Eunice Shriver National Institute of Child Health & Human Development, National Institutes of Health, Programs on Physical Biology, Bethesda, Maryland 20892, USA.
J Biomed Opt. 2012 Dec;17(12):125004. doi: 10.1117/1.JBO.17.12.125004.
Fluorescence correlation spectroscopy (FCS) is increasingly being used to assess the movement of particles diffusing in complex, optically dense surroundings, in which case measurement conditions may complicate data interpretation. It is considered how a single-photon FCS measurement can be affected if the sample properties result in scattering of the incident light. FCS autocorrelation functions of Atto 488 dye molecules diffusing in solutions of polystyrene beads are measured, which acted as scatterers. Data indicated that a scattering-linked increase in the illuminated volume, as much as two fold, resulted in minimal increase in diffusivity. To analyze the illuminated beam profile, Monte-Carlo simulations were employed, which indicated a larger broadening of the beam along the axial than the radial directions, and a reduction of the incident intensity at the focal point. The broadening of the volume in the axial direction has only negligible effect on the measured diffusion time, since intensity fluctuations due to diffusion events in the radial direction are dominant in FCS measurements. Collectively, results indicate that multiple scattering does not result in FCS measurement artifacts and thus, when sufficient signal intensity is attainable, single-photon FCS can be a useful technique for measuring probe diffusivity in optically dense media.
荧光相关光谱学（FCS）越来越多地被用于评估在复杂的、光密度高的环境中扩散的粒子的运动，在这种情况下，测量条件可能会使数据解释复杂化。如果样品的特性导致入射光散射，那么单光子 FCS 测量会受到怎样的影响呢？本文对在聚苯乙烯珠溶液中扩散的 Atto 488 染料分子的 FCS 自相关函数进行了测量，这些聚苯乙烯珠起到了散射体的作用。数据表明，散射导致的照射体积增加了两倍，对扩散率的影响最小。为了分析照射光束的轮廓，本文采用了蒙特卡罗模拟，结果表明，光束在轴向的展宽比径向的展宽更大，并且在焦点处的入射强度降低。由于在 FCS 测量中，径向方向上的扩散事件引起的强度波动占主导地位，因此轴向体积的展宽对测量的扩散时间几乎没有影响。总的来说，结果表明，多次散射不会导致 FCS 测量出现伪影，因此，在能够获得足够的信号强度的情况下，单光子 FCS 可以成为测量光密度介质中探针扩散率的有用技术。
