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Ratiometric Imaging

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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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  • Widefield Calcium Imaging with Calcium Indicator Fura2

    In eukaryotic cells Ca2+ is one of the most widespread second messengers used in signal transduction pathways. Intracellular levels of Ca2+ are usually kept low, as Ca2+ often forms insoluble complexes with phosphorylated and carboxylated compounds. Typically cytosolic Ca2+ concentrations are in the range of 100 nM. In response to stimuli Ca2+ may either be released from external medium or internal stores to raise the Ca2+ concentration.
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  • Ratiometric Imaging Needs a Specialized Setup

    Ratiometric imaging is widely used to study highly dynamic intracellular ion, voltage or pH changes. The most common application, however, is calcium imaging. Ratiometric imaging is also used for investigating cellular networks, where e.g. relative calcium concentrations are passed among cells or different cell types dynamically change ion, voltage or pH levels. Also FRET assays can considerably benefit from ratiometric imaging, as the signal-to-noise-ratio is greatly improved.
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  • Ratiometric Imaging

    Many fundamental functions of a cell strongly depend on delicate, but nevertheless dynamic balances of ions (e.g. calcium, magnesium), voltage potentials and pH between the cell’s cytosol and the surrounding extracellular space. Ratiometric imaging allows reliable estimations of ion concentrations and pH or voltage changes by measuring fluorophore emission shifts.
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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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