Publications by authors named "Niels de Jonge"

85 Publications

Synthesis of 3,4-Dihydro-2H-pyrroles from Ketones, Aldehydes, and Nitro Alkanes via Hydrogenative Cyclization.

Chemistry 2022 May 31. Epub 2022 May 31.

Anorganische Chemie II, Katalysatordesign, Chemie, Universitätsstraße 30, 95440, Bayreuth, GERMANY.

Syntheses of N-heterocyclic compounds that permit a flexible introduction of various substitution patterns 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.
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http://dx.doi.org/10.1002/chem.202201307DOI Listing
May 2022

Nanoscale Faceting and Ligand Shell Structure Dominate the Self-Assembly of Nonpolar Nanoparticles into Superlattices.

Adv Mater 2022 May 17;34(20):e2109093. Epub 2022 Apr 17.

INM - Leibniz Institute for New Materials, 66123, Saarbrücken, Germany.

Self-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.
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http://dx.doi.org/10.1002/adma.202109093DOI Listing
May 2022

Quantification of EGFR-HER2 Heterodimers in HER2-Overexpressing Breast Cancer Cells Using Liquid-Phase Electron Microscopy.

Cells 2021 11 19;10(11). Epub 2021 Nov 19.

INM-Leibniz Institute for New Materials, 66123 Saarbrücken, Germany.

Currently, 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 , 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 , 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.
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http://dx.doi.org/10.3390/cells10113244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8623301PMC
November 2021

High temporal-resolution scanning transmission electron microscopy using sparse-serpentine scan pathways.

Sci Rep 2021 11 22;11(1):22722. Epub 2021 Nov 22.

INM - Leibniz Institute for New Materials, 66123, Saarbrucken, Germany.

Scanning 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 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.
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http://dx.doi.org/10.1038/s41598-021-02052-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8608981PMC
November 2021

The influence of chromatic aberration on the dose-limited spatial resolution of transmission electron microscopy.

Ultramicroscopy 2021 11 18;230:113383. Epub 2021 Aug 18.

INM-Leibniz Institute for New Materials, Saarbrücken 66123, Germany; Department of Physics, Saarland University, Saarbrücken 66123, Germany. Electronic address:

The effect of chromatic aberration (CC) on the spatial resolution in transmission electron microscopy (TEM) was studied in thick specimens in which the sample becomes the limiting factor in the resolution. The sample influences the energy spread of the electron beam, allows only a limited electron dose, and modulates electron scattering events. The experimental set-up consisted of a thin silicon nitride membrane and a silicon wedge containing gold nanoparticles. The resolution was measured as a function of electron dose and sample thickness for different sample configurations and for different microscopy modalities including regular TEM, energy filtered TEM (EFTEM) and CC-corrected TEM. Comparison with an analytical model aided the understanding of the experimental data applied over varied conditions. The general trend for all microscopy modalities was a transition from a noise-limited resolution at low electron dose to a CC-limited resolution at high-dose in the absence of beam blurring. EFTEM required an accurate energy slit offset and an optimal energy spread to energy-slit width ratio to surpass regular TEM. The key advantage of CC correction appeared to be the best possible resolution for larger sample thickness at low electron dose outperforming EFTEM by about fifty percent. Several hypothetical sample configurations relevant to liquid phase electron microscopy were evaluated as well to demonstrate the capabilities of the analytical model and to determine the most optimal microscopy modality for this type of experiment. The analytical model included an automated optimization of the EFTEM settings and may aid in optimizing the sample-limited resolution for experimental analysis and planning.
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http://dx.doi.org/10.1016/j.ultramic.2021.113383DOI Listing
November 2021

Verification of water presence in graphene liquid cells.

Micron 2021 10 30;149:103109. Epub 2021 Jun 30.

