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- ItemEosinophils and Neutrophils-Molecular Differences Revealed by Spontaneous Raman, CARS and Fluorescence Microscopy(Basel : MDPI, 2020) Dorosz, Aleksandra; Grosicki, Marek; Dybas, Jakub; Matuszyk, Ewelina; Rodewald, Marko; Meyer, Tobias; Popp, Jürgen; Malek, Kamilla; Baranska, MalgorzataLeukocytes are a part of the immune system that plays an important role in the host's defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells' types. To prove this hypothesis, UV-Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process.
- ItemThe 2018 correlative microscopy techniques roadmap(Bristol : IOP Publishing, 2018) Ando, Toshio; Bhamidimarri, Satya Prathyusha; Brending, Niklas; Colin-York, H; Collinson, Lucy; De Jonge, Niels; de Pablo, P J; Debroye, Elke; Eggeling, Christian; Franck, Christian; Fritzsche, Marco; Gerritsen, Hans; Giepmans, Ben N G; Grunewald, Kay; Hofkens, Johan; Hoogenboom, Jacob P; Janssen, Kris P F; Kaufmann, Rainer; Klumpermann, Judith; Kurniawan, Nyoman; Kusch, Jana; Liv, Nalan; Parekh, Viha; Peckys, Diana B; Rehfeldt, Florian; Reutens, David C; Roeffaers, Maarten B J; Salditt, Tim; Schaap, Iwan A T; Schwarz, Ulrich S; Verkade, Paul; Vogel, Michael W; Wagner, Richard; Winterhalter, Mathias; Yuan, Haifeng; Zifarelli, GiovanniDevelopments 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.
- 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.
- ItemA guide to super-resolution fluorescence microscopy(New York, NY : Rockefeller Univ. Press, 2010) Schermelleh, L.; Heintzmann, R.; Leonhardt, H.For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells. © 2010 Schermelleh et al.
- 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.
- ItemVinculin binding angle in podosomes revealed by high resolution microscopy(San Francisco, CA : Public Library of Science, 2014) Walde, M.; Monypenny, J.; Heintzmann, R.; Jones, G.E.; Cox, S.Podosomes are highly dynamic actin-rich adhesive structures formed predominantly by cells of the monocytic lineage, which degrade the extracellular matrix. They consist of a core of F-actin and actin-regulating proteins, surrounded by a ring of adhesion-associated proteins such as vinculin. We have characterised the structure of podosomes in macrophages, particularly the structure of the ring, using three super-resolution fluorescence microscopy techniques: stimulated emission depletion microscopy, structured illumination microscopy and localisation microscopy. Rather than being round, as previously assumed, we found the vinculin ring to be created from relatively straight strands of vinculin, resulting in a distinctly polygonal shape. The strands bind preferentially at angles between 116° and 135°. Furthermore, adjacent vinculin strands are observed nucleating at the corners of the podosomes, suggesting a mechanism for podosome growth.
- ItemThe cytosolic domain of Pex22p stimulates the Pex4p-dependent ubiquitination of the PTS1-receptor(San Francisco, CA : Public Library of Science, 2014) El Magraoui, Fouzi; Schrötter, Andreas; Brinkmeier, Rebecca; Kunst, Lena; Mastalski, Thomas; Müller, Thorsten; Marcus, Katrin; Meyer, Helmut E.; Girzalsky, Wolfgang; Erdmann, Ralf; Platta, Harald W.Peroxisomal biogenesis is an ubiquitin-dependent process because the receptors required for the import of peroxisomal matrix proteins are controlled via their ubiquitination status. A key step is the monoubiquitination of the import receptor Pex5p by the ubiquitin-conjugating enzyme (E2) Pex4p. This monoubiquitination is supposed to take place after Pex5p has released the cargo into the peroxisomal matrix and primes Pex5p for the extraction from the membrane by the mechano-enzymes Pex1p/Pex6p. These two AAA-type ATPases export Pex5p back to the cytosol for further rounds of matrix protein import. Recently, it has been reported that the soluble Pex4p requires the interaction to its peroxisomal membrane-anchor Pex22p to display full activity. Here we demonstrate that the soluble C-terminal domain of Pex22p harbours its biological activity and that this activity is independent from its function as membrane-anchor of Pex4p. We show that Pex4p can be functionally fused to the trans-membrane segment of the membrane protein Pex3p, which is not directly involved in Pex5p-ubiquitination and matrix protein import. However, this Pex3(N)-Pex4p chimera can only complement the double-deletion strain pex4Δ/pex22Δ and ensure optimal Pex5p-ubiquitination when the C-terminal part of Pex22p is additionally expressed in the cell. Thus, while the membrane-bound portion Pex22(N)p is not required when Pex4p is fused to Pex3(N)p, the soluble Pex22(C)p is essential for peroxisomal biogenesis and efficient monoubiquitination of the import receptor Pex5p by the E3-ligase Pex12p in vivo and in vitro. The results merge into a picture of an ubiquitin-conjugating complex at the peroxisomal membrane consisting of three domains: the ubiquitin-conjugating domain (Pex4p), a membrane-anchor domain (Pex22(N)p) and an enhancing domain (Pex22(C)p), with the membrane-anchor domain being mutually exchangeable, while the Ubc- and enhancer-domains are essential.
