Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands.

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.