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.

  • Article
  • Published:

Highly specific label-free molecular imaging with spectrally tailored excitation-stimulated Raman scattering (STE-SRS) microscopy

Nature Photonics volume 5, pages 103–109 (2011)Cite this article

Abstract

Label-free microscopy that has chemical contrast and high acquisition speeds up to video rates has recently been made possible using stimulated Raman scattering (SRS) microscopy. SRS imaging offers high sensitivity, but the spectral specificity of the original narrowband implementation is limited, making it difficult to distinguish chemical species with overlapping Raman bands. Here, we present a highly specific imaging method that allows mapping of a particular chemical species in the presence of interfering species, based on tailored multiplex excitation of its vibrational spectrum. This is implemented by spectral modulation of a broadband pump beam at a high frequency (>1 MHz), allowing detection of the SRS signal of the narrowband Stokes beam with high sensitivity. Using the scheme, we demonstrate quantification of cholesterol in the presence of lipids, and real-time three-dimensional spectral imaging of protein, stearic acid and oleic acid in live Caenorhabditis elegans.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Principle of SRS.
Figure 2: Excitation schemes of SRS.
Figure 3: Spectral modulation scheme.
Figure 4: STE-SRS microscopy setup.
Figure 5: Characterization of STE-SRS.
Figure 6: Imaging of lipid storage in C. elegans.

Similar content being viewed by others

References

  1. Zumbusch, A., Holtom, G. R. & Xie, X. S. Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering. Phys. Rev. Lett. 82, 4142–4145 (1999).

    Article  ADS  Google Scholar 

  2. Cheng, J. X. & Xie, X. S. Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications. J. Phys. Chem. B 108, 827–840 (2004).

    Article  Google Scholar 

  3. Evans, C. L. & Xie, X. S. Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine. Annu. Rev. Anal. Chem. 1, 883–909 (2008).

    Article  Google Scholar 

  4. Ploetz, E., Laimgruber, S., Berner, S., Zinth, W. & Gilch, P. Femtosecond stimulated Raman microscopy. Appl. Phys. B 87, 389–393 (2007).

    Article  ADS  Google Scholar 

  5. Freudiger, C. W. et al. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 322, 1857–1861 (2008).

    Article  ADS  Google Scholar 

  6. Ozeki, Y., Dake, F., Kajiyama, S., Fukui, K. & Itoh, K. Analysis and experimental assessment of the sensitivity of stimulated Raman scattering microscopy. Opt. Express 17, 3651–3658 (2009).

    Article  ADS  Google Scholar 

  7. Nandakumar, P., Kovalev, A. & Volkmer, A. Vibrational imaging based on stimulated Raman scattering microscopy. New J. Phys. 11, 033026 (2009).

    Article  ADS  Google Scholar 

  8. Evans, C. L. et al. Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy. Proc. Natl Acad. Sci. USA 102, 16807–16812 (2005).

    Article  ADS  Google Scholar 

  9. Saar, B. G. et al. Video-rate molecular imaging in vivo with stimulated Raman scattering. Science 330, 1368–1370 (2010).

    Article  ADS  Google Scholar 

  10. Levenson, M. D. & Kano, S. S. Introduction to Nonlinear Laser Spectroscopy (Academic Press, 1988).

  11. Kukura, P., McCamant, D. W. & Mathies, R. A. Femtosecond stimulated Raman spectroscopy. Annu. Rev. Phys. Chem. 58, 461–488 (2007).

    Article  ADS  Google Scholar 

  12. Wurpel, G. W. H., Schins, J. M. & Muller, M. Chemical specificity in three-dimensional imaging with multiplex coherent anti-Stokes Raman scattering microscopy. Opt. Lett. 27, 1093–1095 (2002).

    Article  ADS  Google Scholar 

  13. Cheng, J. X., Volkmer, A., Book, L. D. & Xie, X. S. Multiplex coherent anti-Stokes Raman scattering microspectroscopy and study of lipid vesicles. J. Phys. Chem. B 106, 8493–8498 (2002).

