Nano-Science Center & Department of Chemistry, University of Copenhagen Universitetsparken 5, 2100 Copenhagen, Denmark.
Chemical Physics & NanoLund, Department of Chemistry, Lund University, Box 124, 22100 Lund, Sweden.
J Am Chem Soc. 2021 Jan 27;143(3):1377-1385. doi: 10.1021/jacs.0c10457. Epub 2021 Jan 11.
Increasing demand for detecting single molecules in challenging environments has raised the bar for the fluorophores used. To achieve better resolution and/or contrast in fluorescence microscopy, it is now essential to use bright and stable dyes with tailored photophysical properties. While long fluorescence lifetime fluorophores offer many advantages in time-resolved imaging, their inherently lower molar absorption coefficient has limited applications in single molecule imaging. Here we propose a generic approach to prepare bright, long fluorescence lifetime dyad fluorophores comprising an absorbing antenna chromophore with high absorption coefficient linked to an acceptor emitter with a long fluorescence lifetime. We introduce a dyad consisting of a perylene antenna and a triangulenium emitter with 100% energy transfer from donor to acceptor. The dyad retained the long fluorescence lifetime (∼17 ns) and high quantum yield (75%) of the triangulenium emitter, while the perylene antenna increased the molar absorption coefficient (up to 5 times) in comparison to the free triangulenium dye. These triangulenium based dyads with significantly improved brightness can now be detected at the single molecule level and easily discriminated from bright autofluorescence by time-gated and other lifetime-based detection schemes.
在具有挑战性的环境中检测单分子的需求不断增加，这对所用荧光团的性能提出了更高的要求。为了在荧光显微镜中实现更好的分辨率和/或对比度，现在必须使用具有定制光物理特性的明亮且稳定的染料。虽然长荧光寿命荧光团在时间分辨成像中具有许多优势，但它们固有的较低摩尔消光系数限制了它们在单分子成像中的应用。在这里，我们提出了一种通用的方法来制备亮的、长荧光寿命的二聚体荧光团，它由具有高吸收系数的吸收天线色团和具有长荧光寿命的受体给体组成。我们引入了一个由苝天线和三嗪烯给体组成的二聚体，其中能量从供体 100%转移到受体。与游离三嗪烯染料相比，二聚体保留了三嗪烯给体的长荧光寿命（~17 ns）和高光量子产率（75%），而苝天线则增加了摩尔消光系数（高达 5 倍）。这些基于三嗪烯的二聚体具有显著提高的亮度，现在可以在单分子水平上检测到，并可以通过时间门控和其他基于寿命的检测方案轻松与明亮的自发荧光区分开来。
