Braeckmans Kevin, Stubbe Barbara G, Remaut Katrien, Demeester Joseph, De Smedt Stefaan C
Ghent University, Laboratory of General Biochemistry and Physical Pharmacy, Harelbekestraat 72, 9000 Ghent, Belgium.
J Biomed Opt. 2006 Jul-Aug;11(4):044013. doi: 10.1117/1.2337531.
In this study we examine the implications of excitation saturation on fluorescence recovery after photobleaching (FRAP) experiments. In particular we present both experimental and theoretical evidence that fluorescein, one of the most frequently used fluorophores in FRAP, does not always comply with the basic assumptions that are made in many FRAP models: an invariant bleaching illumination intensity distribution (BID) in combination with first-order photobleaching kinetics. High light intensity levels, which are typical for the photobleaching phase of FRAP experiments, can cause excitation saturation of fluorescein in the excited triplet state. We show by experiments and computer simulations that under such saturating conditions the higher-order diffraction maxima of the BID substantially contribute to the photobleaching process and can no longer be neglected. As a result, the bleached regions are larger than expected theoretically from the FRAP models. Although this effect is not always directly evident from the FRAP experiments, neglecting it may shift the calculated diffusion coefficient by as much as over one order of magnitude. We present a discussion on the implications of this saturation effect on various types of FRAP models.
在本研究中，我们探讨了激发饱和对光漂白后荧光恢复（FRAP）实验的影响。特别是，我们给出了实验和理论证据，表明荧光素作为FRAP中最常用的荧光团之一，并不总是符合许多FRAP模型所做的基本假设：不变的漂白光照强度分布（BID）与一级光漂白动力学相结合。FRAP实验漂白阶段典型的高光强度水平，可导致处于激发三重态的荧光素发生激发饱和。我们通过实验和计算机模拟表明，在这种饱和条件下，BID的高阶衍射极大值对光漂白过程有显著贡献，不能再被忽略。结果，漂白区域比FRAP模型理论预期的要大。尽管这种效应在FRAP实验中并不总是直接明显的，但忽略它可能会使计算出的扩散系数偏移多达一个数量级以上。我们讨论了这种饱和效应对各种类型FRAP模型的影响。
