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  • Introduction to Mammalian Cell Culture

    Mammalian cell culture is one of the basic pillars of life sciences. Without the ability to grow cells in the lab, the fast progress in disciplines like cell biology, immunology, or cancer research would be unthinkable. This article gives an overview of mammalian cell culture systems. Mainly, they can be categorized according to their morphology, as well as cell type and organization. Moreover, you can find basic information about the correct growth conditions and what kind of microscope you need to watch your cells.
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  • 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.
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  • Chronic Inflammation Under the Microscope

    In the course of chronic inflammation certain body areas are recurrently inflamed. This goes along with many human diseases. With the help of widefield light microscopy, the underlying processes can be examined from a cellular level to whole organisms. This article presents several widefield microscopy applications such as immunofluorescence, live-cell imaging, histology, and ratiometric analysis to get insight into the development of chronic inflammation, the related diseases, and their treatment.
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  • 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.
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  • Webinar: Imaging CRISPR

    CRISPR has become one of the biologist’s favorite ways for deleting, replacing, or editing DNA, and much of the conversation about CRISPR-Cas9 has revolved around its potential for gene editing in health and disease. This webinar will showcase how CRISPR has also begun to revolutionize our understanding of how genomes work and will discuss the potential of CRISPR imaging tools to study genetic elements within living cells. Two leaders in this field, Gene Yeo from UCSD and Bo Huang from UCSF, discuss techniques, technology, and insights on CRISPR imaging.
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  • Discovery of Novel Peptides Targeting Pro-Atherogenic Endothelium in Disturbed Flow Regions -Targeted siRNA Delivery to Pro-Atherogenic Endothelium in vivo

    Application example of HyVolution Super-Resolution - Atherosclerosis occurs preferentially in arterial regions exposed to disturbed blood flow. Targeting these pro-atherogenic regions is a potential anti-atherogenic therapeutic approach, but it has been extremely challenging. Here, using in vivo phage display approach and the partial carotid ligation model of flow-induced atherosclerosis in mouse, we identified novel peptides that specifically bind to endothelial cells (ECs) exposed to disturbed flow condition in pro-atherogenic regions.
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  • Webinar: Introduction to Fluorescence Microscopy

    In this seminar we will provide an overview about the latest advances in fluorescence microscopy. You will learn how you can use widefield and confocal microscopes to help you understand life’s questions down to tiny details, at high speed and state-of-the-art image quality both in living and fixed samples.
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  • Adeno-associated Viral Vectors do not Efficiently Target Muscle Satellite Cells

    Adeno-associated viral (AAV) vectors are becoming an important tool for gene therapy of numerous genetic and other disorders. Several recombinant AAV vectors (rAAV) have the ability to transduce striated muscles in a variety of animals following intramuscular and intravascular administration, and have attracted widespread interest for therapy of muscle disorders such as the muscular dystrophies. Here we examined the relative ability of rAAV vectors derived from AAV6 to target myoblasts, myocytes, and myotubes in culture and satellite cells and myofibers in vivo. AAV vectors are able to transduce proliferating myoblasts in culture, albeit with reduced efficiency relative to postmitotic myocytes and myotubes. In contrast, quiescent satellite cells are refractory to transduction in adult mice.
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  • Highly Selective Fluorescent and Colorimetric Probe for Live-cell Monitoring of Sulphide Based on Bioorthogonal Reaction

    H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S are desired. Azide fluorogenic probes are popular because they can take place bioorthogonal reactions. In this work, by employing a fluorescein derivative as the fluorophore and an azide group as the recognition unit, we reported a new probe 5-azidofluorescein for H2S with improved sensitivity and selectivety.
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  • Label-free in vivo Imaging of Myelinated Axons in Health and Disease with Spectral Confocal Reflectance Microscopy

    We report a new technique for high-resolution in vivo imaging of myelinated axons in the brain, spinal cord and peripheral nerve that requires no fluorescent labeling. This method, based on spectral confocal reflectance microscopy (SCoRe), uses a conventional laser scanning confocal system to generate images by merging the simultaneously reflected signals from multiple lasers of different wavelengths.
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  • Individual Macromolecule Motion in a Crowded Living Cell

    There is solid evidence for analyzing fluorescence correlation and dual color fluorescence crosscorrelation spectroscopy data ( FCS and dual color FCCS) in cellular applications by equations based on anomalous subdiffusion. Using equations based on normal diffusion causes artifacts of the fitted biological system response parameters and of the interpretations of the FCS and dual color FCCS data in the crowded environment of living cells. Equations based on normal diffusion are not valid in living cells. The original article embraces the status of the experimental situation and touches obstacles that still hinder the applications of single molecules in the cellular environment.
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  • 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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  • What Makes sCMOS Microscope Cameras so Popular?

    sCMOS cameras are more sensitive and are capable of much higher acquisition speed than cameras with other sensor types. Even though CCD cameras are widely used in live cell imaging and time-lapse recordings, researchers are often concerned that their camera does not detect faint signals. In this interview, Dr. Karin Schwab, Product Manager at Leica Microsystems, talks about the characteristics of sCMOS cameras and how researchers benefit from the latest camera sensor technology.
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  • How to do a Proper Cell Culture Quick Check

    In order to successfully work with mammalian cell lines, they must be grown under controlled conditions and require their own specific growth medium. In addition, to guarantee consistency their growth must be monitored at regular intervals. This article describes a typical workflow for subculturing an adherent cell line with detailed illustrations of all of the necessary steps.
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  • HyVolution – the Smart Path to Confocal Super-Resolution

