title: Development of a reversibly switchable fluorescent protein for super-resolution optical fluctuation imaging (SOFI).
abstract: 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.
authors.affiliation: †Institute of Entomology, School of Life Sciences, Central China Normal University, Wuhan 430079, China.|‡Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Zhang
authors.affiliation: §Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China.|∥State Key Laboratory of Biomembrane and Membrane Biotechnology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.
authors.forename: Xuanze
authors.initials: X
authors.lastname: Chen
authors.affiliation: §Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China.
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: ‡Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Mingshu
authors.initials: M
authors.lastname: Zhang
authors.affiliation: ∥State Key Laboratory of Biomembrane and Membrane Biotechnology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.
authors.forename: Yujie
authors.initials: Y
authors.lastname: Sun
authors.affiliation: §Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Xi
authors.affiliation: †Institute of Entomology, School of Life Sciences, Central China Normal University, Wuhan 430079, China.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Peng
authors.affiliation: ‡Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Pingyong
authors.initials: P
authors.lastname: Xu
mesh_terms: D045744:Cell Line, Tumor; D002966:Clathrin / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D009154:Mutation; D061848:Optical Imaging / Q000379:methods*; D014404:Tubulin / Q000378:metabolism
chemical_list: D002966:Clathrin; D014404:Tubulin; D049452:Green Fluorescent Proteins
keywords: SOFI; Skylan-S; live-cell imaging; protein engineering; reversibly switchable fluorescent protein; super-resolution microscopy