MOBILITY MEASUREMENT BY ANALYSIS OF FLUORESCENCE PHOTOBLEACHING RECOVERY KINETICS
MOBILITY MEASUREMENT BY ANALYSIS OF FLUORESCENCE PHOTOBLEACHING RECOVERY KINETICS
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DOI：
10.1016/s0006-3495(76)85755-4
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发表时间：
1976-01-01
影响因子：
3.4
通讯作者：
WEBB, WW
中科院分区：
文献类型：
--
作者：
AXELROD, D;KOPPEL, DE;WEBB, WW
荧光光漂白恢复（FPR）表示用于测量荧光颗粒的二维横向迁移率的方法，例如，荧光标记的分子在λ中的运动。单个细胞表面的10 μ m2区域。荧光表面上的小斑点通过短暂暴露于强聚焦激光束而光漂白，并且随后的荧光恢复由相同但衰减的激光束监测。通过从周围表面的横向运输在漂白斑点中补充完整的荧光团而发生恢复。简单，严格的FPR实验分析的理论基础和一些实际的指导方针。可从FPR实验获得的信息包括传输过程类型的识别，即，随机扩散和均匀定向流动的混合;绝对迁移率系数的确定，即，扩散常数和/或流速;以及总荧光团中移动的的部分。为了说明实验方法和验证扩散理论，一些模型实验罗丹明6 G的水溶液进行了说明。
Fluorescence photobleaching recovery (FPR) denotes a method for measuring 2-dimensional lateral mobility of fluorescent particles, e.g., the motion of fluorescently labeled molecules in .apprx. 10 .mu.m2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. The theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments are presented. Information obtainable from FPR experiments includes identification of transport process type, i.e., the admixture of random diffusion and uniform directed flow; determination of the absolute mobility coefficient, i.e., the diffusion constant and/or flow velocity; and the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, some model experiments on aqueous solutions of rhodamine 6G are described.