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

Journal name:
Nature Chemistry
Volume:
6,
Pages:
681–689
Year published:
DOI:
doi:10.1038/nchem.2002
Received
Accepted
Published online

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.

At a glance

Figures

  1. SLM with spontaneously blinking fluorophore.
    Figure 1: SLM with spontaneously blinking fluorophore.

    a, SLM sequence using a conventional fluorophore and the spontaneously blinking fluorophore. SLM using the spontaneously blinking fluorophore does not require any additive or high-intensity laser irradiation before recording, although both are required in dSTORM using conventional fluorophores. b, Thermal equilibrium of intramolecular spirocyclization between the fluorescent open form and the non-fluorescent closed form. pKcycl is the equilibrium constant for intramolecular spirocyclization and τ is the lifetime of the open form (the duration until the open form reverts to the closed form). c, Schematic pH titration curves of rhodamine derivatives with pKcycl of 6.0 (blue) and 8.5 (red). For SLM, most of the individual fluorophores should be in the closed form at the physiological pH of 7.4 to avoid overlapping signals.

  2. Effect of intramolecular nucleophiles and fluorophores on thermal equilibrium of intramolecular spirocyclization.
    Figure 2: Effect of intramolecular nucleophiles and fluorophores on thermal equilibrium of intramolecular spirocyclization.

    Rhodamine derivatives bearing intramolecular nucleophiles show pH-dependent thermal equilibrium of intramolecular spirocyclization between coloured open forms and colourless closed forms. The closed forms tend to be stabilized with more nucleophilic intramolecular nucleophiles and more electrophilic fluorophores, resulting in decreased pKcycl values. a, Effect of intramolecular nucleophiles on intramolecular spirocyclization. b, Effect of fluorophores as electrophiles on intramolecular spirocyclization. Measured in 0.2 M sodium phosphate buffer at various values of pH in the presence of 0.1% ​DMSO. The normalized absorbance of the ​xanthene moiety of open forms at the wavelengths in parentheses is plotted against pH. pKcycl values are given in parentheses below the chemical structures.

  3. Switching properties of the antibody-bound dyes in the absence of thiols and an oxygen-scavenging system.
    Figure 3: Switching properties of the antibody-bound dyes in the absence of thiols and an ​oxygen-scavenging system.

    In the absence of thiols and an ​oxygen scavenger, Alexa647 (a widely used conventional fluorophore for dSTORM) and 2MeSiR (an SiR derivative without any intramolecular nucleophile) were bleached irreversibly within a few tens of seconds without blinking or after blinking only a few times. In contrast, HMSiR showed reversible and spontaneous fluorescence blinking over a hundred times due to the thermal equilibrium of its intramolecular spirocyclization, even under much lower-power laser irradiation than that used for general dSTORM. ac, Single-molecule fluorescence time traces of antibody-bound Alexa647 (a), 2MeSiR (b) and HMSiR (c). d, Distribution of fluorescence-on time of antibody-bound HMSiR. e, Excitation intensity dependency of photon number per switching event (mean ± s.e., N = 100–5,000). Measured in 10 mM phosphate buffer at pH 7.4. Excitation at 647 nm (100 W cm−2 for ac and 40 W cm−2 for d).