    Article  Google Scholar 

  14. Rinia, H. A., Burger, K. N. J., Bonn, M. & Muller, M. Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy. Biophys. J. 95, 4908–4914 (2008).

    Article  ADS  Google Scholar 

  15. Weiner, A. M. Femtosecond pulse shaping using spatial light modulators. Rev. Sci. Instrum. 71, 1929–1960 (2000).

    Article  ADS  Google Scholar 

  16. Wise, B. M. et al. PLS_Toolbox 4.0 - Manual (Eigenvector Research, 2006).

  17. Mullaney, B. C. & Ashrafi, K. C. elegans fat storage and metabolic regulation. Biochim. Biophys. Acta 1791, 474–478 (2009).

    Article  Google Scholar 

  18. Hellerer, T. et al. Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy. Proc. Natl Acad. Sci. USA 104, 14658–14663 (2007).

    Article  ADS  Google Scholar 

  19. Slipchenko, M. N., Le, T. T., Chen, H. T. & Cheng, J. X. High-speed vibrational imaging and spectral analysis of lipid bodies by compound Raman microscopy. J. Phys. Chem. B 113, 7681–7686 (2009).

    Article  Google Scholar 

  20. Mark, H. & Workman, J. Chemometrics in Spectroscopy (Academic Press, 2007).

  21. Perera, P. N. et al. Observation of water dangling OH bonds around dissolved nonpolar groups. Proc. Natl Acad. Sci. USA 106, 12230–12234 (2009).

    Article  ADS  Google Scholar 

  22. Enejder, A. M. K. et al. Raman spectroscopy for noninvasive glucose measurements. J. Biomed. Opt. 10, 031114 (2005).

    Article  ADS  Google Scholar 

  23. Schulmerich, M. V. et al. Noninvasive Raman tomographic imaging of canine bone tissue. J. Biomed. Opt. 13, 020506 (2008).

    Article  ADS  Google Scholar 

  24. Pully, V. V., Lenferink, A. & Otto, C. Raman-fluorescence hybrid microspectroscopy of cell nuclei. Vib. Spectrosc. 53, 12–18 (2010).

    Article  Google Scholar 

  25. Nelson, M. P., Aust, J. F., Dobrowolski, J. A., Verly, P. G. & Myrick, M. L. Multivariate optical computation for predictive spectroscopy. Anal. Chem. 70, 73–82 (1998).

    Article  Google Scholar 

  26. Uzunbajakava, N., de Peinder, P., 't Hooft, G. W. & van Gogh, A. T. M. Low-cost spectroscopy with a variable multivariate optical element. Anal. Chem. 78, 7302–7308 (2006).

    Article  Google Scholar 

  27. Krafft, C. et al. A comparative Raman and CARS imaging study of colon tissue. J. Biophoton. 2, 303–312 (2009).

    Article  Google Scholar 

  28. Rinia, H. A., Bonn, M. & Muller, M. Quantitative multiplex CARS spectroscopy in congested spectral regions. J. Phys. Chem. B 110, 4472–4479 (2006).

    Article  Google Scholar 

  29. Dudovich, N., Oron, D. & Silberberg, Y. Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy. Nature 418, 512–514 (2002).

    Article  ADS  Google Scholar 

  30. van Rhijn, A. C. W., Postma, S., Korterik, J. P., Herek, J. L. & Offerhaus, H. L. Chemically selective imaging by spectral phase shaping for broadband CARS around 3000 cm−1. J. Opt. Soc. Am. B 26, 559–563 (2009).

    Article  ADS  Google Scholar 

  31. Marks, D. L., Geddes, J. B. & Boppart, S. A. Molecular identification by generating coherence between molecular normal modes using stimulated Raman scattering. Opt. Lett. 34, 1756–1758 (2009).

    Article  ADS  Google Scholar 

  32. Oron, D., Dudovich, N. & Silberberg, Y. All-optical processing in coherent nonlinear spectroscopy. Phys. Rev. A 70, 23415 (2004).

