Direct evaluation of self-quenching behavior of fluorophores at high concentrations using an evanescent field.
Direct evaluation of self-quenching behavior of fluorophores at high concentrations using an evanescent field.
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DOI：
10.1371/journal.pone.0247326
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Jeong C
中科院分区：
文献类型：
--
作者：
Bae W;Yoon TY;Jeong C
当两个或多个相同的荧光团彼此非常接近时，荧光团的量子产率就会降低。蛋白质折叠或颗粒聚集的研究可以基于上述现象--称为自猝灭。然而，由于内滤光效应，包括激发光的耗尽和发射光的重新吸收，很难表征高浓度下荧光团的自猝灭。为此，提出了一种直接评价荧光团自猝灭行为的新方法。利用物镜全内反射荧光(TIRF)显微镜产生的消失场，将激发光和发射光的光程缩短到~100 nm，从而抑制了内滤波效应。直接测量了磺胺荷丹明B、异硫氰酸荧光素和钙黄绿素溶液在1μM到50 mM浓度范围内的荧光强度，以评估1000倍自猝灭所需的浓度，并研究了荧光团发生自猝灭的不同机制。
The quantum yield of a fluorophore is reduced when two or more identical fluorophores are in close proximity to each other. The study of protein folding or particle aggregation is can be done based on this above-mentioned phenomenon—called self-quenching. However, it is challenging to characterize the self-quenching of a fluorophore at high concentrations because of the inner filter effect, which involves depletion of excitation light and re-absorption of emission light. Herein, a novel method to directly evaluate the self-quenching behavior of fluorophores was developed. The evanescent field from an objective-type total internal reflection fluorescence (TIRF) microscope was used to reduce the path length of the excitation and emission light to ~100 nm, thereby supressing the inner filter effect. Fluorescence intensities of sulforhodamine B, fluorescein isothiocyanate (FITC), and calcein solutions with concentrations ranging from 1 μM to 50 mM were directly measured to evaluate the concentration required for 1000-fold degree of self-quenching and to examine the different mechanisms through which the fluorophores undergo self-quenching.
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