Institut für Chemie und Biochemie, Freie Universität Berlin, Thielallee 63, 14195, Berlin, Germany.
Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, 14195, Berlin, Germany.
Angew Chem Int Ed Engl. 2023 Jul 10;62(28):e202302318. doi: 10.1002/anie.202302318. Epub 2023 May 26.
Expansion microscopy (ExM) is a recently developed technique that allows for the resolution of structures below the diffraction limit by physically enlarging a hydrogel-embedded facsimile of the biological sample. The target structure is labeled and this label must be retained in a relative position true to the original, smaller state before expansion by linking it into the gel. However, gel formation and digestion lead to a significant loss in target-delivered label, resulting in weak signal. To overcome this problem, we have here developed an agent combining targeting, fluorescent labeling and gel linkage in a single small molecule. Similar approaches in the past have still suffered from significant loss of label. Here we show that this loss is due to insufficient surface grafting of fluorophores into the hydrogel and develop a solution by increasing the amount of target-bound monomers. Overall, we obtain a significant improvement in fluorescence signal retention and our new dye allows the resolution of nuclear pores as ring-like structures, similar to STED microscopy. We furthermore provide mechanistic insight into dye retention in ExM.
扩展显微镜技术（ExM）是一种新兴的技术，通过物理放大生物样本的水凝胶嵌入副本，可以将分辨率提高到衍射极限以下。目标结构被标记，并且在扩展之前，必须通过将其链接到凝胶中来保持该标记与原始较小状态的相对位置。然而，凝胶形成和消化会导致目标传递的标记物大量丢失，从而导致信号较弱。为了解决这个问题，我们在这里开发了一种将靶向、荧光标记和凝胶连接结合在单个小分子中的试剂。过去类似的方法仍然存在标记物大量丢失的问题。在这里，我们表明这种损失是由于荧光团在水凝胶中的表面接枝不足引起的，并通过增加目标结合单体的数量来开发一种解决方案。总的来说，我们在荧光信号保留方面有了显著的提高，我们的新染料允许将核孔分辨为类似 STED 显微镜的环状结构。我们还提供了有关 ExM 中染料保留的机制见解。
