Ratiometric Imaging

Research activities are increasingly focusing on the identification and the spatial and temporal distribution of e.g. local “hot spots” for dynamic changes in ion concentration, voltage or pH in a cell or a cellular network. Such “hot spots” are often localized in specialized parts of a cell or in certain cells in a cellular network. Additionally, these areas often have different properties compared to the rest of the specimen in terms of cell metabolism or structure. Conventional fluorophores used to investigate dynamic physiological states change their emission intensity upon ion binding, pH change or voltage change (e.g. fluo-4 has increased emission upon calcium binding). However, these markers do not take into account that differences in structure, diameter or marker uptake/expression can cause changes in the quantity of emitted light that are not in correlation with the actual ion concentration, voltage or pH. To quantitatively and comparably detect the changes in cellular structures or different cells, a method insensitive to structure diameter and fluorophore concentration is needed. In contrast to non-ratiometric imaging methods, ratiometric imaging offers the opportunity to reproducibly measure absolute intracellular ion, voltage and pH levels/changes with respect to cell diameter, fluorophore concentration and optical properties of the imaging setup. However, ratiometric imaging depends on a fast change of excitation wavelength or the detected wavelength, a strong light source, excellent transmission of optical components and fast signal detection. The recent development of ultrafast filter wheels, UV-light optimized objectives, highly sensitive fluorophores and new

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