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.

  • Perspective
  • Published:

Two-photon absorption properties of fluorescent proteins

Nature Methods volume 8, pages 393–399 (2011)Cite this article

Subjects

Abstract

Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-photon applications.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: One- and two-photon absorption spectra of fluorescent proteins with different chromophores.
Figure 2: Structure of the two-photon absorption spectrum of a fluorescent protein.
Figure 3: One-photon absorption of the 'Fruit' proteins does not predict which is the brightest two-photon probe.
Figure 4: Two-photon absorption is highly sensitive to the electric field in the protein environment.
Figure 5: Matching of two-photon excitation spectra of red fluorescent proteins with the optimum tissue transparency and with the wavelengths of some short-pulse laser systems.

Similar content being viewed by others

References

  1. Xu, C., Zipfel, W., Shear, J.B., Williams, R.M. & Webb, W.W. Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996).

    Article  CAS  Google Scholar 

  2. Zipfel, W.R., Williams, R.M. & Webb, W.W. Nonlinear magic: multiphoton microscopy in the biosciences. Nat. Biotechnol. 21, 1369–1377 (2003).

    Article  CAS  Google Scholar 

  3. Tsai, P.S. et al. All-optical histology using ultrashort laser pulses. Neuron 39, 27–41 (2003).

    Article  CAS  Google Scholar 

  4. Herz, J. et al. Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator. Biophys. J. 98, 715–723 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Chudakov, D.M., Matz, M.V., Lukyanov, S. & Lukyanov, K.A. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. and references therein 90, 1103–1163 (2010).

    Article  CAS  Google Scholar 

  7. Adam, V. et al. Data storage based on photochromic and photoconvertible fluorescent proteins. J. Biotechnol. 149, 289–298 (2010).

    Article  CAS  Google Scholar 

  8. Moneron, G. & Hell, S.W. Two-photon excitation STED microscopy. Opt. Express 17, 14567–14573 (2009).

    Article  CAS  Google Scholar 

  9. Vaziri, A., Tang, J., Shroff, H. & Shank, C.V. Multilayer three-dimensional super resolution imaging of thick biological samples. Proc. Natl. Acad. Sci. USA 105, 20221–20226 (2008).

    Article  CAS  Google Scholar 

  10. Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

    Article  CAS  Google Scholar 

  11. Drobizhev, M., Tillo, S., Makarov, N.S., Hughes, T.E. & Rebane, A. Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins. J. Phys. Chem. B 113, 855–859 (2009).

    Article  CAS  Google Scholar 

  12. Tillo, S.E., Hughes, T.E., Makarov, N.S., Rebane, A. & Drobizhev, M. A new approach to dual-color two-photon microscopy with fluorescent proteins. BMC Biotechnol. 10, 6 (2010).

    Article  Google Scholar 

  13. Makarov, N.S., Drobizhev, M. & Rebane, A. Two-photon absorption standards in the 550–1600 nm excitation wavelength range. Opt. Express 16, 4029–4047 (2008).

    Article  CAS  Google Scholar 

  14. Drobizhev, M., Makarov, N.S., Hughes, T. & Rebane, A. Resonance enhancement of two-photon absorption in fluorescent proteins. J. Phys. Chem. B 111, 14051–14054 (2007).

    Article  CAS  Google Scholar 

  15. Blab, G.A., Lommerse, P.H.M., Cognet, L., Harms, G.S. & Schmidt, T. Two-photon excitation action cross-sections of the autofluorescent proteins. Chem. Phys. Lett. 350, 71–77 (2001).

    Article  CAS  Google Scholar 

  16. Nifosi, R. & Luo, Y. Predictions of novel two-photon absorption bands in fluorescent proteins. J. Phys. Chem. B 111, 14043–14050 (2007).

    Article  CAS  Google Scholar 

  17. Nguyen, Q.T. et al. An in vivo biosensor for neurotransmitter release and in situ receptor activity. Nat. Neurosci. 13, 127–132 (2010).

