Influence of fluorescence anisotropy on fluorescence intensity and lifetime measurement: Theory, simulations and experiments
Influence of fluorescence anisotropy on fluorescence intensity and lifetime measurement: Theory, simulations and experiments
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DOI：
10.1109/tbme.2006.873539
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发表时间：
2006-06-01
影响因子：
4.6
通讯作者：
Deutsch, Mordechai
中科院分区：
文献类型：
--
作者：
Fixler, Dror;Namer, Yaniv;Deutsch, Mordechai
荧光强度和寿命测量中的荧光各向异性的意义，以及由于其忽视而导致的错误测量和解释，进行了彻底的讨论，制定和量化。在所有与荧光相关的测量中-包括激发和发射光谱、相对荧光强度（FI）、荧光寿命（FLT）、荧光共振能量转移（FRET）等，除了荧光偏振和各向异性之外，通常荧光各向异性越高，荧光测量的失真就越大。当在不考虑荧光各向异性的影响的情况下进行荧光测量时，会发生可量化的失真。在这里，这种影响被描述，通过许多新开发的数学表达式，利用具有不同光谱特性的荧光团的单一和二元荧光溶液进行模拟和实验证实。结果表明，理论与实验数据吻合得很好，表明本文提出的物理假设和数学表达式是正确的。实践和指导意义进行了讨论。下列发现具有特殊的应用价值：1）存在无穷多对幻角; 2）具有不同荧光各向异性的两个同样发荧光的颗粒之间的偏差; 3）当在发射和激发偏振器的各种设置下测量时，检测到的荧光强度和各向异性之间的关系; 4）单指数衰减荧光团的人工归一化稳态权重对其荧光各向异性的依赖性。
The significance of fluorescence anisotropy in fluorescence intensity and lifetime measurements, and erroneous measurements and interpretations resulting from its disregard, are thoroughly discussed, formulated and quantified. In all fluorescence-related measurements-including excitation and emission spectra, relative fluorescence intensity (FI), fluorescenc life time (FLT), fluorescence resonance energy transfer (FRET), etc., with the exception of fluorescence polarization and anisotropy-it is generally true that the higher the fluorescence anisotropy, the greater the distortion of fluorescence measurements. Quantifiable distortions occur when fluorescence measurements are conducted without considering the influence of fluorescence anisotropy. Here, this influence is described, by numerous newly developed mathematical expressions which are simulated and experimentally confirmed utilizing single and binary fluorescent solutions of fluorophores with different spectroscopic characteristics. A marked agreement is shown between the theory and experimental data, clearly indicating the legitimacy of the physical suppositions and the mathematical expressions presented in this paper. Practical and instructive implications are discussed. The following findings are of special applicative importance: 1) the existence of an infinite number of couples of Magic Angles; 2). the deviation between two equally fluorescing particles having different fluorescence anisotropies; 3) the relation between the detected fluorescence intensity and anisotropy when measured under various setups of emission and excitation polarizers; 4) the dependence of the artificial normalized, steady-state weight of a single-exponentially decaying fluorophore on its fluorescence anisotropy.