An improved strip FRAP method for estimating diffusion coefficients: correcting for the degree of photobleaching.
An improved strip FRAP method for estimating diffusion coefficients: correcting for the degree of photobleaching.
复制标题
用于估计扩散系数的改进条带 FRAP 方法：校正光漂白程度。
DOI：
10.1111/j.1365-2818.2009.03347.x
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发表时间：
2010
影响因子：
2
通讯作者：
Yang J
中科院分区：
文献类型：
--
作者：
Yang J
光漂白后的荧光恢复是一种广泛建立的方法，用于估计扩散系数，带漂白与相关的恢复曲线分析是最简单的技术之一。然而，其实施需要在感兴趣的区域中接近100%的漂白，而外部的荧光损失可以忽略不计，对于快速扩散的分子，这两个约束很难同时实现。我们证明，当这些要求不满足时，扩散系数D的估计存在误差，根据哪个假设被违反得最多，估计不足或过高。我们提出了一个简单的修正恢复曲线分析纳入相对漂白质量的概念，给出了一个修正的恢复时间参数τ= m ~ 2 w ~ 2/4π D的宽度w的条带。这种修改后的模型消除了在感兴趣的区域中100%漂白的要求，并允许在漂白期间荧光团的有限扩散。我们通过估计FITC标记的IgG在60%甘油溶液中的（体积）扩散系数D= 4.09 ± 0.21 μ m2 s −1，以及在人B细胞系表面表达的绿色荧光蛋白标记的I类MHC蛋白的（表面）扩散系数D= 0.32 ± 0.03 μ m2 s − 1来验证我们的方法。
Fluorescence recovery after photobleaching is a widely established method for the estimation of diffusion coefficients, strip bleaching with an associated recovery curve analysis being one of the simplest techniques. However, its implementation requires near 100% bleaching in the region of interest with negligible fluorescence loss outside, both constraints being hard to achieve concomitantly for fast diffusing molecules. We demonstrate that when these requirements are not met there is an error in the estimation of the diffusion coefficientD, either an under‐ or overestimation depending on which assumption is violated the most. We propose a simple modification to the recovery curve analysis incorporating the concept of the relative bleached massmgiving a revised recovery time parametrization τ=m2w2/4πDfor a strip of widthw. This modified model removes the requirement of 100% bleaching in the region of interest and allows for limited diffusion of the fluorophore during bleaching. We validate our method by estimating the (volume) diffusion coefficient of FITC‐labelled IgG in 60% glycerol solution,D= 4.09 ± 0.21 μm2s−1, and the (surface) diffusion coefficient of a green‐fluorescent protein‐tagged class I MHC protein expressed at the surface of a human B cell line,D= 0.32 ± 0.03 μm2s−1for a population of cells.