INM - Leibniz Institute for New Materials, Campus D2-2, D-66123, Saarbrücken, Germany; Department of Physics, Saarland University, Campus D2-2, D-66123, Saarbrücken, Germany. Electronic address:

Graphene liquid cells (GLCs) present the thinnest possible sample enclosures for liquid phase electron microscopy. However, the actual presence of liquid within a GLC is not always guaranteed. Of key importance is to reliably test the presence of the liquid, which is most frequently water or saline. Here, the commonly used methods for verifying the presence of water were evaluated. It is shown that depending on the type of sample, applying a single criterion does not always conclusively verify the presence of water. Testing liquid filling for a specific GLC sample preparation protocol should thus be considered critically. The most reliable method is direct observation of the water exciton peak using electron energy loss spectroscopy (EELS). But if this method cannot be carried out, water filling of the GLC can be verified from a combination of higher contrast in the image, the presence of bubbles, and an oxygen signal in the EEL spectrum, which can be accomplished at a high electron dose in spot mode. Nanoparticle movement does not always occur in a GLC.
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http://dx.doi.org/10.1016/j.micron.2021.103109DOI Listing
October 2021

Supra-Molecular Assemblies of ORAI1 at Rest Precede Local Accumulation into Puncta after Activation.

Int J Mol Sci 2021 Jan 14;22(2). Epub 2021 Jan 14.

INM-Leibniz Institute for New Materials, 66123 Saarbrücken, Germany.

The Ca selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins form the core of the channel complex mediating store operated Ca 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.
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http://dx.doi.org/10.3390/ijms22020799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831003PMC
January 2021

Environmental Liquid Cell Technique for Improved Electron Microscopic Imaging of Soft Matter in Solution.

Microsc Microanal 2021 Feb;27(1):44-53

Max Planck Institute for the Structure and Dynamics of Matter, Luruper Chaussee 149, Geb. 99 (CFEL), 22761Hamburg, Germany.

Liquid-phase transmission electron microscopy is a technique for simultaneous imaging of the structure and dynamics of specimens in a liquid environment. The conventional sample geometry consists of a liquid layer tightly sandwiched between two Si3N4 windows with a nominal spacing on the order of 0.5 μm. We describe a variation of the conventional approach, wherein the Si3N4 windows are separated by a 10-μm-thick spacer, thus providing room for gas flow inside the liquid specimen enclosure. Adjusting the pressure and flow speed of humid air inside this environmental liquid cell (ELC) creates a stable liquid layer of controllable thickness on the bottom window, thus facilitating high-resolution observations of low mass-thickness contrast objects at low electron doses. We demonstrate controllable liquid thicknesses in the range 160 ± 34 to 340 ± 71 nm resulting in corresponding edge resolutions of 0.8 ± 0.06 to 1.7 ± 0.8 nm as measured for immersed gold nanoparticles. Liquid layer thickness 40 ± 8 nm allowed imaging of low-contrast polystyrene particles. Hydration effects in the ELC have been studied using poly-N-isopropylacrylamide nanogels with a silica core. Therefore, ELC can be a suitable tool for in situ investigations of liquid specimens.
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http://dx.doi.org/10.1017/S1431927620024654DOI Listing
February 2021

EGFR Expression in HER2-Driven Breast Cancer Cells.

Int J Mol Sci 2020 Nov 27;21(23). Epub 2020 Nov 27.

INM-Leibniz Institute for New Materials, 66123 Saarbrücken, Germany.

The 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) × 10 QDs/µm, whereas labeled HER2 expression was higher ranging from (2-10) × 10 QDs/µm. 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.
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http://dx.doi.org/10.3390/ijms21239008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729501PMC
November 2020

Determining the Efficiency of Single Molecule Quantum Dot Labeling of HER2 in Breast Cancer Cells.

Nano Lett 2020 11 9;20(11):7948-7955. Epub 2020 Oct 9.

Department of Physics, Saarland University, 66123 Saarbrücken, Germany.

Quantum dots exhibit unique properties compared to other fluorophores, such as bright fluorescence and lack of photobleaching, resulting in their widespread utilization as fluorescent protein labels in the life sciences. However, their application is restricted to relative quantifications due to lacking knowledge about the labeling efficiency. We here present a strategy for determining the labeling efficiency of quantum dot labeling of HER2 in overexpressing breast cancer cells. Correlative light- and liquid-phase electron microscopy of whole cells was used to convert fluorescence intensities into the underlying molecular densities of the quantum dots. The labeling procedure with small affinity proteins was optimized yielding a maximal labeling efficiency of 83%, which was applicable to the high amount of ∼1.5 × 10 HER2 per cell. With the labeling efficiency known, it is now possible to derive the absolute protein expression levels in the plasma membrane and its variation within a cell and between cells.
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http://dx.doi.org/10.1021/acs.nanolett.0c02644DOI Listing
November 2020

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy.

