Bassnett S, Reinisch L, Beebe D C
Department of Anatomy, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Am J Physiol. 1990 Jan;258(1 Pt 1):C171-8. doi: 10.1152/ajpcell.1990.258.1.C171.
In the present paper, laser spectroscopy was used to evaluate the utility of a new fluorochrome, carboxyseminaphthorhodafluor-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.
在本论文中，激光光谱法被用于评估一种新型荧光染料羧基半萘罗丹明氟-1（Snarf-1）用于细胞内pH值（pHi）单激发-双发射比率测量的效用。Snarf-1的发射光谱显示出明显的pH依赖性位移，并且从640和587nm最大值计算出的发射比率是pH的敏感指标。当在康宁培养室中照射时，Snarf-1溶液迅速被漂白，并且在pH 7.3或更高时，这种漂白导致640/587nm发射比率降低。在晶状体胚胎上皮细胞的细胞内测量中，在捕获的染料迅速被漂白的条件下也观察到了这些比率变化。随着激光剂量的降低（通过增加采样点之间的步长），漂白可以降低到非常低的水平，并且在这些条件下，比率保持恒定。使用尼日利亚菌素对加载到晶状体上皮外植体中的Snarf-1进行细胞内校准。细胞内校准曲线比体外曲线向更碱性的值移动。细胞内校准允许对pHi进行估计，这些估计与先前发表的晶状体组织值合理一致。讨论了由差异光漂白和细胞内-体外校准引起的潜在假象。
