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Issue 3, 2009
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Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands

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Abstract

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 ± 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 ± 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.

Graphical abstract: Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands

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Publication details

The article was received on 08 Aug 2008, accepted on 28 Nov 2008 and first published on 05 Jan 2009


Article type: Paper
DOI: 10.1039/B813797H
Citation: Photochem. Photobiol. Sci., 2009,8, 319-327
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    Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands

    C. Hille, M. Lahn, H. Löhmannsröben and C. Dosche, Photochem. Photobiol. Sci., 2009, 8, 319
    DOI: 10.1039/B813797H

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