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  • Video Talk by Roger Tsien: Fluorescent Protein Indicators

    In this talk, Roger Tsien discusses how fluorescent proteins have been turned into indicators for a wide variety of biological molecules, including pH, ions, redox potential, and signaling molecules like phosphoinositides. The talk also covers reporters used to measure the activity of enzymes like kinases, phosphatases, and proteases. It covers both single proteins whose intensity or wavelength change, as well as reporters using resonance energy transfer (FRET).
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  • Fluorescent Proteins Illuminate Cell Biology

    Green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria and GFP-like fluorescent proteins from other animals have had an important role in the technical innovations that have driven these advances. This poster provides a comprehensive user's guide to fluorescent proteins and sensors , their key properties and the cell biological questions to which they can be applied.
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  • How to Measure FRET

    Here, I will expand, including what to measure when doing FRET. There are a number of approaches to FRET quantification: 1. Sensitized Emission – This two-channel imaging technique uses an algorithm that corrects for excitation and emission crosstalk.
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  • You May Not Know Theodor Förster but You Know His Work: FRET

    If you think FRET stands for Fluorescence Resonance Energy Transfer, you are wrong … in good company but wrong. FRET actually stands for Förster Resonance Energy Transfer. Find out why and more about FRET in this article.
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  • Universal PAINT – Dynamic Super-Resolution Microscopy

    Super-resolution microscopy techniques have revolutionized biology for the last ten years. With their help cellular components can now be visualized at the size of a protein. Nevertheless, imaging living cells is a challenge for most of the super-resolution principles.
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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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  • Novel Fluorescent Carbonic Nanomaterials for Sensing and Imaging

    Small brightly fluorescent carbon nanoparticles have emerged as a new class of materials important for sensing and imaging applications. We analyze comparatively the properties of nanodiamonds, graphene and graphene oxide ‘dots’, of modified carbon nanotubes and of diverse carbon nanoparticles known as ‘C-dots’ obtained by different methods.
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  • FRET with FLIM

    FLIM combines lifetime measurements with imaging: lifetimes obtained for each image pixel are color-coded to produce additional image contrast. Thus, FLIM delivers information about the spatial distribution of a fluorescent molecule together with information about its biochemical status or nano-environment. A typical application of FLIM is FLIM-FRET. FRET is a well-established technique to study molecular interactions. It scrutinizes protein binding and estimates intermolecular distances on an Angström scale as well.
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  • Quantitative Fluorescence

    Seeing is believing – and measuring is knowing. Microscopes generate images that are not only used for illustration, but are also subject to quantification. More advanced techniques use illumination patterns (without image formation) or do not generate an image at all – but are still microscopical techniques. These F-techniques are becoming increasingly important in current biosciences.
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  • Live-cell Imaging Techniques

    The understanding of complex and/or fast cellular dynamics is an important step for exploring biological processes. Therefore, today’s life science research is increasingly focusing on dynamic processes like cell migration, morphological changes of cells, organs or whole animals and physiological (e.g. changes of intracellular ion composition) events in living specimens in real time.
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  • Modern Fluorescent Proteins and their Biological Applications

    Here we present two review articles on fluorescent proteins and their biological applications. These first article reviews our current knowledge of blue, green, and red chromophore formation in permanently emitting FPs, photoactivatable FPs, and fluorescent timers. The second article focuses on novel monomeric RFPs and their application for studying gene expression, nuclear localization, and dynamics using advanced imaging.
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  • Förster Resonance Energy Transfer (FRET)

    The Förster Resonance Energy Transfer (FRET) phenomenon offers techniques that allow studies of interactions in dimensions below the optical resolution limit. FRET describes the transfer of the energy from an excited state of a donor molecule to an acceptor molecule. Unlike absorption or emission of photons, FRET is a non-radiative energy exchange and consequently not a variation of light-matter interactions.
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  • FLIM-FRET in Solutions

    FRET efficiency can be measured based on fluorescence lifetime microscopy (FLIM). FLIM-FRET allows analysis of molecular interactions both in vitro and in vivo. This article describes the use of FLIM in the time domain (TCSPC) to measure FRET in vitro in a biochemical assay using a Cerulean-Citrine construct.
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  • Webinar: Principles and Applications of Multiphoton Imaging

    Basics of multiphoton microscopy. The advent of multiphoton microscopy has enabled researchers to image deeper, with greater resolution and less background, leading to new scientific insights into topics ranging from stem cell biology to the cellular effects of human disease. In this webinar, you will learn about the latest approaches and applications of multiphoton imaging.
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  • A mTurquoise-Based cAMP Sensor for Both FLIM and Ratiometric Read-Out Has Improved Dynamic Range

    FRET-based sensors for cyclic Adenosine Mono Phosphate (cAMP) have revolutionized the way in which this important intracellular messenger is studied. The currently prevailing sensors consist of the cAMP-binding protein Epac1, sandwiched between suitable donor- and acceptor fluorescent proteins (FPs).
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  • Webinar: Principles and Applications of 
Live Cell Imaging

    Live cell imaging has become a cornerstone of modern cell biology, as well as a technique routinely employed in other fields such as developmental biology, stem cell biology and neuroscience. In this webinar, you will learn about the effective use of live cell imaging for a growing number of applications.
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  • Choose Your Excitation Wavelength

    Although time correlated single photon counting (TCSPC) is the method of choice for fluorescence lifetime quantification, it requires dedicated instrumentation including a pulsed laser source, a photon counting card, and a fast detector.
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  • ATP Changes the Fluorescence Lifetime of Cyan Fluorescent Protein via an Interaction with His148

    Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions.
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  • The First Supercontinuum Confocal that Adapts to the Sample

    Until now, biological and medical research fluorescence imaging in multi-user facilities or institutes has been limited by the type or number of dyes that could be excited. The Leica TCS SP5 X supercontinuum confocal unites the broadband capabilities of the Leica TCS SP5 AOBS® and the freedom and flexibility to select any excitation line within the continuous range of 470 to 670 nm.
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  • A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP Sensor as an Example

    We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker.
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  • FRET Measurements on Fuzzy Fluorescent Nanostructures

    In the last decade, fluorescence resonance energy transfer (FRET) has become a useful technique for studying intermolecular interactions applied to the analysis of biological systems. Although FRET measurements may be very helpful in the comprehension of different cellular processes, it can be difficult to obtain quantitative results, hence the necessity of studying FRET on controllable systems.
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  • FRET Sensitized Emission Wizard Confocal

    Fluorescence Resonance Energy Transfer (FRET) is a technique, which allows insight into the interactions between proteins or molecules in proximities beyond light microscopic resolution.
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  • Correcting Confocal Acquisition to Optimize Imaging of Fluorescence Resonance Energy Transfer by Sensitized Emission

    Imaging of fluorescence resonance energy transfer (FRET) between suitable fluorophores is increasingly being used to study cellular processes with high spatiotemporal resolution. The genetically encoded Cyan (CFP) and Yellow (YFP) variants of Green Fluorescent Protein have become the most popular donor and acceptor pair in cell biology. FRET between these fluorophores can be imaged by detecting sensitized emission. This technique, for which CFP is excited and transfer is detected as emission of YFP, is sensitive, fast, and straightforward, provided that proper corrections are made. In this study, the detection of sensitized emission between CFP and YFP by confocal microscopy is optimized.
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