J Vis Exp 2020 09 21(163). Epub 2020 Sep 21.

INM-Leibniz Institute for New Materials; Department of Physics, Saarland University;

A 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.
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http://dx.doi.org/10.3791/61458DOI Listing
September 2020

Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin.

Front Cell Dev Biol 2020 30;8:521. Epub 2020 Jun 30.

INM - Leibniz Institute for New Materials, Saarbrücken, Germany.

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.
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http://dx.doi.org/10.3389/fcell.2020.00521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344305PMC
June 2020

Blood sampling after COVID-19 - How to organize large scale phlebotomy services in the post SARS CoV-2 era.

Clin Chem Lab Med 2020 08 15;58(9):e155-e157. Epub 2020 Jul 15.

Laboratory of General Clinical Chemistry, Amsterdam UMC, Amsterdam, The Netherlands.

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http://dx.doi.org/10.1515/cclm-2020-0671DOI Listing
August 2020

Anti-correlation of HER2 and focal adhesion complexes in the plasma membrane.

PLoS One 2020 8;15(6):e0234430. Epub 2020 Jun 8.

INM - Leibniz Institute for New Materials, Saarbrücken, Germany.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234430PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279600PMC
August 2020

Liquid-Phase Electron Microscopy for Soft Matter Science and Biology.

Adv Mater 2020 Jun 17;32(25):e2001582. Epub 2020 May 17.

INM - Leibniz Institute for New Materials, Saarbrücken, 66123, Germany.

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.
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http://dx.doi.org/10.1002/adma.202001582DOI Listing
June 2020

Detecting single ORAI1 proteins within the plasma membrane reveals higher-order channel complexes.

J Cell Sci 2020 01 3;133(1). Epub 2020 Jan 3.

INM - Leibniz Institute for New Materials, 66123 Saarbrücken, Germany

ORAI1 proteins form highly selective Ca channels in the plasma membrane. Crystallographic data point towards a hexameric stoichiometry of ORAI1 channels, whereas optical methods postulated ORAI1 channels to reside as dimers at rest, and other data suggests that they have a tetrameric configuration. Here, liquid-phase scanning transmission electron microscopy (STEM) and quantum dot (QD) labeling was utilized to study the conformation of ORAI1 proteins at rest. To address the question of whether ORAI1 was present as a dimer, experiments were designed using single ORAI1 monomers and covalently linked ORAI1 dimers with either one or two label-binding positions. The microscopic data was statistically analyzed via the pair correlation function. Label pairs were found in all cases, even for concatenated dimers with one label-binding position, which is only possible if a significant fraction of ORAI1 was assembled in larger order oligomers than dimers, binding at least two QDs. This interpretation of the data was consistent with Blue Native PAGE analysis showing that ORAI1 is mainly present as a complex of an apparent molecular mass larger than that calculated for a dimer.
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http://dx.doi.org/10.1242/jcs.240358DOI Listing
January 2020

Mathematical modeling of drug-induced receptor internalization in the HER2-positive SKBR3 breast cancer cell-line.

Sci Rep 2019 09 3;9(1):12709. Epub 2019 Sep 3.

INM - Leibniz Institute for New Materials, 66123, Saarbrücken, Germany.

About 20% of breast cancer tumors over-express the HER2 receptor. Trastuzumab, an approved drug to treat this type of breast cancer, is a monoclonal antibody directly binding at the HER2 receptor and ultimately inhibiting cancer cell growth. The goal of our study was to understand the early impact of trastuzumab on HER2 internalization and recycling in the HER2-overexpressing breast cancer cell line SKBR3. To this end, fluorescence microscopy, monitoring the amount of HER2 expression in the plasma membrane, was combined with mathematical modeling to derive the flux of HER2 receptors from and to the membrane. We constructed a dynamic multi-compartment model based on ordinary differential equations. To account for cancer cell heterogeneity, a first, dynamic model was expanded to a second model including two distinct cell phenotypes, with implications for different conformational states of HER2, i.e. monomeric or homodimeric. Our mathematical model shows that the hypothesis of fast constitutive HER2 recycling back to the plasma membrane does not match the experimental data. It conclusively describes the experimental observation that trastuzumab induces sustained receptor internalization in cells with membrane ruffles. It is also concluded that for rare, non-ruffled (flat) cells, HER2 internalization occurs three orders of magnitude slower than for the bulk, ruffled cell population.
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http://dx.doi.org/10.1038/s41598-019-49019-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722142PMC
September 2019

Visualisation of HER2 homodimers in single cells from HER2 overexpressing primary formalin fixed paraffin embedded tumour tissue.

