A general strategy for measuring the energy-transfer efficiencies in multifluorophore assemblies is demonstrated. The present method is based on spectral shaping of the excitation light with molecular solutions representing donor and acceptor fluorophores, which causes a suppressed excitation of the respective donor and acceptor molecules in the sample. The changes in the acceptor emission resulting from spectral shaping of the excitation light are then used to determine the energy-transfer efficiencies (E_x) associated with all participating donor–acceptor pairs. Here, the technique is demonstrated through energy-transfer (ET) measurements in a 4-fluorophore construct featuring a DNA supported assembly of three donor/donor-relay (Cy3, Cy3.5, and Cy5) and one acceptor (Cy5.5) molecules. The resulting E_x were validated using the standard photoluminescence (PL) quenching approach as well as measurements of partial 2- and 3-dye assemblies. The present work highlights general benefits of the spectrally shaped excitation approach to measuring donor–acceptor energetics, including the ability to resolve the spectral cross talk between multiple fluorophores and to exclude charge transfer contributions into donor PL quenching.

中文翻译：

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消除多荧光团装配中的光谱串扰

演示了一种用于测量多荧光团组件中能量转移效率的通用策略。本方法基于具有代表供体和受体荧光团的分子溶液的激发光的光谱整形，这导致样品中各个供体和受体分子的激发被抑制。然后，由激发光的光谱整形导致的受体发射的变化可用于确定能量转移效率（E _x）与所有参与的供体-受体对相关联。在此，通过在4-荧光团构建体中进行能量转移（ET）测量来证明该技术，该构建体具有三个供体/供体-中继体（Cy3，Cy3.5和Cy5）和一个受体（Cy5.5）的DNA支持组装分子。使用标准的光致发光（PL）淬灭方法以及部分2和3染料组装的测量结果验证了所得的E _x。本工作着重介绍了用于测量施主-受主能量的光谱形状激发方法的一般优势，包括解决多个荧光团之间的光谱串扰以及排除电荷转移到施主PL猝灭中的能力。