Fluorescence of dyes in solutions with high absorbance. Inner filter effect correction.
Fluorescence of dyes in solutions with high absorbance. Inner filter effect correction.
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DOI：
10.1371/journal.pone.0103878
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Turoverov KK
中科院分区：
文献类型：
--
作者：
Fonin AV;Sulatskaya AI;Kuznetsova IM;Turoverov KK
荧光是所有知识领域（包括生物学和医学）的成熟工具。其使用中的一个显著障碍是由所谓的初级内滤波器效应引起的荧光强度对荧光团浓度的依赖性的非线性。用于校正荧光强度的现有方法在实践中难以实施;因此，通常认为最好使用稀溶液。我们表明，必须始终进行校正。此外，高浓度溶液（高吸光度）是研究荧光染料的物理化学性质和生物大分子功能重要相互作用的固有条件。我们提出了一种易于使用的方法来校正实验记录的总荧光强度，并表明荧光强度的信息分量在数值上等于吸光度和荧光量子产率的产品的对象。研究表明，如果染料分子之间不发生相互作用，没有重吸收（如NATA）和荧光分光光度计提供了检测到的荧光强度与吸收光部分的比例（对于具有水平狭缝的荧光分光光度计来说是可能的）则实验检测的染料的总荧光强度对其吸光度的依赖性与计算的依赖性和校正一致可根据溶液吸光度计算消除一次内滤效应的因子。实验表明，NATA荧光的吸光度范围很宽（至少高达60）。对于ATTO-425，其荧光和吸收光谱重叠，消除初级和次级滤波器效应和额外的光谱分析允许得出结论，实验检测到的荧光强度对溶液吸光度的依赖性与计算的依赖性的偏差的最可能的原因是染料分子自猝灭，其伴随共振无辐射激发能量转移。
Fluorescence is a proven tool in all fields of knowledge, including biology and medicine. A significant obstacle in its use is the nonlinearity of the dependence of the fluorescence intensity on fluorophore concentration that is caused by the so-called primary inner filter effect. The existing methods for correcting the fluorescence intensity are hard to implement in practice; thus, it is generally considered best to use dilute solutions. We showed that correction must be performed always. Furthermore, high-concentration solutions (high absorbance) are inherent condition in studying of the photophysical properties of fluorescent dyes and the functionally significant interactions of biological macromolecules. We proposed an easy to use method to correct the experimentally recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object. It is shown that if dye molecules do not interact with each other and there is no reabsorption (as for NATA) and spectrofluorimeter provides the proportionality of the detected fluorescence intensity to the part of the absorbed light (that is possible for spectrofluorimeter with horizontal slits) then the dependence of experimentally detected total fluorescence intensity of the dye on its absorbance coincides with the calculated dependence and the correction factor for eliminating the primary inner filter effect can be calculated on the basis of solution absorbance. It was experimentally shown for NATA fluorescence in the wide range of absorbance (at least up to 60). For ATTO-425, which fluorescence and absorption spectra overlap, the elimination of the primary and secondary filter effects and additional spectral analysis allow to conclude that the most probable reason of the deviation of experimentally detected fluorescence intensity dependence on solution absorbance from the calculated dependence is the dye molecules self-quenching, which accompanies resonance radiationless excitation energy transfer.
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影响因子：
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10.1016/j.saa.2004.08.014
发表时间：
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影响因子：
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