Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins
Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins
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DOI：
10.1016/j.bpj.2009.09.053
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发表时间：
2010-01-06
影响因子：
3.4
通讯作者：
Hell, Stefan W.
中科院分区：
文献类型：
--
作者：
Hein, Birka;Willig, Katrin I.;Hell, Stefan W.
我们展示了在活的哺乳动物细胞内用有机染料标记的不同结构元素的远场荧光纳米显微镜。利用受激发射耗尽技术，远场光学显微镜的性能优于衍射势垒限制。我们使用的标签蛋白hAGT（SNAP标签），它共价结合benzy 1鸟嘌呤取代的有机染料，标记。四甲基罗丹明被用来成像的细胞骨架（波形蛋白和微管相关蛋白2），以及位于细胞膜的结构（小窝蛋白和连接蛋白-43）的分辨率低至40 nm。与黄色荧光蛋白Citrine标记的结构的比较验证了这种标记方法。显示连接蛋白-43簇在细胞膜上移动的纳米级电影证明了这种技术观察纳米级结构随时间变化的能力。用于激发和受激发射损耗的脉冲或连续波激光器在活细胞中产生类似分辨率的图像。因此，结合修饰的有机染料的融合蛋白大大扩展了活细胞远场荧光纳米显微镜的应用范围。
We show far-field fluorescence nanoscopy of different structural elements labeled with an organic dye within living mammalian cells. The diffraction barrier limiting far-field light microscopy is outperformed by using stimulated emission depletion. We used the tagging protein hAGT (SNAP-tag), which covalently binds benzy1guanine-substituted organic dyes, for labeling. Tetramethy1rhodamine was used to image the cytoskeleton (vimentin and microtubule-associated protein 2) as well as structures located at the cell membrane (caveolin and connexin-43) with a resolution down to 40 nm. Comparison with structures labeled with the yellow fluorescent protein Citrine validates this labeling approach. Nanoscopic movies showing the movement of connexin-43 clusters across the cell membrane evidence the capability of this technique to observe structural changes on the nanoscale over time. Pulsed or continuous-wave lasers for excitation and stimulated emission depletion yield images of similar resolution in living cells. Hence fusion proteins that bind modified organic dyes expand widely the application range of far-field fluorescence nanoscopy of living cells.