Leica Science Lab - Tag : STED https://www.leica-microsystems.com//science-lab/tag/tags/sted/show/Tag/ Article tagged with STED en-US https://www.leica-microsystems.com/24942 Quantitative Fluorescence Phasor Analysis for FLIM (Fluorescence Lifetime Imaging Microscopy) The Phasor analysis approach to analyze fluorescence lifetime does not require any fitting. Phasor FLIM (fluorescence lifetime imaging microscopy) provides a 2D graphical view of lifetime distributions. This graphical view enables any observer to distinguish and separate different lifetime populations within a FLIM image rapidly. The interpretation of phasor FLIM distributions is straightforward. Multiple molecular species are resolved within a single pixel, because every species has a specific phasor. https://www.leica-microsystems.com//science-lab/phasor-analysis-for-flim-fluorescence-lifetime-imaging-microscopy/ Tue, 07 May 2019 22:00:00 +0000 Dr. Giulia Ossato https://www.leica-microsystems.com/24669 Super-Resolution Extending Nanoscopy Possibilities with STED and exchangeable fluorophores When it comes to STED Nanoscopy, keeping high signal-to-noise is key to achieve the best possible resolution in fixed and living cells. This can be challenging in the case of experiments in 3D and/or with time series, where the sample undergoes many rounds of image acquisition and photobleaching becomes an issue. If fluorophores were completely immune to photobleaching, it should be possible to perform STED indefinitely using the same molecules over and over. In practice, one performs STED with the best available fluorophores in terms of brightness and photostability (Grimm, Muthusamy et al. 2017), and at high labeling densities. However, there is a clever alternative to come closer to the ideal situation: if “fresh” fluorophores replenish the sample in each round of STED, imaging will take place with intact fluorophores every time. https://www.leica-microsystems.com//science-lab/extending-nanoscopy-possibilities-with-sted-and-exchangeable-fluorophores/ Tue, 12 Feb 2019 23:00:00 +0000 Dr. Julia Roberti https://www.leica-microsystems.com/24508 Super-Resolution Confocal Microscopy Live-Cell Imaging Simultaneously Measuring Image Features and Resolution in Live-Cell STED Images Reliable interpretation and quantification of cellular features in fluorescence microscopy requires an accurate estimate of microscope resolution. This is typically obtained by measuring the image of a nonbiological proxy for a point-like object, such as a fluorescent bead. Although appropriate for confocal microscopy, bead-based measurements are problematic for stimulated emission depletion microscopy and similar techniques where the resolution depends critically on the choice of fluorophore and acquisition parameters. In this article, we demonstrate that for a known geometry (e.g., tubules), the resolution can be measured in situ by fitting a model that accounts for both the point spread function (PSF) and the fluorophore distribution. https://www.leica-microsystems.com//science-lab/simultaneously-measuring-image-features-and-resolution-in-live-cell-sted-images/ Mon, 12 Nov 2018 23:00:00 +0000 https://www.leica-microsystems.com/20511 Image Restoration and Deconvolution Super-Resolution How to extract Image Information by Adaptive Deconvolution Confocal Laser Scannning Microscopy (CLSM) is the standard for true 3D resolved fluorescence imaging. Fast optical sectioning using flexible scanning strategies in combination with simultaneous multi-colour, high sensitivity and low noise signal detection provides maximum resolution in the spatial and temporal domain. In combination with modern approaches to image information extraction this helps the researcher to mine as much information as possible from the images acquired. Image information extraction refers to intelligent procedures for image enhancement using a priori knowledge from the imaging system. From simple glare control and optical development to intelligent and ingenious model extraction, there are many ways to see more than just the image. https://www.leica-microsystems.com//science-lab/how-to-extract-image-information-by-adaptive-deconvolution/ Tue, 18 Sep 2018 22:00:00 +0000 Dr. Jürgen Reymann https://www.leica-microsystems.com/20475 Super-Resolution Image Restoration and Deconvolution Observing Malaria Infection at the Right Spot in the Human Host Malaria is a life-threatening disease transmitted through the bites of mosquitoes infected with protozoan parasites. The most common and dangerous type of malaria is caused by the parasite Plasmodium falciparum. Malaria raises serious concerns because half of the world’s population is at risk and there are no effective vaccines available to date. WHO reported 216 million cases in 2016 [1], a half-million deaths, and an economic burden exceeding the billion dollar range. https://www.leica-microsystems.com//science-lab/observing-malaria-infection-at-the-right-spot-in-the-human-host/ Wed, 29 Aug 2018 22:00:00 +0000 Dr. Julia Roberti https://www.leica-microsystems.com/20357 Super-Resolution Free Webinar On-Demand: Super-resolved STED spectroscopy Molecular interactions are key in cellular signalling. They are often ruled or rendered by the mobility of the involved molecules. https://www.leica-microsystems.com//science-lab/free-webinar-on-demand-super-resolved-sted-spectroscopy/ Mon, 06 Aug 2018 22:00:00 +0000 Prof. Christian Eggeling https://www.leica-microsystems.com/20478 Super-Resolution California NanoSystems Institute at UCLA Publications A list of the published scientific articles which include work done in the ALMS/MSI Facilities. https://www.leica-microsystems.com//science-lab/cnsi-publication-list/ Sun, 14 Jan 2018 23:00:00 +0000 https://www.leica-microsystems.com/19872 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/abstracts-of-the-7th-european-super-resolution-user-club-meeting/ Sun, 17 Dec 2017 23:00:00 +0000 Prof. DrSc. Pavel Hozák, Dr. Jindřiška Fišerová, Dr. Eugene Katrukha, Dr. Susan Cox, Prof. Christian Eggeling, Ph.D. Marc van Zandvoort, Dr. Jordi Andilla, Dr. Martin Offterdinger, Prof. Dr. Torsten Ochsenreiter, Dr. Camille Boutin https://www.leica-microsystems.com/19342 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/axial-tubule-junctions-control-rapid-calcium-signaling-in-atria/ Wed, 31 May 2017 09:21:00 +0000 https://www.leica-microsystems.com/18278 Super-Resolution Live-Cell Imaging 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. https://www.leica-microsystems.com//science-lab/super-resolution-optical-microscopy-of-lipid-plasma-membrane-dynamics/ Fri, 23 Dec 2016 14:16:00 +0000 Prof. Christian Eggeling https://www.leica-microsystems.com/18876 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/abstracts-of-the-6th-european-super-resolution-user-club-meeting/ Tue, 18 Oct 2016 08:59:00 +0000 Dr. Timo Zimmermann, Dr. Pablo Loza-Alvarez, Dr. Alberto Lleó, Dr. Gražvydas Lukinavicius, Prof. Philip Tinnefeld, Prof. Hans-Georg Kräusslich, Dr. Steffen Dietzel, Dr. Valeria Caiolfa, Lorenzo Albertazzi https://www.leica-microsystems.com/18801 Super-Resolution Fluorescence Microscopy Image Restoration and Deconvolution 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. https://www.leica-microsystems.com//science-lab/measuring-the-3d-sted-psf-with-a-new-type-of-fluorescent-beads/ Wed, 21 Sep 2016 06:54:00 +0000 PhD Jürgen J. Schmied, MSc Remko Dijkstra, Ph.D. Max B. Scheible, Ph.D. Giulia M. R. De Luca, PhD Jochen J. Sieber https://www.leica-microsystems.com/19026 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/mirror-enhanced-super-resolution-microscopy/ Thu, 18 Aug 2016 09:29:00 +0000 https://www.leica-microsystems.com/18102 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/the-actin-cytoskeleton-modulates-the-activation-of-inkt-cells-by-segregating-cd1d-nanoclusters-on-antigen-presenting-cells/ Wed, 10 Aug 2016 13:28:00 +0000 https://www.leica-microsystems.com/18088 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/translation-microscopy-tram-for-super-resolution-imaging/ Fri, 22 Jul 2016 17:51:00 +0000 https://www.leica-microsystems.com/18271 Super-Resolution Quantitative Fluorescence Live-Cell Imaging 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. https://www.leica-microsystems.com//science-lab/sted-flcs-an-advanced-tool-to-reveal-spatiotemporal-heterogeneity-of-molecular-membrane-dynamics/ Mon, 11 Jul 2016 08:53:00 +0000 Ph.D. Giuseppe Vicidomini https://www.leica-microsystems.com/17991 Super-Resolution Image Restoration and Deconvolution 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. https://www.leica-microsystems.com//science-lab/two-photon-excitation-sted-microscopy-with-time-gated-detection/ Tue, 21 Jun 2016 06:39:00 +0000 https://www.leica-microsystems.com/17957 Super-Resolution Confocal Microscopy Light Sheet Microscopy Neuroscience 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. https://www.leica-microsystems.com//science-lab/super-resolution-mapping-of-neuronal-circuitry-with-an-index-optimized-clearing-agent/ Wed, 27 Apr 2016 10:07:00 +0000 https://www.leica-microsystems.com/17673 Confocal Microscopy Super-Resolution Image Restoration and Deconvolution 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. https://www.leica-microsystems.com//science-lab/hyvolution-super-resolution-imaging-with-a-confocal-microscope/ Fri, 01 Apr 2016 07:19:00 +0000 Dr. Rolf T. Borlinghaus, Dr. Constantin Kappel https://www.leica-microsystems.com/17507 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/how-to-combine-sted-and-clarity/ Fri, 29 Jan 2016 14:46:00 +0000 https://www.leica-microsystems.com/16796 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/video-fluorescence-is-a-state-of-mind/ Thu, 07 Jan 2016 20:45:00 +0000 Prof. Dr. Dr. h.c. Stefan Hell https://www.leica-microsystems.com/16593 Super-Resolution Neuroscience 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. https://www.leica-microsystems.com//science-lab/super-resolution-microscopy-of-the-synaptic-active-zone/ Tue, 15 Dec 2015 10:15:00 +0000 Nadine Ehmann https://www.leica-microsystems.com/16591 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/lytic-immune-synapse-function-requires-filamentous-actin-deconstruction-by-coronin-1a/ Thu, 10 Dec 2015 10:21:00 +0000 Ph.D. Emily M. Mace, Ph.D., MD Jordan S. Orange https://www.leica-microsystems.com/16565 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/cross-strand-binding-of-tfam-to-a-single-mtdna-molecule-forms-the-mitochondrial-nucleoid/ Tue, 17 Nov 2015 17:20:00 +0000 Dr. Christian Kukat https://www.leica-microsystems.com/16521 Super-Resolution Live-Cell Imaging Fluorescence Microscopy 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). https://www.leica-microsystems.com//science-lab/probes-that-fit-rna/ Tue, 22 Sep 2015 17:25:00 +0000 PhD Imre Gaspar https://www.leica-microsystems.com/16097 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/localization-of-hdac1-using-super-resolution-sted-microscopy/ Wed, 02 Sep 2015 15:57:00 +0000 PhD Karin Abarca Heidemann, PhD Sarah Crowe, MS, PhD Tobias Jacob, PhD Carl A. Ascoli https://www.leica-microsystems.com/15928 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/gated-sted-microscopy-with-time-gated-single-photon-avalanche-diode/ Tue, 25 Aug 2015 17:24:00 +0000 https://www.leica-microsystems.com/16033 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/sted-nanoscopy-with-fluorescent-quantum-dots/ Thu, 13 Aug 2015 09:24:00 +0000 https://www.leica-microsystems.com/16176 Super-Resolution Quantitative Fluorescence 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. https://www.leica-microsystems.com//science-lab/a-straightforward-approach-for-gated-sted-fcs-to-investigate-lipid-membrane-dynamics/ Mon, 03 Aug 2015 10:29:00 +0000 https://www.leica-microsystems.com/16174 Super-Resolution Quantitative Fluorescence Live-Cell Imaging 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. https://www.leica-microsystems.com//science-lab/cortical-actin-networks-induce-spatio-temporal-confinement-of-phospholipids-in-the-plasma-membrane-a-minimally-invasive-investigation-by-sted-fcs/ Fri, 24 Jul 2015 13:02:00 +0000 Débora Machado Andrade https://www.leica-microsystems.com/15239 Super-Resolution Quantitative Fluorescence 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. https://www.leica-microsystems.com//science-lab/a-lipid-bound-actin-meshwork-organizes-liquid-phase-separation-in-model-membranes/ Wed, 15 Jul 2015 13:49:00 +0000 https://www.leica-microsystems.com/15241 Super-Resolution Live-Cell Imaging 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. https://www.leica-microsystems.com//science-lab/multi-protein-assemblies-underlie-the-mesoscale-organization-of-the-plasma-membrane/ Thu, 09 Jul 2015 13:44:00 +0000 https://www.leica-microsystems.com/16019 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/abstracts-of-the-5th-european-super-resolution-user-club-meeting/ Tue, 23 Jun 2015 13:23:00 +0000 PhD Kees Jalink, Luca Lanzanò, PhD Imre Gaspar, PhD Luc Reymond, MSc Leila Nahidiazar, Robert Nieuwenhuizen, MSc Daniela Leyton Puig, PhD David Williamson, PhD Jonas Ries, PhD Jürgen J. Schmied, Ivan Michel Antolovic https://www.leica-microsystems.com/14796 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/multilayered-polyelectrolyte-microcapsules-interaction-with-the-enzyme-cytochrome-c-oxidase/ Tue, 05 May 2015 17:31:00 +0000 https://www.leica-microsystems.com/15511 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/encoding-and-decoding-spatio-temporal-information-for-super-resolution-microscopy/ Fri, 17 Apr 2015 10:35:00 +0000 https://www.leica-microsystems.com/15347 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/sted-nanoscopy-a-glimpse-into-the-future/ Mon, 23 Mar 2015 18:08:00 +0000 Paolo Bianchini https://www.leica-microsystems.com/15237 Super-Resolution Live-Cell Imaging 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. https://www.leica-microsystems.com//science-lab/pathways-to-optical-sted-microscopy/ Thu, 12 Mar 2015 15:49:00 +0000 https://www.leica-microsystems.com/15223 Super-Resolution Neuroscience Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. https://www.leica-microsystems.com//science-lab/super-resolution-imaging-of-the-natural-killer-cell-immunological-synapse-on-a-glass-supported-planar-lipid-bilayer/ Thu, 12 Feb 2015 09:26:00 +0000 https://www.leica-microsystems.com/14980 Super-Resolution Image Restoration and Deconvolution Multi-Images Deconvolution Improves Signal-to-Noise Ratio on Gated Stimulated Emission Depletion Microscopy Time-gated detection, namely, only collecting the fluorescence photons after a time-delay from the excitation events, reduces complexity, cost, and illumination intensity of a stimulated emission depletion (STED) microscope. In the gated continuous-wave- (CW-) STED implementation, the spatial resolution improves with increased time-delay, but the signal-to-noise ratio (SNR) reduces. https://www.leica-microsystems.com//science-lab/multi-images-deconvolution-improves-signal-to-noise-ratio-on-gated-stimulated-emission-depletion-microscopy/ Fri, 23 Jan 2015 12:22:00 +0000 https://www.leica-microsystems.com/15079 Super-Resolution Confocal Microscopy Image Restoration and Deconvolution 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). https://www.leica-microsystems.com//science-lab/super-resolution-on-a-heuristic-point-of-view-about-the-resolution-of-a-light-microscope/ Wed, 21 Jan 2015 16:00:00 +0000 Dr. Rolf T. Borlinghaus https://www.leica-microsystems.com/14918 Super-Resolution Live-Cell Imaging Image Restoration and Deconvolution 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. https://www.leica-microsystems.com//science-lab/a-bright-dye-for-live-cell-sted-microscopy/ Mon, 19 Jan 2015 15:47:00 +0000 PhD Stefan Pitsch, PhD Isabelle Köster https://www.leica-microsystems.com/15059 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/finding-defining-and-breaking-the-diffraction-barrier-in-microscopy-a-historical-perspective/ Mon, 12 Jan 2015 15:42:00 +0000 https://www.leica-microsystems.com/13911 Super-Resolution Live-Cell Imaging 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. https://www.leica-microsystems.com//science-lab/nanoscale-protein-diffusion-by-sted-based-pair-correlation-analysis/ Fri, 21 Nov 2014 09:36:00 +0000 Paolo Bianchini https://www.leica-microsystems.com/14451 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/super-resolution-microscopy-get-your-free-e-book-for-download/ Wed, 19 Nov 2014 15:10:00 +0000 https://www.leica-microsystems.com/7277 Super-Resolution Quick Guide to STED Sample Preparation This document is intended as a quick reference guide for the most common questions regarding the preparation of samples for the Leica SP8 TCS STED 3X, by briefly explaining the theoretical basics required from samples for stimulated emission depletion. It provides information about the most common immuno-fluorescence labeling techniques, working mounting media, basic quality optimization procedures and experiment designs. It also contains a detailed list of reagents, antibodies and disposables frequently and successfully used in super-resolution STED microscopy. In summary, this guide is meant to present the necessary knowledge, including some tips and tricks, to users that are preparing their first super-resolution microscopy sample. https://www.leica-microsystems.com//science-lab/quick-guide-to-sted-sample-preparation/ Wed, 12 Nov 2014 12:11:00 +0000 https://www.leica-microsystems.com/14658 Super-Resolution Neuroscience A New Probe for Super-Resolution Imaging of Membranes