Enhancement, Equal Fluorescence Efficiency, and Quenching in the Interpretation of Fluorescence Anisotropy Data
Enhancement, Equal Fluorescence Efficiency, and Quenching in the Interpretation of Fluorescence Anisotropy Data
复制标题
荧光各向异性数据解释中的增强、等荧光效率和猝灭
DOI：
10.1016/j.bpj.2013.11.3767
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发表时间：
2014
影响因子：
3.4
通讯作者：
Zolmajd-Haghighi Z
中科院分区：
文献类型：
--
作者：
Zolmajd-Haghighi Z
Homo-FRET过程中荧光各向异性的降低可以报告蛋白质上的染料数量或簇的大小。当组装成簇时，荧光染料表现出从荧光增强到猝灭的一系列行为。增强和/或猝灭的程度取决于接近的染料的数量。在解释各向异性时，普遍采用等荧光强度的假设。这一假设预测，例如，一个簇内的3个荧光团与簇外的3个相同的荧光团具有相同的荧光强度。这种假设在发生荧光猝灭或增强的情况下会给出不正确的预测。此外，现有的理论通常假设一个簇中的所有位置都是等价的。这些假设的应用在两个方面影响着解释。它会倾向于低估单个物种随机混合的各向异性，并根据染料系统是猝灭的还是增强的，或者低估或过度预测集群的大小。
Decreases in fluorescence anisotropy from homo-FRET processes can report on the number of dyes on a protein or the size of a cluster. Fluorescent dyes exhibit a range of behaviours from fluorescence enhancement to quenching when assembled into clusters. The degree of enhancement and/or quenching depends on the number of dyes in close proximity. When interpreting anisotropy, an assumption of equal fluorescence intensity is widely applied. This assumption predicts, for example, that 3 fluorophores in a cluster have the same fluorescence intensity as the same three fluorophores outside of a cluster. This assumption will give incorrect predictions in cases where either quenching or enhancement of fluorescence occurs. Additionally, existing theory typically assumes that all positions within a cluster are equivalent.Application of these assumptions affects interpretation in two ways. It will tend to under-predict the anisotropy of a stochastic mix of individual species and, depending on whether the dye system is quenched or enhanced, will either under-predict or over-predict cluster sizes.