A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging
A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging
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DOI：
10.1038/nchem.2002
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发表时间：
2014-08-01
期刊：
影响因子：
21.8
通讯作者：
Urano, Yasuteru
中科院分区：
文献类型：
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作者：
Uno, Shin-nosuke;Kamiya, Mako;Urano, Yasuteru
单分子定位显微镜用于构建超分辨率图像，但通常需要事先进行强激光照射，并且在某些情况下需要添加剂（例如硫醇）来诱导荧光团的开关。这些要求限制了该方法的潜在应用。在这里，我们报告了一种基于分子内螺环化反应的一流自发闪烁荧光团。分子内亲核试剂和基于罗丹明的荧光团（亲电子试剂）的优化为荧光开放形式提供了合适的寿命，以及开放形式和非荧光封闭形式之间的平衡。我们证明，这种自发闪烁的荧光团适用于细胞深处的单分子定位显微镜成像以及跟踪活细胞中结构的运动。我们进一步证明了这种荧光团相对于现有方法的优势，通过使用转盘共聚焦显微镜将其应用于位于盖玻片上方远处的核孔结构，并对活细胞中的微管进行长达 1 小时的重复延时超分辨率成像。
Single-molecule localization microscopy is used to construct super-resolution images, but generally requires prior intense laser irradiation and in some cases additives, such as thiols, to induce on-off switching of fluorophores. These requirements limit the potential applications of this methodology. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramolecular spirocyclization reaction. Optimization of the intramolecular nucleophile and rhodamine-based fluorophore (electrophile) provide a suitable lifetime for the fluorescent open form, and equilibrium between the open form and the non-fluorescent closed form. We show that this spontaneously blinking fluorophore is suitable for single-molecule localization microscopy imaging deep inside cells and for tracking the motion of structures in living cells. We further demonstrate the advantages of this fluorophore over existing methodologies by applying it to nuclear pore structures located far above the coverslip with a spinning-disk confocal microscope and for repetitive time-lapse super-resolution imaging of microtubules in live cells for up to 1 h.