Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A dendritic single-molecule fluorescent probe that is monovalent, photostable and minimally blinking

Subjects

Abstract

Single-molecule fluorescence techniques have emerged as a powerful approach to understanding complex biological systems. However, a challenge researchers still face is the limited photostability of nearly all organic fluorophores, including the cyanine and Alexa dyes. We report a new, monovalent probe that emits in the far-red region of the visible spectrum with properties desirable for single-molecule optical imaging. This probe is based on a ring-fused boron-dipyrromethene (BODIPY) core that is conjugated to a polyglycerol dendrimer (PGD). The dendrimer makes the hydrophobic fluorophore water-soluble. This probe exhibits excellent brightness, with an emission maximum of 705 nm. We have observed strikingly long and stable emission from individual PGD–BODIPY probes, even in the absence of anti-fading agents such as Trolox, a combined oxidizing–reducing agent often used in single-molecule studies for improving the photostability of common imaging probes. These interesting properties greatly simplify use of the fluorophore.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Schematic representation of the synthesis of water-soluble and monofunctional polyglycerol-dendronized fluorophores.
Figure 2: Synthesis of biotinylated PGD–BODIPY 12.
Figure 3: MALDI-TOF mass spectra of 712.
Figure 4: Photophysical properties of 12.
Figure 5: Photostability comparison of 12 with Cy5.

References

  1. 1

    Giepmans, B. N. G., Adams, S. R., Ellisman, M. H. & Tsien, R. Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).

    CAS  PubMed  Google Scholar 

  2. 2

    Lavis, L. D. & Raines, R. T. Bright ideas for chemical biology. ACS Chem. Biol. 3, 142–155 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Kobayashi, H., Ogawa, M., Alford, R., Choyke, P. L. & Urano, Y. New strategies for fluorescent probe design in medical diagnostic imaging. Chem. Rev. 110, 2620–2640 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Cordes, T., Vogelsang, J. & Tinnefeld, P. On the mechanism of trolox as antiblinking and antibleaching reagent. J. Am. Chem. Soc. 131, 5018–5019 (2009).

    CAS  PubMed  Google Scholar 

  5. 5

    Berezin, M. Y. & Achilefu, S. Fluorescence lifetime measurements and biological imaging. Chem. Rev. 110, 2641–2684 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J. & Waggoner, A. S. Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconj. Chem. 4, 105–111 (1993).

    CAS  Google Scholar 

  7. 7

    Funatsu, T., Harada, Y., Tokunaga, M., Saito, K. & Yanagida, T. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374, 555–559 (1995).

    CAS  PubMed  Google Scholar 

  8. 8

    Heilemann, M., Margeat, E., Kasper, R., Sauer, M. & Tinnefeld, P. Carbocyanine dyes as efficient reversible single-molecule optical switch. J. Am. Chem. Soc. 127, 3801–3806 (2005).

    CAS  PubMed  Google Scholar 

  9. 9

    Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nature Methods 5, 507–516 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Weiss, S. Fluorescence spectroscopy of single biomolecules. Science 283, 1676–1683 (1999).

    CAS  PubMed  Google Scholar 

  11. 11

    Lu, H. P., Xun, L. & Xie, X. S. Single-molecule enzymatic dynamics. Science 282, 1877–1882 (1998).

    CAS  PubMed  Google Scholar 

  12. 12

    Moerner, W. E. & Fromm, D. P. Methods of single-molecule fluorescence spectroscopy and microscopy. Rev. Sci. Instrum. 74, 3597–3619 (2003).

    CAS  Google Scholar 

  13. 13

    Ha, T. Single-molecule fluorescence resonance energy transfer. Methods 25, 78–86 (2001).

    CAS  PubMed  Google Scholar 

  14. 14

    Ha, T. & Tinnefeld, P. Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. Annu. Rev. Phys. Chem. 63, 595–617 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Giloh, H. & Sedat, J. W. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217, 1252–1255 (1982).