Mol Med 2019 08 28;25(1):42. Epub 2019 Aug 28.

INM - Leibniz Institute for New Materials, Campus D2-2, 66123, Saarbrücken, Germany.

Background: HER2 is considered as one of the most important, predictive biomarkers in oncology. The diagnosis of HER2 positive cancer types such as breast- and gastric cancer is usually based on immunohistochemical HER2 staining of tumour tissue. However, the current immunohistochemical methods do not provide localized information about HER2's functional state. In order to generate signals leading to cell growth and proliferation, the receptor spontaneously forms homodimers, a process that can differ between individual cancer cells.

Materials And Methods: HER2 overexpressing tumour cells were dissociated from formalin-fixed paraffin-embedded (FFPE) patient's biopsy sections, subjected to a heat-induced antigen retrieval procedure, and immobilized on microchips. HER2 was specifically labelled via a two-step protocol involving the incubation with an Affibody-biotin compound followed by the binding of a streptavidin coated quantum dot (QD) nanoparticle. Cells with membrane bound HER2 were identified using fluorescence microscopy, coated with graphene to preserve their hydrated state, and subsequently examined by scanning transmission electron microscopy (STEM) to obtain the locations at the single molecule level. Label position data was statistically analysed via the pair correlation function, yielding information about the presence of HER2 homodimers.

Results: Tumour cells from two biopsies, scored HER2 3+, and a HER2 negative control sample were examined. The specific labelling protocol was first tested for a sectioned tissue sample of HER2-overexpressing tumour. Subsequently, a protocol was optimized to study HER2 homodimerization in single cells dissociated from the tissue section. Electron microscopy data showed membrane bound HER2 in average densities of 201-689 proteins/μm. An automated, statistical analysis of well over 200,000 of measured protein positions revealed the presence of HER2 homodimers in 33 and 55% of the analysed images for patient 1 and 2, respectively.

Conclusions: We introduced an electron microscopy method capable of measuring the positions of individually labelled HER2 proteins in patient tumour cells from which information about the functional status of the receptor was derived. This method could take HER2 testing a step further by examining HER2 homodimerization directly out of tumour tissue and may become important for adjusting a personalized antibody-based drug therapy.
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http://dx.doi.org/10.1186/s10020-019-0108-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6712713PMC
August 2019

Liquid-Phase Electron Microscopy with Controllable Liquid Thickness.

Nano Lett 2019 Jul 19;19(7):4608-4613. Epub 2019 Jun 19.

INM - Leibniz Institute for New Materials , D-66123 Saarbrücken , Germany.

Liquid-phase electron microscopy (LPEM) is capable of imaging nanostructures and processes in a liquid environment. The spatial resolution achieved with LPEM critically depends on the thickness of the liquid layer surrounding the object of interest. An excessively thick liquid results in broadening of the electron beam and a high background signal that decreases the resolution and contrast of the object in an image. The liquid thickness in a standard liquid cell, consisting of two liquid enclosing membranes separated by spacers, is mainly defined by the deformation of the SiN membrane windows toward the vacuum side, and the effective thickness may differ from the spacer height. Here, we present a method involving a pressure controller setup to balance the pressure difference over the membrane windows, thus manipulating the shape profiles of the used silicon nitride membrane windows. Electron energy loss spectroscopy (EELS) measurements to determine the liquid thickness showed that it is possible to control the thickness precisely during an LPEM experiment by regulating the interior pressure of the liquid cell. We demonstrated atomic resolution on gold nanoparticles and the phase contrast using silica nanoparticles in liquid with controlled thickness.
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http://dx.doi.org/10.1021/acs.nanolett.9b01576DOI Listing
July 2019

The 2018 correlative microscopy techniques roadmap.

J Phys D Appl Phys 2018 Nov 31;51(44):443001. Epub 2018 Aug 31.

Institute for Theoretical Physics and BioQuant, Heidelberg University, Heidelberg, Germany.

Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell-cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure-function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints.
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http://dx.doi.org/10.1088/1361-6463/aad055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372154PMC
November 2018

Preface to the special issue on liquid-phase electron microscopy.

