Department of Applied Physics, Experimental Biomolecular Physics, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.
Anal Chem. 2010 Jul 1;82(13):5646-51. doi: 10.1021/ac1006409.
Inverse-fluorescence correlation spectroscopy (iFCS) was recently introduced as an alternative version of FCS that does not require labeling of the analyzed particles or biomolecules. In iFCS, the signal from a medium surrounding the particles is analyzed, as opposed to a signal from the studied particles themselves. As unlabeled particles diffuse through the detection volume, they displace a fraction of the fluorescent medium, causing transient dips in the detected signal which give information about the mobility and concentration of the analyzed particles. Here inverse-fluorescence cross-correlation spectroscopy (iFCCS) is introduced as an extension of iFCS. In iFCCS, labeled particles/biomolecules are analyzed and their fluorescence signal is cross-correlated with the signal from the surrounding medium. When labeled particles are analyzed, a direct estimate of the volume of the particles is obtained or, alternatively, an estimate of the size of the detection volume. Another possibility is to analyze the interaction of small, labeled molecules with unlabeled particles, resulting in cross-correlation as an indication of binding, even though only one binding partner is labeled. This also enables accurate estimation of the degree of labeling, since the amounts of labeled and unlabeled particles are estimated independently.
反荧光相关光谱学（iFCS）最近被引入作为 FCS 的替代版本，它不需要对分析的粒子或生物分子进行标记。在 iFCS 中，分析的是粒子周围介质的信号，而不是研究粒子本身的信号。当未标记的粒子扩散通过检测体积时，它们会取代荧光介质的一部分，导致检测信号的瞬态下降，从而提供有关分析粒子的迁移率和浓度的信息。这里引入了反荧光互相关光谱学（iFCCS）作为 iFCS 的扩展。在 iFCCS 中，分析标记的粒子/生物分子，并且将它们的荧光信号与周围介质的信号进行互相关。当分析标记的粒子时，可以直接估计粒子的体积，或者估计检测体积的大小。另一种可能性是分析小的标记分子与未标记粒子的相互作用，从而导致作为结合指示的互相关，即使只有一个结合伴侣被标记。这也可以实现对标记程度的准确估计，因为标记和未标记粒子的量是独立估计的。
