Septinus M, Berthold T, Naujok A, Zimmermann H W
Histochemistry. 1985;82(1):51-66. doi: 10.1007/BF00502091.
The hydrophobic fluorescence dye 10-n-nonyl-acridinium-orange-chloride, NAO, stains specifically the mitochondria of living HeLa-cells. A dye concentration of 1 X 10(-8) M is sufficient for vital staining and at 5 X 10(-7) M an incubation time less than 1 min is enough to generate the bright green fluorescence of the mitochondria. The retention of NAO by the mitochondria is longer than 7 days. The dye accumulation is not affected by the ionophores valinomycin, nigericin, gramicidin, the uncoupling agents DNP, CCCP or by ouabain. In contrast to Rh 123 the trans-membrane potential is not the driving force of the NAO accumulation. We assume that NAO is bound to the hydrophobic lipids and proteins in the mitochondrial membranes by hydrophobic interaction. With valinomycin, 500 ng/ml, 10 min, the mitochondria in HeLa-cells swell. Now it is possible to observe some details in the enlarged mitochondria by light microscopy. After vital staining with NAO, 5 X 10(-7) M, 10 min, the periphery of the swollen mitochondria shows an intense green fluorescence, the inner part is dark. Obviously the dye is bound to the membranes. By electron microscopy it can be shown that the valinomycin treated and NAO stained mitochondria have outer and inner membranes and cristae. They differ from untreated mitochondria mainly in the size. After incubation of the HeLa-cells with relatively high NAO concentrations, 5 X 10(-6) M, 10 min, the mitochondria show a weak orange fluorescence. It is generated by the dimers D of NAO. Therefore the dye concentration in the mitochondrial membranes is locally very high and causes dye dimerisation. The weak orange fluorescence is instable and disappears within a few seconds. Instead we observe a green fluorescence with growing intensity that is generated by the monomers M of NAO. The intensity has its maximum value after a few seconds. Using low NAO concentrations for incubation, 1 X 10(-7) M, 10 min, we observe only the green fluorescence with increasing intensity. In this case the orange fluorescence is too weak for observation (concentration quenching). It can be shown by experiments and quantum mechanics that the orange fluorescence is assigned to an optical forbidden, the green fluorescence to an allowed electronic transition of D or M respectively. Our results indicate a dissoziation of D in 2 M by irradiation of the mitochondria under the fluorescence microscope.(ABSTRACT TRUNCATED AT 400 WORDS)
疏水性荧光染料10 - 正壬基 - 吖啶鎓 - 橙 - 氯化物（NAO）可特异性地对活的HeLa细胞的线粒体进行染色。1×10⁻⁸ M的染料浓度足以进行活体染色，在5×10⁻⁷ M时，孵育时间少于1分钟就足以产生线粒体的亮绿色荧光。NAO在线粒体中的保留时间超过7天。染料的积累不受离子载体缬氨霉素、尼日利亚菌素、短杆菌肽、解偶联剂二硝基苯酚（DNP）、羰基氰氯苯腙（CCCP）或哇巴因的影响。与罗丹明123（Rh 123）不同，跨膜电位不是NAO积累的驱动力。我们假设NAO通过疏水相互作用与线粒体膜中的疏水性脂质和蛋白质结合。用500 ng/ml的缬氨霉素处理10分钟后，HeLa细胞中的线粒体肿胀。现在可以通过光学显微镜观察放大的线粒体中的一些细节。用5×10⁻⁷ M的NAO进行10分钟的活体染色后，肿胀线粒体的周边呈现强烈的绿色荧光，内部较暗。显然，染料与膜结合。通过电子显微镜可以看出，经缬氨霉素处理并经NAO染色的线粒体有外膜、内膜和嵴。它们与未处理的线粒体的主要区别在于大小。用相对较高浓度（5×10⁻⁶ M）的NAO孵育HeLa细胞10分钟后，线粒体呈现微弱的橙色荧光。这是由NAO的二聚体D产生的。因此，线粒体膜中的染料浓度在局部非常高，导致染料二聚化。微弱的橙色荧光不稳定，在几秒钟内消失。相反，我们观察到由NAO的单体M产生的强度不断增加的绿色荧光。几秒钟后强度达到最大值。使用低浓度（1×10⁻⁷ M）的NAO孵育10分钟，我们只观察到强度不断增加的绿色荧光。在这种情况下，橙色荧光太弱而无法观察到（浓度猝灭）。实验和量子力学表明，橙色荧光对应于光学禁阻跃迁，绿色荧光分别对应于D或M的允许电子跃迁。我们的结果表明，在荧光显微镜下照射线粒体时，D在2 M中发生解离。（摘要截短至400字）
