Hüve Jana, Wesselmann Ramona, Kahms Martin, Peters Reiner
Institute of Medical Physics and Biophysics, and Center for Nanotechnology (CeNTech), University of Münster, Münster, Germany.
Biophys J. 2008 Jul;95(2):877-85. doi: 10.1529/biophysj.107.127449. Epub 2008 Mar 28.
To explore whether super-resolution fluorescence microscopy is able to resolve topographic features of single cellular protein complexes, a two-photon 4Pi microscope was used to study the nuclear pore complex (NPC). The microscope had an axial resolution of 110-130 nm and a two-color localization accuracy of 5-10 nm. In immune-labeled HeLa cells, NPCs could be resolved much better by 4Pi than by confocal microscopy. When two epitopes of the NPC, one localized at the tip of the cytoplasmic filaments and the other at the ring of the nuclear basket, were immune-labeled, they could be clearly resolved in single NPCs, with the distance between them determined to be 152 +/- 30 nm. In cells expressing a green fluorescent protein construct localized at the NPC center, the distances between the ring of the nuclear filaments and the NPC center was 76 +/- 12 (Potorous tridactylus cells) or 91 +/- 21 nm (normal rat kidney cells), whereas the distance between the NPC center and the tips of the cytoplasmic filaments was 84 +/- 18 nm, all values in good agreement with previous electron or single-molecule fluorescence estimates. We conclude that super-resolution fluorescence microscopy is a powerful method for analyzing single protein complexes and the cellular nanomachinery in general.
为了探究超分辨率荧光显微镜是否能够分辨单细胞蛋白质复合物的拓扑特征，使用双光子4Pi显微镜研究核孔复合体（NPC）。该显微镜的轴向分辨率为110 - 130纳米，双色定位精度为5 - 10纳米。在免疫标记的HeLa细胞中，4Pi显微镜对NPC的分辨效果比共聚焦显微镜好得多。当对NPC的两个表位进行免疫标记时，一个位于细胞质丝的末端，另一个位于核篮的环上，它们在单个NPC中能够清晰分辨，两者之间的距离确定为152±30纳米。在表达定位在NPC中心的绿色荧光蛋白构建体的细胞中，核丝环与NPC中心之间的距离为76±12（长鼻袋狸细胞）或91±21纳米（正常大鼠肾细胞），而NPC中心与细胞质丝末端之间的距离为84±18纳米，所有这些值与先前的电子或单分子荧光估计值吻合良好。我们得出结论，超分辨率荧光显微镜是一种用于分析单个蛋白质复合物以及一般细胞纳米机器的强大方法。
