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.

Higher-order coherent anti-Stokes Raman scattering microscopy realizes label-free super-resolution vibrational imaging

Abstract

Coherent Raman scattering (for example, coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering) microscopy has emerged as a powerful tool for label-free biomolecular imaging in biological and biomedical systems, but its spatial resolution is diffraction limited. Here, we report a higher-order coherent anti-Stokes Raman scattering (HO-CARS) microscopy to break the diffraction limit for label-free, super-resolution vibrational imaging. The resolution enhancement of HO-CARS microscopy has been analysed and demonstrated in biological samples (for example, live HeLa and buccal cells). The HO-CARS technique provides an inherent high resonant to non-resonant background ratio compared with conventional CARS microscopy. We affirm that under a tight focusing, the HO-CARS signal originating from the higher-order nonlinear process (χ(5), χ(7)) dominates over the cascaded lower-order nonlinear process (χ(3)), yielding much richer spectroscopic information. This study illustrates that HO-CARS microscopy can be an appealing tool for label-free, super-resolution imaging in biological and biomedical systems with high image contrast.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Principle of HO-CARS.
Fig. 2: Experimental observation of HO-CARS.
Fig. 3: Characteristics of HO-CARS processes.
Fig. 4: Super-resolution HO-CARS images of a DPBD crystal.
Fig. 5: Super-resolution HO-CARS images of live unstained HeLa and buccal cells at 2,845 cm−1 (CH2 stretching of lipids), and buccal cells at 2,935 cm−1 (CH3 stretching of proteins and lipids).

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.

References

  1. 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).

    ADS  Google Scholar 

  2. 2.

    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).

    Google Scholar 

  3. 3.

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

    ADS  Google Scholar 

  4. 4.

    Cheng, J. X. & Xie, X. S. Vibrational spectroscopic imaging of living systems: an emerging platform for biology and medicine. Science 350, aaa8870 (2015).

    Google Scholar 

  5. 5.

    Lu, F., Zheng, W., Sheppard, C. & Huang, Z. Interferometric polarization coherent anti-Stokes Raman scattering (IP-CARS) microscopy. Opt. Lett. 33, 602–604 (2008).

    ADS  Google Scholar 

  6. 6.

    Lin, J., Lu, F., Zheng, W. & Huang, Z. Annular aperture-detected coherent anti-Stokes Raman scattering microscopy for high contrast vibrational imaging. Appl. Phys. Lett. 97, 083701 (2010).

    ADS  Google Scholar 

  7. 7.

    Wang, Z., Zheng, W. & Huang, Z. Lock-in-detection-free line-scan stimulated Raman scattering microscopy for near video-rate Raman imaging. Opt. Lett. 41, 3960–3963 (2016).

    ADS  Google Scholar 

  8. 8.

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

    ADS  Google Scholar 

  9. 9.

    Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

    Google Scholar 

  10. 10.

    Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    ADS  Google Scholar 

  11. 11.

    Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

    Google Scholar 

  12. 12.

    Fujita, K., Kobayashi, M., Kawano, S., Yamanaka, M. & Kawata, S. High-resolution confocal microscopy by saturated excitation of fluorescence. Phys. Rev. Lett. 99, 228105 (2007).

    ADS  Google Scholar 

  13. 13.

    Prince, R. C. & Potma, E. O. Going visible: high-resolution coherent Raman imaging of cells and tissues. Light Sci. Appl. 8, 10 (2019).

    Google Scholar 

  14. 14.

    Beeker, W. P. et al. A route to sub-diffraction-limited CARS microscopy. Opt. Express 17, 22632–22638 (2009).

    ADS  Google Scholar 

  15. 15.

    Cleff, C. et al. Ground-state depletion for subdiffraction-limited spatial resolution in coherent anti-Stokes Raman scattering microscopy. Phys. Rev. A 86, 023825 (2012).

    ADS  Google Scholar 

  16. 16.