    CAS  PubMed  Google Scholar 

  16. 16

    Lichtman, J. W. & Conchello, J-A. Fluorescence microscopy. Nature Methods 2, 910–919 (2005).

    CAS  PubMed  Google Scholar 

  17. 17

    Rasnik, I., McKinney, S. A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nature Methods 3, 891–893 (2006).

    CAS  PubMed  Google Scholar 

  18. 18

    Vogelsang, J. et al. A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew. Chem. Int. Ed. 47, 5465–5469 (2008).

    CAS  Google Scholar 

  19. 19

    Dave, R., Terry, D. S., Munro, J. B. & Blanchard, S. C. Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging. Biophys. J. 96, 2371–2381 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Altman, R. B. et al. Cyanine fluorophore derivatives with enhanced photostability. Nature Methods 9, 68–71 (2012).

    CAS  Google Scholar 

  21. 21

    Loudet, A. & Burgess, K. BODIPY dyes and their derivatives: syntheses and spectroscopic properties. Chem. Rev. 107, 4891–4932 (2007).

    CAS  PubMed  Google Scholar 

  22. 22

    Ulrich, G., Ziessel, R. & Harriman, A. The chemistry of fluorescent bodipy dyes: versatility unsurpassed. Angew. Chem. Int. Ed. 47, 1184–1201 (2008).

    CAS  Google Scholar 

  23. 23

    Benniston, A. C. & Copley, G. Lighting the way ahead with boron dipyrromethene (Bodipy) dyes. Phys. Chem. Chem. Phys. 11, 4124–4131 (2009).

    CAS  PubMed  Google Scholar 

  24. 24

    Le Gall, A. et al. Improved photon yield from a green dye with a reducing and oxidizing system. ChemPhysChem 12, 1657–1660 (2011).

    CAS  PubMed  Google Scholar 

  25. 25

    Johnson, I. & Spence, M. T. Z. The Handbook: A Guide to Fluorescent Probes and Labeling Technologies 11th edn (Molecular Probes, 2010).

    Google Scholar 

  26. 26

    Hinkeldey, B., Schmitt, A., & Jung, G. Comparative photostability studies of BODIPY and fluorescein dyes by using fluorescence correlation spectroscopy. ChemPhysChem 9, 2019–2027 (2008).

  27. 27

    Chen, J., Burghart, A., Derecskei-Kovacs, A. & Burgess, K. 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes modified for extended conjugation and restricted bond rotations. J. Org. Chem. 65, 2900–2906 (2000).

    CAS  PubMed  Google Scholar 

  28. 28

    Zhao, W. & Carreira, E. M. Conformationally restricted aza-bodipy: a highly fluorescent, stable, near-infrared-absorbing dye. Angew. Chem. Int. Ed. 44, 1677–1679 (2005).

    CAS  Google Scholar 

  29. 29

    Ulrich, G., Goeb, S., De Nicola, A., Retailleau, P. & Ziessel, R. Synthesis of bisisoindolomethene dyes bearing anisole or ethylthiophene residues for red and near-IR fluorescence. Synlett 2007, 1517–1520 (2007).

    Google Scholar 

  30. 30

    Atilgan, S., Ozdemir, T. & Akkaya, E. U. A sensitive and selective ratiometric near IR fluorescent probe for zinc ions based on the distyryl–bodipy fluorophore. Org. Lett. 10, 4065–4067 (2008).

    CAS  PubMed  Google Scholar 

  31. 31

    Zeng, L. et al. Anthracene-fused BODIPYs as near-infrared dyes with high photostability. Org. Lett. 13, 6026–6029 (2011).

    CAS  PubMed  Google Scholar 

  32. 32

    Umezawa, K., Nakamura, Y., Makino, H., Citterio, D. & Suzuki, K. Bright, color-tunable fluorescent dyes in the visible–near-infrared region. J. Am. Chem. Soc. 130, 1550–1551 (2008).

