期刊
ACS NANO
卷 9, 期 3, 页码 2659-2667出版社
AMER CHEMICAL SOC
DOI: 10.1021/nn5064387
关键词
reversibly switchable fluorescent protein; super-resolution microscopy; Skylan-S; protein engineering; live-cell imaging; SOFI
类别
资金
- National Basic Research Program (973 Program) of China [2013CB910103, 2010CB933901, 2011CB809101]
- National Instrument Development Special Program [2013YQ03065102]
- National Natural Science Foundation of China [31170818, 31370851, 61178076, 31327901, 61475010, 31300612]
- Chinese Academy of Sciences [XDB08030202]
- Beijing Natural Science Foundation, China [7131011]
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 SOH imaging with high spatial-temporal resolution.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据