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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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  • 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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  • The Molecular Architecture of Hemidesmosomes as Revealed by Super-Resolution Microscopy

    Hemidesmosomes have been extensively studied by immunofluorescence microscopy, but due to its limited resolution, their precise organization remained poorly understood. We studied hemidesmosome organization in cultured keratinocytes by 2- and 3-color super-resolution microscopy. We observed that in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that β4 is distributed along rather than under keratin filaments. By applying innovative methods to quantify molecular distances, we demonstrate that the hemidesmosomal plaque protein plectin interacts simultaneously and asymmetrically with β4 and keratin.
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  • 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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  • Quantitative Analysis of PPT1 Interactome in Human Neuroblastoma Cells

    Application example of HyVolution Super-Resolution - Mutations in the CLN1 gene that encodes Palmitoyl protein thioesterase 1 (PPT1) or CLN1, cause Infantile NCL (INCL, MIM#256730). PPT1 removes long fatty acid chains such as palmitate from modified cysteine residues of proteins. The data shown here result from isolated protein complexes from PPT1-expressing SH-SY5Y stable cells that were subjected to single step affinity purification coupled to mass spectrometry (AP-MS). Prior to the MS analysis, we utilised a modified filter-aided sample preparation (FASP) protocol. Based on label free quantitative analysis of the data by SAINT, 23 PPT1 interacting partners (IP) were identified.
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  • Methods to Calibrate and Scale Axial Distances in Confocal Microscopy as a Function of Refractive Index

    Application example of HyVolution Super-Resolution - Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high-end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented.
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  • Evaluation of Zebrafish as a Model to Study the Pathogenesis of the Opportunistic Pathogen Cronobacter Turicensis

    Application example of HyVolution Super-Resolution - Bacteria belonging to the genus Cronobacter spp. have been recognized as causative agents of life-threatening systemic infections, primarily in premature, low-birth weight and/or immune-compromised neonates. Knowledge remains scarce regarding the underlying molecular mechanisms of disease development. In this study, we evaluated the use of a zebrafish model to study the pathogenesis of Cronobacter turicensis LMG 23827T, a clinical isolate responsible for two fatal sepsis cases in neonates.
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  • Discovery of Novel Peptides Targeting Pro-Atherogenic Endothelium in Disturbed Flow Regions -Targeted siRNA Delivery to Pro-Atherogenic Endothelium in vivo

    Application example of HyVolution Super-Resolution - Atherosclerosis occurs preferentially in arterial regions exposed to disturbed blood flow. Targeting these pro-atherogenic regions is a potential anti-atherogenic therapeutic approach, but it has been extremely challenging. Here, using in vivo phage display approach and the partial carotid ligation model of flow-induced atherosclerosis in mouse, we identified novel peptides that specifically bind to endothelial cells (ECs) exposed to disturbed flow condition in pro-atherogenic regions.
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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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  • Practical Guide for Excellent GSDIM Super-Resolution Images

    Do you know that most protists and bacteria lack in one feature that each of our body cell has? Our cells are touch and communicate with one another. They send and receive a variety of signals that coordinate their behavior to act together as a functional multicellular organism. Exploring the way of cellular communication and the ways how the cell surface interacts to organize tissues and body structures is of great interest. Kees Jalink and his team of scientists at the Netherlands Cancer Institute (NKI) in Amsterdam obtained new scientific insights into the molecular architecture of hemidesmosomes, cytoskeletal components, cell surface receptors and vesicular proteins with the help of Ground-State-Depletion (GSD)/ dSTORM microscopy. In this interview, Kees Jalink comments on their developments in imaging chambers, buffer conditions and image analysis to get the perfect super resolution image.
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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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  • Visualizing Tropoelastin in a Long-Term Human Elastic Fibre Cell Culture Model

    Elastin is an essential protein found in a variety of tissues where resilience and flexibility are needed, such as the skin and the heart. When aiming to engineer suitable implants, elastic fibres are needed to allow adequate tissue renewal. However, the visualization of human elastogenesis remains in the dark. To date, the visualization of human tropoelastin (TE) production in a human cell context and its fibre assembly under live cell conditions has not been achieved. Here, we present a long-term cell culture model of human dermal fibroblasts expressing fluorescence-labelled human TE. We employed a lentiviral system to stably overexpress Citrine-labelled TE to build a fluorescent fibre network. Using immunofluorescence, we confirmed the functionality of the Citrine-tagged TE. Furthermore, we visualized the fibre assembly over the course of several days using confocal microscopy. Applying super resolution microscopy, we were able to investigate the inner structure of the elastin–fibrillin-1 fibre network.
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  • P53- and Mevalonate Pathway–Driven Malignancies Require Arf6 for Metastasis and Drug Resistance

    Application example of HvYolution Super-Resolution - Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial–mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program.
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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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  • 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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  • 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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  • 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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  • Quantifying the Resolution of a Leica SR GSD 3D Localization Microscopy System with 2D and 3D Nanorulers

    DNA origami based nanorulers produced by GATTAquant are common standards to test the achievable spatial resolution of super-resolution microscopes. Recently the nanorulers were used to test the performance of the Leica SR GSD 3D microscope.
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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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  • HyVolution – the Smart Path to Confocal Super-Resolution

    Super-resolution refers to any device or method that can resolve better than the classical Abbe limit. Apart from infinite super-resolution techniques such as STED (stimulated emission depletion) and SMLM (single-molecule localization methods) that can theoretically resolve to any detail, there are also methods for limited super-resolution. Here we present HyVolution by Leica, which merges optical super-resolution and computational super-resolution. The optical part is provided by confocal microscopy, and the computational part by deconvolution. Lateral resolution of 140 nm is demonstrated. HyVolution offers multiple fluorescence recording in truly simultaneous mode.
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  • 4Pi-RESOLFT Nanoscopy

    Here we apply the 4Pi scheme to RESOLFT nanoscopy using two-photon absorption for the on-switching of fluorescent proteins. We show that in this combination, the lobes are so low that low-light level, 3D nanoscale imaging of living cells becomes possible. Our method thus offers robust access to densely packed, axially extended cellular regions that have been notoriously difficult to super-resolve. Our approach also entails a fluorescence read-out scheme that translates molecular sensitivity to local off-switching rates into improved signal-to-noise ratio and resolution.
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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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