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- ItemStudying dynamic processes of nano-sized objects in liquid using scanning transmission electron microscopy(Cambridge : JoVE, 2017) de Jong, Niels; Hermannsdörfer, JustusSamples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.
- ItemCathodoluminescence and TEM investigations of structural and optical properties of AlGaN on epitaxial laterally overgrown AlN/sapphire templates(Milton Park : Taylor & Francis, 2013) Zeimer, U.; Mogilatenko, A.; Kueller, V.; Knauer, A.; Weyers, M.Surface steps as high as 15 nm on up to 10 μm thick AlN layers grown on patterned AlN/sapphire templates play a major role for the structural and optical properties of AlxGa1−xN layers with x ≥ 0.5 grown subsequently by metalorganic vapour phase epitaxy. The higher the Ga content in these layers is, the stronger is the influence of the surface morphology on their properties. For x = 0.5 not only periodic inhomogeneities in the Al content due to growth of Ga-rich facets are observed by cathodoluminescence, but these facets give rise to additional dislocation formation as discovered by annular dark-field scanning transmission electron microscopy. For AlxGa1−xN layers with x = 0.8 the difference in Al content between facets and surrounding material is much smaller. Therefore, the threading dislocation density (TDD) is only defined by the TDD in the underlying epitaxially laterally overgrown (ELO) AlN layer. This way high quality Al0.8Ga0.2N with a thickness up to 1.5 μm and a TDD ≤ 5x108 cm−2 was obtained.
- ItemGaN-based radial heterostructure nanowires grown by MBE and ALD(Bristol : Institute of Physics Publishing, 2013) Lari, L.; Ross, I.M.; Walther, T.; Black, K.; Cheze, C.; Geelhaar, L.; Riechert, H.; Chalker, P.R.A combination of molecular beam epitaxy (MBE) and atomic layer deposition (ALD) was adopted to fabricate GaN-based core/shell NW structures. ALD was used to deposit a HfO2 shell of onto the MBE grown GaN NWs. Electron transparent samples were prepared by focussed ion beam methods and characterized using state-of-the-art analytical transmission and scanning transmission electron microscopy. The polycrystalline coating was found to be uniform along the whole length of the NWs. Photoluminescence and Raman spectroscopy analysis confirms that the HfO2 ALD coating does not add any structural defect when deposited on the NWs.
- ItemStudying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy([S.l.] : [s.n.], 2015) Peckys, Diana B.; de Jonge, NielsThis protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.
- ItemNanoscale spectroscopic imaging of GaAs-AlGaAs quantum well tube nanowires: Correlating luminescence with nanowire size and inner multishell structure(Berlin : De Gruyter, 2019) Prete, P.; Wolf, D.; Marzo, F.; Lovergine, N.The luminescence and inner structure of GaAs-AlGaAs quantum well tube (QWT) nanowires were studied using lowerature cathodoluminescence (CL) spectroscopic imaging, in combination with scanning transmission electron microscopy (STEM) tomography, allowing for the first time a robust correlation between the luminescence properties of these nanowires and their size and inner 3D structure down to the nanoscale. Besides the core luminescence and minor defects-related contributions, each nanowire showed one or more QWT peaks associated with nanowire regions of different diameters. The values of the GaAs shell thickness corresponding to each QWT peak were then determined from the nanowire diameters by employing a multishell growth model upon validation against experimental data (core diameter and GaAs and AlGaAs shell thickness) obtained from the analysis of the 3D reconstructed STEM tomogram of a GaAs-AlGaAs QWT nanowire. We found that QWT peak energies as a function of thus-estimated (3-7 nm) GaAs shell thickness are 40-120 meV below the theoretical values of exciton recombination for uniform QWTs symmetrically wrapped around a central core. However, the analysis of the 3D tomogram further evidenced azimuthal asymmetries as well as (azimuthal and axial) random fluctuations of the GaAs shell thickness, suggesting that the red-shift of QWT emissions is prominently due to carrier localization. The CL mapping of QWT emission intensities along the nanowire axis allowed to directly image the nanoscale localization of the emission, supporting the above picture. Our findings contribute to a deeper understanding of the luminescence-structure relationship in QWT nanowires and will foster their applications as efficient nanolaser sources for future monolithic integration onto silicon.
- ItemEditors' Choice - Precipitation of Suboxides in Silicon, their Role in Gettering of Copper Impurities and Carrier Recombination(Pennington, NJ : ECS, 2020) Kissinger, G.; Kot, D.; Huber, A.; Kretschmer, R.; Müller, T.; Sattler, A.This paper describes a theoretical investigation of the phase composition of oxide precipitates and the corresponding emission of self-interstitials at the minimum of the free energy and their evolution with increasing number of oxygen atoms in the precipitates. The results can explain the compositional evolution of oxide precipitates and the role of self-interstitials therein. The formation of suboxides at the edges of SiO2 precipitates after reaching a critical size can explain several phenomena like gettering of Cu by segregation to the suboxide region and lifetime reduction by recombination of minority carriers in the suboxide. It provides an alternative explanation, based on minimized free energy, to the theory of strained and unstrained plates. A second emphasis was payed to the evolution of the morphology of oxide precipitates. Based on the comparison with results from scanning transmission electron microscopy the sequence of morphology evolution of oxide precipitates was deduced. It turned out that it is opposite to the sequence assumed until now. © 2020 The Author(s). Published on behalf of The Electrochemical Society by IOP Publishing Limited.
- ItemVisualisation of HER2 homodimers in single cells from HER2 overexpressing primary formalin fixed paraffin embedded tumour tissue(London : BioMed Central, 2019) Peckys, D.B.; Hirsch, D.; Gaiser, T.; De, Jonge, N.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/μm2. 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. © 2019 The Author(s).
- 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.