Hasselt University, Biomedical Research Institute, Agoralaan Building C, B-3590 Diepenbeek, Belgium.
J Biomed Opt. 2013 Jul;18(7):76012. doi: 10.1117/1.JBO.18.7.076012.
Fluorescence recovery after photobleaching (FRAP) is a common technique to probe mobility of fluorescently labeled proteins in biological membranes by monitoring the time-dependence of the spatially integrated fluorescence signals after a bleaching pulse. Discrimination by FRAP between free diffusion with an immobile fraction (FDIM) and the phenomenological model for anomalous diffusion based on the time-dependent diffusion coefficient (TDDC) is a challenging problem, requiring extremely long observation times for differentiation. Recently, rectangular FRAP (rFRAP) has been introduced for normal diffusion by considering not only the temporal but also spatial information, taking the effective point spread function of the optical system into account. In this work we provide an extension of rFRAP toward anomalous diffusion according to the continuous time random walk (CTRW). We explore whether the spatial information in rFRAP allows for enhanced discrimination between FDIM, TDDC, and CTRW in a single experiment within a feasible time window. Simulations indicate that rFRAP can indeed differentiate the different models by evaluating the spatial autocorrelation of the differences between the measured and fitted pixel values. Hence, rFRAP offers a tool that is capable of discriminating different types of diffusion at shorter time scales than in the case where spatial information is discarded.
荧光漂白后恢复（FRAP）是一种常用的技术，通过监测漂白脉冲后空间积分荧光信号的时间依赖性来探测生物膜中荧光标记蛋白的流动性。FRAP 通过区分具有无定形分数的自由扩散（FDIM）和基于时变扩散系数的异常扩散的唯象模型（TDDC）来区分，这是一个具有挑战性的问题，需要非常长的观察时间来区分。最近，矩形 FRAP（rFRAP）已被引入用于正常扩散，不仅考虑了时间信息，还考虑了空间信息，考虑了光学系统的有效点扩散函数。在这项工作中，我们根据连续时间随机行走（CTRW）将 rFRAP 扩展到异常扩散。我们探索了在可行的时间窗口内，rFRAP 中的空间信息是否允许在单次实验中增强 FDIM、TDDC 和 CTRW 之间的区分。模拟表明，rFRAP 确实可以通过评估测量值和拟合像素值之间的差异的空间自相关来区分不同的模型。因此，rFRAP 提供了一种工具，能够在比丢弃空间信息的情况下更短的时间尺度上区分不同类型的扩散。
