Controlled Grafting Expansion Microscopy

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. We developed a trifunctional probe to combine covalent binding to the target protein, gel linkage and fluorescent labeling in one small molecule. Addition of the crosslinker AcX further increases the monomer density on the target surface. This results in increased grafting and thus higher retention of the fluorescent signal compared to a commercial dye that lacks the monomer group.