Yi Jason, Manna Asit, Barr Valarie A, Hong Jennifer, Neuman Keir C, Samelson Lawrence E
Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Mol Biol Cell. 2016 Nov 7;27(22):3591-3600. doi: 10.1091/mbc.E16-05-0330. Epub 2016 Oct 5.
Investigation of heterogeneous cellular structures using single-molecule localization microscopy has been limited by poorly defined localization accuracy and inadequate multiplexing capacity. Using fluorescent nanodiamonds as fiducial markers, we define and achieve localization precision required for single-molecule accuracy in dSTORM images. Coupled with this advance, our new multiplexing strategy, madSTORM, allows accurate targeting of multiple molecules using sequential binding and elution of fluorescent antibodies. madSTORM is used on an activated T-cell to localize 25 epitopes, 14 of which are on components of the same multimolecular T-cell receptor complex. We obtain an average localization precision of 2.6 nm, alignment error of 2.0 nm, and <0.01% cross-talk. Combining these technical advances affords the ability to move beyond obtaining superresolved structures to defining spatial relationships among constituent molecules within structures. Probing the molecular topology of complex signaling cascades and other heterogeneous networks is feasible with madSTORM.
使用单分子定位显微镜对异质细胞结构进行研究，一直受到定位精度定义不明确和多重分析能力不足的限制。我们使用荧光纳米金刚石作为基准标记，定义并实现了dSTORM图像中单分子精度所需的定位精度。在此进展的基础上，我们的新多重分析策略madSTORM，允许通过荧光抗体的顺序结合和洗脱对多个分子进行精确靶向。madSTORM应用于活化的T细胞，以定位25个表位，其中14个位于同一多分子T细胞受体复合物的组分上。我们获得了2.6 nm的平均定位精度、2.0 nm的对齐误差和<0.01%的串扰。结合这些技术进步，使得我们有能力超越获得超分辨结构，进而定义结构内组成分子之间的空间关系。利用madSTORM探究复杂信号级联反应和其他异质网络的分子拓扑结构是可行的。
