OPTICAL PROBES OF MEMBRANE-POTENTIAL
OPTICAL PROBES OF MEMBRANE-POTENTIAL
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DOI：
10.1007/bf01869143
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发表时间：
1976-01-01
影响因子：
2.4
通讯作者：
WAGGONER, A
中科院分区：
文献类型：
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作者：
WAGGONER, A
花菁、花菁和羟基酚染料的荧光和吸收变化有两种基本不同的机理。这意味着染料(菁染料和羟基酚染料，带有离域电荷)是通过依赖于电位的积累机制工作的。这些染料在细胞悬浮液中表现出很大(高达80%)的荧光和吸收变化，这些变化在几秒钟内完成。非意指染料(带有局域电荷的花青染料)和意指染料也显示出在几毫秒内发生的光学变化。快速的光学变化相对较小(.5.泰晤士报。10-3)，但在单细胞实验中往往很容易检测到。这种快速的、非累积的光学变化是由膜局部的染料运动引起的。菁染料吸收的变化是由于染料在膜的高浓度侧的膜和相邻的水溶液区域之间的电势依赖的分配。在变化过程中，在水溶液区域形成了二聚体和更大的聚集体。花菁染料也可能通过同样的机制起作用。DIS-C3-(5)是目前测量悬浮液中细胞、细胞器和囊泡膜电位的最好的染料，但其他几种花菁也有类似的效果。对于每个体系，必须改变染料与膜的比例，直到找到最佳的荧光变化。必须为每个系统获得单独的校准曲线。在测量单细胞的荧光和/或吸收变化时，花菁540和DIBA-C4-(5)很好地工作，但在高强度光照下会产生一些光动力损伤。罗丹宁花菁(WW-375)具有很大的吸收变化，在强光照射下不会损伤组织。随着光学变化机理的阐明，设计和合成更灵敏、毒性更低、更容易校准的染料应该是可能的，这些染料可能有助于研究可激发膜的结构和动力学。
There are 2 basically different mechanisms for the fluorescence and absorption changes of merocyanine, cyanine and oxonol dyes. The permeant dyes (cyanine and oxonol dyes, with delocalized charges) work by a potential-dependent accumulation mechanism. These dyes show large (up to 80%) fluorescence and absorption changes with suspensions of cells, and the changes are complete in seconds. The impermeant dyes (merocyanine dyes, with localized charges) and the permeant dyes also show optical changes that take place in fractions of milliseconds. The rapid optical changes are relatively small (.ltoreq. 5 .times. 10-3) but can often be easily detected in experiments with single cells. The rapid, nonaccumulative, optical changes result from membrane-localized dye movements. Cyanine dye-absorption changes occur because of a potential-dependent partition of dye between the membrane and the adjacent aqueous region at the high dye-concentration side of the membrane. Dimers and larger aggregates are formed in the aqueous region during the change. Merocyanine dyes may also work by the same mechanism. DiS-C3-(5) is presently the best dye for measuring membrane potentials of cells, organelles and vesicles in suspension, but several other cyanines work nearly as well. For each system, the ratio of dye to membrane must be varied until the optimum fluorescence change is found. A separate calibration curve must be obtained for each system. For measuring fluorescence and/or absorption changes in single cells, merocyanine 540 and diBA-C4-(5) work well but produce some photodynamic damage with high intensity illumination. A rhodanine merocyanine (WW-375) gives very large absorption changes and does not damage tissue during strong illumination. As the mechanisms of the optical changes are worked out, it should be possible to design and synthesize more sensitive, less toxic dyes that are easier to calibrate, and these dyes may be useful for studying the structure and dynamics of excitable membranes.