Superresolution Microscopy on the Basis of Engineered Dark States
Superresolution Microscopy on the Basis of Engineered Dark States
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DOI：
10.1021/ja806590m
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发表时间：
2008-12-17
影响因子：
15
通讯作者：
Tinnefeld, Philip
中科院分区：
文献类型：
--
作者：
Steinhauer, Christian;Forthmann, Carsten;Tinnefeld, Philip
通过使用可光切换或可光激活荧光团对单分子进行后续定位的超分辨率荧光显微镜新概念正在迅速出现，并提供了解析超出衍射极限的结构的新方法。在这里，我们证明，通过控制其发射特性，几乎可以对每种单分子兼容的合成荧光团进行超分辨率成像。我们通过去除氧来准备暗态，将三线态寿命延长到几毫秒。我们使用电子转移反应进一步增加关闭状态的持续时间，以创建寿命延长数倍的自由基离子状态，对固定细胞中的单分子、肌动蛋白丝和微管进行成像以及模拟表明，由此创建的暗状态足够长，足以实现类似于 50 nm 的分辨率。
New concepts for superresolution fluorescence microscopy by subsequent localization of single molecules using photoswitchable or photoactivatable fluorophores are rapidly emerging and provide new ways to resolve structures beyond the diffraction limit. Here, we demonstrate that superresolution imaging can be carried out with practically every single-molecule compatible, synthetic fluorophore by controlling their emission properties. We prepare dark states by removing oxygen that extends the triplet state lifetime to several milliseconds. We further increase the duration of the off-states using electron transfer reactions to create radical ion states of severalfold longer lifetimes, Imaging single molecules, actin filaments, and microtubules in fixed cells as well as simulations demonstrate that the thus created dark states are sufficiently long for resolution of similar to 50 nm.