Meseth U, Wohland T, Rigler R, Vogel H
Department of Chemistry, LCPPM, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland.
Biophys J. 1999 Mar;76(3):1619-31. doi: 10.1016/S0006-3495(99)77321-2.
The resolution limit of fluorescence correlation spectroscopy for two-component solutions is investigated theoretically and experimentally. The autocorrelation function for two different particles in solution were computed, statistical noise was added, and the resulting curve was fitted with a least squares fit. These simulations show that the ability to distinguish between two different molecular species in solution depends strongly on the number of photons detected from each particle, their difference in size, and the concentration of each component in solution. To distinguish two components, their diffusion times must differ by at least a factor of 1.6 for comparable quantum yields and a high fluorescence signal. Experiments were conducted with Rhodamine 6G and Rhodamine-labeled bovine serum albumin. The experimental results support the simulations. In addition, they show that even with a high fluorescence signal but significantly different quantum yields, the diffusion times must differ by a factor much bigger than 1.6 to distinguish the two components. Depending on the quantum yields and the difference in size, there exists a concentration threshold for the less abundant component below which it is not possible to determine with statistical means alone that two particles are in solution.
从理论和实验两方面研究了荧光相关光谱法对双组分溶液的分辨率极限。计算了溶液中两种不同粒子的自相关函数，加入统计噪声，并用最小二乘法对所得曲线进行拟合。这些模拟结果表明，区分溶液中两种不同分子物种的能力在很大程度上取决于从每个粒子检测到的光子数量、它们的大小差异以及溶液中各组分的浓度。对于相当的量子产率和高荧光信号，要区分两种组分，它们的扩散时间必须至少相差1.6倍。用罗丹明6G和罗丹明标记的牛血清白蛋白进行了实验。实验结果支持了模拟结果。此外，结果表明，即使具有高荧光信号但量子产率显著不同，扩散时间也必须相差远大于1.6倍才能区分这两种组分。根据量子产率和大小差异，对于含量较少的组分存在一个浓度阈值，低于该阈值，仅通过统计方法无法确定溶液中存在两种粒子。