  4. SLM with HMSiR in vitro and in fixed cells.
    Figure 4: SLM with HMSiR in vitro and in fixed cells.

    Both SLM with TIRFM and SLM deep inside cells with a spinning-disk confocal microscope were successfully achieved using our spontaneously blinking fluorophore, which does not require either additive or prior intense laser irradiation. a,b, In vitro SLM of ​RecA filaments polymerized on ΦX174 RFII DNA with TIRFM: averaged image (a) and super-resolution image (b) obtained with HMSiR. FWHM = 51.4 ± 3.2 nm (mean ± s.e., N = 10). Excitation at 647 nm (500 W cm−2). Measured in 50 mM Tris-HCl buffer at pH 7.4 containing 100 mM ​NaCl, 7 mM ​MgCl2 and 1.5 mM ​ATP-γ-S. Scale bars, 500 nm. c, Transverse profiles of localizations corresponding to the region boxed in yellow in b. df, SLM of NPC on the apical side of the nucleus with a spinning-disk confocal microscope. HeLa cells expressing ​POM121-GFP or ​Nup107-GFP fusion protein were fixed and immunostained using HMSiR-labelled antibody. Measured in PBS (pH 7.4). With a spinning-disk confocal microscope, each divided illumination beam is too weak to drive conventional fluorescent molecules into the dark state, whereas HMSiR shows spontaneous blinking regardless of the laser intensity. In d, the NPC is shown on the basal side of the nucleus. In e, representative ring structures of the NPC are shown on the apical side of the nucleus, located 4 µm above the coverslip. f, Box-and-whisker plot of the distance from the centre to the nuclear pore proteins: 48.5 ± 1.4 nm for ​POM121 and 38.6 ± 1.2 nm for ​Nup107. (mean ± s.e., N = 100). Scale bars: 500 nm (d) and 50 nm (e).

  5. Live-cell time-lapse SLM with HMSiR.
    Figure 5: Live-cell time-lapse SLM with HMSiR.

    β-Tubulin–Halo fusion proteins expressed in Vero cells were labelled with HMSiR–Halo. HMSiR can blink in the intracellular environment without ​oxygen depletion or thiol addition and at much lower-power laser intensity than that used for dSTORM or GSDIM, which enables us to perform time-lapse imaging of microtubule dynamics with minimal photobleaching and photodamage for 1 h at 10 min intervals. a,b, Averaged image (a) and super-resolution image (b) obtained with HMSiR. FWHM = 563.1 ± 13.1 nm and 79.8 ± 3.3 nm (mean ± s.e., N = 10) for averaged image and super-resolution image, respectively. c, Sequential acquisition of super-resolution images of microtubules at 0 min (white), 31 min (yellow) and 63 min (green). Excitation at 647 nm (40 W cm−2). Each super-resolution image was reconstructed from 1,000 images (30 ms/frame) corresponding to the acquisition time of 30 s. Measured in Leibovitz's L-15 medium after re-plating. d, Transverse profiles of fluorescence intensity and localizations corresponding to regions boxed in yellow in a and b. Scale bars, 2 µm.