Elucidates Trafficking Pathways The molecular composition of the organelles involved in membrane recycling is difficult to establish as a result of the absence of suitable labeling tools. We introduce in this paper a novel probe, named membrane-binding fluorophore-cysteine-lysine-palmitoyl group (mCLING), which labels the plasma membrane and is taken up during endocytosis. https://www.leica-microsystems.com//science-lab/a-new-probe-for-super-resolution-imaging-of-membranes-elucidates-trafficking-pathways/ Fri, 17 Oct 2014 12:16:00 +0000 https://www.leica-microsystems.com/14647 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/3d-sted-stimulated-emission-depletion-microscopy/ Tue, 14 Oct 2014 14:03:00 +0000 PhD Christopher Vega https://www.leica-microsystems.com/14623 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/nobel-prize-in-chemistry-for-achievements-in-super-resolution-microscopy/ Thu, 09 Oct 2014 13:25:00 +0000 Nobel Media AB 2014 https://www.leica-microsystems.com/13795 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/video-interview-with-stefan-hell-the-inventor-of-super-resolution/ Wed, 08 Oct 2014 18:14:00 +0000 Prof. Dr. Dr. h.c. Stefan Hell, Dipl. oec.-troph. Anja Schué, PhD Isabelle Köster https://www.leica-microsystems.com/13883 Super-Resolution 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." https://www.leica-microsystems.com//science-lab/video-interview-with-timo-zimmermann/ Wed, 10 Sep 2014 09:19:00 +0000 Dr. Timo Zimmermann, Dipl. oec.-troph. Anja Schué, PhD Isabelle Köster https://www.leica-microsystems.com/14246 Super-Resolution Neuroscience 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. https://www.leica-microsystems.com//science-lab/correlated-optical-and-isotopic-nanoscopy/ Fri, 22 Aug 2014 09:53:00 +0000 https://www.leica-microsystems.com/13950 Super-Resolution Neuroscience STED Microscopy of Living Cells – New Frontiers in Membrane and Neurobiology Recent developments in fluorescence far-field microscopy such as STED microscopy have accomplished observation of the living cell with a spatial resolution far below the diffraction limit. Here, we briefly review the current approaches to super-resolution optical microscopy and present the implementation of STED microscopy for novel insights into live cell mechanisms, with a focus on neurobiology and plasma membrane dynamics. https://www.leica-microsystems.com//science-lab/sted-microscopy-of-living-cells-new-frontiers-in-membrane-and-neurobiology/ Tue, 12 Aug 2014 12:11:00 +0000 Prof. Christian Eggeling https://www.leica-microsystems.com/14017 Super-Resolution Image Restoration and Deconvolution Abstracts of the 4th European Super-Resolution User-Club Meeting The 4th Super-Resolution User Club Meeting was held in collaboration with Christian Eggeling and the Weatherall Institute of Molecular Medicine in Oxford, UK. Here we present the abstracts of the talks and interviews with participants. https://www.leica-microsystems.com//science-lab/abstracts-of-the-4th-european-super-resolution-user-club-meeting/ Fri, 08 Aug 2014 08:09:00 +0000 Prof. Christian Eggeling, Dr. Timo Zimmermann, Dr. Katrin Willig, PhD Kees Jalink, Dr. Marko Lampe, Prof. Mark Neil, Dr. Sergi Padilla-Parra, MSc Leila Nahidiazar, Ph.D. Giuseppe Vicidomini, Ph.D. Marc van Zandvoort https://www.leica-microsystems.com/13877 Super-Resolution Neuroscience Super-Resolution Microscopy Helped to Create the First 3D Model of a Synapse A research team from Göttingen, led by Prof. Silvio O. Rizzoli, managed to determine the copy numbers and positions of all important building blocks of a synapse for the first time. This allowed them to reconstruct the first scientifically accurate 3D model of a synapse. https://www.leica-microsystems.com//science-lab/super-resolution-microscopy-helped-to-create-the-first-3d-model-of-a-synapse/ Tue, 01 Jul 2014 14:59:00 +0000 https://www.leica-microsystems.com/13527 Super-Resolution Video Talk on Super-Resolution: Overview and Stimulated Emission Depletion (STED) Microscopy Historically, light microscopy has been limited in its ability to resolve closely spaced objects, with the best microscopes only able to resolve objects separated by 200 nm or more. This limit is known as the diffraction limit. In the last twenty years, a number of techniques have been developed that allow resolution beyond the diffraction limit. https://www.leica-microsystems.com//science-lab/video-talk-on-super-resolution-overview-and-stimulated-emission-depletion-sted-microscopy/ Thu, 05 Jun 2014 17:05:00 +0000 Prof. Dr. Dr. h.c. Stefan Hell https://www.leica-microsystems.com/13740 Super-Resolution Neuroscience Interview with Dr. Yasushi Okada Yasushi Okada, team leader at Riken Quantitative Biology Center in Osaka, Japan, investigates vesicular transport mechanisms in neuronal cells. As the size of transported vesicles is below 100 nm and the diameter of microtubules is about 25 nm, he uses super-resolution techniques to study the sophisticated machinery of neuronal transport. https://www.leica-microsystems.com//science-lab/interview-with-dr-yasushi-okada/ Tue, 03 Jun 2014 15:38:00 +0000 Dipl. oec.