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      저자 : Lohmannsroben, Hans-Gerd (검색결과 1 건)
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        Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands

        Hille, Carsten,Lahn, Mattes,Lohmannsroben, Hans-Gerd,Dosche, Carsten Korean Society of Photoscience 2009 Photochemical & photobiological sciences Vol.8 No.3

        Although chloride plays an important role in many cellular processes, there is a lack of data about intracellular chloride concentrations $[Cl^-]_i$, particularly due to technical problems. To overcome that, in this study fluorescence lifetime imaging microscopy in the time-domain by using time-correlated single-photon counting was combined with two-photon excitation (2P-FLIM). This 2P-FLIM setup has been successfully used with the $Cl^-$-sensitive fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxy-quinolinium bromide (MQAE) in order to measure $[Cl^-]_i$ in cockroach salivary glands, a well-established model system for studying epithelial ion transport processes. MQAE was well suitable for two-photon excitation, when loaded into cells, and displayed a sufficient dynamic range of its fluorescence decay time changes in response to variation of $[Cl^-]_i$ according to the Stern-Volmer relationship. On this basis a uniform $[Cl^-]_i$ in the range of 42.80 mM with a mean value of $59\;mM{\pm}1\;mM$ was found in resting cockroach salivary ducts, indicating active $Cl^-$ accumulation. However, exposure to $Cl^-$-free saline caused only a moderate $[Cl^-]_i$ drop to $48\;mM{\pm}4\;mM$, suggesting a relatively low basolateral $Cl^-$ permeability in ducts, at least under resting conditions. Additionally, bath application of the biogenic amine dopamine, known to stimulate the saliva modification in the ducts, caused no significant $[Cl^-]_i$ changes. These results suggest a more complex scenario of $[Cl^-]_i$ homeostasis in cockroach salivary ducts. In conclusion, 2P-FLIM seems to be a suitable technique for quantitative $[Cl^-]_i$ measurements in many biological systems.

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