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  • Researchers Find a “Digital” Mechanism Behind Neuronal Changes from Learning

    Researchers Find a “Digital” Mechanism Behind Neuronal Changes from Learning

    Neurons react to learning and memory by activating synaptic connections. The mechanisms behind this fundamental process are complex and poorly understood. Researchers at Thomas Jefferson University have found that neuron plasticity operates in a “digital” fashion through nanomodules of discrete size that multiply and strengthen neuronal connections upon stimulation. This breakthrough was published on April 23rd in the journal Nature Neuroscience.
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  • Basal Body Positioning and Anchoring in the Multiciliated Cell Paramecium Tetraurelia: Roles of OFD1 and VFL3

    Basal Body Positioning and Anchoring in the Multiciliated Cell Paramecium Tetraurelia: Roles of OFD1 and VFL3

    The development of a ciliary axoneme requires the correct docking of the basal body at cytoplasmic vesicles or plasma membrane. In the multiciliated cell Paramecium, three conserved proteins, FOR20, Centrin 2, and Centrin 3 participate in this process, FOR20 and Centrin 2 being involved in the assembly of the transition zone. We investigated the function of two other evolutionary conserved proteins, OFD1 and VFL3, likely involved in this process.
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  • Abstracts of the 7th European Super-Resolution User-Club Meeting

    Abstracts of the 7th European Super-Resolution User-Club Meeting

    The 7th Super-Resolution User Club Meeting was held in collaboration with Prof Pavel Hozák , at the Institute of Molecular Genetics of the ASCR in Prague. Keeping the event close to science is one of the founding principles of the event, allowing all participants to network, share and explore exciting new super-resolution and nanoscopy applications. Central to this are the scientific talks given during the meeting, with this cutting-edge microscopy technique as their central theme. A wide selection of topics were covered, prompting interesting discussions during the workshops.
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  • Free Webinar On-Demand: Enhance Your Imaging with Super-Resolution

    Free Webinar On-Demand: Enhance Your Imaging with Super-Resolution

    This webinar features the scientific results of two scientists Dr. Sebastian Haensch of the Center for Advanced Imaging at the Heinrich Heine-University in Düsseldorf, Germany and Dr. Imre Gaspar of the European Molecular Biology Laboratory, Developmental Biology Unit, Heidelberg, Germany. Both researchers explain how they used the Leica TCS SP8 and HyVolution 2 to dramatically enhance their imaging results.
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  • Actin-Dependent Vacuolar Occupancy of the Cell Determines Auxin-Induced Growth Repression

    Actin-Dependent Vacuolar Occupancy of the Cell Determines Auxin-Induced Growth Repression

    The cytoskeleton is an early attribute of cellular life, and its main components are composed of conserved proteins. The actin cytoskeleton has a direct impact on the control of cell size in animal cells, but its mechanistic contribution to cellular growth in plants remains largely elusive. Here, we reveal a role of actin in regulating cell size in plants. The actin cytoskeleton shows proximity to vacuoles, and the phytohormone auxin not only controls the organization of actin filaments but also impacts vacuolar morphogenesis in an actin-dependent manner.
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  • Super-Resolution Optical Microscopy of Lipid Plasma Membrane Dynamics

    Super-Resolution Optical Microscopy of Lipid Plasma Membrane Dynamics

    Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid–protein interactions and the traditional lipid ‘raft’ theory.
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  • Abstracts of the 6th European Super-Resolution User-Club Meeting

    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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  • Measuring the 3D STED-PSF with a new Type of Fluorescent Beads

    Measuring the 3D STED-PSF with a new Type of Fluorescent Beads

    A new type of fluorescent bead is presented by GATTAquant. These beads, called GATTA-Beads, are characterized by a small diameter (23 nm), high intensity and size uniformity. In combination with state-of the-art STED microscopes such as the Leica TCS SP8 STED 3X and high-end image restoration methods available in the Huygens Software, it is shown that these new beads can be used for accurate STED PSF characterization in 3D. Furthermore, it is shown that the measured 3D STED-PSF can be used to improve image restoration quality in combination with STED deconvolution methods available in the Huygens Software.
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  • Mirror-Enhanced Super-Resolution Microscopy

    Mirror-Enhanced Super-Resolution Microscopy

    Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes.
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  • The Actin Cytoskeleton Modulates the Activation of iNKT Cells by Segregating CD1d Nanoclusters on Antigen-Presenting Cells

    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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  • Translation Microscopy (TRAM) for Super-Resolution Imaging

    Translation Microscopy (TRAM) for Super-Resolution Imaging

    Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane.
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  • STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics

    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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  • Two-Photon Excitation STED Microscopy with Time-Gated Detection

    Two-Photon Excitation STED Microscopy with Time-Gated Detection

    We report on a novel two-photon excitation stimulated emission depletion (2PE-STED) microscope based on time-gated detection. The time-gated detection allows for the effective silencing of the fluorophores using moderate stimulated emission beam intensity. This opens the possibility of implementing an efficient 2PE-STED microscope with a stimulated emission beam running in a continuous-wave.
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  • TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion

    TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion

    Collagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export.
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  • Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent

    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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  • How to Combine STED and CLARITY

    How to Combine STED and CLARITY

    Previously, the preferred way to study the subtlest elements of the kidney, such as foot processes and the slit diaphragm has been by the use of electron microscopy. Using STED microscopy, we show that the nanoscale localization of slit diaphragm proteins can now be resolved using light microscopy. Even if the nanoscopic resolution has been available for a decade, light microscopy studies of the slit diaphragm are not found in the literature. This is likely due to the difficulties of achieving the high quality of fluorescent labelling needed for super-resolution microscopy. By applying an optical clearing protocol based on the CLARITY technique, we found that the immunostaining quality in kidney tissue can be improved. The improvement is likely due to the removal of lipids, resulting in a higher availability of binding epitopes in cleared tissue, as compared to PFA fixed non-cleared tissue.
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  • Video: Fluorescence is a State of Mind

    Video: Fluorescence is a State of Mind

    How to break a fundamental law of physics and win a Nobel Prize to boot. Stefan Hell explains super-resolved fluorescence microscopy for which he shared the 2014 Nobel Prize in chemistry.
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  • Super-Resolution Microscopy of the Synaptic Active Zone

    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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  • Lytic Immune Synapse Function Requires Filamentous Actin Deconstruction by Coronin 1A

    Lytic Immune Synapse Function Requires Filamentous Actin Deconstruction by Coronin 1A

    Natural killer (NK) cells are cytolytic effector cells of the innate immune system. Here, we show that deconstruction of synaptic cortical filamentous (F)-actin by Coronin 1A (Coro1A) is required for NK cell cytotoxicity through the remodeling of F-actin to enable lytic granule secretion. We define this requirement for remodeling using superresolution nanoscopy and Coro1A-deficient NK cells. In addition, we use NK cells from a patient with a rare Coro1A mutation, thus illustrating a critical link between Coro1A function and human health.
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  • Cross-strand Binding of TFAM to a Single mtDNA Molecule Forms the Mitochondrial Nucleoid

    Cross-strand Binding of TFAM to a Single mtDNA Molecule Forms the Mitochondrial Nucleoid

    Scientists from three Max Planck Institutes have gained fundamental insights into the organization of mitochondrial DNA (mtDNA). The researchers observed in high-resolution images gained with nobel prize-winning microscopy techniques that single copies of mtDNA are packaged by a specialized protein into slightly elongated structures of circa 100 nm in length.
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  • Probes that FIT RNA

    Probes that FIT RNA

    We have been developing new tools based on fluorogenic forced intercalation (FIT) probes for RNA detection quantification and interference in biological samples. Upon duplex formation with target nucleic acids, the base surrogates TO dye increases its quantum yield and brightness substantially (>10 fold).
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  • A Straightforward Approach for Gated STED-FCS to Investigate Lipid Membrane Dynamics

    A Straightforward Approach for Gated STED-FCS to Investigate Lipid Membrane Dynamics

    Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity.
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  • Super-Resolution – On a Heuristic Point of View About the Resolution of a Light Microscope

    Super-Resolution – On a Heuristic Point of View About the Resolution of a Light Microscope

    Since super-resolution has become one of the most favored methods in biomedical research, the term has become increasingly popular. Still, there is much of confusion about what is super-resolution and what is resolution at all. Here, the classical view of microscopic resolution is discussed and some techniques that resolve better than classical are briefly introduced. The picture on the right shows the intensity distribution of an image of two points whose distance is just the Rayleigh criterion (false color coding).
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  • A Bright Dye for Live-Cell STED Microscopy

    A Bright Dye for Live-Cell STED Microscopy

    The aim of cell biology is to study smallest details on a cellular level preferably in a live cell experiment. By providing fast and direct super-resolution, STED (Stimulated Emission Depletion) microscopy is the perfect tool for studying cellular details in the nanometer range in vivo.
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  • 3D STED (Stimulated Emission Depletion) Microscopy

    3D STED (Stimulated Emission Depletion) Microscopy

    The resolution needed to image subcellular architecture and dynamics in light microscopy is hindered by the diffraction limits as described by Ernst Abbe. Simply stated, structures smaller than 200 nanometers are lost in a blur. However, the field of super-resolution microscopy has produced methods to obtain resolution beyond this limit. Leica Microsystems has pioneered this field and offers the Leica TCS SP8 STED 3X for 3D Stimulated Emission Depletion microscopy. STED instantly produces super-resolution images, compatible with the dynamics of living cells, without the need for post-processing.
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