    Article  ADS  Google Scholar 

  33. Roy, S., Wrzesinski, P., Pestov, D., Dantus, M. & Gord, J. R. Single-beam coherent anti-Stokes Raman scattering (CARS) spectroscopy of gas-phase CO2 via phase and polarization shaping of a broadband continuum. J. Raman Spectrosc. 41, 1194–1199 (2010).

    Article  ADS  Google Scholar 

  34. Evans, C. L., Potma, E. O. & Xie, X. S. N. Coherent anti-Stokes Raman scattering spectral interferometry: determination of the real and imaginary components of nonlinear susceptibility χ(3) for vibrational microscopy. Opt. Lett. 29, 2923–2925 (2004).

    Article  ADS  Google Scholar 

  35. Jurna, M., Korterik, J. P., Otto, C., Herek, J. L. & Offerhaus, H. L. Background free CARS imaging by phase sensitive heterodyne CARS. Opt. Express 16, 15863–15869 (2008).

    Article  ADS  Google Scholar 

  36. Cheng, J. X. & Xie, X. S. Green's function formulation for third-harmonic generation microscopy. J. Opt. Soc. Am. B 19, 1604–1610 (2002).

    Article  ADS  MathSciNet  Google Scholar 

  37. Fu, D., Ye, T., Matthews, T. E., Yurtsever, G. & Warren, W. S. Two-color, two-photon, and excited-state absorption microscopy. J. Biomed. Opt. 12, 054004 (2007).

    Article  ADS  Google Scholar 

  38. Fu, D. et al. Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin. J. Biomed. Opt. 13, 054036 (2008).

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  40. Jones, D. J. et al. Synchronization of two passively mode-locked, picosecond lasers within 20 fs for coherent anti-Stokes Raman scattering microscopy. Rev. Sci. Instrum. 73, 2843–2848 (2002).

    Article  ADS  Google Scholar 

  41. Hopt, A. & Neher, E. Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys. J. 80, 2029–2036 (2001).

    Article  ADS  Google Scholar 

  42. Nan, X. L., Potma, E. O. & Xie, X. S. Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-stokes Raman scattering microscopy. Biophys. J. 91, 728–735 (2006).

    Article  ADS  Google Scholar 

  43. Fu, Y., Wang, H. F., Shi, R. Y. & Cheng, J. X. Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy. Opt. Express 14, 3942–3951 (2006).

    Article  ADS  Google Scholar 

Download references

Acknowledgements

The authors thank Linjiao Luo and Aravinthan Samuel for providing the C. elegans sample for initial testing, B. Saar and Sijia Lu for helpful discussions and comments on the manuscript, and Xu Zhang for assisting in the final concentration measurements. C.W.F. acknowledges Boehringer Ingelheim Fonds for a PhD Fellowship. This work was supported by the National Institutes of Health (NIH) Director's Pioneer Award and NIH TR01 grant 1R01EB010244-01.

Author information

Authors and Affiliations

  1. Department of Physics, Harvard University, Cambridge, 02138, Massachusetts, USA

    Christian W. Freudiger

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 02138, Massachusetts, USA

    Christian W. Freudiger, Wei Min, Gary R. Holtom & X. Sunney Xie

  3. Biophotonic Solutions Inc, East Lansing, 48823, Michigan, USA

    Bingwei Xu

  4. Department of Chemistry and Department of Physics, Michigan State University, East Lansing, 48824, Michigan, USA

    Marcos Dantus

Authors
  1. Christian W. Freudiger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei Min
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gary R. Holtom
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bingwei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marcos Dantus
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. X. Sunney Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.W.F., W.M. and X.S.X. conceived the idea and drafted the manuscript. C.W.F. and G.R.H. built the instrument, B.X. and M.D. designed and built the pulse-shaper, and C.W.F. conducted the experiments.

Corresponding author

Correspondence to X. Sunney Xie.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Freudiger, C., Min, W., Holtom, G. et al. Highly specific label-free molecular imaging with spectrally tailored excitation-stimulated Raman scattering (STE-SRS) microscopy. Nature Photon 5, 103–109 (2011). https://doi.org/10.1038/nphoton.2010.294

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nphoton.2010.294

This article is cited by

Search

Advanced 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