Lopez A, Dupou L, Altibelli A, Trotard J, Tocanne J F
Centre de Recherche de Biochimie et Génétique Cellulaires du Centre National de la Recherche Scientifique, Toulouse, France.
Biophys J. 1988 Jun;53(6):963-70. doi: 10.1016/S0006-3495(88)83177-1.
A simple fluorescence recovery after photobleaching (FRAP) apparatus using a fluorescence microscope with a conventional mercury arc lamp, working under conditions of "uniform disk illumination" is described. This set-up was designed essentially for the use of anthracene as fluorescent probe, which is bleached (photodimerization reaction) by illumination in the near ultraviolet range (360 nm). It is shown that the lateral diffusion coefficients D can be readily calculated from fluorescence recovery curves using a finite differentiate method in combination with statistical analysis of the data. In contrast to the analytical solutions so far described, this numerical approach is particularly versatile. With a minimization algorithm, D and the probe mobile fraction can be readily calculated for any recovery time under various experimental conditions. These include different probe concentration profiles in the illuminated area after the bleaching step, and situations of infinite or noninfinite reservoir in the diffusion area outside the illuminated area.
描述了一种简单的光漂白后荧光恢复（FRAP）装置，该装置使用配备传统汞弧灯的荧光显微镜，在“均匀圆盘照明”条件下工作。此装置主要设计用于将蒽用作荧光探针，蒽在近紫外范围（360 nm）光照下会发生漂白（光二聚反应）。结果表明，结合数据的统计分析，使用有限差分法可轻松从荧光恢复曲线计算横向扩散系数D。与迄今为止描述的解析解不同，这种数值方法具有特别的通用性。通过最小化算法，可在各种实验条件下，针对任何恢复时间轻松计算D和探针移动分数。这些条件包括漂白步骤后照明区域中不同的探针浓度分布，以及照明区域外扩散区域中存在无限或非无限储存库的情况。
