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A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy

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Abstract

Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm2) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy.

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Figure 1: Photophysical characteristics of Kohinoor.
Figure 2: Kohinoor localization and photoswitching.
Figure 3: RESOLFT imaging of a live HeLa cell expressing vimentin-Kohinoor.

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Acknowledgements

This work was supported by a grant-in-aid for Scientific Research on Innovative Areas, 'Spying minority in biological phenomena (no. 3306)', from the Ministry of Education, Culture, Sports, Science and Technology, Japan (no. 23115003) (to T.N.), Cooperative Research Program of 'Network Joint Research Center for Materials and Devices' (to K.F.) and a postdoctoral fellowship from the Japan Society for the Promotion of Science (to D.K.T.).

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Authors and Affiliations

  1. The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan

    Dhermendra K Tiwari, Yoshiyuki Arai, Tomoki Matsuda, Masakazu Agetsuma, Masahiro Nakano & Takeharu Nagai

  2. Department of Applied Physics, Osaka University, Osaka, Japan

    Masahito Yamanaka & Katsumasa Fujita

Authors
  1. Dhermendra K Tiwari
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  2. Yoshiyuki Arai
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  3. Masahito Yamanaka
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  4. Tomoki Matsuda
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  5. Masakazu Agetsuma
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  6. Masahiro Nakano
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  7. Katsumasa Fujita
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  8. Takeharu Nagai
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Contributions

T.N. conceived the project. D.K.T. constructed Kohinoor and characterized it with contributions from T.M. and M.N. M.Y. and K.F. set up the RESOLFT microscopy. D.K.T., M.Y. and Y.A. performed RESOLFT imaging. M.A. and Y.A. performed two-photon imaging. All authors discussed and commented on the results and wrote the manuscript.

Corresponding author

Correspondence to Takeharu Nagai.

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Competing interests

The authors have filed an international patent application for Kohinoor (no. PCT-JP2014-074121).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–17 and Supplementary Tables 1–4 (PDF 12752 kb)

Repeated photoswitching of Kohinoor-β-actin in a live HeLa cell

Photoswitching was repeated 500 times by the alternative excitation of 405-nm and 488-nm lasers in a confocal laserscanning microscope. The cell did not show any morphological changes during photoswitching, indicating the lack of phototoxicity of the 405-nm light. (MP4 15346 kb)

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Tiwari, D., Arai, Y., Yamanaka, M. et al. A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy. Nat Methods 12, 515–518 (2015). https://doi.org/10.1038/nmeth.3362

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