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  • Human NK Cell Development Requires CD56-mediated Motility and Formation of the Developmental Synapse
    Science Lab Topic: Confocal Microscopy

    Human NK Cell Development Requires CD56-mediated Motility and Formation of the Developmental Synapse

    While distinct stages of natural killer (NK) cell development have been defined, the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which, through contact-dependent mechanisms, promote the generation of mature, functional human NK cells from CD34+ precursors. We show that developing NK cells undergo unique, developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells, and that the relative motility of NK cells increases as they move through development in vitro and ex vivo.
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  • Actin-Dependent Vacuolar Occupancy of the Cell Determines Auxin-Induced Growth Repression
    Science Lab Topic: 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.
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  • Visualizing Tropoelastin in a Long-Term Human Elastic Fibre Cell Culture Model
    Science Lab Topic: Super-Resolution

    Visualizing Tropoelastin in a Long-Term Human Elastic Fibre Cell Culture Model

    Elastin is an essential protein found in a variety of tissues where resilience and flexibility are needed, such as the skin and the heart. When aiming to engineer suitable implants, elastic fibres are needed to allow adequate tissue renewal. However, the visualization of human elastogenesis remains in the dark. To date, the visualization of human tropoelastin (TE) production in a human cell context and its fibre assembly under live cell conditions has not been achieved. Here, we present a long-term cell culture model of human dermal fibroblasts expressing fluorescence-labelled human TE. We employed a lentiviral system to stably overexpress Citrine-labelled TE to build a fluorescent fibre network. Using immunofluorescence, we confirmed the functionality of the Citrine-tagged TE. Furthermore, we visualized the fibre assembly over the course of several days using confocal microscopy. Applying super resolution microscopy, we were able to investigate the inner structure of the elastin–fibrillin-1 fibre network.
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  • P53- and Mevalonate Pathway–Driven Malignancies Require Arf6 for Metastasis and Drug Resistance
    Science Lab Topic: Super-Resolution

    P53- and Mevalonate Pathway–Driven Malignancies Require Arf6 for Metastasis and Drug Resistance

    Application example of HvYolution Super-Resolution - Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial–mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program.
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  • Mirror-Enhanced Super-Resolution Microscopy
    Science Lab Topic: 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.
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  • Time Gating of Chloroplast Autofluorescence Allows Clearer Fluorescence Imaging In Planta
    Science Lab Topic: White Confocal

    Time Gating of Chloroplast Autofluorescence Allows Clearer Fluorescence Imaging In Planta

    Chloroplast, an organelle facilitating photosynthesis, exhibits strong autofluorescence, which is an undesired background signal that restricts imaging experiments with exogenous fluorophore in plants. In this study, the autofluorescence was characterized in planta under confocal laser microscopy, and it was found that the time-gated imaging technique completely eliminates the autofluorescence. As a demonstration of the technique, a clearer signal of fluorescent protein-tagged phototropin, a blue-light photoreceptor localized at the chloroplast periphery, was visualized in planta.
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  • The Bimodally Expressed MicroRNA miR‐142 Gates Exit from Pluripotency
    Science Lab Topic: Confocal Microscopy

    The Bimodally Expressed MicroRNA miR‐142 Gates Exit from Pluripotency

    A stem cell's decision to self‐renew or differentiate is thought to critically depend on signaling cues provided by its environment. It is unclear whether stem cells have the intrinsic capacity to control their responsiveness to environmental signals that can be fluctuating and noisy. Using a novel single‐cell microRNA activity reporter, we show that miR‐142 is bimodally expressed in embryonic stem cells, creating two states indistinguishable by pluripotency markers.
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  • From Light to Mind: Sensors and Measuring Techniques in Confocal Microscopy
    Science Lab Topic: Confocal Microscopy

    From Light to Mind: Sensors and Measuring Techniques in Confocal Microscopy

    This article outlines the most important sensors used in confocal microscopy. By confocal microscopy, we mean "True Confocal Scanning", i.e. the technique that illuminates and measures one single point only. The aim is not to impart in-depth specialist knowledge, but to give the user a small but clear overview of the differences between the various technologies and to advise on which sensor may be most suitable for which applications.
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  • Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points
    Science Lab Topic: Multiphoton Microscopy

    Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

    Cancer research unsing multiphoton microscopy and 3D electron microscopy. Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task.
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  • "Leica is always flexible and dynamic" - Interview with Audrey Salles, Pasteur Institute, Paris
    Science Lab Topic: Confocal Microscopy

    "Leica is always flexible and dynamic" - Interview with Audrey Salles, Pasteur Institute, Paris

    Audrey Salles is a specialist for confocal and super-resolution microscopy at Pasteur Institute, Imagopole, PFID, Paris, France. Her research interests are cytokine signaling and skeleton organization of human TCD4-cells.
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  • Multiple Microscopy Modes in a Single Sweep with Supercontinuum White Light
    Science Lab Topic: White Confocal

    Multiple Microscopy Modes in a Single Sweep with Supercontinuum White Light

    Lasers have been critical to the advancement on confocal microscopy, and the white light laser (WLL) offers particular advantages. Finessing WLL output for bioimaging is a complex task, though, and traditional approaches retain key limitations. But acousto-optical beamsplitting enables smoother operation, leading to enhanced microscopy capabilities.
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  • Detectors for Sensitive Detection: HyD
    Science Lab Topic: Confocal Microscopy

    Detectors for Sensitive Detection: HyD

    This article discusses detectors (more precisely: sensors), that are employed in single point, i.e. true confocal scanning microscopes. The sensors in such systems are usually photomultiplier tubes. Also, the silicon pendants of PMTs are used for particular applications, especially single-molecule measurements. A new development has led to chimeric devices called hybrid detector (HyD) which unite benefits of both technologies.
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  • Sensors for True Confocal Scanning
    Science Lab Topic: Confocal Microscopy

    Sensors for True Confocal Scanning

    In this article, advantages and disadvantages of different types of sensors for single point true confocal scanning devices are discussed. Traditionally, photomultiplier tubes have been employed in such systems. For some cases, avalanche photodiodes have proven to fit best. A new development uniting vacuum and silicon technology has led to chimeric sensors, called hybrid detectors (HyD). They benefit from both technologies.
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  • Step by Step Guide to Hybrid Detection and Photon Counting
    Science Lab Topic: Live-Cell Imaging

    Step by Step Guide to Hybrid Detection and Photon Counting

    This tutorial explains the underlying hybrid detection technology and compares it to photomultiplier technology. The implications of hybrid detection design for imaging and photon counting are discussed. The tutorial closes with a brief summary of photon counting in the context of imaging.
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