INTRACELLULAR PH MEASUREMENT USING SINGLE EXCITATION-DUAL EMISSION FLUORESCENCE RATIOS
INTRACELLULAR PH MEASUREMENT USING SINGLE EXCITATION-DUAL EMISSION FLUORESCENCE RATIOS
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DOI：
10.1152/ajpcell.1990.258.1.c171
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发表时间：
1990-01-01
影响因子：
--
通讯作者：
BEEBE, DC
中科院分区：
文献类型：
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作者：
BASSNETT, S;REINISCH, L;BEEBE, DC
在本文中，激光光谱法被用来评估一种新的荧光染料，羧基-萘酚或-1（Snarf-1），用于单激发双发射比测量细胞内pH值（pHi）的实用性。Snarf-1的发射光谱显示出明显的pH值依赖性的变化，从640和587 nm的最大值计算的发射比是一个敏感的pH值指标。当照射在坎宁安室，解决方案的Snarf-1迅速漂白，并在pH值7.3或更高，这种漂白导致640/587 nm的发射比下降。这些比例的变化也观察到在细胞内测量透镜胚胎上皮细胞的条件下，其中包埋的染料迅速漂白。随着激光剂量的减少（通过增加采样点之间的步长），漂白可以减少到非常低的水平，并且在这些条件下，比率保持恒定。使用尼日利亚菌素在细胞内校准加载到透镜上皮外植体中的Snarf-1。细胞内校准曲线比体外曲线更碱性。细胞内校准允许与先前发表的透镜组织值合理一致的pHi估计值。潜在的文物所产生的差异光漂白和细胞内体外校准进行了讨论。
In the present paper, laser spectroscopy was used to evaluate the utility of a new fluorochrome, carboxy-seminaphthorhodafluor-1 (Snarf-1), for single excitation-dual emission ratio measurement of intracellular pH (pHi). The emission spectrum of Snarf-1 showed clear pH-dependent shifts, and emission ratios calculated from the 640 and 587 nm maxima were a sensitive indicator of pH. When irradiated in Cunningham chambers, solutions of Snarf-1 were rapidly bleached, and at pH 7.3 or higher, this bleaching led to a decrease in the 640/587 nm emission ratio. These ratio changes were also observed in intracellular measurements on lens embryonic epithelial cells under conditions in which the entrapped dye was rapidly bleached. As the laser dosage was reduced (by increasing the step size between sample points), bleaching could be reduced to very low levels, and under these conditions, the ratio remained constant. Snarf-1 loaded into lens epithelial explants was calibrated intracellularly using nigericin. Intracellular calibration curves were shifted to more alkaline values than in vitro curves. Intracellular calibration allowed estimates of pHi that were in reasonable agreement with previously published values for lens tissue. Potential artifacts arising from differential photobleaching and intracellular-in vitro calibration are discussed.