Pieper Christoph, Weiß Kerstin, Gregor Ingo, Enderlein Jörg
Third Institute of Physics-Biophysics, Georg-August-University Göttingen, Göttingen, Germany.
Methods Enzymol. 2013;518:175-204. doi: 10.1016/B978-0-12-388422-0.00008-X.
This chapter introduces into the technique of dual-focus fluorescence correlation spectroscopy or 2fFCS. In 2fFCS, the fluorescence signals generated in two laterally shifted but overlapping focal regions are auto- and crosscorrelated. The resulting correlation curves are then used to determine diffusion coefficients of fluorescent molecules or particles in solutions or membranes. Moreover, the technique can also be used for noninvasively measuring flow-velocity profiles in three dimensions. Because the distance between the focal regions is precisely known and not changed by most optical aberrations, this provides an accurate and immutable external length scale for determining diffusivities and velocities, making 2fFCS the method of choice for accurately measuring absolute values of these quantities at pico- to nanomolar concentration.
本章介绍双焦点荧光相关光谱技术（2fFCS）。在2fFCS中，在两个横向偏移但重叠的焦点区域中产生的荧光信号进行自相关和互相关。然后，所得的相关曲线用于确定溶液或膜中荧光分子或颗粒的扩散系数。此外，该技术还可用于三维非侵入性测量流速分布。由于焦点区域之间的距离是精确已知的，并且不受大多数光学像差的影响，这为确定扩散率和速度提供了一个准确且不变的外部长度尺度，使得2fFCS成为在皮摩尔到纳摩尔浓度下精确测量这些量绝对值的首选方法。