Micron 2019 04 21;119:117-118. Epub 2018 Dec 21.

Leibniz Institute for New Materials (INM), Saarbrücken D-66123, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.micron.2018.12.005DOI Listing
April 2019

Dynamics of gold nanoparticle clusters observed with liquid-phase electron microscopy.

Micron 2019 02 23;117:68-75. Epub 2018 Nov 23.

INM - Leibniz Institute for New Materials, Campus D2 -2, D-66123, Saarbrücken, Germany; Department of Physics, Saarland University, Campus D2 -2, D-66123, Saarbrücken, Germany. Electronic address:

The dynamics of processes of nanoparticles such as diffusion, attraction and repulsion, and self-assembly of structures of nanoparticles at the solid-liquid interfaces differ significantly from those occurring for bulk conditions and their fundamental physical rules are still unknown. Here, we used liquid phase scanning transmission electron microscopy (LP-STEM) to study several aspects of nanoparticle dynamics of colloidal chitosan coated gold nanoparticle (TCHIT-AuNP) clusters in a liquid layer enclosed between two SiN membranes. We found that upon beam irradiation using an electron flux of 0.9 e/sÅ, the AuNPs assembled in clusters that shifted and rotated with time. The newly formed clusters could join and form larger clusters via a mechanism of oriented attachment. By increasing the electron flux to 6.2 e/sÅ, we observed the fragmentation of some of the clusters and TCHIT-AuNPs were exchanged between clusters. At the highest electron flux studied 25 e/sÅ, we observed AuNPs moving at a very slow speed compared to Brownian motion in liquid even though they were not directly attached or pinned to the liquid-enclosing membrane. Experiments using branched polyethylenimine (BPEI) coated AuNPs were carried out for comparison.
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http://dx.doi.org/10.1016/j.micron.2018.11.006DOI Listing
February 2019

Reduced Radiation Damage in Transmission Electron Microscopy of Proteins in Graphene Liquid Cells.

Nano Lett 2018 12 15;18(12):7435-7440. Epub 2018 Nov 15.

Leibniz Institute for New Materials (INM) , Saarbrücken D-66123 , Germany.

Liquid-phase electron microscopy (LPEM) is capable of imaging native (unstained) protein structure in liquid, but the achievable spatial resolution is limited by radiation damage. This damaging effect is more pronounced when targeting small molecular features than for larger structures. The matter is even more complicated because the critical dose that a sample can endure before radiation damage not only varies between proteins but also critically depends on the experimental conditions. Here, we examined the effect of the electron beam on the observed protein structure for optimized conditions using a liquid sample enclosure assembled from graphene sheets. It has been shown that graphene can reduce the damaging effect of electrons on biological materials. We used radiation sensitive microtubule proteins and investigated the radiation damage on these structures as a function of the spatial frequencies of the observed features with transmission electron microscopy (TEM). Microtubule samples were also examined using cryo-electron microscopy (cryo-TEM) for comparison. We used an electron flux of 11 ± 1-16 ± 1 e/Ås and obtained a series of images from the same sample region. Our results show that graphene-encapsulated microtubules can maintain their structural features of spatial frequencies of up to 0.20 nm (5 nm), reflecting protofilaments for electron densities of up to 7.2 ± 1.4 × 10 e/Å, an order of magnitude higher than measured for frozen microtubules in amorphous ice.
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http://dx.doi.org/10.1021/acs.nanolett.8b02490DOI Listing
December 2018

Corrigendum to: "Theory of the spatial resolution of (scanning) transmission electron microscopy in liquid water or ice layers" [Ultramicroscopy 187 (2018) 113-125].

Authors:
Niels de Jonge

Ultramicroscopy 2019 01 18;196:129-130. Epub 2018 Oct 18.

Department of Physics, INM - Leibniz Institute for New Materials, Saarland University, Saarbrücken 66123, Germany. Electronic address:

Several errors are present in the equations of the article Ultramicroscopy 187 (2018) 113-125. Yet, the calculations described in the paper and used for the figures were conducted using the correct equations, so that the results and the conclusions are based on correct calculations.
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http://dx.doi.org/10.1016/j.ultramic.2018.10.006DOI Listing
January 2019

Linear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron Microscopy.

Biophys J 2018 08 18;115(3):503-513. Epub 2018 Jun 18.

