Skip to main content

Thank you for visiting 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.

Room-temperature ultrafast nonlinear spectroscopy of a single molecule


Single-molecule spectroscopy aims to unveil often hidden but potentially very important contributions of single entities to a system’s ensemble response. Albeit contributing tremendously to our ever growing understanding of molecular processes, the fundamental question of temporal evolution, or change, has thus far been inaccessible, thus painting a static picture of a dynamic world. Here, we finally resolve this dilemma by performing ultrafast time-resolved transient spectroscopy on a single molecule. By tracing the femtosecond evolution of excited electronic state spectra of single molecules over hundreds of nanometres of bandwidth at room temperature, we reveal their nonlinear ultrafast response in an effective three-pulse scheme with fluorescence detection. A first excitation pulse is followed by a phase-locked de-excitation pulse pair, providing spectral encoding with 25 fs temporal resolution. This experimental realization of true single-molecule transient spectroscopy demonstrates that two-dimensional electronic spectroscopy of single molecules is experimentally within reach.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Concept of transient absorption spectroscopy and single-molecule implementation.
Fig. 2: Experimental implementation of spectral modulation spectroscopy and proof-of-concept experiments on single molecules.
Fig. 3: Single-molecule transient fluorescence spectroscopy and transient stimulated emission spectra.
Fig. 4: Ultrafast broadband single-molecule transient absorption spectroscopy.


  1. 1.

    Musser, A. J. et al. Evidence for conical intersection dynamics mediating ultrafast singlet exciton fission. Nat. Phys. 11, 352–357 (2015).

    Article  Google Scholar 

  2. 2.

    Schoenlein, R. W., Peteanu, L. A., Mathies, R. A. & Shank, C. V. The first step in vision: femtosecond isomerization of rhodopsin. Science 254, 412–415 (1991).

    ADS  Article  Google Scholar 

  3. 3.

    Rose, T. S., Rosker, M. J. & Zewail, A. H. Femtosecond realtime observation of wave packet oscillations (resonance) in dissociation reactions. J. Chem. Phys. 88, 6672–6673 (1988).

    ADS  Article  Google Scholar 

  4. 4.

    Dostál, J., Pšenčík, J. & Zigmantas, D. Mapping the energy flow through the entire photosynthetic apparatus in situ. Nat. Chem. 8, 705–710 (2015).

    Article  Google Scholar 

  5. 5.

    Shank, C. V. Measurement of ultrafast phenomena in the femtosecond time domain. Science 219, 1027–1031 (1983).

    ADS  Article  Google Scholar 

  6. 6.

    Kukura, P., Celebrano, M., Renn, A. & Sandoghdar, V. Single-molecule sensitivity in optical absorption at room temperature. J. Phys. Chem. Lett. 1, 3323–3327 (2010).

    Article  Google Scholar 

  7. 7.

    Chong, S., Min, W. & Xie, X. S. Ground-state depletion microscopy: detection sensitivity of single-molecule optical absorption at room temperature. J. Phys. Chem. Lett. 1, 3316–3322 (2010).

    Article  Google Scholar 

  8. 8.

    Gaiduk, A., Yorulmaz, M., Ruijgrok, P. V. & Orrit, M. Room-temperature detection of a single molecule’s absorption by photothermal contrast. Science 330, 353–356 (2010).

    ADS  Article  Google Scholar 

  9. 9.

    Min, W. et al. Imaging chromophores with undetectable fluorescence by stimulated emission microscopy. Nature 461, 1105–1109 (2009).

    ADS  Article  Google Scholar 

  10. 10.

    Moerner, W. E. & Kador, L. Optical detection and spectroscopy of single molecules in a solid. Phys. Rev. Lett. 62, 2535–2538 (1989).

    ADS  Article  Google Scholar 

  11. 11.

    Maser, A., Gmeiner, B., Utikal, T., Götzinger, S. & Sandoghdar, V. Few-photon coherent nonlinear optics with a single molecule. Nat. Photon. 10, 450–453 (2016).

    ADS  Article  Google Scholar 

  12. 12.

    Orrit, M. & Bernhard, J. Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal. Phys. Rev. Lett. 65, 2716–2719 (1990).

    ADS  Article  Google Scholar 

  13. 13.

    Nie, S., Chiu, D. T., Zare, R. N. & Zaret, R. N. Probing individual molecules with confocal fluorescence microscopy. Science 266, 1018–1021 (1994).

    ADS  Article  Google Scholar 

  14. 14.

