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- ItemCombining super-resolution microcopy with neuronal network recording using magnesium fluoride thin films as cover layer for multi-electrode array technology(Berlin : Nature Publishing, 2019) Schmidl, Lars; Schmidl, Gabriele; Gawlik, Annett; Dellith, Jan; Hübner, Uwe; Tympel, Volker; Schmidl, Frank; Plentz, Jonathan; Geis, Christian; Haselmann, HolgerWe present an approach for fabrication of reproducible, chemically and mechanically robust functionalized layers based on MgF2 thin films on thin glass substrates. These show great advantages for use in super-resolution microscopy as well as for multi-electrode-array fabrication and are especially suited for combination of these techniques. The transparency of the coated substrates with the low refractive index material is adjustable by the layer thickness and can be increased above 92%. Due to the hydrophobic and lipophilic properties of the thin crystalline MgF2 layers, the temporal stable adhesion needed for fixation of thin tissue, e.g. cryogenic brain slices is given. This has been tested using localization-based super-resolution microscopy with currently highest spatial resolution in light microscopy. We demonstrated that direct stochastic optical reconstruction microscopy revealed in reliable imaging of structures of central synapses by use of double immunostaining of post- (homer1 and GluA2) and presynaptic (bassoon) marker structure in a 10 µm brain slice without additional fixing of the slices. Due to the proven additional electrical insulating effect of MgF2 layers, surfaces of multi-electrode-arrays were coated with this material and tested by voltage-current-measurements. MgF2 coated multi-electrode-arrays can be used as a functionalized microscope cover slip for combination with live-cell super-resolution microscopy.
- ItemVideo-rate multi-color structured illumination microscopy with simultaneous real-time reconstruction(Berlin : Nature Publishing, 2019) Markwirth, A; Lachetta, Mario; Mönkemöller, V.; Heintzmann, Rainer; Hübner, Wolfgang; Huser, Thomas; Müller, MarcelSuper-resolved structured illumination microscopy (SR-SIM) is among the fastest fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-build instruments are able to deliver two-fold resolution enhancement with high acquisition speed. SR-SIM is usually a two-step process, with raw-data acquisition and subsequent, time-consuming post-processing for image reconstruction. In contrast, wide-field and (multi-spot) confocal techniques produce high-resolution images instantly. Such immediacy is also possible with SR-SIM, by tight integration of a video-rate capable SIM with fast reconstruction software. Here we present instant SR-SIM by VIGOR (Video-rate Immediate GPU-accelerated Open-Source Reconstruction). We demonstrate multi-color SR-SIM at video frame-rates, with less than 250 ms delay between measurement and reconstructed image display. This is achieved by modifying and extending high-speed SR-SIM image acquisition with a new, GPU-enhanced, network-enabled image-reconstruction software. We demonstrate high-speed surveying of biological samples in multiple colors and live imaging of moving mitochondria as an example of intracellular dynamics.
- ItemStructural insights into heme binding to IL-36α proinflammatory cytokine(Berlin : Nature Publishing, 2019) Wißbrock, Amelie; Goradia, Nishit; Kumar, Amit; Paul George, Ajay Abisheck; Kühl, Toni; Bellstedt, Peter; Ramachandran, Ramadurai; Hoffmann, Patrick; Galler, Kerstin; Popp, Jürgen; Neugebauer, Ute; Hampel, Kornelia; Zimmermann, Bastian; Adam, Susanne; Wiendl, Maximilian; Krönke, Gerhard; Hamza, Iqbal; Heinemann, Stefan H.; Frey, Silke; Hueber, Axel J.; Ohlenschläger, Oliver; Imhof, DianaCytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.
