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  • Interview with Dr. Shigeki Watanabe on Research in Synaptic Membrane Dynamics

    Dr. Shigeki Watanabe, principle investigator of the department of Cell Biology at the Johns Hopkins University School of Medicine in Baltimore, held a workshop in Zürich, Switzerland on methods to study synaptic dynamics with millisecond precision. In collaboration with Dr. Andres Käch from the University of Zurich all workshop attendees enjoyed presentations and hands-on sessions on the EM ICE by Leica Microsystems with Light and Electrical Stimulation, revealing the latest developments in brain research. During this workshop Dr. Bernd Sägmüller from Leica Microsystems had the chance for an interview with Dr. Watanabe.
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  • Human NK Cell Development Requires CD56-mediated Motility and Formation of the Developmental Synapse

    While distinct stages of natural killer (NK) cell development have been defined, the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which, through contact-dependent mechanisms, promote the generation of mature, functional human NK cells from CD34+ precursors. We show that developing NK cells undergo unique, developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells, and that the relative motility of NK cells increases as they move through development in vitro and ex vivo.
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  • Visualization of Membrane Dynamics with Millisecond Temporal Resolution

    Application Note for Leica EM ICE, Leica EM AFS2 - Electrical stimulation of neurons combined with high-pressure freezing allows physiological activation of synaptic activity and precise control over the time frame of the induced synaptic activity.
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  • Botulinum Neurotoxin Type-A Enters a Non-Recycling Pool of Synaptic Vesicles

    Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity.
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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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  • Freeze-Fracture Replication of Pyramidal Cells

    Application Note for Leica EM HPM100 - Frozen samples (90 μm thick slices frozen by HPM100) were inserted into a double replica table and then fractured into two pieces at –130°C (after insertion of the tissue into BAF 060 the samples should be left in the chamber for 20 min to reach the –130°C).
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  • Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent

    Super-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we report an optimized optical clearing agent for high-resolution fluorescence imaging (SeeDB2). SeeDB2 matches the refractive indices of fixed tissues to that of immersion oil (1.518), thus minimizing both light scattering and spherical aberrations.
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  • Super-Resolution Microscopy of the Synaptic Active Zone

    At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM techniques can be used to obtain information on the organization of AZ proteins.
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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 Werner Zuschratter

    Werner Zuschratter's personal focus is on analyzing the neuronal network, meaning the contacts between nerve cells. Out of this reason he started doing super-resolution microscopy: “It gives us deeper insight into the synapses, into the synaptic machinery, into the molecules we would like to see. Before we could only do electron microscopy and now, with super-resolution, we also have access by light microscopy to the deeper structures inside the nerve system.”
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  • Webinar: Exploring Neurons and Synapses: Imaging Tools and Techniques

    In the coming years, considerable effort and resources will be directed at understanding the neural connections of the brain. During this webinar, we will examine many of the tools being used to study how neurons interact with one another at their synapses.
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  • Super-Resolution Microscopy Helped to Create the First 3D Model of a Synapse

    A research team from Göttingen, led by Prof. Silvio O. Rizzoli, managed to determine the copy numbers and positions of all important building blocks of a synapse for the first time. This allowed them to reconstruct the first scientifically accurate 3D model of a synapse.
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  • Video Interview with Stephan Sigrist

    Stephan Sigrist is professor for biology at the Freie Universität Berlin in Germany. His research focus are synapses, synaptic information transfer and processing between neurons in the developing drosophila larva. His aim is to understand how synapses actually get diversified in our brains.
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  • Webinar: "Flash-and-Freeze" Time-resolved Electron Microscopy

    Electron microscopy only captures a static image of a cell. What is the cell doing? What is the true sequence of events in a cellular process? We can make flip books from our micrographs that tell a story, but their arrangement can be influenced by the story we want to tell.
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  • Synaptic Vesicle Recycling: Steps and Principles

    Synaptic vesicle recycling is one of the best‐studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole.
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  • Capturing Cellular Dynamics with Millisecond Temporal Resolution

    The combination of two powerful techniques: optogenetics and high-pressure freezing now makes it possible to visualize a dynamic cellular activity with temporal resolution of 5 milliseconds. By coupling a flash of light with high-pressure freezing, the process of vesicle recycling at the synapses can now be imaged by electron microscopy.
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  • Webinar: Super-Resolution Imaging of Neurons

    In this webinar, Daniel Choquet, Xiaowei Zhuang, and Stephan Sigrist will discuss how super-resolution imaging can elucidate the inner workings of neurons at the single-molecule and macro-molecular levels using specialized probes and optical techniques they have helped design and pioneer in the field of neuroscience.
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  • Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

    Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact.
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  • Abstracts of the 3rd European Super-Resolution User-Club Meeting

    The 3rd meeting of the Leica Super-Resolution User Club was held from June 17th to 19th, 2013 in collaboration with Alberto Diaspro and the Italian Institute of Technology (IIT) in Genoa. Confocal and widefield super-resolution users from ten European countries took three days’ out to deepen their knowledge on super-resolution techniques and applications and make use of an opportunity for full exchange of experiences.
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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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  • Abstracts of the 2nd European Super-Resolution User-Club Meeting

    The 2nd meeting of the Leica Super-resolution User club was held from September 25 to 27, 2012 in collaboration with the Science for Life Laboratory at the Karolinska Institute, Stockholm, Sweden. With a mixture of engaging talks by key experts in the field of super-resolution microscopy and stimulating discussion sessions, the meeting proved as popular as last year’s event, attracting a wide range of scientists interested in both confocal and widefield super-resolution and sample preparation techniques.
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  • Sharp Live Images from the Mouse Brain

    To explore the most intricate structures of the brain in order to decipher how it functions – Stefan Hell’s team of researchers at the Max Planck Institute for Biophysical Chemistry in Göttingen has made a significant step closer to this goal. Using the STED microscopy developed by Hell, the scientists have, for the first time, managed to record detailed live images inside the brain of a living mouse.
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  • Two-color STED Microscopy of Living Synapses using a Single Laser-beam Pair

    The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores.
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  • Abstracts of the First European Super-Resolution User Club Meeting

    The first European Super-resolution User Club meeting took place from October 27 to 29 in Göttingen, Germany. Prof. Stefan Hell, the inventor of the STED technology, has hosted this first meeting. The user club is aimed at pioneering researchers from the European scientific community, who are early adopters and developers of super-resolution techniques.
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  • STED Nanoscopy of Actin Dynamics in Synapses deep inside Living Brain Slices

    It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo.
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  • A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms

    Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce ‘"miniSOG"’ (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2.
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  • Mapping Billions of Synapses with Microscopy and Mathematics

    A combination of widefield imaging techniques and image segmentation analysis enable researchers to map learning-induced functional changes in individual synapses throughout the hippocampus.
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  • The Missing Link to the Nanocosm of Life

    Fully understanding the functionality and complexity of the human central nervous system remains one of the major open questions in modern science. Stimulated emission depletion microscopy (STED) can be the method to reveal biological nanostructures
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  • Restless Receptors

    Synapses are the switch-points in our brain for information transmission, learning and memory. News studies and developments of imaging techniques have provided new insights into the dynamics of glutamate receptors. The use of superresolution technologies is making an essential contribution to this research.
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  • The Fate of Synaptic Vesicle Components upon Fusion

    Neurotransmitter release relies on the fusion of synaptic vesicles with the plasma membrane of synaptic boutons, which is followed by the recycling of vesicle components and formation of new vesicles. It is not yet clear whether upon fusion the vesicles persist as multimolecular patches in the plasma membrane, or whether they segregate into individual components.
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