    Moerner, W. E. & Orrit, M. Illuminating single molecules in condensed matter. Science 283, 1670–1676 (1999).

    ADS  Article  Google Scholar 

  15. 15.

    Van Dijk, E. et al. Single-molecule pump–probe detection resolves ultrafast pathways in individual and coupled quantum systems. Phys. Rev. Lett. 94, 078302 (2005).

    ADS  Article  Google Scholar 

  16. 16.

    Hernando, J. et al. Effect of disorder on ultrafast exciton dynamics probed by single molecule spectroscopy. Phys. Rev. Lett. 97, 216403 (2006).

    ADS  Article  Google Scholar 

  17. 17.

    Tian, P. F., Keusters, D., Suzaki, Y. & Warren, W. S. Femtosecond phase-coherent two-dimensional spectroscopy. Science 300, 1553–1555 (2003).

    ADS  Article  Google Scholar 

  18. 18.

    Brinks, D. et al. Ultrafast dynamics of single molecules. Chem. Soc. Rev. 43, 2476–2491 (2014).

    Article  Google Scholar 

  19. 19.

    Toninelli, C. et al. Near-infrared single-photons from aligned molecules in ultrathin crystalline films at room temperature. Opt. Express 18, 6577–6582 (2010).

    ADS  Article  Google Scholar 

  20. 20.

    Sanders, J. N. et al. Compressed sensing for multidimensional electronic spectroscopy experiments. J. Phys. Chem. Lett. 3, 2697–2702 (2012).

    Article  Google Scholar 

  21. 21.

    Scherer, N. F. et al. Fluorescence-detected wave packet interferometry: time resolved molecular spectroscopy with sequences of femtosecond phase-locked pulses. J. Chem. Phys. 95, 1487–1511 (1991).

    ADS  Article  Google Scholar 

  22. 22.

    Albrecht, A. W., Hybl, J. D., Gallagher Faeder, S. M. & Jonas, D. M. Experimental distinction between phase shifts and time delays: implications for femtosecond spectroscopy and coherent control of chemical reactions. J. Chem. Phys. 111, 10934–10956 (1999).

    ADS  Article  Google Scholar 

  23. 23.

    Brinks, D. et al. Visualizing and controlling vibrational wave packets of single molecules. Nature 465, 905–908 (2010).

    ADS  Article  Google Scholar 

  24. 24.

    Hildner, R., Brinks, D. & van Hulst, N. F. Femtosecond coherence and quantum control of single molecules at room temperature. Nat. Phys. 7, 172–177 (2011).

    Article  Google Scholar 

  25. 25.

    Piatkowski, L., Gellings, E. & van Hulst, N. F. Broadband single-molecule excitation spectroscopy. Nat. Commun. 7, 10411 (2016).

    ADS  Article  Google Scholar 

  26. 26.

    Weigel, A., Sebesta, A. & Kukura, P. Shaped and feedback-controlled excitation of single molecules in the weak-field limit. J. Phys. Chem. Lett. 6, 4023–4037 (2015).

    Article  Google Scholar 

  27. 27.

    Kennis, J. T. M. et al. Ultrafast protein dynamics of bacteriorhodopsin probed by photon echo and transient absorption spectroscopy. J. Phys. Chem. B 106, 6067–6080 (2002).

    Article  Google Scholar 

  28. 28.

    Monmayrant, A., Weber, S. & Chatel, B. A newcomer’s guide to ultrashort pulse shaping and characterization. J. Phys. B 43, 103001 (2010).

    ADS  Article  Google Scholar 

  29. 29.

    Ernst, R. R., Bodenhausen, G. & Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions (Oxford Univ., New York, 1990).

    Google Scholar 

  30. 30.

    Mukamel, S. & Biggs, J. D. Communication: comment on the effective temporal and spectral resolution of impulsive stimulated Raman signals. J. Chem. Phys. 134, 161101 (2011).

    ADS  Article  Google Scholar 

  31. 31.

    Polli, D., Brida, D., Mukamel, S., Lanzani, G. & Cerullo, G. Effective temporal resolution in pump–probe spectroscopy with strongly chirped pulses. Phys. Rev. A 82, 053809 (2010).

    ADS  Article  Google Scholar 

  32. 32.

    Nicolet, A. A. L., Hofmann, C., Kol’chenko, M. A., Kozankiewicz, B. & Orrit, M. Single dibenzoterrylene molecules in an anthracene crystal: spectroscopy and photophysics. Chem. Phys. Chem. 8, 1215–1220 (2007).

