Irion G, Ochsenfeld L, Naujok A, Zimmermann H W
Institut für Physikalische Chemie der Universität, Freiburg, Germany.
Histochemistry. 1993 Jan;99(1):75-83. doi: 10.1007/BF00268024.
Lipophilic cationic fluorescent dyes (D) specifically stain the mitochondria of living cells. A perfusion chamber for cell cultures is described, which can be used to determine the kinetics of vital staining of the mitochondria of single selected cells in situ. In these experiments styrylpyridinium dyes and cultures of HeLa cells were used. The dyes differ strongly in their lipophilic properties; Rm values and the partition coefficients Po/w between n-octanol (o) and water (w) were determined in order to characterize their lipophilicity. In the thermostat-regulated chamber the concentration of the dye CD can be increased from CD = 0 to CD > 0 within a few seconds (concentration jump). Thus, the time t = 0 for the beginning of the vital staining and the dye concentration in the cell medium during the staining experiment, CD = const., are unambiguously defined. The concentration of the dye, Cb, which is bound to the mitochondria (b), is proportional to the intensity of the fluorescence Ib. On the other hand, the free dye molecules (f) in the aqueous medium exhibit practically no fluorescence, I(f) << Ib. The intensity of the fluorescence I = Ib was measured as a function of time t; the measured values were corrected for photobleaching. The fluorescence intensity I(t) at first increases linearly with t and reaches a saturation value for t-->infinity. In the linear range of I(t) the flow J(o) = (dI/dt)o of the dye into the cell depends strongly on the dye concentration and increases linearly with CD. The concentration range CD = 10(-9)-10(-5) M at 37 degrees C was investigated. From the linear correlation between J(o) and CD it follows that the kinetics of the vital staining of mitochondria is controlled by diffusion. At t = 0 the flow of the xenobiotic agent through the cell membrane determines the rate of staining. The slope dJ(o)/dCD of the plot J(o) vs CD describes the efficiency of dye accumulation at the mitochondria and strongly increases with increasing lipophilicity of the dye molecules. Thus lipophilic dyes pass through the cell membrane more easily than less lipophilic molecules.
亲脂性阳离子荧光染料（D）能特异性地对活细胞的线粒体进行染色。本文描述了一种用于细胞培养的灌注室，它可用于原位测定单个选定细胞线粒体的活体染色动力学。在这些实验中，使用了苯乙烯基吡啶染料和HeLa细胞培养物。这些染料的亲脂性差异很大；测定了它们的Rm值以及正辛醇（o）与水（w）之间的分配系数Po/w，以表征其亲脂性。在恒温调节室中，染料CD的浓度可在几秒钟内从CD = 0增加到CD > 0（浓度跃升）。因此，活体染色开始的时间t = 0以及染色实验期间细胞培养基中的染料浓度CD = const.都被明确界定。与线粒体结合的染料Cb的浓度与荧光强度Ib成正比。另一方面，水相中游离的染料分子（f）几乎不产生荧光，I(f) << Ib。测量荧光强度I = Ib随时间t的变化；对测量值进行光漂白校正。荧光强度I(t)起初随t线性增加，并在t趋于无穷大时达到饱和值。在I(t)的线性范围内，染料流入细胞的流量J(o) = (dI/dt)o强烈依赖于染料浓度，并随CD线性增加。研究了37℃下CD = 10(-9)-10(-5) M的浓度范围。从J(o)与CD之间的线性相关性可知，线粒体活体染色的动力学受扩散控制。在t = 0时，异生物质通过细胞膜的流量决定了染色速率。J(o)对CD作图的斜率dJ(o)/dCD描述了染料在线粒体上的积累效率，并随染料分子亲脂性的增加而显著增加。因此，亲脂性染料比亲脂性较低的分子更容易穿过细胞膜。
