We present phalloidin-based points accumulation for imaging in nanoscale topography (phalloidin-PAINT), enabling quantitative superresolution imaging of filamentous actin (F-actin) in the cell body and delicate membrane protrusions. We demonstrate that the intrinsic phalloidin dissociation enables PAINT superresolution microscopy in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-PAINT are its ability to consistently quantify F-actin at the nanoscale throughout the entire cell and its enhanced preservation of fragile cellular structures. In a proof-of-concept study, we employed phalloidin-PAINT to superresolve F-actin structures in U2OS and dendritic cells (DCs). We demonstrate more consistent F-actin quantification in the cell body and structurally delicate membrane protrusions of DCs compared with direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse DCs as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane protrusions on the culture glass surface after lipopolysaccharide exposure. The concept of our work opens new possibilities for quantitative protein-specific PAINT using commercially available reagents.

中文翻译：

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Phalloidin-PAINT：F-肌动蛋白的增强定量纳米级成像


我们提出了基于鬼笔环肽的点积累，用于纳米级地形成像（鬼笔环肽-PAINT），从而能够对细胞体和精致的膜突起中的丝状肌动蛋白（F-肌动蛋白）进行定量超分辨率成像。我们证明，内在的鬼笔环肽解离使得在含有低浓度染料结合的鬼笔环肽的成像缓冲液中进行PAINT超分辨率显微镜观察成为可能。我们进一步展示了通过化学促进鬼笔环肽解离来增强单分子标记。鬼笔环肽-PAINT 的两个优点是能够在整个细胞的纳米尺度上一致地定量 F-肌动蛋白，以及增强对脆弱细胞结构的保存。在一项概念验证研究中，我们使用鬼笔环肽-PAINT 来超解析 U2OS 和树突状细胞 (DC) 中的 F-肌动蛋白结构。与直接随机光学重建显微镜 (dSTORM) 相比，我们证明了细胞体中 F-肌动蛋白的定量更加一致，并且 DC 的结构精致的膜突起。使用 DC2.4 小鼠树突状细胞作为模型系统，我们显示了脂多糖暴露后，F-肌动蛋白从足小体重新分布到肌动蛋白丝，并改变了培养玻璃表面上 F-肌动蛋白相关膜突出的发生率。我们的工作概念为使用市售试剂定量蛋白质特异性 PAINT 开辟了新的可能性。