Contact & Support

GSD

RSS feed
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy

    Co-localization analysis is a widely used tool to seek evidence for functional interactions between molecules in different color channels in microscopic images. Here we extend the basic co-localization analysis by including the orientations of the structures on which the molecules reside. We refer to the combination of co-localization of molecules and orientational alignment of the structures on which they reside as co-orientation. Because the orientation varies with the length scale at which it is evaluated, we consider this scale as a separate informative dimension in the analysis. Additionally we introduce a data driven method for testing the statistical significance of the co-orientation and provide a method for visualizing the local co-orientation strength in images. We demonstrate our methods on simulated localization microscopy data of filamentous structures, as well as experimental images of similar structures acquired with localization microscopy in different color channels.
    Read article
  • Webinar: Applications and Methodology of Super-Resolution Microscopy with Leica SR GSD 3D

    This webinar, sponsored by Leica Microsystems, will highlight applications in super-resolution microscopy. Researchers from the University of Washington and the federal University of São Paulo will share their experiences using the Ground State Depletion (GSD) method of super-resolution imaging to address diverse research topics such as the role of calcium signaling and excitation in cardiac muscle, and the structure and functional relationship of biological macromolecules.
    Read article
  • Detailed Morphological Characterisation of Hendra Virus Infection of Different Cell Types Using Super-Resolution and Conventional Imaging

    Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells.
    Read article
  • Universal PAINT – Dynamic Super-Resolution Microscopy

    Super-resolution microscopy techniques have revolutionized biology for the last ten years. With their help cellular components can now be visualized at the size of a protein. Nevertheless, imaging living cells is a challenge for most of the super-resolution principles.
    Read article
  • Finding, Defining and Breaking the Diffraction Barrier in Microscopy – A Historical Perspective

    Diffraction plays a crucial role in microscopy as it prevents the recording of arbitrarily sharp images with conventional light microscopes. Many names are connected with the notion of diffraction and the definition of resolution. An overview over the contributions of the different scientists to the recognition and definition of the diffraction barrier in the past centuries is given and the recent developments that led to breaking this barrier are portrayed.
    Read article
  • Video Interview with Jean-Luc Vonesch

    Jean-Luc Vonesch is head of the imaging facility at the Institute of Genetics and Molecular and Cellular Biology (IGBMC), Strasburg, France. 23 years ago he was the founder of this facility which nowadays serves more than 850 scientists distributed among 47 working groups. Looking deeply into the cells is of a special interest Vonesch states. And with super-resolution microscopy he pretends it is easier to identify the regions of interest for subsequent electron microscopy: “And so we can gain time thanks to the super-resolution” he says.
    Read article
  • JC Virus Inclusions in Progressive Multifocal Leukoencephalopathy: Scaffolding Promyelocytic Leukemia Nuclear Bodies Grow With Cell Cycle Transition Through an S-to-G2–Like State in Enlarging Oligodendrocyte Nuclei

    In progressive multifocal leukoencephalopathy, JC virus–infected oligodendroglia display 2 distinct patterns of intranuclear viral inclusions: full inclusions in which progeny virions are present throughout enlarged nuclei and dot-shaped inclusions in which virions are clustered in subnuclear domains termed “promyelocytic leukemia nuclear bodies” (PML-NBs). Promyelocytic leukemia nuclear bodies may serve a scaffolding role in viral progeny production.
    Read article
  • 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.”
    Read article
  • 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.
    Read article
  • Sample Preparation for GSDIM Localization Microscopy – Protocols and Tips

    The widefield super-resolution technique GSDIM (Ground State Depletion followed by individual molecule return) is a localization microscopy technique that is capable of resolving details as small as 20 nanometers. GSDIM is suitable for a wide range of samples.
    Read article
  • Webinar: New Dimensions in Super-Resolution Microscopy

    This webinar will highlight ground-breaking techniques in microscopy. Two scientists will present the impressive results they have achieved using confocal and widefield 3D super-resolution methods. Dr. Timo Zimmermann, Head of the Advanced Light Microscopy Unit, CRG – Centre for Genomic Regulation, Barcelona, Spain, will present biological applications for the next generation of STED microscopy systems. Dr. Eric Hosy from CNRS, University of Bordeaux, France, will talk about live cell localization microscopy with Leica SR GSD 3D.
    Read article
  • The Force of the Dark Side – Embedding Media for GSDIM Super-Resolution Localization Microscopy

