新功能、新界面、新体验,扫描即可下载生物谷APP!
首页 » 基金与期刊 » 小蛋白,大功能!一种可用于超高分辨显微成像的新型荧光蛋白

小蛋白,大功能!一种可用于超高分辨显微成像的新型荧光蛋白

来源:生物谷 2015-03-03 09:31

2015年3月3日讯 /生物谷BIOON/--最近中科院生物物理所的徐平勇课题组在著名期刊ACS NANO上发表了题为Development of a Reversibly Switchable Fluorescent Protein for Super-Resolution Optical Fluctuation Imaging (SOFI)的文章,报告了一种可用作高速活细胞超分辨率显微成像的新型反复光激活绿色荧光蛋白kylan-S。

绿色荧光蛋白(GFP)的发明因其能够提供对于活细胞和活体动物的靶向基因修饰标记而获得2008年诺贝尔化学奖。进一步,由基因改造的光激活荧光蛋白(PAGFP)能够提供单分子特性,而实现了超分辨显微成像,使得这一技术获得2014年诺贝尔化学奖。

随后,超高分辨显微成像技术的发展向着活细胞动态超高时空分辨率显微成像迈进。其中,光学波动超高分辨成像技术(SOFI)是一种简单可行的活细胞超高分辨显微成像新技术,它利用特殊荧光蛋白(或者染料)的反复光开关特性以及成像像素点随时间的波动和相关特性进行成像,因其可以突破单分子发光限制,同时实现多个荧光分子高速成像而迅速成为国际关注的热点。

kylan-S与现有的适用于SOFI成像的Dronpa相比具有突出的优点:在波动(fluctuation)状态下的荧光亮度比Dronpa高6-8倍,波动动态范围高4倍,同时具有极高的光学稳定性。Skylan-S是一种单体,因此能够用于活细胞成像而不影响目标蛋白的定位和功能。基于这些特性,Skylan-S应用于SOFI超分辨成像中具有极高的时空分辨率。在SOFI成像中,获得了1.5秒的超高时空分辨率成像(每帧3毫秒,500帧进行SOFI重构),可利用4阶SOFI获得clathrin-coated pits(CCP)的环状结构结构,空间分辨率好于100nm。

同时,Skylan-S能够动态观察亚细胞结构60s以上。而Dronpa在第一帧就无法实现这一高分辨率,在30s中超过一半基本被漂白。利用Skylan-S和Dronpa分别标记细胞的肌动蛋白丝,Skylan-S能够以丰富灰阶显示不同标记浓度的蛋白丝的精细结构,而Dronpa则仅能提供信噪比非常有限的图像。另外,Skylan-S在较高能量的488纳米激光照射下具有很好的单分子特性,适用于PALM成像。同时,它也广泛适用于传统的共聚焦和双光子成像。(生物谷Bioon.com)

本文系生物谷原创编译整理,欢迎转载!转载请注明来源并附原文链接。更多资讯请下载生物谷APP

生物谷推荐的英文摘要:

ACS NANO  DOI: 10.1021/nn5064387

Development of a Reversibly Switchable Fluorescent Protein for Super-Resolution Optical Fluctuation Imaging (SOFI)

Xi Zhang, Xuanze Chen, Zhiping Zeng, Mingshu Zhang, Yujie Sun, Peng Xi, Jianxin Peng, and Pingyong Xu

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.

温馨提示:87%用户都在生物谷APP上阅读,扫描立刻下载! 天天精彩!


相关标签

最新会议 培训班 期刊库