Leica Science Lab - Tag : gated STED https://www.leica-microsystems.com//science-lab/tag/tags/gated-sted/show/Tag/ Article tagged with gated STED en-US https://www.leica-microsystems.com/30582 Super-Resolution Time-resolved STED microscopy Introduced more than 30 years ago, stimulated emission depletion (STED) microscopy has raised to a standard and widely used method for imaging in the life sciences. Thanks to continuous technological progress, STED microscopy can now provide effective sub-diffraction spatial resolution, while preserving most of the useful aspects of fluorescence microscopy, such as optical sectioning, molecular specificity and sensitivity, and live-cell compatibility. https://www.leica-microsystems.com//science-lab/time-resolved-sted-microscopy/ Thu, 03 Sep 2020 12:04:00 +0000 Ph.D. Giuseppe Vicidomini 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/18098 Super-Resolution Actin-Dependent Vacuolar Occupancy of the Cell Determines Auxin-Induced Growth Repression The cytoskeleton is an early attribute of cellular life, and its main components are composed of conserved proteins. The actin cytoskeleton has a direct impact on the control of cell size in animal cells, but its mechanistic contribution to cellular growth in plants remains largely elusive. Here, we reveal a role of actin in regulating cell size in plants. The actin cytoskeleton shows proximity to vacuoles, and the phytohormone auxin not only controls the organization of actin filaments but also impacts vacuolar morphogenesis in an actin-dependent manner. https://www.leica-microsystems.com//science-lab/actin-dependent-vacuolar-occupancy-of-the-cell-determines-auxin-induced-growth-repression/ Fri, 03 Feb 2017 08:14:00 +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 Imaging 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 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/18164 Super-Resolution TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion Collagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export. https://www.leica-microsystems.com//science-lab/tfg-promotes-organization-of-transitional-er-and-efficient-collagen-secretion/ Tue, 14 Jun 2016 08:36:00 +0000 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/16176 Super-Resolution Quantitative Imaging 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/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/14980 Super-Resolution 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/14656 Confocal Microscopy Super-Resolution CLEM Live-Cell Imaging Quantitative Imaging ICln: A New Regulator of Non-Erythroid 4.1R Localisation and Function To optimise the efficiency of cell machinery, cells can use the same protein (often called a hub protein) to participate in different cell functions by simply changing its target molecules. There are large data sets describing protein-protein interactions ("interactome") but they frequently fail to consider the functional significance of the interactions themselves. https://www.leica-microsystems.com//science-lab/icln-a-new-regulator-of-non-erythroid-41r-localisation-and-function/ Mon, 03 Nov 2014 18:02:00 +0000 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/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/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