- ItemA new human adipocyte model with PTEN haploinsufficiency(Abingdon : Taylor and Francis Inc., 2020) Kässner F.; Kirstein A.; Händel N.; Schmid G.L.; Landgraf K.; Berthold A.; Tannert A.; Schaefer M.; Wabitsch M.; Kiess W.; Körner A.; Garten A.Few human cell strains are suitable and readily available as in vitro adipocyte models. We used resected lipoma tissue from a patient with germline phosphatase and tensin homolog (PTEN) haploinsufficiency to establish a preadipocyte cell strain termed LipPD1 and aimed to characterize cellular functions and signalling pathway alterations in comparison to the established adipocyte model Simpson-Golabi-Behmel-Syndrome (SGBS) and to primary stromal-vascular fraction cells. We found that both cellular life span and the capacity for adipocyte differentiation as well as adipocyte-specific functions were preserved in LipPD1 and comparable to SGBS adipocytes. Basal and growth factor-stimulated activation of the PI3 K/AKT signalling pathway was increased in LipPD1 preadipocytes, corresponding to reduced PTEN levels in comparison to SGBS cells. Altogether, LipPD1 cells are a novel primary cell model with a defined genetic lesion suitable for the study of adipocyte biology. © 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
- ItemComputational tissue staining of non-linear multimodal imaging using supervised and unsupervised deep learning(Washington, DC : OSA, 2021) Pradhan, Pranita; Meyer, Tobias; Vieth, Michael; Stallmach, Andreas; Waldner, Maximilian; Schmitt, Michael; Popp, Juergen; Bocklitz, ThomasHematoxylin and Eosin (H&E) staining is the 'gold-standard' method in histopathology. However, standard H&E staining of high-quality tissue sections requires long sample preparation times including sample embedding, which restricts its application for 'real-time' disease diagnosis. Due to this reason, a label-free alternative technique like non-linear multimodal (NLM) imaging, which is the combination of three non-linear optical modalities including coherent anti-Stokes Raman scattering, two-photon excitation fluorescence and second-harmonic generation, is proposed in this work. To correlate the information of the NLM images with H&E images, this work proposes computational staining of NLM images using deep learning models in a supervised and an unsupervised approach. In the supervised and the unsupervised approach, conditional generative adversarial networks (CGANs) and cycle conditional generative adversarial networks (cycle CGANs) are used, respectively. Both CGAN and cycle CGAN models generate pseudo H&E images, which are quantitatively analyzed based on mean squared error, structure similarity index and color shading similarity index. The mean of the three metrics calculated for the computationally generated H&E images indicate significant performance. Thus, utilizing CGAN and cycle CGAN models for computational staining is beneficial for diagnostic applications without performing a laboratory-based staining procedure. To the author's best knowledge, it is the first time that NLM images are computationally stained to H&E images using GANs in an unsupervised manner.
- ItemTowards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes(New York, NY [u.a.] : Hindawi Publ. Corp., 2017) Menzel, Sarah; Finocchiaro, Nicole; Donay, Christine; Thiebes, Anja Lena; Hesselmann, Felix; Arens, Jutta; Djeljadini, Suzana; Wessling, Matthias; Schmitz-Rode, Thomas; Jockenhoevel, Stefan; Cornelissen, Christian GabrielIn patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.