Development of a Reversibly Switchable Fluorescent Protein for Super-Resolution Optical Fluctuation Imaging (SOFI)
Development of a Reversibly Switchable Fluorescent Protein for Super-Resolution Optical Fluctuation Imaging (SOFI)
复制标题
开发用于超分辨率光学波动成像 (SOFI) 的可逆可切换荧光蛋白。
DOI：
10.1021/nn5064387
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发表时间：
2015-03-01
期刊：
影响因子：
17.1
通讯作者：
Xu, Pingyong
中科院分区：
文献类型：
--
作者：
Zhang, Xi;Chen, Xuanze;Xu, Pingyong
可逆开关荧光蛋白（RSFPs）可以有效地用于基于单分子开关和波动的超分辨率光学波动成像（SOFI）。RSFP的几个属性强烈影响SOFI图像的质量。这些性质包括（i）波动状态下的平均荧光强度，（ii）开/关对比度，（iii）光稳定性，和（iv）低聚倾向。前三个性质决定了成像像素和SOFI信号的波动范围，这对空间分辨率至关重要，最后一个可能导致目标蛋白质的人工聚集。目前用于SOFI的RSFP在波动状态下的平均荧光强度、光稳定性和开/关对比度方面较低，从而限制了SOFI在生物超分辨率成像中的应用范围。在这项研究中，我们开发了一种新的单体绿色RSFP称为Skylan-S，它具有非常高的光稳定性，对比度，和平均荧光强度的波动状态。利用Skylan-S优异的光学性能，与Dronpa相比，成像像素的波动范围提高了4倍，SOFI分辨率也更高。此外，超分辨率成像的肌动蛋白或微管蛋白的结构和网格蛋白包被的坑（CCP）在活的U2 OS细胞标记与Skylan-S使用SOFI技术证明。总体而言，Skylan-S开发具有出色的光化学性能是有前途的长时间SOFI成像与高时空分辨率。
Reversibly switchable fluorescent proteins (RSFPs) can be effectively used for super-resolution optical fluctuation imaging (SOFI) based on the switching and fluctuation of single molecules. Several properties of RSFPs strongly influence the quality of SOFI images. These properties include (i) the averaged fluorescence intensity in the fluctuation state, (ii) the on/off contrast ratio, (iii) the photostability, and (iv) the oligomerization tendency. The first three properties determine the fluctuation range of the imaged pixels and the SOFI signal, which are of essential importance to the spatial resolution, and the last may lead to artificial aggregation of target proteins. The RSFPs that are currently used for SOFI are low in averaged fluorescence intensity in the fluctuation state, photostability, and on/off contrast ratio, thereby limiting the range of application of SOFI in biological super-resolution imaging. In this study, we developed a novel monomeric green RSFP termed Skylan-S, which features very high photostability, contrast ratio, and averaged fluorescence intensity in the fluctuation state. Taking advantage of the excellent optical properties of Skylan-S, a 4-fold improvement in the fluctuation range of the imaged pixels and higher SOFI resolution can be obtained compared with Dronpa. Furthermore, super-resolution imaging of the actin or tubulin structures and clathrin-coated pits (CCPs) in living U2OS cells labeled with Skylan-S was demonstrated using the SOFI technique. Overall, Skylan-S developed with outstanding photochemical properties is promising for long-time SOFI imaging with high spatial-temporal resolution.