-troph. Anja Schué, MD, PhD Yasushi Okada, PhD Isabelle Köster https://www.leica-microsystems.com/13128 Super-Resolution Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton. https://www.leica-microsystems.com//science-lab/visualization-of-the-immunological-synapse-by-dual-color-time-gated-stimulated-emission-depletion-sted-nanoscopy/ Fri, 28 Mar 2014 16:34:00 +0000 Ph.D. Emily M. Mace, Ph.D., MD Jordan S. Orange https://www.leica-microsystems.com/13103 Super-Resolution A New Filtering Technique for Removing Anti-Stokes Emission Background in Gated CW-STED Microscopy Stimulated emission depletion (STED) microscopy is a prominent approach of super-resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time-gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub-diffraction spatial resolution. https://www.leica-microsystems.com//science-lab/a-new-filtering-technique-for-removing-anti-stokes-emission-background-in-gated-cw-sted-microscopy/ Tue, 25 Mar 2014 20:34:00 +0000 https://www.leica-microsystems.com/11693 Live-Cell Imaging Super-Resolution Confocal Microscopy Neuroscience Fluorescence Microscopy Live-Cell Imaging Evolves to Find New Niches Since its introduction in the 1600s, improvements in microscope technology have continually broadened the types of cells and cellular processes that can be studied. Advances in automation have made this already-simple tool faster and more capable, and time-lapse imaging reveals function and dynamics in addition to structure. Live-cell imaging has enabled us to witness incredible moments in biology in unprecedented detail. Even embryogenesis – the process of cell division and cellular differentiation that occurs at the earliest stages of life – has recently been captured. https://www.leica-microsystems.com//science-lab/live-cell-imaging-evolves-to-find-new-niches/ Mon, 25 Nov 2013 10:15:00 +0000 https://www.leica-microsystems.com/11089 Super-Resolution Super-resolution Microscopy and the Third Dimension Optical imaging devices have a finite depth of field and diffraction limited resolution. The depth of field problem was tackled first with confocal microscopes, diffraction unlimited resolution is available since a few years with super-resolution microscopes. Super-resolution microscopes with a solved depth of field problem are now available. https://www.leica-microsystems.com//science-lab/super-resolution-microscopy-and-the-third-dimension/ Mon, 04 Nov 2013 09:55:00 +0000 Dr. Rolf T. Borlinghaus https://www.leica-microsystems.com/11024 Super-Resolution Interview with Stefan Hell: Breaking Nanoscale Boundaries Stefan Hell is credited with having conceived and applied the first viable concept for breaking Abbe’s diffraction limit in a light-focusing microscope. He is a scientific member of the Max Planck Society and a director at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, where he currently leads the department of nanobiophotonics. https://www.leica-microsystems.com//science-lab/interview-with-stefan-hell-breaking-nanoscale-boundaries/ Wed, 09 Oct 2013 13:29:00 +0000 Dr. Peter Andersen https://www.leica-microsystems.com/10826 Super-Resolution STED Microscopy: A New Chapter in Light Imaging When Stefan Hell invented a technique that pushed beyond the diffraction limit of light for the first time, a new field of nanoscale imaging was born. Now laboratories around the world are using STED microscopy to smash through more boundaries in fundamental science. https://www.leica-microsystems.com//science-lab/sted-microscopy-a-new-chapter-in-light-imaging/ Mon, 16 Sep 2013 10:28:00 +0000 https://www.leica-microsystems.com/10730 Super-Resolution Confocal Microscopy Image Restoration and Deconvolution Towards Real-time Image Deconvolution: Application to Confocal and STED Microscopy Although deconvolution can improve the quality of any type of microscope, the high computational time required has so far limited its massive spreading. Here we demonstrate the ability of the scaled-gradient-projection (SGP) method to provide accelerated versions of the most used algorithms in microscopy. To achieve further increases in efficiency, we also consider implementations on graphic processing units (GPUs). https://www.leica-microsystems.com//science-lab/towards-real-time-image-deconvolution-application-to-confocal-and-sted-microscopy/ Mon, 02 Sep 2013 07:51:00 +0000 https://www.leica-microsystems.com/10216 Super-Resolution Multiphoton Microscopy Single-Wavelength Two-Photon Excitation-stimulated Emission Depletion (SW2PE-STED) Superresolution Imaging Two-photon microscopy, multiphoton microcopy and super-resolution imaging. We developed a new class of two-photon excitation–stimulated emission depletion (2PE-STED) optical microscope. In this work, we show the opportunity to perform superresolved fluorescence imaging, exciting and stimulating the emission of a fluorophore by means of a single wavelength. https://www.leica-microsystems.com//science-lab/single-wavelength-two-photon-excitation-stimulated-emission-depletion-sw2pe-sted-superresolution-imaging/ Fri, 23 Aug 2013 11:02:00 +0000 Paolo Bianchini https://www.leica-microsystems.com/10653 Super-Resolution Live-Cell Imaging Watching Molecule Movements in Live Cells The newly developed STED-RICS microscopy method records rapid movements of molecules in live samples. By combining raster image correlation spectroscopy (RICS) with STED fluorescence microscopy, researchers of Karlsruhe Institute of Technology (KIT) opened up new applications in medical research, e.g. analyzing the dynamics of cell membranes at high protein concentrations. https://www.leica-microsystems.com//science-lab/watching-molecule-movements-in-live-cells/ Thu, 22 Aug 2013 13:28:00 +0000 https://www.leica-microsystems.com/10290 Super-Resolution Live-Cell Imaging Neuroscience New Labeling Tools Can Help to Realize the Full Potential of Super-Resolution Microscopy Since super-resolution microscopy techniques revolutionized the concept of light microscopy by overcoming the physical diffraction limit, STED microscopy and other super-resolution techniques have aroused considerable interest. The diffraction limit imposes no more constraints on resolution. New microscopes with ever-decreasing resolution limits are being developed, for instance by the inventor of STED microscopy, Prof. Stefan Hell, now director at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany. https://www.leica-microsystems.com//science-lab/new-labeling-tools-can-help-to-realize-the-full-potential-of-super-resolution-microscopy/ Fri, 02 Aug 2013 10:55:00 +0000 Dr. Matthias Schauen, Dr. Felipe Opazo, Prof. Silvio