PDF(Pubmed)
Abstract：
Super-resolution microscopy has promoted the development of cell biology, but imaging proteins with low copy numbers in cellular structures remains challenging. The limited number of designated proteins within nuclear pore complexes (NPCs) impedes continuous observation in live cells, although they are often used as a standard for evaluating various SR methods. To address this issue, we tagged POM121 with Halo-SiR and imaged it using structured illumination microscopy with sparse deconvolution (Sparse-SIM). Remarkably, POM121-SiR exhibited more than six-fold fluorescence intensity and four-fold enhanced contrast compared to the same protein labeled with tandem-linked mCherry, while showing negligible photo-bleaching during SR imaging for 200 frames. Using this technique, we discovered various types of NPCs, including ring-like and cluster-like structures, and observed dynamic remodeling along with the sequential appearance of different Nup compositions. Overall, Halo-SiR with Sparse-SIM is a potent tool for extended SR imaging of dynamic structures of NPCs in live cells, and it may also help visualize proteins with limited numbers in general.
摘要：
超分辨率显微镜促进了细胞生物学的发展,但是成像在细胞结构中具有低拷贝数的蛋白质仍然具有挑战性。核孔复合物（NPC）中指定蛋白质的数量有限，阻碍了在活细胞中的连续观察。尽管它们通常被用作评估各种SR方法的标准。为了解决这个问题，我们用Halo-SiR标记POM121，并使用带有稀疏反卷积的结构化照明显微镜（Sparse-SIM）对其进行成像。值得注意的是,与用串联连接的mCherry标记的相同蛋白质相比，POM121-SiR表现出超过六倍的荧光强度和四倍的增强对比度，同时在200帧的SR成像过程中显示出可忽略不计的光漂白。使用这种技术，我们发现了各种类型的NPC,包括环状和簇状结构，并观察到动态重构以及不同Nup组合物的顺序出现。总的来说,Halo-SiR与稀疏-SIM是一个强大的工具，用于扩展SR成像的动态结构的NPC在活细胞中，它也可能有助于可视化数量有限的蛋白质。