    CAS  PubMed  Google Scholar 

  33. 33

    Umezawa, K., Matsui, A., Nakamura, Y., Citterio, D. & Suzuki, K. Bright, color-tunable fluorescent dyes in the Vis/NIR region: establishment of new ‘tailor-made’ multicolor fluorophores based on borondipyrromethene. Chem. Eur. J. 15, 1096–1106 (2008).

    Google Scholar 

  34. 34

    Atilgan, S., Ekmekci, Z., Dogan, A. L., Guc, D. & Akkaya, E. U. Water soluble distyryl-boradiazaindacenes as efficient photosensitizers for photodynamic therapy. Chem. Commun. 4398–4400 (2006).

  35. 35

    Li, L., Han, J., Nguyen, B. & Burgess, K. Syntheses and spectral properties of functionalized, water-soluble BODIPY derivatives. J. Org. Chem. 73, 1963–1970 (2008).

    CAS  PubMed  Google Scholar 

  36. 36

    Komatsu, T. et al. Development of 2,6-carboxy-substituted boron dipyrromethene (BODIPY) as a novel scaffold of ratiometric fluorescent probes for live cell imaging. Chem. Commun. 7015–7017 (2009).

  37. 37

    Niu, S. L. et al. Water-soluble BODIPY derivatives. Org. Lett. 11, 2049–2052 (2009).

    CAS  PubMed  Google Scholar 

  38. 38

    Bura, T. & Ziessel, R. Water-soluble phosphonate-substituted BODIPY derivatives with tunable emission channels. Org. Lett. 13, 3072–3075 (2011).

    CAS  PubMed  Google Scholar 

  39. 39

    Yang, S. K. et al. Monovalent, clickable, uncharged, water-soluble perylenediimide-cored dendrimers for target-specific fluorescent biolabeling. J. Am. Chem. Soc. 133, 9964–9967 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Heek, T. et al. Highly fluorescent water-soluble polyglycerol-dendronized perylene bisimide dyes. Chem. Commun. 46, 1884–1886 (2010).

    CAS  Google Scholar 

  41. 41

    Schlick, K. H., Morgan, J. R., Weiel, J. J., Kelsey, M. S. & Cloninger, M. J. Clusters of ligands on dendrimer surfaces. Bioorg. Med. Chem. Lett. 21, 5078–5083 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Elmer, S. L., Man, S. & Zimmerman, S. C. Synthesis of polyglycerol, porphyrin-cored dendrimers using click chemistry. Eur. J. Org. Chem. 2008, 3845–3851 (2008).

    Google Scholar 

  43. 43

    Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40, 2004–2021 (2001).

    CAS  Google Scholar 

  44. 44

    Zill, A. T. et al. Clickable polyglycerol hyperbranched polymers and their application to gold nanoparticles and acid-labile nanocarriers. Chem. Commun. 47, 1279–1281 (2011).

    CAS  Google Scholar 

  45. 45

    Shi, X., Lim, J. & Ha, T. Acidification of the oxygen scavenging system in single-molecule fluorescence studies: in situ sensing with a ratiometric dual-emission probe. Anal. Chem. 82, 6132–6138 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Shi, X. et al. Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging. Nature Methods 9, 499–503 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors acknowledge the National Institutes of Health (GM087448 to S.C.Z., GM065367 and AI083025 to T.H.) and the National Science Foundation (PHY-0822613) for financial support.

Author information

Affiliations

Authors

Contributions

S.K.Y. and S.C.Z. conceived the study. S.K.Y. designed and synthesized the polyglycerol-dendronized fluorophores. X.S. performed the single-molecule experiments. X.S. and S.P. performed the microtubule labelling experiments. S.K.Y., X.S., T.H. and S.C.Z. wrote the manuscript.

Corresponding authors

Correspondence to Taekjip Ha or Steven C. Zimmerman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 2226 kb)

Supplementary Movie

Supplementary Movie (MOV 4396 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yang, S., Shi, X., Park, S. et al. A dendritic single-molecule fluorescent probe that is monovalent, photostable and minimally blinking. Nature Chem 5, 692–697 (2013). https://doi.org/10.1038/nchem.1706

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing