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  • TIRF Publication List

    This monthly updated references list presents current papers using Leica AM TIRF in the major application fields for TIRF microscopy.
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  • Initiation of Lamellipodia and Ruffles Involves Cooperation Between mDia1 and the Arp2/3 Complex

    Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by the Arp2/3 complex. Although regulation of Arp2/3 complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here, we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles.
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  • Abstracts of the 6th European Super-Resolution User-Club Meeting

    The 6th European Super-Resolution User Club Meeting was held in collaboration with Dr. Timo Zimmermann, CRG, and Dr. Pablo Loza-Alvarez, ICFO, Barcelona. According to the founding principle of the club of keeping close to science, both imaging facilities at the CRG and the ICFO opened their doors to the User Club members, allowing them to explore exciting super-resolution and and nanoscopy applications. The meeting agenda covered highly relevant talks around this year’s central theme “Core Facilities and Super-Resolution Microscopy”, as well as plenty of opportunities to network amongst super-resolution users from different European countries. Here we present the abstracts of the talks held during the meeting.
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  • The Actin Cytoskeleton Modulates the Activation of iNKT Cells by Segregating CD1d Nanoclusters on Antigen-Presenting Cells

    The ability of invariant natural killer T (iNKT) cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton.
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  • STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics

    Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS ) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells.
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  • A Lipid Bound Actin Meshwork Organizes Liquid Phase Separation in Model Membranes

    The eukaryotic cell membrane is connected to a dense actin rich cortex. We present FCS and STED experiments showing that dense membrane bound actin networks have severe influence on lipid phase separation. Our results reveal a mechanism how cells may prevent macroscopic demixing of their membrane components, while at the same time regulate the local membrane composition.
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  • Video Talk by Daniel Axelrod: Total Internal Reflection Fluorescence (TIRF) Microscopy

    Total Internal Reflection Fluorescence (TIRF) Microscopy is a technique that only illuminates dye molecules near a surface. In this video, the pioneer of TIRF Microscopy describes what this technique is used for, explains the principles of the evanescent wave, gives many examples of different microscope configurations used in TIRF, and shows how polarized light TIRF can be used to image membrane orientation.
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  • Universal PAINT – Dynamic Super-Resolution Microscopy

    Super-resolution microscopy techniques have revolutionized biology for the last ten years. With their help cellular components can now be visualized at the size of a protein. Nevertheless, imaging living cells is a challenge for most of the super-resolution principles.
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  • Influencing Factors and Applicability of the Viability EMA-qPCR for a Detection and Quantification of Campylobacter Cells from Water Samples

    In recent years, increasing numbers of human campylobacteriosis cases caused by contaminated water have been reported. As the culture-based detection of Campylobacter is time consuming and can yield false-negative results, the suitability of a quantitative real-time PCR method in combination with an ethidium monoazide pretreatment of samples (EMA-qPCR) for the rapid, quantitative detection of viable Campylobacter cells from water samples was investigated.
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  • ICln: A New Regulator of Non-Erythroid 4.1R Localisation and Function

    To optimise the efficiency of cell machinery, cells can use the same protein (often called a hub protein) to participate in different cell functions by simply changing its target molecules. There are large data sets describing protein-protein interactions ("interactome") but they frequently fail to consider the functional significance of the interactions themselves.
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  • A New Probe for Super-Resolution Imaging of Membranes Elucidates Trafficking Pathways

    The molecular composition of the organelles involved in membrane recycling is difficult to establish as a result of the absence of suitable labeling tools. We introduce in this paper a novel probe, named membrane-binding fluorophore-cysteine-lysine-palmitoyl group (mCLING), which labels the plasma membrane and is taken up during endocytosis.
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  • Video Interview with William Hughes

    William Hughes works at the Garvan Institute of Medical Research, Sydney (Australia). In his Lab Head position he is interested in the causes of diabetes particularly looking at changes in exocytic behavior of pancreatic beta cells as well as fat and muscle cells. TIRF microscopy is predestined for researchers looking at cellular processes near the cytoplasmic membrane.
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  • Glucose-Stimulated Calcium Dynamics in Islets of Langerhans in Acute Mouse Pancreas Tissue Slices

