A Generalization of Theory for Two-Dimensional Fluorescence Recovery after Photobleaching Applicable to Confocal Laser Scanning Microscopes
A Generalization of Theory for Two-Dimensional Fluorescence Recovery after Photobleaching Applicable to Confocal Laser Scanning Microscopes
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DOI：
10.1016/j.bpj.2009.06.017
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发表时间：
2009-09-02
影响因子：
3.4
通讯作者：
DiBenedetto, Emmanuele
中科院分区：
文献类型：
--
作者：
Kang, Minchul;Day, Charles A.;DiBenedetto, Emmanuele
利用激光共聚焦扫描显微镜进行荧光漂白后恢复(FRAP)已成为研究生物分子在细胞内扩散的一种有价值的技术。然而，二维共焦FRAP有时会产生随实验设置而异的结果，例如不同的漂白方案和漂白点大小。此外，当共焦FRAP用于测量快速扩散分子的扩散系数(D)时，其D值通常比理论预测的或用其他方法(如荧光相关光谱)测量的D值小一个或两个数量级。最近，有研究表明，这种低估D的情况可以通过考虑光漂白过程中的扩散来修正。然而，目前关于共焦FRAP的理论还没有达成共识，也没有努力统一传统FRAP和共焦FRAP的理论。为此，我们推广了传统的FRAP理论来考虑光漂白过程中的扩散，使得传统的FRAP理论对圆形感兴趣区域的分析很容易适用于共焦FRAP。最后，我们通过在甘油水溶液中以及在COS7细胞的胞浆和胞核中测量可溶性增强绿色荧光蛋白的D来证明这些新公式的准确性。
Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells. However, two-dimensional confocal FRAP sometimes yields results that vary with experimental setups, such as different bleaching protocols and bleaching spot sizes. In addition, when confocal FRAP is used to measure diffusion coefficients (D) for fast diffusing molecules, it often yields D-values that are one or two orders-of-magnitude smaller than that predicted theoretically or measured by alternative methods such as fluorescence correlation spectroscopy. Recently, it was demonstrated that this underestimation of D can be corrected by taking diffusion during photobleaching into consideration. However, there is currently no consensus on confocal FRAP theory, and no efforts have been made to unify theories on conventional and confocal FRAP. To this end, we generalized conventional FRAP theory to incorporate diffusion during photobleaching so that analysis by conventional FRAP theory for a circular region of interest is easily applicable to confocal FRAP. Finally, we demonstrate the accuracy of these new (to our knowledge) formulae by measuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells.