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/26264 Super-Resolution The Guide to STED Sample Preparation This guide is intended to help users optimize sample preparation for stimulated emission depletion (STED) nanoscopy, specifically when using the TCS SP8 STED 3X nanoscope from Leica Microsystems. It gives an overview of fluorescent labels used for single color STED imaging and a ranking of their performance. Fluorescent label combinations for dual and triple color STED imaging that minimize cross-talk during detection are recommended. There is a discussion of considerations for immunofluorescence labeling and a detailed protocol to obtain high quality images, with a high signal/noise (S/N) ratio, of interesting structures in a specimen. Important details for sample mounting and substrates that enable optimal imaging, minimizing aberrations and autofluorescence due to the mounting medium, are reviewed. Finally, for STED imaging of live-cells, the most appropriate fluorescent labels are mentioned, both fluorescent proteins (FPs) and organic fluorophores which give the best performance. https://www.leica-microsystems.com//science-lab/the-guide-to-sted-sample-preparation/ Mon, 22 Jul 2019 17:00:00 +0000 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, Prof. Dr. Torsten Ochsenreiter, Dr. Martin Offterdinger, Dr. Jordi Andilla, Ph.D. Marc van Zandvoort, Prof. Christian Eggeling, Dr. Susan Cox, Dr. Eugene Katrukha, Dr. Jindřiška Fišerová, 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