Subdiffraction super‐resolution fluorescence microscopy, or nanoscopy, has seen remarkable developments in the last two decades. Yet, for the visualization of plant cells, nanoscopy is still rarely used. In this study, we established RESOLFT nanoscopy on living green plant tissue. Live‐cell RESOLFT nanoscopy requires and utilizes comparatively low light doses and intensities to overcome the diffraction barrier. We generated a transgenic Arabidopsis thaliana plant line expressing the reversibly switchable fluorescent protein rsEGFP2 fused to the mammalian microtubule‐associated protein 4 (MAP4) in order to ubiquitously label the microtubule cytoskeleton. We demonstrate the use of RESOLFT nanoscopy for extended time‐lapse imaging of cortical microtubules in Arabidopsis leaf discs. By combining our approach with fluorescence lifetime gating, we were able to acquire live‐cell RESOLFT images even close to chloroplasts, which exhibit very strong autofluorescence. The data demonstrate the feasibility of subdiffraction resolution imaging in transgenic plant material with minimal requirements for sample preparation.

中文翻译：

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转基因拟南芥的活细胞RESOLFT纳米显微镜

在过去的二十年中，亚衍射超分辨率荧光显微镜或纳米显微镜已取得了令人瞩目的发展。然而，对于可视化植物细胞，纳米显微镜仍然很少使用。在这项研究中，我们建立了绿色活体植物组织的RESOLFT纳米显微镜。活细胞RESOLFT纳米显微镜需要并利用相对较低的光剂量和强度来克服衍射障碍。我们生成了一种转基因拟南芥植物系，该植物系表达可逆转换的荧光蛋白rsEGFP2与哺乳动物微管相关蛋白4（MAP4）融合，以便普遍标记微管细胞骨架。我们展示了RESOLFT纳米显微镜在拟南芥中皮层微管的延时成像中的应用叶盘。通过将我们的方法与荧光寿命门控相结合，我们甚至可以获得接近于显示非常强的自发荧光的叶绿体的活细胞RESOLFT图像。数据表明亚衍射分辨率成像在转基因植物材料中的可行性，而样品制备的要求最低。