INM-Leibniz Institute for New Materials, Saarbrücken, Germany; Department of Physics, Saarland University, Saarbrücken, Germany. Electronic address:

The spatial distribution of the human epidermal growth factor 2 (HER2) receptor in the plasma membrane of SKBR3 and HCC1954 breast cancer cells was studied. The receptor was labeled with quantum dot nanoparticles, and fixed whole cells were imaged in their native liquid state with environmental scanning electron microscopy using scanning transmission electron microscopy detection. The locations of individual HER2 positions were determined in a total plasma membrane area of 991 μm for several SKBR3 cells and 1062 μm for HCC1954 cells. Some of the HER2 receptors were arranged in a linear chain with interlabel distances of 40 ± 7 and 32 ± 10 nm in SKBR3 and HCC1954 cells, respectively. The finding was tested against randomly occurring linear chains of six or more positions, from which it was concluded that the experimental finding is significant and did not arise from random label distributions. Because the measured interlabel distance in the HER2 chains is similar to the 36-nm helix-repetition distance of actin filaments, it is proposed that a linking mechanism between HER2 and actin filaments leads to linearly aligned oligomers.
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http://dx.doi.org/10.1016/j.bpj.2018.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6084255PMC
August 2018

Strategies for Preparing Graphene Liquid Cells for Transmission Electron Microscopy.

Nano Lett 2018 06 25;18(6):3313-3321. Epub 2018 May 25.

INM, Leibniz Institute for New Materials , D-66123 Saarbrücken , Germany.

A graphene liquid cell for transmission electron microscopy (TEM) uses one or two graphene sheets to separate the liquid from the vacuum in the microscope. In principle, graphene is an excellent material for such an application because it allows the highest possible spatial resolution, provides a flexible covering foil, and effectively protects the liquid from evaporating. Examples in open literature have demonstrated atomic-resolution TEM using small liquid pockets and the coverage of whole biological cells with graphene sheets. A total of three different basic types of liquid cells are discerned: (i) one graphene sheet is used to cover a liquid sample supported by a thin membrane of another material (for example, silicon nitride, SiN), (ii) two graphene sheets pressed together leaving liquid pockets with graphene at both sides, and (iii) a spacer material with liquid pockets covered at both sides by graphene. A total of four different process flows are available for liquid cell assembly, but there is not yet a consensus on the best routes, and a number of variations exist. The key step is the transfer of graphene to a liquid sample, which is complicated by practical issues that arise from imperfections in the graphene sheets, such as cracks. This review provides an overview of these different approaches to assembling graphene liquid cells and discusses the main obstacles and ideas to overcome them with the prospect of developing the nanoscale technology needed for graphene liquid cells so that they become available on a routine basis for electron microscopy in liquid. It also provides guidance in selecting the appropriate type of graphene liquid cell and the best assembly method for a specific experiment.
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http://dx.doi.org/10.1021/acs.nanolett.8b01366DOI Listing
June 2018

The Influence of Beam Broadening on the Spatial Resolution of Annular Dark Field Scanning Transmission Electron Microscopy.

Microsc Microanal 2018 02;24(1):8-16

3Department of Materials Engineering,McGill University,Montreal,QC H3A 0C5,Canada.

The spatial resolution of aberration-corrected annular dark field scanning transmission electron microscopy was studied as function of the vertical position z within a sample. The samples consisted of gold nanoparticles (AuNPs) positioned in different horizontal layers within aluminum matrices of 0.6 and 1.0 µm thickness. The highest resolution was achieved in the top layer, whereas the resolution was reduced by beam broadening for AuNPs deeper in the sample. To examine the influence of the beam broadening, the intensity profiles of line scans over nanoparticles at a certain vertical location were analyzed. The experimental data were compared with Monte Carlo simulations that accurately matched the data. The spatial resolution was also calculated using three different theoretical models of the beam blurring as function of the vertical position within the sample. One model considered beam blurring to occur as a single scattering event but was found to be inaccurate for larger depths of the AuNPs in the sample. Two models were adapted and evaluated that include estimates for multiple scattering, and these described the data with sufficient accuracy to be able to predict the resolution. The beam broadening depended on z 1.5 in all three models.
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http://dx.doi.org/10.1017/S1431927618000077DOI Listing
February 2018

Theory of the spatial resolution of (scanning) transmission electron microscopy in liquid water or ice layers.