    Gong, L. & Wang, H. Breaking the diffraction limit by saturation in stimulated-Raman-scattering microscopy: a theoretical study. Phys. Rev. A 90, 013818 (2014).

    ADS  Google Scholar 

  17. 17.

    Gong, L. & Wang, H. Suppression of stimulated Raman scattering by an electromagnetically-induced-transparency-like scheme and its application for super-resolution microscopy. Phys. Rev. A 92, 023828 (2015).

    ADS  Google Scholar 

  18. 18.

    Yonemaru, Y. et al. Super-spatial- and -spectral-resolution in vibrational imaging via saturated coherent anti-Stokes Raman scattering. Phys. Rev. Appl. 4, 014010 (2015).

    ADS  Google Scholar 

  19. 19.

    Silva, W. R., Graefe, C. T. & Frontiera, R. R. Toward label-free super-resolution microscopy. ACS Photon. 3, 79–86 (2016).

    Google Scholar 

  20. 20.

    Choi, D. S. et al. Selective suppression of CARS signal with three-beam competing stimulated Raman scattering processes. Phys. Chem. Chem. Phys. 20, 17156–17170 (2018).

    Google Scholar 

  21. 21.

    Kim, D. et al. Selective suppression of stimulated Raman scattering with another competing stimulated Raman scattering. J. Phys. Chem. Lett. 8, 6118–6123 (2017).

    Google Scholar 

  22. 22.

    Gong, L., Zheng, W., Ma, Y. & Huang, Z. Saturated stimulated Raman scattering microscopy for far-field super-resolution bioimaging. Phys. Rev. Appl. 11, 034041 (2019).

    ADS  Google Scholar 

  23. 23.

    Gong, L. et al. Supercritical focusing coherent anti-Stokes Raman scattering microscopy for high-resolution vibrational imaging. Opt. Lett. 43, 5615–5618 (2018).

    ADS  Google Scholar 

  24. 24.

    Kim, H., Bryant, G. W. & Stranick, S. J. Superresolution four-wave mixing microscopy. Opt. Express 20, 6042–6051 (2012).

    ADS  Google Scholar 

  25. 25.

    Boyd, R. W. Nonlinear Optics 3rd edn (Academic Press, Elsevier, 2010).

    Google Scholar 

  26. 26.

    Kawashima, Y. & Katagiri, G. Fundamentals, overtones, and combinations in the Raman spectrum of graphite. Phys. Rev. B 52, 10053–10059 (1995).

    ADS  Google Scholar 

  27. 27.

    Compaan, A., Wiener-Avnear, E. & Chandra, S. Second-order coherent Raman scattering. Phys. Rev. A 17, 1083–1092 (1978).

    ADS  Google Scholar 

  28. 28.

    Mukamel, S. Multidimensional femtosecond correlation spectroscopies of electronic and vibrational excitations. Annu. Rev. Phys. Chem. 51, 691–729 (2000).

    ADS  Google Scholar 

  29. 29.

    Pelegati, V. B., Kyotoku, B. B. C., Padilha, L. A. & Cesar, C. L. Six-wave mixing coherent anti-Stokes Raman scattering microscopy. Biomed. Opt. Express 9, 2407–2417 (2018).

    Google Scholar 

  30. 30.

    Blank, D. A., Kaufman, L. J. & Fleming, G. R. Fifth-order two-dimensional Raman spectra of CS2 are dominated by third-order cascades. J. Chem. Phys. 111, 3105–3114 (1999).

    ADS  Google Scholar 

  31. 31.

    Kano, H. & Hamaguchi, H. Cascading third-order Raman process studied by six-wave mixing broadband multiplex coherent anti-Stokes Raman scattering spectroscopy. J. Chem. Phys. 118, 4556–4562 (2003).

    ADS  Google Scholar 

  32. 32.

