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  • 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.
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  • 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.
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  • 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.
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  • 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.
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  • 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.
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  • 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.
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  • Pathways to Optical STED Microscopy

    STED nanoscopy has evolved to a highly versatile tool for the observation of the living cell, more and more finding its way into state-of-the-art optical imaging facilities in biomedical research institutes.
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  • 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.
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  • 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.
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  • Huygens STED Deconvolution Quick Guide

    This document is intended to give Leica STED users a brief introduction to deconvolving images on Huygens Professional using images acquired with the Leica TCS SP8 STED 3X microscope. For a more detailed description of Huygens Professional, including additional features and tools, please visit the Manuals section of SVI (www.svi.nl).
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  • 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
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