    In endocrine cells within islets of Langerhans calcium ions couple cell stimulation to hormone secretion. Since the advent of modern fluorimetry, numerous in vitro studies employing primarily isolated mouse islets have investigated the effects of various secretagogues on cytoplasmic calcium, predominantly in insulin-secreting beta cells. Due to technical limitations, insights of these studies are inherently limited to a rather small subpopulation of outermost cells.
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  • Functional Connectivity in Islets of Langerhans from Mouse Pancreas Tissue Slices

    We propose a network representation of electrically coupled beta cells in islets of Langerhans. Beta cells are functionally connected on the basis of correlations between calcium dynamics of individual cells, obtained by means of confocal laser-scanning calcium imaging in islets from acute mouse pancreas tissue slices.
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  • Capturing Neurotransmitter Receptors and Ion Channels

    Neurotransmitter receptors and ion channels in the central nervous system are localized to synaptic and extrasynaptic membrane compartments of pre- and postsynaptic elements of neurons. The impact of the activation of these proteins on synaptic integration and regulation of transmitter release depends on their precise location relative to synapses, as well as on the density and coupling of molecules in microcompartments of the cells. High-resolution qualitative and quantitative visualization of membranebound receptors and ion channels is, therefore, essential for understanding their roles in cell communication.
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  • Protein Transport Processes at the Apical Membrane of Polarized Epithelial Cells

    Due to their special role in organ function and the exchange of biological components some body cells developed certain polarization characteristics. These are reflected in differences of their plasma membrane composition. The essential and fascinating task of polarized protein transport in epithelial cells is to get the right protein into the right membrane.
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  • Total Internal Reflection Fluorescence (TIRF) Microscopy

    Total internal reflection fluorescence (TIRF) is a special technique in fluorescence microscopy developed by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s. TIRF microscopy delivers images with an outstandingly high axial resolution below 100 nm. This allows the observation of membrane-associated processes.
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  • Applications of TIRF Microscopy in Life Science Research

    The special feature of TIRF microscopy is the employment of an evanescent field for fluorophore excitation. Unlike standard widefield fluorescence illumination procedures with arc lamps, LEDs or lasers, the evanescent field only penetrates the specimen by about 100 nm starting from the coverslip/medium interface.
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  • The Patch-Clamp Technique

    Especially in neuroscience, the physiology of ion channels has always been a major topic of interest. The development of the patch-clamp technique in the late 1970s has given electrophysiologists new prospects. It allows high-resolution current recordings not only of whole cells, but also of excised cellular patches. Even single-channel opening events can be investigated. However, with its complex technical, physical and biological background, the need for highly sensitive equipment and the huge amount of skills required of the experimenter, electrophysiology is still one of the most challenging methods in daily laboratory work.
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  • TIRF Microscopy of the Apical Membrane of Polarized Epithelial Cells

    Application of TIRF microscopy (Total Internal Reflection Fluorescence) allows the visualization of structures at the apical surface of polarized epithelial cells that have been hidden in conventional fluorescence microscopy images. Hence, the approach reveals new insights into the composition of this characteristic cell pole that elucidate processes in apical protein trafficking.
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  • Apical Cargo Traverses Endosomal Compartments on the Passage to the Cell Surface

    Epithelial polarity is based on intracellular sorting machinery that maintains the asymmetric distribution of lipids and proteins to the cell surface. Dependent on their lipid raft affinity, newly synthesized apical polypeptides are segregated into distinct vesicle populations subsequent to the passage through the Golgi apparatus.
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  • Exploring Cell Logistics

    Using TIRF microscopy, scientists have been able to take a closer look at intracellular transport processes with the example of the galactose-binding protein Galectin-3, which has been identified as a potential apical sorting receptor.
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