Hille Carsten, Lahn Mattes, Löhmannsröben Hans-Gerd, Dosche Carsten
Physical Chemistry/Applied Laser Sensing, Institute of Chemistry & Interdisciplinary Center for Photonics, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476, Potsdam-Golm, Germany.
Photochem Photobiol Sci. 2009 Mar;8(3):319-27. doi: 10.1039/b813797h. Epub 2009 Jan 5.
Although chloride plays an important role in many cellular processes, there is a lack of data about intracellular chloride concentrations Cl(-), 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(-) 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(-) according to the Stern-Volmer relationship. On this basis a uniform Cl(-) 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(-) 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(-) changes. These results suggest a more complex scenario of Cl(-) homeostasis in cockroach salivary ducts. In conclusion, 2P-FLIM seems to be a suitable technique for quantitative Cl(-) measurements in many biological systems.
尽管氯离子在许多细胞过程中发挥着重要作用，但关于细胞内氯离子浓度[Cl⁻]i的数据却很缺乏，尤其是由于技术问题。为了克服这一问题，在本研究中，将时域中的荧光寿命成像显微镜与时间相关单光子计数相结合，并采用双光子激发（2P-FLIM）。这种2P-FLIM装置已成功用于与Cl⁻敏感荧光染料N-(乙氧羰基甲基)-6-甲氧基喹啉溴化物（MQAE）结合，以测量蟑螂唾液腺中的[Cl⁻]i，蟑螂唾液腺是研究上皮离子转运过程的成熟模型系统。当MQAE加载到细胞中时，它非常适合双光子激发，并根据斯特恩-沃尔默关系，其荧光衰减时间变化对[Cl⁻]i变化显示出足够的动态范围。在此基础上，在静息的蟑螂唾液导管中发现[Cl⁻]i均匀分布在42-80 mM范围内，平均值为59 mM±1 mM，表明存在活跃的Cl⁻积累。然而，暴露于无Cl⁻盐溶液中仅导致[Cl⁻]i适度下降至48 mM±4 mM，这表明导管中基底外侧Cl⁻通透性相对较低，至少在静息条件下如此。此外，浴用已知能刺激导管中唾液修饰的生物胺多巴胺，并未引起[Cl⁻]i的显著变化。这些结果表明蟑螂唾液导管中[Cl⁻]i稳态的情况更为复杂。总之，2P-FLIM似乎是在许多生物系统中定量测量[Cl⁻]i的合适技术。
