Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.

多色单分子跟踪（SMT）提供了一个功能强大的工具，可以机械地探测活细胞中的分子相互作用。然而，由于当前可用的荧光团的光学和化学性质以及多蛋白标记策略的局限性，细胞内多色SMT对于一般研究仍具有挑战性。在这里，我们介绍一种实用的方法，利用纳米孔电穿孔（NanoEP）技术将多种有机染料标记的蛋白质传递到活细胞中进行成像。它可以在商用显微镜中轻松扩展到三个通道，也可以与其他原位标记方法结合使用。利用NanoEP，我们展示了三色SMT用于细胞溶质和膜蛋白。特别，我们同时监测了活细胞中EGFR信号通路下游的单分子事件。结果提供了Grb2和SOS募集到激活的EGFR以及产生的Ras激活的空间定位和动力学的详细解析。