Powerful optical tools have revolutionized science and technology. The prevalent fluorescence detection offers superb sensitivity down to single molecules but lacks sufficient chemical information1,2,3. In contrast, Raman-based vibrational spectroscopy provides exquisite chemical specificity about molecular structure, dynamics and coupling, but is notoriously insensitive3,4,5. Here, we report a hybrid technique of stimulated Raman excited fluorescence (SREF) that integrates superb detection sensitivity and fine chemical specificity. Through stimulated Raman pumping to an intermediate vibrational eigenstate, followed by an upconversion to an electronic fluorescent state, SREF encodes vibrational resonance into the excitation spectrum of fluorescence emission. By harnessing the narrow vibrational linewidth, we demonstrated multiplexed SREF imaging in cells, breaking the ‘colour barrier’ of fluorescence. By leveraging the superb sensitivity of SREF, we achieved all-far-field single-molecule Raman spectroscopy and imaging without plasmonic enhancement, a long-sought-after goal in photonics. Thus, through merging Raman and fluorescence spectroscopy, SREF would be a valuable tool for chemistry and biology.
Subscribe to Journal
Get full journal access for 1 year
only $14.08 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.
Lakowicz, J. R. Principles of Fluorescence Spectroscopy 3rd edn (Springer US, New York, 2007).
Moerner, W. & Orrit, M. Illuminating single molecules in condensed matter. Science 283, 1670–1676 (1999).
Schatz, G. C. & Ratner, M. A. Quantum Mechanics in Chemistry (Courier Corp., New York, 1993).
Herzberg, G. Infrared and Raman Spectra of Polyatomic Molecules Vol. 2 (D. Van Nostrand, New York, 1945).
Nie, S. & Emory, S. R. Probing single molecules and single nanoparticles by surface-enhanced Raman scattering. Science 275, 1102–1106 (1997).
Seilmeier, A., Kaiser, W., Laubereau, A. & Fischer, S. A novel spectroscopy using ultrafast two-pulse excitation of large polyatomic molecules. Chem. Phys. Lett. 58, 225–229 (1978).
Hübner, H.-J., Wörner, M., Kaiser, W. & Seilmeier, A. Subpicosecond vibrational relaxation of skeletal modes in polyatomic molecules. Chem. Phys. Lett. 182, 315–320 (1991).
Mastron, J. N. & Tokmakoff, A. Two-photon-excited fluorescence-encoded infrared spectroscopy. J. Phys. Chem. A 120, 9178–9187 (2016).
Cheng, J. & Xie, X. Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications. J. Phys. Chem. B 108, 827–840 (2004).
Min, W., Freudiger, C. W., Lu, S. & Xie, X. S. Coherent nonlinear optical imaging: beyond fluorescence microscopy. Annu. Rev. Phys. Chem. 62, 507–530 (2011).
Lee, S., Nguyen, D. & Wright, J. Double resonance excitation of fluorescence by stimulated Raman scattering. Appl. Spectrosc. 37, 472–474 (1983).
Shim, S., Stuart, C. M. & Mathies, R. A. Resonance Raman cross‐sections and vibronic analysis of Rhodamine 6G from broadband stimulated Raman spectroscopy. ChemPhysChem 9, 697–699 (2008).
Wei, L. & Min, W. Electronic preresonance stimulated Raman scattering microscopy. J. Phys. Chem. Lett. 9, 4294–4301 (2018).
Etchegoin, P. G., Le, Ru,E. C. & Meyer, M. Evidence of natural isotopic distribution from single-molecule SERS. J. Am. Chem. Soc. 131, 2713–2716 (2009).
Min, W. et al. Imaging chromophores with undetectable fluorescence by stimulated emission microscopy. Nature 461, 1105–1109 (2009).
Sperber, P., Spangler, W., Meier, B. & Penzkofer, A. Experimental and theoretical investigation of tunable picosecond pulse generation in longitudinally pumped dye laser generators and amplifiers. Opt. Quant. Electron. 20, 395–431 (1988).
Wei, L. et al. Super-multiplex vibrational imaging. Nature 544, 465–470 (2017).
Dean, K. M. & Palmer, A. E. Advances in fluorescence labeling strategies for dynamic cellular imaging. Nat. Chem. Biol. 10, 512–523 (2014).
Kneipp, K. et al. Single molecule detection using surface-enhanced Raman scattering (SERS). Phys. Rev. Lett. 78, 1667–1670 (1997).
Sonntag, M. D. et al. Single-molecule tip-enhanced Raman spectroscopy. J. Phys. Chem. C 116, 478–483 (2011).
Yampolsky, S. et al. Seeing a single molecule vibrate through time-resolved coherent anti-Stokes Raman scattering. Nat. Photon. 8, 650–656 (2014).
Zhang, Y. et al. Coherent anti-Stokes Raman scattering with single-molecule sensitivity using a plasmonic Fano resonance. Nat. Commun. 5, 4424 (2014).
Mahmoudi, M. et al. Protein–nanoparticle interactions: opportunities and challenges. Chem. Rev. 111, 5610–5637 (2011).
Macklin, J., Trautman, J., Harris, T. & Brus, L. Imaging and time-resolved spectroscopy of single molecules at an interface. Science 272, 255–258 (1996).
Nie, S., Chiu, D. T. & Zare, R. N. Probing individual molecules with confocal fluorescence microscopy. Science 266, 1018–1021 (1994).
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).
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).
Gaiduk, A., Yorulmaz, M., Ruijgrok, P. & Orrit, M. Room-temperature detection of a single molecule’s absorption by photothermal contrast. Science 330, 353–356 (2010).
Zrimsek, A. et al. Single-molecule chemistry with surface- and tip-enhanced Raman spectroscopy. Chem. Rev. 117, 7583–7613 (2017).
Winterhalder, M., Zumbusch, A., Lippitz, M. & Orrit, M. Toward far-field vibrational spectroscopy of single molecules at room temperature. J. Phys. Chem. B 115, 5425–5430 (2011).
Brinks, D. et al. Ultrafast dynamics of single molecules. Chem. Soc. Rev. 43, 2476–2491 (2014).
Kukura, P., McCamant, D. W. & Mathies, R. A. Femtosecond stimulated Raman spectroscopy. Annu. Rev. Phys. Chem. 58, 461–488 (2007).
Siegman, A. E. Lasers. (University Science Books, Mill Valley, CA, 1986).
Wright, J. C. Double resonance excitation of fluorescence in the condensed phase—an alternative to Infrared, Raman, and fluorescence spectroscopy. Appl. Spectrosc. 34, 151–157 (1980).
Zipfel, W. R., Williams, R. M. & Webb, W. W. Nonlinear magic: multiphoton microscopy in the biosciences. Nat. Biotechnol. 21, 1369–1377 (2003).
We are grateful for discussions with L. E. Brus and X. Y. Zhu. This work was supported by grant R01GM128214 from the NIH, and by the Camille and Henry Dreyfus Foundation.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Xiong, H., Shi, L., Wei, L. et al. Stimulated Raman excited fluorescence spectroscopy and imaging. Nat. Photonics 13, 412–417 (2019). https://doi.org/10.1038/s41566-019-0396-4
Applied Sciences (2021)
Journal of the American Chemical Society (2021)
Background-free imaging of chemical bonds by a simple and robust frequency-modulated stimulated Raman scattering microscopy
Optics Express (2020)
Journal of Biophotonics (2020)