Rizzoli https://www.leica-microsystems.com/10135 Super-Resolution Widefield Microscopy Image Restoration and Deconvolution 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. https://www.leica-microsystems.com//science-lab/abstracts-of-the-3rd-european-super-resolution-user-club-meeting/ Mon, 08 Jul 2013 10:27:00 +0000 Prof. Alberto Diaspro, Ph.D. Illaria Testa, Prof. Silvio Rizzoli, Ph.D. Giuseppe Vicidomini, Prof. Ralf Jacob, Ph.D. Eric Hosy, Prof. Colin Sheppard, Zeno Lavagnino https://www.leica-microsystems.com/10208 Super-Resolution STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used to substantially improve the effective spatial resolution in STED nanoscopy using time-gated detection, pulsed excitation and continuous wave (CW) STED beams. https://www.leica-microsystems.com//science-lab/sted-nanoscopy-with-time-gated-detection-theoretical-and-experimental-aspects/ Mon, 08 Jul 2013 08:48:00 +0000 Ph.D. Giuseppe Vicidomini https://www.leica-microsystems.com/9043 Super-Resolution 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. https://www.leica-microsystems.com//science-lab/sted-and-gsdim-diffraction-unlimited-resolution-for-all-types-of-fluorescence-imaging/ Thu, 28 Feb 2013 23:00:00 +0000 Dr. Rolf T. Borlinghaus https://www.leica-microsystems.com/7988 Confocal Microscopy Super-Resolution Gates Open for Improved Confocal Fluorescence and Super-Resolution STED True confocal microscope systems feature single-point illumination and single-point detection. The method is called "optical sectioning" since the generated image contains only information from the focal plane. The serial detection offers highly efficient and low-noise sensors for signal conversion. Although the nonparallel detection is not conducive to high-speed imaging, modern scanning concepts allow frame rates above 400 frames per second at reasonable noise levels. This is by far enough for most applications, including the monitoring of fast ion-transport phenomena in living material. https://www.leica-microsystems.com//science-lab/gates-open-for-improved-confocal-fluorescence-and-super-resolution-sted/ Wed, 23 Jan 2013 23:00:00 +0000 Dr. Rolf T. Borlinghaus https://www.leica-microsystems.com/7903 Super-Resolution Mitochondrial DNA Molecules are Packaged Individually At the Max Planck Institute for Biology of Ageing in Cologne founded in 2008, three departments are working on the molecular, physiological and evolutionary mechanisms of the ageing of cells, tissue and organisms. The long-term aim of the research is to help people age more healthily. Here, Dr. Christian Kukat from the department of Professor Nils-Göran Larsson is examining the role played by mitochondria in the ageing process. In close collaboration with Dr. Christian Wurm from the research group of Professor Stefan Jakobs at the Max Planck Institute for Biophysical Chemistry in Göttingen and researchers at the University of Gothenburg, Sweden, they harnessed super-resolution STED microscopy to find out new and surprising facts about the mitochondrial nucleoid. https://www.leica-microsystems.com//science-lab/mitochondrial-dna-molecules-are-packaged-individually/ Sun, 06 Jan 2013 23:00:00 +0000 Dr. Matthias Schauen, Dr. Christian Kukat https://www.leica-microsystems.com/7735 Super-Resolution Widefield Microscopy Image Restoration and Deconvolution 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. https://www.leica-microsystems.com//science-lab/abstracts-of-the-2nd-european-super-resolution-user-club-meeting/ Thu, 01 Nov 2012 23:00:00 +0000 Prof. Hjalmar Brismar, Prof. Dr. Dr. h.c. Stefan Hell, Prof. Christian Eggeling, Dr. Anna Szymborska, Prof. Alberto Diaspro, Ph.D. Eric Hosy, Ph.D. Giuseppe Vicidomini, Hans van der Voort https://www.leica-microsystems.com/5815 Super-Resolution Gated STED Microscopy with CW-STED Lasers Among the major steps in the development of Stimulated Emission Depletion (STED) microscopy, the demonstration of the use continuous wave lasers (CW-STED) was certainly contributing the most to a wide dissemination of the method due to the affordability and elegant simplicity of this implementation. Nevertheless, CW-STED was so far not reaching the same spatial resolution as pulsed-lasers STED configurations. A recent investigation on the time-course of the fluorescence emission probability in CW-STED has revealed the benefit of using a gated fluorescence detection (gSTED) to improve further the resolution of C-S https://www.leica-microsystems.com//science-lab/gated-sted-microscopy-with-cw-sted-lasers/ Sun, 01 Apr 2012 22:00:00 +0000 Ph.D. Giuseppe Vicidomini, Gael Moneron https://www.leica-microsystems.com/5391 Super-Resolution Visualization of the Natural Killer Cell Immune Synapse by Super-Resolution Nanoscopy Natural killer (NK) cells are innate immune effectors that recognize and kill virally infected and tumorigenic cells. Central to their effector function is the formation of an immunological synapse with a target cell followed by directed secretion of cytolytic granules. https://www.leica-microsystems.com//science-lab/visualization-of-the-natural-killer-cell-immune-synapse-by-super-resolution-nanoscopy/ Thu, 08 Mar 2012 23:00:00 +0000 Ph.D. Emily M. Mace, Ph.D., MD Jordan S. Orange https://www.leica-microsystems.com/5331 Neuroscience Super-Resolution Sharp Live Images from the Mouse Brain To explore the most intricate structures of the brain in order to decipher how it functions – Stefan Hell’s team of researchers at the Max Planck Institute for Biophysical Chemistry in Göttingen has made a significant step closer to this goal. Using the STED microscopy developed by Hell, the scientists have, for the first time, managed to record detailed live images inside the brain of a living mouse. https://www.leica-microsystems.com//science-lab/sharp-live-images-from-the-mouse-brain/ Sun, 04 Mar 2012 23:00:00 +0000 Dr. Sebastian