Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy
Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy
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DOI：
10.1529/biophysj.107.108811
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发表时间：
2008-02-15
影响因子：
3.4
通讯作者：
Schwille, Petra
中科院分区：
文献类型：
--
作者：
Petrasek, Zdenek;Schwille, Petra
我们采用扫描荧光相关光谱 (sFCS) 来精确测定溶液中荧光分子的扩散系数。分子被激发并检测荧光的测量体积以 0.5-2 kHz 的频率在半径与其大小相当的圆中进行扫描。通过仔细校准以高精度确定的扫描半径 R 提供了确定扩散系数 D 所需的空间测量，而无需知道测量体积的确切尺寸。确定测量体积大小的困难限制了具有固定测量体积的标准FCS在相对测量中的应用，其中扩散系数是通过与标准比较来确定的。我们通过几种常见荧光染料的示例证明，sFCS 可用于高精度测量 D，而不需要标准。在存在弱光漂白且修改测量体积大小时可以确定 D 的正确值，表明该方法的稳健性。通过测量 HeLa 细胞细胞质中 eGFP 的扩散系数，证明了所提出的 sFCS 实施对生物系统的适用性。借助模拟，我们找到了实验的扫描半径 R 的最佳值。
We have implemented scanning fluorescence correlation spectroscopy (sFCS) for precise determination of diffusion coefficients of fluorescent molecules in solution. The measurement volume where the molecules are excited, and from which the fluorescence is detected, was scanned in a circle with radius comparable to its size at frequencies 0.5-2 kHz. The scan radius R, determined with high accuracy by careful calibration, provides the spatial measure required for the determination of the diffusion coefficient D, without the need to know the exact size of the measurement volume. The difficulties in the determination of the measurement volume size have limited the application of standard FCS with fixed measurement volume to relative measurements, where the diffusion coefficient is determined by comparison with a standard. We demonstrate, on examples of several common fluorescent dyes, that sFCS can be used to measure D with high precision without a need for a standard. The correct value of D can be determined in the presence of weak photobleaching, and when the measurement volume size is modified, indicating the robustness of the method. The applicability of the presented implementation of sFCS to biological systems in demonstrated on the measurement of the diffusion coefficient of eGFP in the cytoplasm of HeLa cells. With the help of simulations, we find the optimal value of the scan radius R for the experiment.