Bright G R, Fisher G W, Rogowska J, Taylor D L
J Cell Biol. 1987 Apr;104(4):1019-33. doi: 10.1083/jcb.104.4.1019.
Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, 1984, J. Cell Biol., 98:717-724) has been used to measure the spatial variations in cytoplasmic pH of individual quiescent and nonquiescent Swiss 3T3 cells. Fundamental issues of ratio imaging that permit precise and accurate temporal and spatial measurements have been addressed including: excitation light levels, lamp operation, intracellular probe concentrations, methods of threshold selection, photobleaching, and spatial signal-to-noise ratio measurements. Subcellular measurements can be measured accurately (less than 3% coefficient of variation) in an area of 3.65 microns 2 with the present imaging system. Quiescent Swiss 3T3 cells have a measured cytoplasmic pH of 7.09 (0.01 SEM), whereas nonquiescent cells have a pH of 7.35 (0.01 SEM) in the presence of bicarbonate buffer. A unimodal distribution of mean cytoplasmic pH in both quiescent and nonquiescent cells was identified from populations of cells measured on a cell by cell basis. Therefore, unlike earlier studies based on cell population averages, it can be stated that cells in each population exhibit a narrow range of cytoplasmic pH. However, the mean cytoplasmic pH can change based on the physiological state of the cells. In addition, there appears to be little, if any, spatial variation in cytoplasmic pH in either quiescent or nonquiescent Swiss 3T3 cells. The pH within the nucleus was always the same as the surrounding cytoplasm. These values will serve as a reference point for investigating the role of temporal and spatial variations in cytoplasmic pH in a variety of cellular processes including growth control and cell movement.
荧光比率成像显微镜技术（塔纳苏甘，L.，P. 麦克尼尔，G. 雷诺兹，以及D. L. 泰勒，1984年，《细胞生物学杂志》，98:717 - 724）已被用于测量单个静止和非静止的瑞士3T3细胞胞质pH值的空间变化。比率成像的一些基本问题已得到解决，这些问题有助于进行精确和准确的时间和空间测量，包括：激发光水平、灯的操作、细胞内探针浓度、阈值选择方法、光漂白以及空间信噪比测量。使用当前的成像系统，可以在3.65平方微米的区域内准确测量亚细胞水平的数据（变异系数小于3%）。在碳酸氢盐缓冲液存在的情况下，静止的瑞士3T3细胞测得的胞质pH值为7.09（标准误0.01），而非静止细胞的pH值为7.35（标准误0.01）。从逐个测量的细胞群体中，确定了静止和非静止细胞中平均胞质pH值的单峰分布。因此，与早期基于细胞群体平均值的研究不同，可以说每个群体中的细胞胞质pH值范围较窄。然而，平均胞质pH值会根据细胞的生理状态而变化。此外，无论是静止还是非静止的瑞士3T3细胞，胞质pH值似乎几乎没有空间变化。细胞核内的pH值始终与周围细胞质相同。这些值将作为一个参考点，用于研究胞质pH值的时间和空间变化在包括生长控制和细胞运动在内的各种细胞过程中的作用。
