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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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  • 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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  • 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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  • 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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  • 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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  • 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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  • 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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  • 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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  • CRISPR/Cas9-mediated Endogenous Protein Tagging for RESOLFT Super-Resolution Microscopy of Living Human Cells

    Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein.
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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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  • Video Talk by Daniel Choquet: Our brain, this black box

    What happens in your brain when you learn something? When you store a memory? In this informative and fascinating talk, Daniel Choquet shares some of the most recent findings regarding those brain functions. Light makes it possible to see what is inside the powerful black box that is the brain, and opens new paths for fighting brain dysfunctions.
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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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  • 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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  • Four questions for Professor Stefan Hell on the subject of FOM 2015

    For Professor Stefan Hell, who won the Nobel Prize for Chemistry in 2014 for the development of super-resolved fluorescence microscopy and the development of STED microscopy, the Focus on Microscopy conference has a very special significance. It was at the very first FOM 1998 in Sydney where Hell gave one of his first lectures on super-resolution, entitled "Super-resolution through 4Pi-confocal microscopy in cellular imaging".
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  • STED Nanoscopy: A Glimpse into the Future

    The well-known saying of "Seeing is believing" became even more apt in biology when stimulated emission depletion (STED) nanoscopy was introduced in 1994 by the Nobel laureate S. Hell and coworkers. This article gives an overview of the various cutting-edge implementations of STED nanoscopy and tries to shine a light into the future: imaging everything faster with unprecedented sensitivity and label-free.
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  • Pathways to Optical STED Microscopy

    STED nanoscopy has evolved to a highly versatile tool for the observation of the living cell, more and more finding its way into state-of-the-art optical imaging facilities in biomedical research institutes.
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  • Towards Digital Photon Counting Cameras for Single-molecule Optical Nanoscopy

    A SPAD array camera with single-photon sensitivity and zero read-out noise allows for the detection of extremely weak signals at ultra-fast imaging speeds. With temporal resolution in the order of micro-seconds, a SPAD array camera offers great potential for live-cell imaging with super-resolution.
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  • Which Super-Resolution Method is Right for You?

    Super-resolution microscopy has dramatically improved our understanding of intracellular dynamics, redefining what is possible in biological research. This infographic gives a compact overview on the different super-resolution techniques such as localization, structured illumination and stimulated emission depletion and will help you to choose the technology that best fulfills your individual research needs.
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  • Video Interview with Rainer Pepperkok

    Rainer Pepperkok is Head of the Advanced Light Microscopy Core Facility and Senior Scientist at the EMBL in Heidelberg (Germany). In the course of his studies he is interested in membrane traffic of the early secretory pathway in mammalian cells which he is trying to analyze with the help of most modern light microcopy techniques.
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  • Nanoscale Protein Diffusion by STED-Based Pair Correlation Analysis

    We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested in systems characterized by high and low signal to noise ratio, i.e. Capsid Like Particles (CLPs) bearing several (>100) active fluorescent proteins and monomeric fluorescent proteins transiently expressed in living Chinese Hamster Ovary cells, respectively.
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  • Super-Resolution Microscopy – Get Your Free e-Book for Download

    Until recently, the diffraction of light had placed a fundamental limit on how far biologists could peer into cells with optical microscopes, preventing them from resolving features less than 250 nm in size, missing critical structures within cells. Over the past 20 years scientists have developed several ingenious techniques allowing them to resolve features as small as 20 nm.
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  • 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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  • Nobel Prize in Chemistry for Achievements in Super-Resolution Microscopy

    On October 8th 2014, The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2014 to Eric Betzig, Stefan W. Hell and William E. Moerner "for the development of super-resolved fluorescence microscopy". For a long time optical microscopy was held back by a presumed limitation: that it would never obtain a better resolution than half the wavelength of light. Helped by fluorescent molecules the Nobel Laureates in Chemistry 2014 ingeniously circumvented this limitation. Their ground-breaking work has brought optical microscopy into the nanodimension.
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  • Video Interview with Stefan Hell, the Inventor of Super-Resolution

    Professor Stefan Hell is director at the Max Planck Institute for Biophysical Chemistry and head of the department of NanoBiophotonics in Goettingen and widely considered as the father of super-resolution. His inventions of 4Pi and STED microscopy were turned into the first commercial super-resolution microscopes available by Leica Microsystems in 2004 and 2007.
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  • Video Interview with Timo Zimmermann

    The first super-resolution image he saw was an eye opener for him: "It was not just structures that got smaller. I was looking at a sample that I specifically had high hopes of seeing another layer of complexity and this actually was there."
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  • Video Interviews with Kees Jalink

    Kees Jalink's group at the Netherlands Cancer Institute in Amsterdam, The Netherlands, explores signal transduction pathways and cell adhesion processes in cancer cells. In his eyes especially the new three-dimensional nanoscopic view of the relevant structure of interest is an essential feature to get the full picture.
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  • Correlated Optical and Isotopic Nanoscopy

    The isotopic composition of different materials can be imaged by secondary ion mass spectrometry. In biology, this method is mainly used to study cellular metabolism and turnover, by pulsing the cells with marker molecules such as amino acids labelled with stable isotopes (15N, 13C). The incorporation of the markers is then imaged with a lateral resolution that can surpass 100 nm.
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