    Article  CAS  Google Scholar 

  18. Kawano, H., Kogure, T., Abe, Y., Mizuno, H. & Miyawaki, A. Two-photon dual-color imaging using fluorescent proteins. Nat. Methods 5, 373–374 (2008).

    Article  CAS  Google Scholar 

  19. Shu, X., Shaner, N.C., Yarbrough, C.A., Tsien, R.Y. & Remington, S.J. Novel chromophores and buried charges control color in mFruits. Biochemistry 45, 9639–9647 (2006).

    Article  CAS  Google Scholar 

  20. Drobizhev, M., Tillo, S., Makarov, N.S., Hughes, T.E. & Rebane, A. Color hues in red fluorescent proteins are due to internal quadratic Stark effect. J. Phys. Chem. B 113, 12860–12864 (2009).

    Article  CAS  Google Scholar 

  21. Wachter, R.M., Elsliger, M.-A., Kallio, K., Hanson, G.T. & Remington, S.J. Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein. Structure 6, 1267–1277 (1998).

    Article  CAS  Google Scholar 

  22. Heikal, A.A., Hess, S.T. & Webb, W.W. Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity. Chem. Phys. 274, 37–45 (2001).

    Article  CAS  Google Scholar 

  23. Heikal, A.A., Hess, S.T., Baird, G.S., Tsien, R.Y. & Webb, W.W. Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc. Natl. Acad. Sci. USA 97, 11996–12001 (2000).

    Article  CAS  Google Scholar 

  24. Hashimoto, H. et al. Measurement of two-photon excitation spectra of fluorescent proteins with nonlinear Fourier-transform spectroscopy. Appl. Opt. 49, 3323–3329 (2010).

    Article  CAS  Google Scholar 

  25. Piatkevich, K.D. et al. Monomeric red fluorescent proteins with a large Stokes shift. Proc. Natl. Acad. Sci. USA 107, 5369–5374 (2010).

    Article  CAS  Google Scholar 

  26. Rizzo, M.A., Springer, G., Segawa, K., Zipfel, W.R. & Piston, D.W. Optimization of pairings and detection conditions for measurements of FRET between cyan and yellow fluorescent proteins. Microsc. Microanal. 12, 238–254 (2006).

    Article  CAS  Google Scholar 

  27. Hosoi, H., Yamaguchi, S., Mizuno, H., Miyawaki, A. & Tahara, T. Hidden electronic excited state of enhanced green fluorescent protein. J. Phys. Chem. B 112, 2761–2763 (2008).

    Article  CAS  Google Scholar 

  28. Oulianov, D.A., Tomov, I.V., Dvornikov, A.S. & Rentzepis, P.M. Observations on the measurement of two-photon absorption cross-section. Opt. Commun. 191, 235–243 (2001).

    Article  CAS  Google Scholar 

  29. Albota, M.A., Xu, C. & Webb, W.W. Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm. Appl. Opt. 37, 7352–7356 (1998).

    Article  CAS  Google Scholar 

  30. Kredel, S. et al. Optimized and far-red-emitting variants of fluorescent protein eqFP611. Chem. Biol. 15, 224–233 (2008).

    Article  CAS  Google Scholar 

  31. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  Google Scholar 

  32. Marchant, J.S., Stutzmann, G.E., Leissring, M.A., LaFerla, F.M. & Parker, I. Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling. Nat. Biotechnol. 19, 645–649 (2001).

    Article  CAS  Google Scholar 

  33. Chen, T.-S., Zeng, S.-Q., Luo, Q.-M., Zhang, Z.-H. & Zhou, W. High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy. Biochem. Biophys. Res. Commun. 291, 1272–1275 (2002).

    Article  CAS  Google Scholar 

  34. Ji, N., Magee, J.C. & Betzig, E. High-speed, low-photodamage nonlinear imaging using passive pulse splitters. Nat. Methods 5, 197–202 (2008).

    Article  CAS  Google Scholar 

  35. Donnert, G., Eggeling, C. & Hell, S.W. Major signal increase in fluorescence microscopy through dark-state relaxation. Nat. Methods 4, 81–86 (2007).

