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.

Major signal increase in fluorescence microscopy through dark-state relaxation

Abstract

We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 μs, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5–25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 μs. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes. Please visit methagora to view and post comments on this article

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: Photobleaching of GFP and Atto532 decreases with inter-pulse break Δt = 1/f for one- and two-photon excitation.
Figure 2: Total fluorescence signal generated by one-photon excitation (G1p) for GFP and Atto532 for a given number of excitation pulses (1.4 × 106).
Figure 3: Energy diagram of a typical organic fluorophore, indicating the major molecular pathways for excitation (Exc), fluorescence (Fl), intersystem crossing (ISC), relaxation (dashed lines) and photobleaching (bleach).
Figure 4: Total fluorescence signal generated by two-photon excitation (G2p) for GFP and Atto532 for a given number of excitation pulses (1.4 × 106).
Figure 5: The pulse duration does not affect the total fluorescence signal G2p of two-photon excitation.
Figure 6: Two-photon fluorescence images of Escherichia coli cells expressing the fluorescent protein Venus are brighter when recorded in the D-Rex mode.

References

  1. 1

    Tsien, R.Y. Imagining imaging's future. Nat. Rev. Mol. Cell Biol. 4 (Suppl.), SS16–SS21 (2003).

    Google Scholar 

  2. 2

    Pawley, J.B. (ed.) Handbook of biological confocal microscopy 3rd edn. (Springer, New York, 2006).

    Book  Google Scholar 

  3. 3

    Donnert, G. et al. Macromolecular-scale resolution in biological fluorescence microscopy. Proc. Natl. Acad. Sci. USA 103, 11440–11445 (2006).

    CAS  Article  Google Scholar 

  4. 4

    Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    CAS  Article  Google Scholar 

  5. 5

    Tsien, R.Y., Ernst, L. & Waggoner, A. Fluorophores for confocal microscopy: photophysics and photochemistry. in Handbook of biological confocal microscopy, 3rd edn. (ed., Pawley, J.B.) 338–352 (Springer, New York, 2006).

    Chapter  Google Scholar 

  6. 6

    Webb, W.W., Wells, K.S., Sandison, D.R. & Strickler, J. Criteria for quantitative dynamical confocal fluorescence imaging. in Optical Microscopy for Biology (eds Herman, B. & Jacobson, K.) 73–108 (Wiley, New York, 1990).

    Google Scholar 

  7. 7

    Conchello, J.-A. & Lichtman, J.W. Optical sectioning microscopy. Nat. Methods 2, 920–931 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Hell, S.W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated emission depletion microscopy. Opt. Lett. 19, 780–782 (1994).

    CAS  Article  Google Scholar 

  9. 9

    Brakenhoff, G.J. et al. Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system. J. Microsc. 181, 253–259 (1996).

    CAS  Article  Google Scholar 

  10. 10

    Beaurepaire, E., Oheim, M. & Mertz, J. Ultra-deep two-photon fluorescence excitation in turbid media. Opt. Commun. 188, 25–29 (2001).

    CAS  Article  Google Scholar 

  11. 11

    Theer, P., Mazahir, H.T. & Denk, W. Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Masters, B.R. et al. Mitigating thermal mechanical damage potential during two-photon dermal imaging. J. Biomed. Opt. 9, 1265–1270 (2004).

    Article  Google Scholar 

  13. 13

    Gustafsson, M.G.L. Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. USA 102, 13081–13086 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Eggeling, C., Widengren, J., Rigler, R. & Seidel, C.A.M. Photostability of fluorescent dyes for single-molecule spectroscopy: mechanisms and experimental methods for estimating photobleaching in aqueous solution. in Applied fluorescence in chemistry, biology and medicine. (eds., Rettig, W., Strehmel, B., Schrader, M. & Seifert, H.) 193–240 (Springer, Berlin, 1999).

    Chapter  Google Scholar 

  15. 15

    Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    CAS  Article  Google Scholar 

  16. 16

    Sanchez, E.J., Novotny, L., Holtom, G.R. & Xie, X.S. Room-temperature fluorescence imaging and spectroscopy of single molecules by two-photon excitation. J. Phys. Chem. A 101, 7019–7023 (1997).

    CAS  Article  Google Scholar 

  17. 17

    Eggeling, C., Widengren, J., Rigler, R. & Seidel, C.A.M. Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis. Anal. Chem. 70, 2651–2659 (1998).

    CAS  Article  Google Scholar 

  18. 18

    Widengren, J., Mets, Ü. & Rigler, R. Fluorescence correlation spectroscopy of triplet states in solution: A theoretical and experimental study. J. Phys. Chem. 99, 13368–13379 (1995).

    CAS  Article  Google Scholar 

  19. 19

    Patterson, G.H. & Piston, D.W. Photobleaching in two-photon excitation microscopy. Biophys. J. 78, 2159–2162 (2000).

    CAS  Article  Google Scholar 

  20. 20

    Eggeling, C., Volkmer, A. & Seidel, C.A.M. Molecular photobleaching kinetics of rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy. ChemPhysChem 6, 791–804 (2005).

    CAS  Article  Google Scholar 

  21. 21

    Kasha, M. Paths of molecular excitation. Radiat. Res. 2 (Suppl.), 243–275 (1960).

    CAS  PubMed  Google Scholar 

  22. 22

    Koester, H.J., Baur, D., Uhl, R. & Hell, S.W. Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. Biophys. J. 77, 2226–2236 (1999).

    CAS  Article  Google Scholar 

  23. 23

    Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank A. Miyawaki (Laboratory for Cell Function and Dynamics, Riken) for supplying the fluorescent protein Venus. We also thank S. Jakobs, S. Verrier and D. Ouw for preparation of the samples, A. Giske, R. Kellner and K. Willig for help with the experimental setup, and V. Westphal and A. Egner for fruitful discussions. Finally, we thank A. Schönle for support with the IMSPECTOR software.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Stefan W Hell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Donnert, G., Eggeling, C. & Hell, S. Major signal increase in fluorescence microscopy through dark-state relaxation. Nat Methods 4, 81–86 (2007). https://doi.org/10.1038/nmeth986

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