- ItemIn Vitro Selection of Specific DNA Aptamers Against the Anti-Coagulant Dabigatran Etexilate(Berlin : Nature Publishing, 2018) Aljohani, Maher M; Chinnappan, Raja; Eissa, Shimaa; Alsager, Omar A; Weber, Karina; Cialla-May, Dana; Popp, Jürgen; Zourob, MohammedDabigatran Etexilate (PRADAXA) is a new oral anticoagulant increasingly used for a number of blood thrombosis conditions, prevention of strokes and systemic emboli among patients with atrial fibrillation. It provides safe and adequate anticoagulation for prevention and treatment of thrombus in several clinical settings. However, anticoagulation therapy can be associated with an increased risk of bleeding. There is a lack of specific laboratory tests to determine the level of this drug in blood. This is considered the most important obstacles of using this medication, particularly for patients with trauma, drug toxicity, in urgent need for surgical interventions or uncontrolled bleeding. In this work, we performed Systematic evolution of ligands by exponential enrichment (SELEX) to select specific DNA aptamers against dabigatran etexilate. Following multiple rounds of selection and enrichment with a randomized 60-mer DNA library, specific DNA aptamers for dabigatran were selected. We investigated the affinity and specificity of generated aptamers to the drug showing dissociation constants (Kd) ranging from 46.8–208 nM. The most sensitive aptamer sequence was selected and applied in an electrochemical biosensor to successfully achieve 0. 01 ng/ml level of detection of the target drug. With further improvement of the assay and optimization, these aptamers would replace conventional antibodies for developing detection assays in the near future.Dabigatran Etexilate (PRADAXA) is a new oral anticoagulant increasingly used for a number of blood thrombosis conditions, prevention of strokes and systemic emboli among patients with atrial fibrillation. It provides safe and adequate anticoagulation for prevention and treatment of thrombus in several clinical settings. However, anticoagulation therapy can be associated with an increased risk of bleeding. There is a lack of specific laboratory tests to determine the level of this drug in blood. This is considered the most important obstacles of using this medication, particularly for patients with trauma, drug toxicity, in urgent need for surgical interventions or uncontrolled bleeding. In this work, we performed Systematic evolution of ligands by exponential enrichment (SELEX) to select specific DNA aptamers against dabigatran etexilate. Following multiple rounds of selection and enrichment with a randomized 60-mer DNA library, specific DNA aptamers for dabigatran were selected. We investigated the affinity and specificity of generated aptamers to the drug showing dissociation constants (Kd) ranging from 46.8–208 nM. The most sensitive aptamer sequence was selected and applied in an electrochemical biosensor to successfully achieve 0. 01 ng/ml level of detection of the target drug. With further improvement of the assay and optimization, these aptamers would replace conventional antibodies for developing detection assays in the near future.
- ItemSurface deep profile synchrotron studies of mechanically modified top-down silicon nanowires array using ultrasoft X-ray absorption near edge structure spectroscopy(Berlin : Nature Publishing, 2019) Turishchev, S.Yu.; Parinova, V.E.; Pisliaruka, Aleksandra; Koyuda, D.A.; Yermukhamed, Dana; Ming, Tingsen; Ovsyannikov, Ruslan; Smirnov, Dmitriy; Makarova, Anna; Sivakov, VladimirAtomic, electronic structure and composition of top-down metal-assisted wet-chemically etched silicon nanowires were studied by synchrotron radiation based X-ray absorption near edge structure technique. Local surrounding of the silicon and oxygen atoms in silicon nanowires array was studied on as-prepared nanostructured surfaces (atop part of nanowires) and their bulk part after, first time applied, in-situ mechanical removal atop part of the formed silicon nanowires. Silicon suboxides together with disturbed silicon dioxide were found in the composition of the formed arrays that affects the electronic structure of silicon nanowires. The results obtained by us convincingly testify to the homogeneity of the phase composition of the side walls of silicon nanowires and the electronic structure in the entire length of the nanowire. The controlled formation of the silicon nanowires array may lead to smart engineering of its atomic and electronic structure that influences the exploiting strategy of metal-assisted wet-chemically etched silicon nanowires as universal matrices for different applications.
