In vivo super-resolution RESOLFT microscopy of Drosophila melanogaster
In vivo super-resolution RESOLFT microscopy of Drosophila melanogaster
复制标题
DOI：
10.7554/elife.15567
复制
发表时间：
2016-06-29
期刊：
影响因子：
7.7
通讯作者：
Jakobs, Stefan
中科院分区：
文献类型：
--
作者：
Schnorrenberg, Sebastian;Grotjohann, Tim;Jakobs, Stefan
尽管衍射无限超分辨显微镜取得了令人瞩目的进展，但组织和模型生物的活体纳米显微镜仍然没有令人满意地建立起来，而且很少实现。RESOLFT纳米显微镜特别适合于活细胞成像，因为它需要相对较低的光线来克服衍射障碍。此前，我们引入了可逆性可切换荧光蛋白rsEGFP2，它有助于快速RESOLFT纳米观察(Grodohann等人，2012年)。在这项研究中，与大多数其他纳米研究一样，只对培养的单个细胞进行了分析。在这里，我们报告使用rsEGFP2对完整的果蝇幼虫和切除的组织的亚细胞结构进行活细胞RESOLFT纳米观察。我们产生了表达α-微管蛋白和rsEGFP2融合蛋白的果蝇，突出了所有细胞中的微管细胞骨架。通过聚焦于完整的幼虫角质层，我们实现了对
Despite remarkable developments in diffraction unlimited super-resolution microscopy, in vivo nanoscopy of tissues and model organisms is still not satisfactorily established and rarely realized. RESOLFT nanoscopy is particularly suited for live cell imaging because it requires relatively low light levels to overcome the diffraction barrier. Previously, we introduced the reversibly switchable fluorescent protein rsEGFP2, which facilitated fast RESOLFT nanoscopy (Grodohann et al., 2012). In that study, as in most other nanoscopy studies, only cultivated single cells were analyzed. Here, we report on the use of rsEGFP2 for live-cell RESOLFT nanoscopy of sub-cellular structures of intact Drosophila melanogaster larvae and of resected tissues. We generated flies expressing fusion proteins of alpha-tubulin and rsEGFP2 highlighting the microtubule cytoskeleton in all cells. By focusing through the intact larval cuticle, we achieved lateral resolution of