    Super-resolution microscopy such as Stimulated Emission Depletion (STED) and single-molecule based techniques rely on the same principle for breaking the diffraction limit: the unwanted fluorescence signals are switched off during the image acquisition process. Consequently, Ground State Depletion followed by Individual Molecule Return (GSDIM) microscopy and related techniques like PALM, STORM and dSTORM use metastable dark states of a fluorophore for temporal separation of single molecules.
    Read article
  • Super-Resolution Microscopy Gives New Insights into Nuclear Pore Complex Organization

    The Nuclear Pore Complex (NPC) is a large complex in the nuclear membrane, representing the gate to the eukaryotic genetic makeup. Because of this outstanding function the structure of the NPC is of great interest. Anna Szymborska, scientist at the EMBL in Heidelberg, comments on her resaerch results and the potential of Ground State Depletion microscopy (GSD) for protein complex analysis in the following interview.
    Read article
  • Three-Dimensional Super-Resolution GSDIM Microscopy

    With the new 3D GSDIM technique structures like the Golgi and the microtubular network are resolved not only laterally, but also in a third dimension. The principle is based on the use of optical astigmatism to determine the accurate lateral and axial position of individual fluorochromes.
    Read article
  • 3D Localization Microscopy With Ground State Depletion (GSD)

    With the latest development of a GSD 3D super-resolution platform, it is now possible to achieve a lateral resolution of down to 20 nm and an axial resolution of 70 nm. The technology is based on an astigmatism approach using a manipulated PSF to localize the molecule in z. This following tutorial describes the basic principles of the 3D GSD technology.
    Read article
  • 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.
    Read article
  • Webinar: From Epifluorescence to Super-Resolution in 3D

    This webinar will illustrate results obtained by biochemical, Epifluorescence, TIRFM, Confocal and GSD techniques. Depending on the aim of experimental question, different imaging techniques deliver insights into varying aspects of intracellular pathways. To achieve "True-to-detail imaging" of the spatial arrangement of proteins and other biomolecules in cells, GSDIM achieves resolutions up to 20 nm in x and y direction – beyond the diffraction limit of light microscopy. But Super-resolution microscopy can be applied in the axial (z-) direction, too. A recent commercial implementation of the astigmatism approach will be discussed in more detail during this webinar.
    Read article
  • STED and GSDIM: Diffraction Unlimited Resolution for all Types of Fluorescence Imaging

    This article gives an overview of two different types of superresolution techniques. Stimulated emission depletion (STED) microscopy is a versatile and fast method that is based on point scanning microscopy – usually an extension of a confocal microscope. Ground state depletion imaging (GSDIM) is a parallel recording widefield approach that explores inherent switching of fluorochromes and typically comes with a TIRF microscope. The two methods use very different approaches to reach the same goal: to see more details in light microscopes than possible when diffraction limited.
    Read article
  • Webinar: From Live Cell Imaging to Super-Resolution

    The beauty of cells, magnified and resolved by light microscopy, has been fascinating investigators since the 19th century. Today, functional research in living cells is often a prerequisite for biological studies. And keeping the cells close to the necessary conditions whilst under microscopical observation is key. In this webinar, Dr. Christoph Bauer will share his experiences of live cell imaging and talk about how the microscopy challenges, such as optical aberrations at 37°C or focus drift in long-term experiments, can be addressed.
    Read article
  • Tubulin Modifications Affect Monolayer Formation and Apical Trafficking in Epithelial Cells

    The development and maintenance of polarized epithelial cells requires the establishment of complicated subcellular machinery. We studied the role of post-translational tubulin modifications within this process. At first, the distribution of detyrosinated microtubules was assessed in MDCK cells via immunofluorescence microscopy. No preferential accumulation of tyrosinated or detyrosinated microtubules could be detected at the apical or basal cell poles in epithelial cell cysts. However, during monolayer formation, the quantities of detyrosinated tubulin increased significantly over time.
    Read article
  • 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.
    Read article
  • Widefield Super-Resolution with GSDIM

    Great advancements in biology have been possible by using fluorescence microscopy. So far, the resolution of the images was limited due to physical constraints. In the past couple of years, new methods evolved circumventing these limitations and bringing fluorescence microscopy to a new level of resolution, boosting the possibilities in science with fluorescence microscopes.
    Read article
  • Video Tutorial: How to Optimize Sample Preparation for GSD Microscopy

    This video tutorial presents a reliable way to produce samples for super-resolution GSD imaging with a special focus on the mounting step. Stable and flat mounting of the coverslip increases the performance of the overall system, leading to an improved resolution of the GSD image.
    Read article
  • 1
  • 2