References

  1. Hell, S. W. Far-field optical nanoscopy. Science 316, 11531158 (2007).
  2. Huang, B., Babcock, H. & Zhuang, X. Breaking the diffraction barrier: super-resolution imaging of cells. Cell 143, 10471058 (2010).
  3. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 16421645 (2006).
  4. Hess, S. T., Girirajan, T. P. K. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 42584272 (2006).
  5. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793796 (2006).
  6. Folling, J. et al. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nature Methods 5, 943945 (2008).
  7. Heilemann, M. et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew. Chem. Int. Ed. 47, 61726176 (2008).
  8. Vogelsang, J. et al. Make them blink: probes for super-resolution microscopy. ChemPhysChem 11, 24752490 (2010).
  9. van de Linde, S., Heilemann, M. & Sauer, M. Live-cell super-resolution imaging with synthetic fluorophores. Annu. Rev. Phys. Chem. 63, 519540 (2012).
  10. Steinhauer, C., Forthmann, C., Vogelsang, J. & Tinnefeld, P. Superresolution microscopy on the basis of engineered dark states. J. Am. Chem. Soc. 130, 1684016841 (2008).
  11. Schwering, M. et al. Far-field nanoscopy with reversible chemical reactions. Angew. Chem. Int. Ed. 50, 29402945 (2011).
  12. van de Linde, S. et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nature Protoc. 6, 9911009 (2011).
  13. Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M. & Zhuang, X. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nature Methods 8, 10271036 (2011).
  14. Wombacher, R. et al. Live-cell super-resolution imaging with trimethoprim conjugates. Nature Methods 7, 717719 (2010).
  15. Klein, T. et al. Live-cell dSTROM with SNAP-tag fusion proteins. Nature Methods 8, 79 (2011).
  16. Lukinavičius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nature Chem. 5, 132139 (2013).
  17. Kim, H. N., Lee, M. H., Kim, H. J., Kim, J. S. & Yoon, J. A new trend in rhodamine-based chemosensors: application of spirolactam ring-opening to sensing ions. Chem. Soc. Rev. 37, 14651472 (2008).
  18. Kenmoku, S., Urano, Y., Kojima, H. & Nagano, T. Development of a highly specific rhodamine-based fluorescence probe for hypochlorous acid and its application to real-time imaging of phagocytosis. J. Am. Chem. Soc. 129, 73137318 (2007).
  19. Koide, Y., Urano, Y., Hanaoka, K., Terai, T. & Nagano, T. Development of an Si-rhodamine-based far-red to near-infrared fluorescence probe selective for hypochlorous acid and its applications for biological imaging. J. Am. Chem. Soc. 133, 56805682 (2011).
  20. Kamiya, M. et al. β-Galactosidase fluorescence probe with improved cellular accumulation based on a spirocyclized rhodol scaffold. J. Am. Chem. Soc. 133, 1296012963 (2011).
  21. Sakabe, M. et al. Rational design of highly sensitive fluorescence probes for protease and glycosidase based on precisely controlled spirocyclization. J. Am. Chem. Soc. 135, 409414 (2013).
  22. Fölling, J. et al. Photochromic rhodamines provide nanoscopy with optical sectioning. Angew. Chem.Int. Ed. 46, 62666270 (2007).
  23. Belov, V. N., Bossi, M. L., Fölling, J., Boyarskiy, V. P. & Hell, S. W. Rhodamine spiroamides for multicolor single-molecule switching fluorescent nanoscopy. Chem. Eur. J. 15, 1076210776 (2009).
  24. Fu, M., Xiao, Y., Qian, X., Zhao, D. & Xu, Y. A design concept of long-wavelength fluorescent analogs of rhodamine dyes: replacement of oxygen with silicon atom. Chem. Commun. 17801782 (2008).
  25. Koide, Y., Urano, Y., Hanaoka, K., Terai, T. & Nagano, T. Evolution of group 14 rhodamines as platforms for near-infrared fluorescence probes utilizing photoinduced electron transfer. ACS Chem. Biol. 6, 600608 (2011).
  26. Corrie, J. E. T. et al. Ring-chain interconversion of sulforhodamine-amine conjugates involves an unusually labile C–N bond and allows measurement of sulfonamide ionization kinetics. J. Phys. Org. Chem. 21, 286298 (2008).
  27. Katayama, H., Yamamoto, A., Mizushima, N., Yoshimori, T. & Miyawaki, A. GFP-like proteins stably accumulate in lysosomes. Cell. Struct. Funct. 33, 112 (2008).
  28. Jones, S. A., Shim, S.-H., He, J. & Zhuang, X. Fast, three-dimensional super-resolution imaging of live cells. Nature Methods 8, 499505 (2011).
  29. Quan, T., Zeng, S. & Huang, Z-L. Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal–oxide semiconductor, and charge-coupled devices for superresolution imaging. J. Biomed. Opt. 15, 066005 (2010).
  30. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nature Methods 5, 159161 (2008).
  31. Löschberger, A. et al. Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. J. Cell. Sci. 125, 570575 (2012).
  32. Szymborska, A. et al. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341, 655658 (2013).
  33. Funakoshi, T. et al. Two distinct human POM121 genes: requirement for the formation of nuclear pore complexes. FEBS Lett. 581, 49104916 (2007).
  34. Maeshima, K. et al. Nuclear pore formation but not nuclear growth is governed by cyclin-dependent kinases (Cdks) during interphase. Nature Struct. Mol. Biol. 17, 10651071 (2010).
  35. Hoelz, A., Debler, E. W. & Blobel, G. The structure of the nuclear pore complex. Annu. Rev. Biochem. 80, 613643 (2011).
  36. Hinner, M. J. & Johnsson, K. How to obtain labeled proteins and what to do with them. Curr. Opin. Biotechnol. 21, 766776 (2010).
  37. Los, G. V. et al. Halotag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373382 (2008).

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Author information

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

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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The authors declare no competing financial interests.

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