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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鬼笔环肽-PAINT：F-肌动蛋白的增强定量纳米成像


我们提出了基于鬼笔环肽的点积累，用于纳米级地形学 （phalloidin-PAINT） 的成像，能够对细胞体内的丝状肌动蛋白 （F-actin） 和精细的膜突起进行定量超分辨率成像。我们证明，内在的鬼笔环肽解离能够在含有低浓度染料偶联鬼笔环肽的成像缓冲液中进行 PAINT 超分辨率显微镜检查。我们进一步显示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 的两个好处是它能够在整个细胞中一致地定量纳米级的 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更一致的 F-肌动蛋白定量和 DC 结构精细的膜突起。使用 DC2.4 小鼠 DC 作为模型系统，我们显示了脂多糖暴露后 F-肌动蛋白从足小体到肌动蛋白丝的重新分布以及培养玻璃表面 F-肌动蛋白相关膜突起的发生率改变。我们的工作理念为使用市售试剂进行定量蛋白质特异性 PAINT 开辟了新的可能性。