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Department of Molecular Physiology

Our interests

fluo-microscopy
Chloride measurement




Fluorescence microscopy is a very useful method for the measurement of intracellular ions. Intracellular chloride measurements by fluorescence dyes have been in use since end of the 1980's. Several chloride-sensitive dyes are commercially available. Their fluorescence is physically quenched by chloride ions so that an increasing chloride concentration causes a decrease of fluorescence intensity. The relationship between fluorescence intensity and chloride concentration is given by the Stern-Volmer equation. In our lab, we use MQAE, which has peak absorption at 350 nm and emission at 460 nm. MQAE has a high sensitivity for chloride ions, a high fluorescence quantum yield and high membrane permeability. MQAE can remain in the cell for about 1 hour after the loading.

To obtain absolute values for the intracellular chloride concentration, the relation between fluorescence intensity and intracellular chloride concentration must be established. Calibration is necessary for each individual cell because the fluorescence intensity depends not only on intracellular chloride concentration but also on the dye concentration in the cells, cell volume and so on. Thus, intracellular calibration is very difficult. A solution for this problem may be fluorescence lifetime analysis. Fluorescence lifetime is not affected by dye concentration. The fluorescence lifetime can replace fluorescence intensity in the Stern-Volmer equation. When the intracellular quenching efficiency is known, fluorescence lifetime, unlike intensity, directly reports the intracellular chloride concentration. Moreover, lifetime imaging can be applied not only to isolated cells but also to intact tissues.



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