- ItemMolecular Characterization and Comparative Genomics of Clinical Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains in Sweden(Berlin : Nature Publishing, 2019) Bai, Xiangning; Zhang, Ji; Ambikan, Anoop; Jernberg, Cecilia; Ehricht, Ralf; Scheutz, Flemming; Xiong, Yanwen; Matussek, AndreasHybrid E. coli pathotypes are representing emerging public health threats with enhanced virulence from different pathotypes. Hybrids of Shiga toxin-producing and enterotoxigenic E. coli (STEC/ETEC) have been reported to be associated with diarrheal disease and hemolytic uremic syndrome (HUS) in humans. Here, we identified and characterized four clinical STEC/ETEC hybrids from diarrheal patients with or without fever or abdominal pain and healthy contact in Sweden. Rare stx2 subtypes were present in STEC/ETEC hybrids. Stx2 production was detectable in stx2a and stx2e containing strains. Different copies of ETEC virulence marker, sta gene, were found in two hybrids. Three sta subtypes, namely, sta1, sta4 and sta5 were designated, with sta4 being predominant. The hybrids represented diverse and rare serotypes (O15:H16, O187:H28, O100:H30, and O136:H12). Genome-wide phylogeny revealed that these hybrids exhibited close relatedness with certain ETEC, STEC/ETEC hybrid and commensal E. coli strains, implying the potential acquisition of Stx-phages or/and ETEC virulence genes in the emergence of STEC/ETEC hybrids. Given the emergence and public health significance of hybrid pathotypes, a broader range of virulence markers should be considered in the E. coli pathotypes diagnostics, and targeted follow up of cases is suggested to better understand the hybrid infection.
- ItemSymmetry breaking-induced magnetic Fano resonances in densely packed arrays of symmetric nanotrimers(Berlin : Nature Publishing, 2019) Wang, Ning; Zeisberger, Matthias; Hübner, Uwe; Giannini, Vincenzo; Schmidt, Markus A.Due to unique properties and great design flexibilities, Fano resonances represent one of the most promising optical features mediated by metallic nanostructures, while the excitation of some Fano modes is impossible due to symmetry reasons. The aim of this work is to show that dense lattice arrangements can have a profound impact on the optical properties of nanostructures and, in particular, can enable the excitation of otherwise dark modes. Here, we demonstrate this concept using the example of rectangular arrays of symmetric trimers packed so densely that the coupling between neighbouring unit cells imposes a symmetry break, enabling the excitation of magnetic Fano resonances. We found that in experiments as well as in simulations, electric and magnetic Fano resonances can be simultaneously formed in cases where the inter-trimer distances are sufficiently small. By analysing the transition from an isolated trimer mode into a regime of strong near-field coupling, we show that by modifying the rectangular unit cell lengths due to the symmetry mismatch between lattice and trimer, two types of Fano resonances can be found, especially magnetic Fano resonances with loop-type magnetic field distributions within the centre of each trimer, which can be either enhanced or suppressed. In addition, the influence of the refractive index environment was measured, showing sensitivity values of approximately 300 nm/RIU. Our work provides fundamental insights into the interaction of the lattice and nanostructure response and paves the way towards the observation of novel optical excitations.
- ItemNew perspectives for viability studies with high-content analysis Raman spectroscopy (HCA-RS)(Berlin : Nature Publishing, 2019) Mondol, Abdullah S.; Töpfer, Natalie; Rüger, Jan; Neugebauer, Ute; Popp, Jürgen; Schie, Iwan W.Raman spectroscopy has been widely used in clinical and molecular biological studies, providing high chemical specificity without the necessity of labels and with little-to-no sample preparation. However, currently performed Raman-based studies of eukaryotic cells are still very laborious and time-consuming, resulting in a low number of sampled cells and questionable statistical validations. Furthermore, the approach requires a trained specialist to perform and analyze the experiments, rendering the method less attractive for most laboratories. In this work, we present a new high-content analysis Raman spectroscopy (HCA-RS) platform that overcomes the current challenges of conventional Raman spectroscopy implementations. HCA-RS allows sampling of a large number of cells under different physiological conditions without any user interaction. The performance of the approach is successfully demonstrated by the development of a Raman-based cell viability assay, i.e., the effect of doxorubicin concentration on monocytic THP-1 cells. A statistical model, principal component analysis combined with support vector machine (PCA-SVM), was found to successfully predict the percentage of viable cells in a mixed population and is in good agreement to results obtained by a standard cell viability assay. This study demonstrates the potential of Raman spectroscopy as a standard high-throughput tool for clinical and biological applications.