    Article  Google Scholar 

  33. 33.

    Dobryakov, A. L., Kovalenko, S. A. & Ernsting, N. P. Coherent and sequential contributions to femtosecond transient absorption spectra of a rhodamine dye in solution. J. Chem. Phys. 123, 044502 (2005).

    ADS  Article  Google Scholar 

  34. 34.

    Cheng, J.-X., Volkmer, A. & Xie, X. S. Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy. J. Opt. Soc. Am. B 19, 1363–1375 (2002).

    ADS  Article  Google Scholar 

  35. 35.

    Weigel, A., Sebesta, A. & Kukura, P. Dark field microspectroscopy with single molecule fluorescence sensitivity. ACS Photon. 1, 848–856 (2014).

    Article  Google Scholar 

  36. 36.

    Jonas, D. M. Two-dimensional femtosecond spectroscopy. Annu. Rev. Phys. Chem. 54, 425–463 (2003).

    ADS  Article  Google Scholar 

  37. 37.

    Shim, S.-H. & Zanni, M. T. How to turn your pump–probe instrument into a multidimensional spectrometer: 2D IR and vis spectroscopies via pulse shaping. Phys. Chem. Chem. Phys. 11, 748–761 (2009).

    Article  Google Scholar 

  38. 38.

    Tekavec, P. F., Lott, G. A. & Marcus, A. H. Fluorescence-detected two-dimensional electronic coherence spectroscopy by acousto-optic phase modulation. J. Chem. Phys. 127, 214307 (2007).

    ADS  Article  Google Scholar 

  39. 39.

    Wende, T., Liebel, M., Schnedermann, C., Pethick, R. J. & Kukura, P. Population controlled impulsive vibrational spectroscopy: background- and baseline-free Raman spectroscopy of excited electronic states. J. Phys. Chem. A 118, 9976–9984 (2014).

    Article  Google Scholar 

  40. 40.

    Liebel, M., Schnedermann, C. & Kukura, P. Vibrationally coherent crossing and coupling of electronic states during internal conversion in β-carotene. Phys. Rev. Lett. 112, 198302 (2014).

    ADS  Article  Google Scholar 

  41. 41.

    De, A. K., Monahan, D., Dawlaty, J. M. & Fleming, G. R. Two-dimensional fluorescence-detected coherent spectroscopy with absolute phasing by confocal imaging of a dynamic grating and 27-step phase-cycling. J. Chem. Phys. 140, 194201 (2014).

    ADS  Article  Google Scholar 

  42. 42.

    Kane, D. J. & Trebino, R. Charaterization of arbitrary femtosecond pulses using frequency-resolved optical gating. IEEE J. Quantum Electron. 29, 571–579 (1993).

    ADS  Article  Google Scholar 

  43. 43.

    Lozovoy, V. V., Pastirk, I. & Dantus, M. Multiphoton intrapulse interference. IV. Ultrashort laser pulse spectral phase characterization and compensation. Opt. Lett. 29, 775–777 (2004).

    ADS  Article  Google Scholar 

Download references


This research was funded by the European Commission (European Research Council (ERC) Advanced Grant 670949-LightNet), the Ministerio de Economía, Industria y Competitividad (MINECO) Severo Ochoa Programme for Centres of Excellence in R&D (SEV-2015-0522, FIS2015-69258-P, FIS2015-72409-EXP), the Catalan Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR; 2014SGR01540), Fundació Privada Cellex and Generalitat de Catalunya through the CERCA programme. M.L. acknowledges financial support from the Marie-Curie International Fellowship, and co-funding of regional, national and international programmes (COFUND). C.T. thanks the COST Action Nanoscale Quantum Optics (MP1403) and acknowledges financial support from MIUR programme Q-Sec and Ente Cassa di Risparmio di Firenze (GRANCASSA). We thank A. Weigel and C. Schnedermann for helpful discussions while preparing the manuscript.

Author information




M.L. proposed the project. M.L. experimentally realized the project and analysed all experimental data. C.T. supplied the DBT samples. N.F.v.H. and M.L. wrote the manuscript. All authors discussed the experimental results and the manuscript.

Corresponding authors

Correspondence to Matz Liebel or Niek F. van Hulst.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplementary Information

Supplementary Sections 1–6

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liebel, M., Toninelli, C. & van Hulst, N.F. Room-temperature ultrafast nonlinear spectroscopy of a single molecule. Nature Photon 12, 45–49 (2018).

Download citation

Further reading


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