijk, Nico; de Jonge, NielsObserving processes of nanoscale materials of low atomic number is possible using liquid phase electron microscopy (LP-EM). However, the achievable spatial resolution (d) is limited by radiation damage. Here, we examine a strategy for optimizing LP-EM experiments based on an analytical model and experimental measurements, and develop a method for quantifying image quality at ultra low electron dose De using scanning transmission electron microscopy (STEM). As experimental test case we study the formation of a colloidal binary system containing 30 nm diameter SiO2 nanoparticles (SiONPs), and 100 nm diameter polystyrene microspheres (PMs). We show that annular dark field (DF) STEM is preferred over bright field (BF) STEM for practical reasons. Precise knowledge of the material's density is crucial for the calculations in order to match experimental data. To calculate the detectability of nano-objects in an image, the Rose criterion for single pixels is expanded to a model of the signal to noise ratio obtained for multiple pixels spanning the image of an object. Using optimized settings, it is possible to visualize the radiation-sensitive, hierarchical low-Z binary structures, and identify both components.
- ItemSupra-Molecular Assemblies of ORAI1 at Rest Precede Local Accumulation into Puncta after Activation(Basel : Molecular Diversity Preservation International, 2021) Peckys, Diana B.; Gaa, Daniel; Alansary, Dalia; Niemeyer, Barbara A.; de Jonge, NielsThe Ca2+ selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins form the core of the channel complex mediating store operated Ca2+ entry (SOCE). Using liquid phase electron microscopy (LPEM), the distribution of ORAI1 proteins was examined at rest and after SOCE-activation at nanoscale resolution. The analysis of over seven hundred thousand ORAI1 positions revealed a number of ORAI1 channels had formed STIM-independent distinct supra-molecular clusters. Upon SOCE activation and in the presence of STIM proteins, a fraction of ORAI1 assembled in micron-sized two-dimensional structures, such as the known puncta at the ER plasma membrane contact zones, but also in divergent structures such as strands, and ring-like shapes. Our results thus question the hypothesis that stochastically migrating single ORAI1 channels are trapped at regions containing activated STIM, and we propose instead that supra-molecular ORAI1 clusters fulfill an amplifying function for creating dense ORAI1 accumulations upon SOCE-activation.
- ItemEGFR Expression in HER2-Driven Breast Cancer Cells(Basel : MDPI, 2020) Weinberg, Florian; Peckys, Diana B.; de Jonge, NielsThe epidermal growth factor receptor HER2 is overexpressed in 20% of breast cancer cases. HER2 is an orphan receptor that is activated ligand-independently by homodimerization. In addition, HER2 is able to heterodimerize with EGFR, HER3, and HER4. Heterodimerization has been proposed as a mechanism of resistance to therapy for HER2 overexpressing breast cancer. Here, a method is presented for the simultaneous detection of individual EGFR and HER2 receptors in the plasma membrane of breast cancer cells via specific labeling with quantum dot nanoparticles (QDs). Correlative fluorescence microscopy and liquid phase electron microscopy were used to analyze the plasma membrane expression levels of both receptors in individual intact cells. Fluorescent single-cell analysis of SKBR3 breast cancer cells dual-labeled for EGFR and HER2 revealed a heterogeneous expression for receptors within both the cell population as well as within individual cells. Subsequent electron microscopy of individual cells allowed the determination of individual receptors label distributions. QD-labeled EGFR was observed with a surface density of (0.5–5) × 101 QDs/µm2, whereas labeled HER2 expression was higher ranging from (2–10) × 102 QDs/µm2. Although most SKBR3 cells expressed low levels of EGFR, an enrichment was observed at large plasma membrane protrusions, and amongst a newly discovered cellular subpopulation termed EGFR-enriched cells.
- ItemGraphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy(Cambridge, MA : JoVE, 2020) Blach, Patricia; Keskin, Sercan; de Jonge, NielsA protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.
- ItemNanoscale Faceting and Ligand Shell Structure Dominate the Self-Assembly of Nonpolar Nanoparticles into Superlattices(Weinheim : Wiley-VCH, 2022) Bo, Arixin; Liu, Yawei; Kuttich, Björn; Kraus, Tobias; Widmer-Cooper, Asaph; de Jonge, NielsSelf-assembly of nanoscale structures at liquid–solid interfaces occurs in a broad range of industrial processes and is found in various phenomena in nature. Conventional theory assumes spherical particles and homogeneous surfaces, but that model is oversimplified, and nanoscale in situ observations are needed for a more complete understanding. Liquid-phase scanning transmission electron microscopy (LP-STEM) is used to examine the interactions that direct the self-assembly of superlattices formed by gold nanoparticles (AuNPs) in nonpolar liquids. Varying the molecular coating of the substrate modulates short-range attraction and leads to switching between a range of different geometric structures, including hexagonal close-packed (hcp), simple hexagonal (sh), dodecahedral quasi-crystal (dqc), and body-centered cubic (bcc) lattices, as well as random distributions. Langevin dynamics simulations explain the experimental results in terms of the interplay between nanoparticle faceting, ligand shell structure, and substrate–NP interactions.