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A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging

Nature Chemistry volume 6, pages 681–689 (2014)Cite this article

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Abstract

Single-molecule localization microscopy is used to construct super-resolution images, but generally requires prior intense laser irradiation and in some cases additives, such as thiols, to induce on–off switching of fluorophores. These requirements limit the potential applications of this methodology. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramolecular spirocyclization reaction. Optimization of the intramolecular nucleophile and rhodamine-based fluorophore (electrophile) provide a suitable lifetime for the fluorescent open form, and equilibrium between the open form and the non-fluorescent closed form. We show that this spontaneously blinking fluorophore is suitable for single-molecule localization microscopy imaging deep inside cells and for tracking the motion of structures in living cells. We further demonstrate the advantages of this fluorophore over existing methodologies by applying it to nuclear pore structures located far above the coverslip with a spinning-disk confocal microscope and for repetitive time-lapse super-resolution imaging of microtubules in live cells for up to 1 h.

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Figure 1: SLM with spontaneously blinking fluorophore.
Figure 2: Effect of intramolecular nucleophiles and fluorophores on thermal equilibrium of intramolecular spirocyclization.
Figure 3: Switching properties of the antibody-bound dyes in the absence of thiols and an oxygen-scavenging system.
Figure 4: SLM with HMSiR in vitro and in fixed cells.
Figure 5: Live-cell time-lapse SLM with HMSiR.

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Acknowledgements

This research was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research (KAKENHI): 20117003 and 23249004 to Y.U., 23113504 and 25113707 to M.K., 23710108 to M.C.T., 24659092, 25113723 and 25293046 to Y.O. and 21121004 to T. F.), by The Daiichi-Sankyo Foundation of Life Science (to Y.U.), by the Uehara memorial Foundation (to Y.O.), by The Mochida Memorial Foundation for Medical and Pharmaceutical Research (to M.K.) and by a JSPS stipend (to S.U.). The authors thank D. Toomre for many useful discussions, K. Johnsson and G. Lukinavičius for β-tubulin–SNAP plasmid, N. Imamoto for HeLa cell lines expressing nuclear pore proteins, R. Wong for POM121 plasmid, J. Asada and S. Dan Xu for the construction of plasmids and experimental assistance, and Y. Arai for the ImageJ script for analysing projection profiles of the localization of super-resolution images. The African green monkey kidney normal cell line Vero (JCRB0111) was obtained from the Japanese Collection of Research Bioresources (JCRB) cell bank.

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Authors and Affiliations

  1. Graduate School of Medicine, The University of Tokyo, Tokyo, 113-0033, Japan

    Shin-nosuke Uno, Mako Kamiya & Yasuteru Urano

  2. Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, 376-8515, Japan

    Toshitada Yoshihara & Seiji Tobita

  3. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, 113-0033, Japan

    Ko Sugawara, Kohki Okabe, Takashi Funatsu & Yasuteru Urano

  4. JST, PRESTO, Saitama, 332-0012, Japan

    Kohki Okabe

  5. Center for International Research on Micronano Mechatronics, Institute of Industrial Science, The University of Tokyo, Tokyo, 153-8505, Japan

    Mehmet C. Tarhan & Hiroyuki Fujita

  6. Laboratory for Cell Polarity Regulation, Quantitative Biology Center, RIKEN, Suita, 565-0874, Japan

    Yasushi Okada

  7. Basic Research Program, Japan Science and Technology Agency, Tokyo, 102-0075, Japan

    Yasuteru Urano

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  1. Shin-nosuke Uno
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Contributions

S.U., M.K., T.Y., Y.O., S.T. and Y.U. conducted experiments and performed analyses. S.U., M.K., Y.O. and Y.U. co-wrote the manuscript. K.S., K.O., M.C.T., T.F. and H.F. contributed materials/analysis tools. M.K. and Y.U. planned and initiated the project, designed experiments, and supervised the entire project.

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Correspondence to Mako Kamiya or Yasuteru Urano.

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Uno, Sn., Kamiya, M., Yoshihara, T. et al. A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging. Nature Chem 6, 681–689 (2014). https://doi.org/10.1038/nchem.2002

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