Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 19107, Philadelphia, Pennsylvania.
J Fluoresc. 1995 Dec;5(4):329-35. doi: 10.1007/BF01152559.
Fluorescent measurements of intracellular H(+) and Na(+) are improved by using whole spectra of the fluorescent indicators BCECF and SBFI, respectively. The extra data in whole spectra enable both an accurate calibration and a ready detection of artifacts which are not possible to identify using a more conventional data analysis that relies upon only two wavelength "windows" in the fluorescence spectra. The whole-spectrum technique is applicable to cell suspensions in a conventional fluorimeter (as is reported here with SBFI), as well as to attached cells using a fluorimeter combined with an inverted epifluorescence microscope. The spectral method was highly reproducible in that pairs of successive pH measurements differed, on average, by only 0.01±0.02 U. Random uncertainty from sample to sample was estimated numerically from the standard deviation of measurements on ionophore-treated cells. When full-spectrum analysis was employed, this scatter showed a two-fold improvement over results obtained using the two-wavelength ratio method. Because SBFI has a relatively narrow dynamic range, whole-spectrum analysis has been applied to improve the accuracy of sodium determinations. The calibrated system measured [Na(+)]i with excellent linearity over the range 2-150 mM and with an accuracy of approximately 5 mM.
荧光测量细胞内 H(+) 和 Na(+) 通过使用全谱的 BCECF 和 SBFI 分别为荧光指示剂。全谱中的额外数据可以进行准确的校准和现成的检测伪影，这是不可能使用更传统的数据分析，只依赖于荧光光谱中的两个波长“窗口”来识别。全谱技术适用于在传统的荧光计中的细胞悬浮液（如这里报道的与 SBFI），以及使用荧光计与倒置相差显微镜组合的附着细胞。光谱法是高度可重复的，即一对连续的 pH 值测量平均相差仅 0.01±0.02 U。从样本到样本的随机不确定性是从离子载体处理细胞的测量值的标准偏差数值估计。当全谱分析被采用时，与使用双波长比值法获得的结果相比，这种分散度提高了两倍。由于 SBFI 具有相对较窄的动态范围，全谱分析已被应用于提高钠测定的准确性。校准系统在 2-150 mM 的范围内以优异的线性度测量 [Na(+)]i，并且精度约为 5 mM。