Berning, Dr. Katrin Willig, Dr. Heinz Steffens, Dr. Payam Dibaj, Prof. Dr. Dr. h.c. Stefan Hell https://www.leica-microsystems.com/10210 Super-Resolution Dual Channel STED Nanoscopy of Lytic Granules on Actin Filaments in Natural Killer Cells Natural killer (NK) cells are innate immune effectors that eliminate diseased and tumorigenic targets through the directed secretion of specialized secretory lysosomes, termed lytic granules. This directed secretion is triggered following the formation of an immunological synapse (IS), which is characterized by actin re-modeling and receptor organization at the interface between the NK cell and its susceptible target. https://www.leica-microsystems.com//science-lab/dual-channel-sted-nanoscopy-of-lytic-granules-on-actin-filaments-in-natural-killer-cells/ Thu, 01 Mar 2012 11:32:00 +0000 Ph.D. Emily M. Mace https://www.leica-microsystems.com/5108 Live-Cell Imaging Confocal Microscopy Live-cell Imaging Techniques The understanding of complex and/or fast cellular dynamics is an important step for exploring biological processes. Therefore, today’s life science research is increasingly focusing on dynamic processes like cell migration, morphological changes of cells, organs or whole animals and physiological (e.g. changes of intracellular ion composition) events in living specimens in real time. https://www.leica-microsystems.com//science-lab/live-cell-imaging-techniques/ Thu, 23 Feb 2012 23:00:00 +0000 Dr. Thomas Veitinger, Dr. Zhongxiang Jiang https://www.leica-microsystems.com/4820 Super-Resolution Videos on STED Microscopy and its Inventor Stefan Hell Here we present an collection of videos telling the story of the invention of nanometer-scale microscopy by Stefan Hell and explaining the basic principle of STED microscopy. https://www.leica-microsystems.com//science-lab/videos-on-sted-microscopy-and-its-inventor-stefan-hell/ Thu, 08 Dec 2011 14:33:00 +0000 https://www.leica-microsystems.com/4867 Super-Resolution Image Restoration and Deconvolution Subcellular Localization of AKT and Tubulin using Super-Resolution Microscopy Stimulated Emission Depletion microscopy, or STED nanoscopy, is a technique that uses the non-linear de-excitation of fluorescent dyes to overcome the resolution limit imposed by diffraction encountered with standard confocal laser scanning microscopes and conventional far-field optical microscopes[1]. Compared to traditional confocal microscopy, STED offers exceptional improvements in resolution allowing visualization of cellular events at unprecedented levels. https://www.leica-microsystems.com//science-lab/subcellular-localization-of-akt-and-tubulin-using-super-resolution-microscopy/ Wed, 30 Nov 2011 23:00:00 +0000 Ph.D. David P. Chimento, Ph.D. Myriam Gastard, PhD Carl A. Ascoli https://www.leica-microsystems.com/10159 Super-Resolution Two-color STED Microscopy of Living Synapses using a Single Laser-beam Pair The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores. https://www.leica-microsystems.com//science-lab/two-color-sted-microscopy-of-living-synapses-using-a-single-laser-beam-pair/ Sun, 20 Nov 2011 15:22:00 +0000 https://www.leica-microsystems.com/4713 Fluorescence Microscopy Modern Fluorescent Proteins and their Biological Applications Here we present two review articles on fluorescent proteins and their biological applications. These first article reviews our current knowledge of blue, green, and red chromophore formation in permanently emitting FPs, photoactivatable FPs, and fluorescent timers. The second article focuses on novel monomeric RFPs and their application for studying gene expression, nuclear localization, and dynamics using advanced imaging. https://www.leica-microsystems.com//science-lab/modern-fluorescent-proteins-and-their-biological-applications/ Fri, 18 Nov 2011 12:50:00 +0000 https://www.leica-microsystems.com/6918 Super-Resolution Webinar: Applications, Labeling Strategies and Fluorophores for Super-Resolution The free online webinar on super-resolution presented by Leica Microsystems in association with Microscopy & Analysis took place on Tuesday, 15 November 2011. Register and view the webinar on demand. https://www.leica-microsystems.com//science-lab/webinar-applications-labeling-strategies-and-fluorophores-for-super-resolution/ Tue, 15 Nov 2011 15:52:00 +0000 Dr. Marko Lampe https://www.leica-microsystems.com/4718 Neuroscience Neurobiology and Microscopy Neurobiology, the science of nerves and the brain, has mainly been driven forward in the last 200 years by microscopic investigations. The structures of cellular and subcellular structures, interaction and the three-dimensional assembly of neurons were made visible by various microscopy techniques. The optical microscope is also a necessary tool for visualizing micropipettes in electrophysiological measurements. Thirdly, many types of functional imaging are performed by means of optical microscopy. https://www.leica-microsystems.com//science-lab/neurobiology-and-microscopy/ Wed, 09 Nov 2011 13:07:00 +0000 Dr. Rolf T. Borlinghaus https://www.leica-microsystems.com/10212 Super-Resolution Natural Killer Cell Lytic Granule Secretion Occurs through a Pervasive Actin Network at the Immune Synapse Accumulation of filamentous actin (F-actin) at the immunological synapse (IS) is a prerequisite for the cytotoxic function of natural killer (NK) cells. Subsequent to reorganization of the actin network, lytic granules polarize to the IS where their contents are secreted directly toward a target cell, providing critical access to host defense. https://www.leica-microsystems.com//science-lab/natural-killer-cell-lytic-granule-secretion-occurs-through-a-pervasive-actin-network-at-the-immune-synapse/ Thu, 15 Sep 2011 11:00:00 +0000 https://www.leica-microsystems.com/10165 Super-Resolution Neuroscience Live-Cell Imaging STED Nanoscopy of Actin Dynamics in