    Article  CAS  Google Scholar 

  36. Kawano, H. et al. Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses. Biochem. Biophys. Res. Commun. 311, 592–596 (2003).

    Article  CAS  Google Scholar 

  37. Field, J.J. et al. Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation. Opt. Express 18, 13661–13672 (2010).

    Article  CAS  Google Scholar 

  38. Patterson, G.H., Knobel, S.M., Sharif, W.D., Kain, S.R. & Piston, D.W. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790 (1997).

    Article  CAS  Google Scholar 

  39. Ritz, J.-P. et al. Optical properties of native and coagulated porcine liver tissue between 400 and 2400 nm. Lasers Surg. Med. 29, 205–212 (2001).

    Article  CAS  Google Scholar 

  40. Ai, H.W., Shaner, N.C., Cheng, Z., Tsien, R.Y. & Campbell, R.E. Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry 46, 5904–5910 (2007).

    Article  CAS  Google Scholar 

  41. Rizzo, M.A., Springer, G.H., Granada, B. & Piston, D.W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).

    Article  CAS  Google Scholar 

  42. Ai, H.W., Henderson, J.N., Remington, S.J. & Campbell, R.E. Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem. J. 400, 531–540 (2006).

    Article  CAS  Google Scholar 

  43. Ai, H.W., Hazelwood, K.L., Davidson, M.W. & Campbell, R.E. Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors. Nat. Methods 5, 401–403 (2008).

    Article  CAS  Google Scholar 

  44. Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. Reducing the environmental sensitivity of yellow fluorescent protein. J. Biol. Chem. 31, 29188–29194 (2001).

    Article  Google Scholar 

  45. Tsutsui, H., Karasawa, S., Okamura, Y. & Miyawaki, A. Improving membrane voltage measurements using FRET with new fluorescent proteins. Nat. Methods 5, 683–685 (2008).

    Article  CAS  Google Scholar 

  46. Merzlyak, E.M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4, 555–557 (2007).

    Article  CAS  Google Scholar 

  47. Yanushevich, Y.G. et al. A strategy for the generation of non-aggregating mutants of Anthozoa fluorescent proteins. FEBS Lett. 511, 11–14 (2002).

    Article  CAS  Google Scholar 

  48. Wang, L., Jackson, W.C., Steinbach, P.A. & Tsien, R.Y. Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc. Natl. Acad. Sci. USA 101, 16745–16749 (2004).

    Article  CAS  Google Scholar 

  49. Shcherbo, D. et al. Far-red fluorescent tags for protein imaging in living tissues. Biochem. J. 418, 567–574 (2009).

    Article  CAS  Google Scholar 

  50. Lin, M.Z. et al. Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem. Biol. 16, 1169–1179 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the US National Institute of General Medical Sciences grant R01 GM086198. We thank B.H. Davis for technical help, and R. Campbell (University of Alberta, Edmonton, Canada), D.M. Chudakov (Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow), M. Lin (Stanford University) and R.Y. Tsien (University of California San Diego) for providing us cDNA of different fluorescent proteins.

Author information

Author notes

  1. Nikolay S Makarov & Shane E Tillo

    Present address: Present addresses: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA (N.S.M.) and Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA (S.E.T.).,

Authors and Affiliations

  1. Department of Physics, Montana State University, Bozeman, Montana, USA

    Mikhail Drobizhev, Nikolay S Makarov & Aleksander Rebane

  2. Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana, USA

    Shane E Tillo & Thomas E Hughes

  3. National Institute of Chemical Physics and Biophysics, Akadeemia tee, Tallinn, Estonia

    Aleksander Rebane

Authors
  1. Mikhail Drobizhev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nikolay S Makarov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shane E Tillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas E Hughes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Aleksander Rebane
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mikhail Drobizhev.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 1–2, Supplementary Methods (PDF 1755 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Drobizhev, M., Makarov, N., Tillo, S. et al. Two-photon absorption properties of fluorescent proteins. Nat Methods 8, 393–399 (2011). https://doi.org/10.1038/nmeth.1596

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1596

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research