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  • Axial Tubule Junctions Control Rapid Calcium Signaling in Atria

    The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface.
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  • 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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  • Webinar: STED Nanoscopy in Combination with Optical Clearing Reveals Localization of Slit Diaphragm Proteins in the Kidney

    In this webinar, we show that optical clearing drastically increases the signal-to-noise ratio and staining quality, thus enabling STED nanoscopy of the subtlest elements of the kidney. In this way we show that optical clearing is not only a sample preparation technique to consider when imaging large mm-scale samples, but could also be fruitful when imaging at the nanoscale. Furthermore, the increased transparency of the optically cleared sample enables volumetric 3D STED imaging at sub-diffraction-limited resolution.
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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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  • 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

    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 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

    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

    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

    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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  • 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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  • HyVolution – Super-Resolution Imaging with a Confocal Microscope

    Since the invention of the microscope, there has been continual discussion about the possibility of showing more detailed features of specimens as compared to just magnifying them. In this article we describe the HyVolution concept and how the combination of confocal multiparameter fluorescence imaging at the confocal super-resolution regime with psf-based real deconvolution allows high-speed multicolor imaging with a resolution down to 140 nm.
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  • 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

    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

    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

    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

    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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  • Webinar: Using FIT Probes and Super-Resolution Microscopy to Decipher Steps of mRNP Assembly in Developing Oocytes

    In this webinar, presented by Dr. Imre Gaspar of the Developmental Biology Unit at EMBL, you will learn: importance of mRNA localization and function of mRNPs, advantages of using fluorogenic FIT probes to visualize mRNPs in vivo and in fixed specimen, and how super-resolution microscopy can identify factors required for mRNP biogenesis.
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  • 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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  • Localization of HDAC1 Using Super-Resolution STED Microscopy

    Here we show staining of HDAC1 in cancer tissue and epidermoid carcinoma cells. These results clearly show that the use of appropriate validated antibodies and STED microscopy are important tools to study subcellular structures beyond the diffraction limit correcting ill-defined images. This is critical in co-localization studies of proteins inside cells.
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  • Gated STED Microscopy with Time-gated Single-photon Avalanche Diode

    The maximization of the useful (within the time gate) photon flux is then an important aspect to obtain super-resolved STED images. Here we show that by using a fast-gated single-photon avalanche diode (SPAD), i.e. a detector able to rapidly (hundreds picoseconds) switch-on and -off can improve significantly the signal-to-noise ratio (SNR) of the gated STED image. In addition to an enhancement of the image SNR, the use of the fast-gated SPAD reduces also the system complexity. We demonstrate these abilities both on calibration and biological sample.
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  • STED Nanoscopy with Fluorescent Quantum Dots

    The widely popular class of quantum-dot molecular labels could so far not be utilized as standard fluorescent probes in STED (stimulated emission depletion) nanoscopy. This is because broad quantum-dot excitation spectra extend deeply into the spectral bands used for STED, thus compromising the transient fluorescence silencing required for attaining super-resolution.
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  • 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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  • Cortical Actin Networks Induce Spatio-temporal Confinement of Phospholipids in the Plasma Membrane – A Minimally Invasive Investigation by STED-FCS

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking.
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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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  • Multi-protein Assemblies Underlie the Mesoscale Organization of the Plasma Membrane

    Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins.
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  • Abstracts of the 5th European Super-Resolution User-Club Meeting

    The 5th Super-Resolution User Club Meeting was held in collaboration with Professor Kees Jalink and The Netherlands Cancer Institute (NKI) in Amsterdam. Having the meeting at a location where super-resolution microscopy is used on a daily basis makes a big difference, offering participants the chance to use live cells for workshops and see systems working in their true environments. Thanks also to the scientists that supported the meeting by coming and giving talks. As super-resolution continues to grow in importance in research, we recognize the need to come together to network, share information and experiences. Here we present the abstracts of the talks.
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  • Webinar: Unleashing the Powers of Super-Resolution Microscopy to Solve Immunological Challenges

    In this webinar, Christian Eggeling and Dongfang Liu will discuss their experiences using STED super-resolution microscopy to explore, uncover and define the intricate machinery involved in immunological pathways and infectious disorders.
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  • Multilayered Polyelectrolyte Microcapsules: Interaction with the Enzyme Cytochrome C Oxidase

    Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components.
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  • Encoding and Decoding Spatio-Temporal Information for Super-Resolution Microscopy

    The challenge of increasing the spatial resolution of an optical microscope beyond the diffraction limit can be reduced to a spectroscopy task by proper manipulation of the molecular states. The nanoscale spatial distribution of the molecules inside the detection volume of a scanning microscope can be encoded within the fluorescence dynamics and decoded by resolving the signal into its dynamics components.
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