- ItemFabrication of self-assembled spherical Gold Particles by pulsed UV Laser Treatment(Berlin : Nature Publishing, 2018) Schmidl, Gabriele; Jia, Guobin; Gawlik, Annett; Kreusch, Jonathan; Schmidl, Frank; Dellith, Jan; Dathe, André; Lin, Zhan-Hong; Huang, Jer-Shing; Plentz, JonathanWe report on the fabrication of spherical Au spheres by pulsed laser treatment using a KrF excimer laser (248 nm, 25 ns) under ambient conditions as a fast and high throughput fabrication technique. The presented experiments were realized using initial Au layers of 100 nm thickness deposited on optically transparent and low cost Borofloat glass or single-crystalline SrTiO3 substrates, respectively. High (111)-orientation and smoothness (RMS ≈ 1 nm) are the properties of the deposited Au layers before laser treatment. After laser treatment, spheres with size distribution ranging from hundreds of nanometers up to several micrometers were produced. Single-particle scattering spectra with distinct plasmonic resonance peaks are presented to reveal the critical role of optimal irradiation parameters in the process of laser induced particle self-assembly. The variation of irradiation parameters like fluence and number of laser pulses influences the melting, dewetting and solidification process of the Au layers and thus the formation of extremely well shaped spherical particles. The gold layers on Borofloat glass and SrTiO3 are found to show a slightly different behavior under laser treatment. We also discuss the effect of substrates.We report on the fabrication of spherical Au spheres by pulsed laser treatment using a KrF excimer laser (248 nm, 25 ns) under ambient conditions as a fast and high throughput fabrication technique. The presented experiments were realized using initial Au layers of 100 nm thickness deposited on optically transparent and low cost Borofloat glass or single-crystalline SrTiO3 substrates, respectively. High (111)-orientation and smoothness (RMS ≈ 1 nm) are the properties of the deposited Au layers before laser treatment. After laser treatment, spheres with size distribution ranging from hundreds of nanometers up to several micrometers were produced. Single-particle scattering spectra with distinct plasmonic resonance peaks are presented to reveal the critical role of optimal irradiation parameters in the process of laser induced particle self-assembly. The variation of irradiation parameters like fluence and number of laser pulses influences the melting, dewetting and solidification process of the Au layers and thus the formation of extremely well shaped spherical particles. The gold layers on Borofloat glass and SrTiO3 are found to show a slightly different behavior under laser treatment. We also discuss the effect of substrates.
- ItemMagnetically induced transparency of a quantum metamaterial composed of twin flux qubits(Berlin : Nature Publishing, 2018) Shulga, Kirill; Il'ichev, Evgeny; Fistul, Mikhail V.; Besedin, I.S.; Butz, Susanne; Astafiev, Oleg; Hübner, Uwe; Ustinov, Alexey V.Quantum theory is expected to govern the electromagnetic properties of a quantum metamaterial, an artificially fabricated medium composed of many quantum objects acting as artificial atoms. Propagation of electromagnetic waves through such a medium is accompanied by excitations of intrinsic quantum transitions within individual meta-atoms and modes corresponding to the interactions between them. Here we demonstrate an experiment in which an array of double-loop type superconducting flux qubits is embedded into a microwave transmission line. We observe that in a broad frequency range the transmission coefficient through the metamaterial periodically depends on externally applied magnetic field. Field-controlled switching of the ground state of the meta-atoms induces a large suppression of the transmission. Moreover, the excitation of meta-atoms in the array leads to a large resonant enhancement of the transmission. We anticipate possible applications of the observed frequency-tunable transparency in superconducting quantum networks.