Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany.
Angew Chem Int Ed Engl. 2023 Sep 25;62(39):e202307538. doi: 10.1002/anie.202307538. Epub 2023 Aug 23.
Super-resolution techniques like single-molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non-covalent, weak affinity-based transient labelling systems. DNA-based hybrid systems are a prominent example among these transient labelling systems, offering excellent opportunities for multi-target fluorescence imaging. However, these techniques suffer from higher background relative to covalently bound fluorophores, originating from unbound fluorophore-labelled single-stranded oligonucleotides. Here, we introduce short-distance self-quenching in fluorophore dimers as an efficient mechanism to reduce background fluorescence signal, while at the same time increasing the photon budget in the bound state by almost 2-fold. We characterise the optical and thermodynamic properties of fluorophore-dimer single-stranded DNA, and show super-resolution imaging applications with STED and SMLM with increased spatial resolution and reduced background.
超分辨率技术，如单分子定位显微镜（SMLM）和受激发射损耗（STED）显微镜，已经通过使用非共价、弱亲和力的瞬时标记系统得到了扩展。在这些瞬时标记系统中，基于 DNA 的杂交系统是一个突出的例子，为多目标荧光成像提供了极好的机会。然而，与共价结合的荧光团相比，这些技术的背景信号更高，这源于未结合的荧光团标记的单链寡核苷酸。在这里，我们引入荧光团二聚体的短程自猝灭作为一种有效的机制，以降低背景荧光信号，同时将结合态的光子预算增加近 2 倍。我们描述了荧光团二聚体单链 DNA 的光学和热力学性质，并展示了使用 STED 和 SMLM 的超分辨率成像应用，具有更高的空间分辨率和更低的背景。
