Koppel D E, Axelrod D, Schlessinger J, Elson E L, Webb W W
Biophys J. 1976 Nov;16(11):1315-29. doi: 10.1016/S0006-3495(76)85776-1.
We have developed an effective experimental system for the characterization of molecular and structural mobility. It incorporates a modified fluorescence microscope geometry and a variety of analytical techniques to measure effective diffusion coefficients ranging over almost six orders of magnitude, from less than 10(-11) cm2/s to greater than 10(-6) cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are employed. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochemically in a microscopic volume by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concentration fluctuations in microscopic sample volumes. The kinetics are analyzed by computing time-correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solutions, lipid bilayer membranes, and mammalian cell plasma membranes.
我们开发了一种有效的实验系统，用于表征分子和结构的流动性。该系统采用了改进的荧光显微镜结构和多种分析技术，可测量有效扩散系数，其范围跨越近六个数量级，从小于10^(-11) cm²/s到大于10^(-6) cm²/s。我们采用了两种主要技术，即荧光相关光谱法（FCS）和荧光光漂白恢复法（FPR）。在FPR技术中，通过监测由短脉冲强激光辐射在微观体积中光化学产生的荧光空间不均匀性的演变来测量平移传输速率。相比之下，FCS使用激光诱导荧光来探测微观样品体积中的自发浓度波动。通过计算测量的荧光强度的随机波动的时间相关函数来分析动力学。描述并讨论了围绕专用小型计算机设计的光学系统和数字光计数相关器。通过对本体溶液、脂质双层膜和哺乳动物细胞质膜进行的研究实例，展示了这些技术的一般效能。