Synapses deep inside Living Brain Slices It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo. https://www.leica-microsystems.com//science-lab/sted-nanoscopy-of-actin-dynamics-in-synapses-deep-inside-living-brain-slices/ Wed, 07 Sep 2011 16:39:00 +0000 https://www.leica-microsystems.com/3911 Super-Resolution Unlimited Resolution - STED STED uses a differential method of two different diffraction patterns, where one pattern excites and the second pattern de-excites fluorochromes. The residual excited area is controllable by intensity down to (theoretically) zero – unlimited resolution. https://www.leica-microsystems.com//science-lab/unlimited-resolution-sted/ Thu, 30 Jun 2011 22:00:00 +0000 Dr. Rolf T. Borlinghaus https://www.leica-microsystems.com/10218 Super-Resolution Live-Cell Imaging Nanoscopy in a Living Multicellular Organism Expressing GFP We report superresolution fluorescence microscopy in an intact living organism, namely Caenorhabditis elegans nematodes expressing green fluorescent protein (GFP)-fusion proteins. We also superresolve, by stimulated emission depletion (STED) microscopy, living cultured cells, demonstrating that STED microscopy with GFP can be widely applied. https://www.leica-microsystems.com//science-lab/nanoscopy-in-a-living-multicellular-organism-expressing-gfp/ Wed, 15 Jun 2011 12:38:00 +0000 https://www.leica-microsystems.com/2727 Neuroscience Super-Resolution Restless Receptors Synapses are the switch-points in our brain for information transmission, learning and memory. News studies and developments of imaging techniques have provided new insights into the dynamics of glutamate receptors. The use of superresolution technologies is making an essential contribution to this research. https://www.leica-microsystems.com//science-lab/restless-receptors/ Mon, 01 Nov 2010 23:00:00 +0000 Dipl. oec.-troph. Anja Schué, PhD Daniel Choquet https://www.leica-microsystems.com/2471 Neuroscience Super-Resolution The Missing Link to the Nanocosm of Life Fully understanding the functionality and complexity of the human central nervous system remains one of the major open questions in modern science. Stimulated emission depletion microscopy (STED) can be the method to reveal biological nanostructures https://www.leica-microsystems.com//science-lab/the-missing-link-to-the-nanocosm-of-life/ Mon, 01 Nov 2010 23:00:00 +0000 https://www.leica-microsystems.com/10214 Super-Resolution Neuroscience The Fate of Synaptic Vesicle Components upon Fusion Neurotransmitter release relies on the fusion of synaptic vesicles with the plasma membrane of synaptic boutons, which is followed by the recycling of vesicle components and formation of new vesicles. It is not yet clear whether upon fusion the vesicles persist as multimolecular patches in the plasma membrane, or whether they segregate into individual components. https://www.leica-microsystems.com//science-lab/the-fate-of-synaptic-vesicle-components-upon-fusion/ Fri, 01 Oct 2010 11:41:00 +0000 Dr. Felipe Opazo https://www.leica-microsystems.com/4411 Super-Resolution A Guide to Super-Resolution Fluorescence Microscopy For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. https://www.leica-microsystems.com//science-lab/a-guide-to-super-resolution-fluorescence-microscopy/ Sun, 18 Jul 2010 22:00:00 +0000 https://www.leica-microsystems.com/2718 Super-Resolution Confocal Nanoscopy Goes Multicolor Scientists strive to understand the architecture of life. They want to learn how biological structures are arranged in respect to one another. Multicolor superresolution imaging allows fundamental questions to be addressed by far-field fluorescence microscopy in unprecedented detail. https://www.leica-microsystems.com//science-lab/confocal-nanoscopy-goes-multicolor/ Thu, 11 Feb 2010 10:06:00 +0000 PhD Jochen J. Sieber https://www.leica-microsystems.com/4138 Super-Resolution Neuroscience Observing Life’s Nanostructures with STED The secrets of life and the causes of many diseases can only be fully explained if we understand the functions of the smallest components of organisms. Using the super high resolution STED microscope, research scientists are now able to observe cellular proteins and molecular structures measuring only a few nanometres. https://www.leica-microsystems.com//science-lab/observing-lifes-nanostructures-with-sted/ Mon, 16 Mar 2009 23:00:00 +0000 Dipl. oec.-troph. Anja Schué, Prof. Dr. Stephan Sigrist, Prof. Silvio Rizzoli, Dr. Gregorz Wilczynski https://www.leica-microsystems.com/9445 Super-Resolution In Vivo Labeling Method Using a Genetic Construct for Nanoscale Resolution Microscopy We demonstrate beam scanning-stimulated emission depletion microscopy with in vivo labeled cells. A red emitting fluorescent dye is introduced into membrane protein fused to a multifunctional reporter protein (HaloTag, Promega, Madison, WI) in live cells. This approach allows superresolution stimulated emission depletion imaging without the limitations of immunofluorescence-based staining. https://www.leica-microsystems.com//science-lab/in-vivo-labeling-method-using-a-genetic-construct-for-nanoscale-resolution-microscopy/ Wed, 07 Jan 2009 12:21:00 +0000 Dr. Jan Schröder, Hélène Benink, Marcus Dyba https://www.leica-microsystems.com/14660 Super-Resolution Interview with Stefan Hell – For me, Pioneering is … Our interview partner on the topic of Pioneering is Prf. Stefan Hell, a scientific member of the Max Planck Society and director at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, where he heads the Department of Nanobiophotonics. In addition to further chairs and memberships, he is also head of the High resolution Optical Microscopy research group, a partnership department of the German Cancer Research Center (DFKZ). https://www.leica-microsystems.com//science-lab/interview-with-stefan-hell-for-me-pioneering-is/ Sun, 31 Aug 2008 22:00:00 +0000