Development of a reversibly switchable fluorescent protein for super-resolution optical fluctuation imaging (SOFI).

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.

[1]  Christian Eggeling,et al.  Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy , 2008, Nature Biotechnology.

[2]  Theo Lasser,et al.  Comparison between SOFI and STORM , 2011, Biomedical optics express.

[3]  S. Weiss,et al.  Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI) , 2009, Proceedings of the National Academy of Sciences.

[4]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[5]  Yongdeng Zhang,et al.  A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications , 2012, Proceedings of the National Academy of Sciences.

[6]  M. Gustafsson.  Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Yongdeng Zhang,et al.  Rational design of true monomeric and bright photoactivatable fluorescent proteins , 2012, Nature Methods.

[8]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[9]  Shimon Weiss,et al.  Superresolution optical fluctuation imaging (SOFI). , 2012, Advances in experimental medicine and biology.

[10]  Shimon Weiss,et al.  Labeling Cytosolic Targets in Live Cells with Blinking Probes. , 2013, The journal of physical chemistry letters.

[11]  Joshua W. Shaevitz,et al.  Spatial Covariance Reconstructive (SCORE) Super-Resolution Fluorescence Microscopy , 2014, PloS one.

[12]  Shimon Weiss,et al.  Superresolution optical fluctuation imaging with organic dyes. , 2010, Angewandte Chemie.

[13]  S. Weiss,et al.  Achieving increased resolution and more pixels with Superresolution Optical Fluctuation Imaging (SOFI) , 2010, Optics express.

[14]  Rafael C. González,et al.  Digital image processing using MATLAB , 2006 .

[15]  Hao Zhang,et al.  Fast Super-Resolution Imaging with Ultra-High Labeling Density Achieved by Joint Tagging Super-Resolution Optical Fluctuation Imaging , 2015, Scientific reports.

[16]  S. Hell.  Far-field optical nanoscopy , 2010 .

[17]  Peter Dedecker,et al.  Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[19]  Peter Dedecker,et al.  Widely accessible method for superresolution fluorescence imaging of living systems , 2012, Proceedings of the National Academy of Sciences.

[20]  Shingo Fukui,et al.  Real-time nanoscopy by using blinking enhanced quantum dots. , 2010, Biophysical journal.

[21]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[22]  Benjamien Moeyaert,et al.  Green-to-red photoconvertible Dronpa mutant for multimodal super-resolution fluorescence microscopy. , 2014, ACS nano.

[23]  S. Weiss,et al.  Resolving the spatial relationship between intracellular components by dual color super resolution optical fluctuations imaging (SOFI) , 2013, Optical Nanoscopy.

[24]  Peng Xi,et al.  Three-dimensional multimodal sub-diffraction imaging with spinning-disk confocal microscopy using blinking/fluctuating probes , 2015, Nano Research.

[25]  Christian Eggeling,et al.  Diffraction-unlimited all-optical imaging and writing with a photochromic GFP , 2011, Nature.

[26]  J. Heuser,et al.  Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation , 1989, The Journal of cell biology.

[27]  T. Lasser,et al.  Mapping molecular statistics with balanced super-resolution optical fluctuation imaging (bSOFI) , 2012, Optical Nanoscopy.