    Guo, Z., Molesky, B. P., Cheshire, T. P. & Moran, A. M. Two-dimensional resonance Raman signatures of vibronic coherence transfer in chemical reactions. Top. Curr. Chem. 375, 87 (2017).

    Google Scholar 

  33. 33.

    Bae, K. et al. Epi-detected hyperspectral stimulated Raman scattering microscopy for label-free molecular subtyping of glioblastomas. Anal. Chem. 90, 10249–10255 (2018).

    Google Scholar 

  34. 34.

    Bae, K., Zheng, W., Ma, Y. & Huang, Z. Real-time monitoring of pharmacokinetics of antibiotics in biofilms with Raman-tagged hyperspectral stimulated Raman scattering microscopy. Theranostics 9, 1348–1357 (2019).

    Google Scholar 

  35. 35.

    Lin, K., Zheng, W., Lim, C. M. & Huang, Z. Real-time in vivo diagnosis of nasopharyngeal carcinoma using rapid fiber-optic Raman spectroscopy. Theranostics 7, 3517–3526 (2017).

    Google Scholar 

  36. 36.

    Wang, H., Fu, Y. & Cheng, J. X. Experimental observation and theoretical analysis of Raman resonance-enhanced photodamage in coherent anti-Stokes Raman scattering microscopy. J. Opt. Soc. Am. B 24, 544–552 (2007).

    ADS  Google Scholar 

  37. 37.

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

    ADS  Google Scholar 

  38. 38.

    König, K., Becker, T. W., Fischer, P., Riemann, I. & Halbhuber, K. J. Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes. Opt. Lett. 24, 113–115 (1999).

    ADS  Google Scholar 

  39. 39.

    Bi, Y. et al. Near-resonance enhanced label-free stimulated Raman scattering microscopy with spatial resolution near 130 nm. Light Sci. Appl. 7, 81 (2018).

    ADS  Google Scholar 

  40. 40.

    Lu, F. K. et al. Label-free DNA imaging in vivo with stimulated Raman scattering microscopy. Proc. Natl Acad. Sci. USA 112, 11624–11629 (2015).

    ADS  Google Scholar 

  41. 41.

    Gough, K. M. & Henry, B. R. Gas-phase overtone spectral investigation of inequivalent aryl and alkyl carbon-hydrogen (C–H) bonds in toluene and the xylenes. J. Phys. Chem. 88, 1298–1302 (1984).

    Google Scholar 

  42. 42.

    Amrein, A., Dübal, H. R. & Quack, M. Multiple anharmonic resonances in the vibrational overtone spectra of CHClF2. Mol. Phys. 56, 727–735 (1985).

    ADS  Google Scholar 

  43. 43.

    Angioni, E. et al. UV spectral properties of lipids as a tool for their identification. Eur. J. Lipid. Sci. Tech. 104, 59–64 (2002).

    Google Scholar 

  44. 44.

    Pace, C. N., Vajdos, F., Fee, L., Grimsley, G. & Gray, T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411–2423 (1995).

    Google Scholar 

Download references

Acknowledgements

This work was supported by the Academic Research Fund (AcRF)-Tier 1 and Tier 2 from Ministry of Education (MOE) (MOE2014-T2-1-010), and the National Medical Research Council (NMRC) (NMRC/TCR/016-NNI/2016), Singapore.

Author information

Affiliations

Authors

Contributions

L.G. and Z.H. conceived the concept and designed the experiments. L.G. performed the experiments. Y.M. performed chemical synthesis. L.G., W.Z. and Z.H. performed the data analysis and wrote the manuscript. Z.H. finalized the manuscript.

Corresponding author

Correspondence to Zhiwei Huang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gong, L., Zheng, W., Ma, Y. et al. Higher-order coherent anti-Stokes Raman scattering microscopy realizes label-free super-resolution vibrational imaging. Nat. Photonics 14, 115–122 (2020). https://doi.org/10.1038/s41566-019-0535-y

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