    Super-resolution refers to any device or method that can resolve better than the classical Abbe limit. Apart from infinite super-resolution techniques such as STED (stimulated emission depletion) and SMLM (single-molecule localization methods) that can theoretically resolve to any detail, there are also methods for limited super-resolution. Here we present HyVolution by Leica, which merges optical super-resolution and computational super-resolution. The optical part is provided by confocal microscopy, and the computational part by deconvolution. Lateral resolution of 140 nm is demonstrated. HyVolution offers multiple fluorescence recording in truly simultaneous mode.
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  • 4Pi-RESOLFT Nanoscopy

    Here we apply the 4Pi scheme to RESOLFT nanoscopy using two-photon absorption for the on-switching of fluorescent proteins. We show that in this combination, the lobes are so low that low-light level, 3D nanoscale imaging of living cells becomes possible. Our method thus offers robust access to densely packed, axially extended cellular regions that have been notoriously difficult to super-resolve. Our approach also entails a fluorescence read-out scheme that translates molecular sensitivity to local off-switching rates into improved signal-to-noise ratio and resolution.
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  • FRAP with TCS SP8 Resonant Scanner

    Fast FRAP experiments need a sufficient number of measurement points for meaningful interpretation and fitting analysis. To study very fast translocational processes, the use of a resonant scanner (RS) is preferred. The advantage in using FRAP with the RS is that statistics are much better in experiments that require fast acquisition: If the half time of recovery is about 0.5 sec you may have only about 3 to 4 data points using the conventional scanner, whereas with the resonant scanner you can get about 20 data points.
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  • Webinar: Dissecting Protein Dynamics in Living Cells by FRAP

    This webinar presented by Dr Marco Fritzsche, University of Oxford, and Jennifer Horner, PhD, Leica Microsystems, you will learn about how to use Fluorescence Recovery After Photo-bleaching (FRAP) microscopy to study protein dynamics.
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  • Webinar: Using FIT Probes and Super-Resolution Microscopy to Decipher Steps of mRNP Assembly in Developing Oocytes

    In this webinar, presented by Dr. Imre Gaspar of the Developmental Biology Unit at EMBL, you will learn: importance of mRNA localization and function of mRNPs, advantages of using fluorogenic FIT probes to visualize mRNPs in vivo and in fixed specimen, and how super-resolution microscopy can identify factors required for mRNP biogenesis.
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  • Light Sheet Microscopy Turned Vertically

    Living cells and organisms often suffer from the high light intensities used for fluorescent imaging. Light sheet microscopy reduces phototoxic effects and bleaching by illuminating a specimen in only a single plane at a time. A new light sheet microscope combines light sheet and confocal microscopy in one system without compromising either functionality and allows the combination of the two methods, e.g. confocal photomanipulation with subsequent light sheet acquisition, for new applications.
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  • 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).
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  • Webinar: Advances in Live Cell Imaging - Skin Cell Motility: Integrins Lead the Way

    In this webinar presented by Leica Microsystems and LabRoots you will learn about efforts to dissect mechanisms that underlie the directed migration of epidermal keratinocytes as they populate wounds or in cancer.
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  • 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.
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  • CRISPR/Cas9-mediated Endogenous Protein Tagging for RESOLFT Super-Resolution Microscopy of Living Human Cells

    Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein.
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  • 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.
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  • Webinar: The Best of Both Worlds: Combining Light Sheet and Confocal Microscopy

    Living cells and organisms often suffer from high light intensities that are used in conventional imaging. Light sheet microscopy reduces phototoxic effects and bleaching, by only illuminating a specimen in a single plane at a time whilst the signal is detected in a perpendicular direction. In combination with high-speed cameras for image acquisition, light sheet microscopy is a very gentle method to observe fast biological processes in sensitive organisms over an extended time period.
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  • Correlative Light and Electron Microscopy – Get Your Free CLEM e-Book for Download

    The urge to go deeper into the microscopic world has led researchers to combine the versatility of the light microscopy (LM) with the resolution power of the electron microscope (EM) to produce Correlative Light and Electron Microscopy (CLEM). CLEM’s most significant and powerful characteristic for cell biology research is an ability to study the same cell using two different microscopy platforms. The Essential Knowledge Briefing describes the basics of CLEM and also reviews potential pitfalls and problems as well as tricks for solving them.
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  • Webinar: Light Sheet Imaging – New Solutions and Their Applications in Zebrafish Embryogenesis

    Living cells and organisms often suffer from high light intensities that are used in conventional imaging. Light sheet microscopy reduces phototoxic effects and bleaching, by only illuminating a specimen in a single plane at a time whilst the signal is detected in a perpendicular direction. In combination with high-speed cameras for image acquisition, light sheet microscopy is a very gentle method to observe fast biological processes in sensitive organisms over an extended time period.
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  • Third Harmonic Generation Microscopy – Label-Free 3D-Tissue Imaging and Blood Flow Characterization

    THG microscopy as special variants of multiphoton microscopy. Third Harmonic Generation (THG) microscopy is a non-fluorescent multi-photon technique that combines the advantages of label-free imaging with restriction of signal generation to the focal spot of the scanning laser. It allows three-dimensional imaging of refraction index mismatches and of hemoglobin.
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