Abstract：
Super-resolution fluorescence imaging has emerged as a potent tool for investigating the nanoscale structure and function of the plasma membrane (PM). Nevertheless, the challenge persists in achieving super-resolution imaging of PM dynamics due to limitations in probe photostability and issues with cell internalization staining. Herein, we report assembly-mediated buffering fluorogenic probes BMP-14 and BMP-16 exhibiting fast PM labeling and extended retention time (over 2 h) on PM. The incorporation of alkyl chains proves effective in promoting the aggregation of BMP-14 and BMP-16 into nonfluorescent nanoparticles to realize fluorogenicity and regulate the buffering capacity to rapidly replace photobleached probes ensuring stable long-term super-resolution imaging of PM. Utilizing these PM-buffering probes, we observed dynamic movements of PM filopodia and continuous shrinkage, leading to the formation of extracellular vesicles (EVs) using structured illumination microscopy (SIM). Furthermore, we discovered two distinct modes of EV fusion: one involving fusion through adjacent lipids and the other through filamentous lipid traction. The entire process of EV fusion outside the PM was dynamically tracked. Additionally, BMP-16 exhibited a unique capability of inducing single-molecule fluorescence blinking when used for cell membrane staining. This property makes BMP-16 suitable for the PAINT imaging of cell membranes.
摘要：
超分辨率荧光成像已成为研究质膜（PM）的纳米级结构和功能的有力工具。然而，由于探针光稳定性的限制和细胞内化染色的问题,在实现PM动力学的超分辨率成像方面仍然存在挑战。在这里，我们报告了组装介导的缓冲荧光探针BMP-14和BMP-16表现出快速的PM标记和在PM上延长的保留时间（超过2小时）。烷基链的掺入证明可有效促进BMP-14和BMP-16聚集成非荧光纳米颗粒以实现荧光性并调节缓冲能力以快速替代光漂白的探针,从而确保PM的稳定的长期超分辨率成像。利用这些PM缓冲探头，我们观察到PM丝足的动态运动和持续收缩，使用结构化照明显微镜（SIM）导致形成细胞外囊泡（EV）。此外，我们发现了两种不同的EV融合模式：一种通过相邻脂质融合，另一种通过丝状脂质牵引融合。动态跟踪PM外EV融合的整个过程。此外,BMP-16在用于细胞膜染色时表现出诱导单分子荧光闪烁的独特能力。该性质使得BMP-16适用于细胞膜的PAINT成像。
