CHARACTERISTICS OF FLUOROPROBES FOR MEASURING INTRACELLULAR PH
CHARACTERISTICS OF FLUOROPROBES FOR MEASURING INTRACELLULAR PH
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DOI：
10.1016/0003-2697(86)90174-0
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发表时间：
1986-07-01
影响因子：
2.9
通讯作者：
PASTORIZAMUNOZ, E
中科院分区：
文献类型：
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作者：
GRABER, ML;DILILLO, DC;PASTORIZAMUNOZ, E
我们评估了四种不同的荧光团，以确定其在测量细胞内pH值的荧光指示剂技术的能力和局限性。在体外，羧基荧光素、二甲基羧基荧光素、双羧基乙基羧基荧光素和4-甲基伞形酮（4 MU）在它们各自的pH α附近都显示出极强的荧光和优异的pH敏感性。价值观主要差异被发现之间的4 MU和荧光素的蛋白结合，浓度效应，漂白率，和细胞内的保留时间。在pH敏感/pH不敏感的激发波长的荧光和荧光比随pH值的所有化合物，和激发光强度的大的变化的比例完全校正。与此相反，由于自猝灭效应和光谱位移，随着染料浓度的增加，该比率显示出大的人为变化。蛋白质的相互作用同样引起光谱位移和比率异常，但钙，镁和氧对荧光比率没有影响。我们的结论是，测量细胞pH值的荧光技术是受自猝灭和蛋白质结合引起的文物。使用荧光比率技术不一定校正这些伪影，特别是比率技术不校正荧光探针浓度的变化。由于主要伪影导致4 MU和荧光素的比率沿相反方向移动，因此如果这两类荧光探针的荧光和比率沿相同方向变化，则可以显示实验操作导致pH的真实变化。
We evaluated four different fluorophores to determine their capabilities and limitations in measuring intracellular pH by the fluorescent indicator technique. In vitro, carboxyfluorescein, dimethylcarboxyfluorescein, biscarboxyethyl carboxyfluorescein, and 4-methylumbelliferone (4MU) all showed comparably intense fluorescence and excellent pH sensitivity near their respective pK.alpha. values. Major differences were found between 4MU and the fluoresceins in terms of protein binding, concentration effects, bleach rates, and the retention time within cells. Both fluorescence and a fluorescence ratio at pH-sensitive/pH-insensitive excitation wavelengths increased with pH for all compounds, and the ratio completely corrected for large changes in the excitation light intensity. In contrast, the ratio showed large artifactual changes as dye concentration increased because of self-quenching effects and spectral shifts. Protein interactions likewise caused spectral shift and ratio aberrancies, but calcium, magnesium, and oxygen had no effect on the fluorescence ratios. We conclude that measurements of cell pH by fluorescence techniques are subject to artifacts induced by self-quenching and protein binding. Use of the fluorescence ratio technique does not necessarily correct for these artifacts, and in particular the ratio technique does not correct for changes in fluoroprobe concentration. Because the major artifacts cause the ratios for 4MU and for the fluoresceins to move in opposite directions, an experimental maneuver can be shown to cause a true change in pH if the fluorescence and ratios change in the same direction for these two classes of fluoroprobes.