Authors:
Niels de Jonge

Ultramicroscopy 2018 04 1;187:113-125. Epub 2018 Feb 1.

INM - Leibniz Institute for New Materials, Department of Physics, Saarland University, Saarbrücken 66123, Germany. Electronic address:

The sample dependent spatial resolution was calculated for transmission electron microscopy (TEM) and scanning TEM (STEM) of objects (e.g., nanoparticles, proteins) embedded in a layer of liquid water or amorphous ice. The theoretical model includes elastic- and inelastic scattering, beam broadening, and chromatic aberration. Different contrast mechanisms were evaluated as function of the electron dose, the detection angle, and the sample configuration. It was found that the spatial resolution scales with the electron dose to the -1/4th power. Gold- and carbon nanoparticles were examined in the middle of water layers ranging from 0.01--10 µm thickness representing relevant classes of experiments in both materials science and biology. The optimal microscope settings differ between experimental configurations. STEM performs the best for gold nanoparticles for all layer thicknesses, while carbon is best imaged with phase-contrast TEM for thin layers but bright field STEM is preferred for thicker layers. The resolution was also calculated for a water layer enclosed between thin membranes. The influence of chromatic aberration correction for TEM was examined as well. The theory is broadly applicable to other types of materials and sample configurations.
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http://dx.doi.org/10.1016/j.ultramic.2018.01.007DOI Listing
April 2018

Low-force spectroscopy on graphene membranes by scanning tunneling microscopy.

Nanoscale 2018 Jan;10(4):2148-2153

Institute of Experimental Physics, Saarland University, Saarbruecken, D-66041, Germany.

Two-dimensional atomically flat sheets with a high mechanical flexibility are very attractive as ultrathin membranes but are also inherently challenging for microscopic investigations. We report on a method using Scanning Tunneling Microscopy (STM) under ultra-high vacuum conditions for non-indenting low-force spectroscopy on micrometer-sized freestanding graphene membranes. The method is based on applying quasi-static voltage ramps with active feedback at low tunneling currents and ultimately relies on the attractive electrostatic force between the tip and the membrane. As a result a bulge-test scenario can be established. The convenience and simplicity of the method relies on the fact that the loading force and the membrane deflection detection are both provided simultaneously by the STM. This permits the continuous measurement of the stress-strain relation. Electrostatic forces applied are typically below 1 nN and the membrane deflection is detected at sub-nanometer resolution. Experiments on single-layer graphene membranes with a strain of 0.1% reveal a two-dimensional elastic modulus E = 220 N m.
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http://dx.doi.org/10.1039/c7nr07300cDOI Listing
January 2018

Involvement of two uptake mechanisms of gold and iron oxide nanoparticles in a co-exposure scenario using mouse macrophages.

Beilstein J Nanotechnol 2017 14;8:2396-2409. Epub 2017 Nov 14.

Adolphe Merkle Institute, Université de Fribourg, Chemin des Verdiers 4, CH 1700, Fribourg, Switzerland.

Little is known about the simultaneous uptake of different engineered nanoparticle types, as it can be expected in our daily life. In order to test such co-exposure effects, murine macrophages (J774A.1 cell line) were incubated with gold (AuNPs) and iron oxide nanoparticles (FeO NPs) either alone or combined. Environmental scanning electron microscopy revealed that single NPs of both types bound within minutes on the cell surface but with a distinctive difference between FeO NPs and AuNPs. Uptake analysis studies based on laser scanning microscopy, transmission electron microscopy, and inductively coupled plasma optical emission spectrometry revealed intracellular appearance of both NP types in all exposure scenarios and a time-dependent increase. This increase was higher for both AuNPs and FeO NPs during co-exposure. Cells treated with endocytotic inhibitors recovered after co-exposure, which additionally hinted that two uptake mechanisms are involved. Cross-talk between uptake pathways is relevant for toxicological studies: Co-exposure acts as an uptake accelerant. If the goal is to maximize the cellular uptake, e.g., for the delivery of pharmaceutical agents, this can be beneficial. However, co-exposure should also be taken into account in the case of risk assessment of occupational settings. The demonstration of co-exposure-invoked pathway interactions reveals that synergetic nanoparticle effects, either positive or negative, must be considered for nanotechnology and nanomedicine in particular to develop to its full potential.
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http://dx.doi.org/10.3762/bjnano.8.239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704759PMC
November 2017
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