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.

High-speed coherent Raman fingerprint imaging of biological tissues

Abstract

An imaging platform based on broadband coherent anti-Stokes Raman scattering has been developed that provides an advantageous combination of speed, sensitivity and spectral breadth. The system utilizes a configuration of laser sources that probes the entire biologically relevant Raman window (500–3,500 cm–1) with high resolution (<10 cm–1). It strongly and efficiently stimulates Raman transitions within the typically weak ‘fingerprint’ region using intrapulse three-colour excitation, and utilizes the non-resonant background to heterodyne-amplify weak Raman signals. We demonstrate high-speed chemical imaging in two- and three-dimensional views of healthy murine liver and pancreas tissues as well as interfaces between xenograft brain tumours and the surrounding healthy brain matter.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Coherent Raman imaging with BCARS microspectroscopy.
Figure 2: CRI of murine liver tissue.
Figure 3: Three-dimensional CRI of murine pancreatic ducts.
Figure 4: Histopathology using broadband CRI.

References

  1. Huang, Z. et al. Near-infrared Raman spectroscopy for optical diagnosis of lung cancer. Int. J. Cancer 107, 1047–1052 (2003).

    Article  Google Scholar 

  2. Haka, A. S. et al. Diagnosing breast cancer by using Raman spectroscopy. Proc. Natl Acad. Sci. USA 102, 12371–12376 (2005).

    Article  ADS  Google Scholar 

  3. Gniadecka, M. et al. Melanoma diagnosis by Raman spectroscopy and neural networks: structure alterations in proteins and lipids in intact cancer tissue. J. Invest. Dermatol. 122, 443–449 (2004).

    Article  Google Scholar 

  4. Meyer, T. et al. Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis. J. Biomed. Opt. 16, 021113 (2011).

    Article  ADS  Google Scholar 

  5. Kirsch, M., Schackert, G., Salzer, R. & Krafft, C. Raman spectroscopic imaging for in vivo detection of cerebral brain metastases. Anal. Bioanal. Chem. 398, 1707–1713 (2010).

    Article  Google Scholar 

  6. Krafft, C., Sobottka, S. B., Schackert, G. & Salzer, R. Raman and infrared spectroscopic mapping of human primary intracranial tumors: a comparative study. J. Raman Spectrosc. 37, 367–375 (2006).

    Article  ADS  Google Scholar 

  7. Koljenović, S. et al. Discriminating vital tumor from necrotic tissue in human glioblastoma tissue samples by Raman spectroscopy. Lab. Invest. 82, 1265–1277 (2002).

    Article  Google Scholar 

  8. Nijssen, A. et al. Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy. J. Invest. Dermatol. 119, 64–69 (2002).

    Article  Google Scholar 

  9. 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 

  10. 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 

  11. 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 

  12. 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 

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

    Article  ADS  Google Scholar 

  14. Bachler, B. R., Fermann, M. E. & Ogilvie, J. P. Multiplex Raman induced Kerr effect microscopy. Opt. Express 20, 835–844 (2012).

    Article  ADS  Google Scholar 

  15. 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 

  16. Rock, W., Bonn, M. & Parekh, S. H. Near shot-noise limited hyperspectral stimulated Raman scattering spectroscopy using low energy lasers and a fast CMOS array. Opt. Express 21, 15113–15120 (2013).

    Article  ADS  Google Scholar 

  17. Fu, D. et al. Quantitative chemical imaging with multiplex stimulated Raman scattering microscopy. J. Am. Chem. Soc. 134, 3623–3626 (2012).

    Article  Google Scholar 

  18. Kong, L. et al. Multicolor stimulated Raman scattering microscopy with a rapidly tunable optical parametric oscillator. Opt. Lett. 38, 145–147 (2013).

    Article  ADS  Google Scholar 

  19. Müller, M. & Schins, J. M. Imaging the thermodynamic state of lipid membranes with multiplex CARS microscopy. J. Phys. Chem. B 106, 3715–3723 (2002).

    Article  Google Scholar 

  20. 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 

  21. Kano, H. & Hamaguchi, H. Femtosecond coherent anti-Stokes Raman scattering spectroscopy using supercontinuum generated from a photonic crystal fiber. Appl. Phys. Lett. 85, 4298–4300 (2004).

    Article  ADS  Google Scholar 

  22. Kee, T. W. & Cicerone, M. T. Simple approach to one-laser, broadband coherent anti-Stokes Raman scattering microscopy. Opt. Lett. 29, 2701–2703 (2004).

    Article  ADS  Google Scholar 

  23. Ploetz, E., Marx, B. & Gilch, P. Disturbing interference patterns in femtosecond stimulated Raman microscopy. J. Raman Spectrosc. 41, 609–613 (2010).

    Article  ADS  Google Scholar 

  24. Müller, M. & Zumbusch, A. Coherent anti-Stokes Raman scattering microscopy. ChemPhysChem 8, 2156–2170 (2007).

    Article  Google Scholar 

  25. Liu, Y., Lee, Y. J. & Cicerone, M. T. Broadband CARS spectral phase retrieval using a time-domain Kramers–Kronig transform. Opt. Lett. 34, 1363–1365 (2009).

    Article  ADS  Google Scholar 

  26. Vartiainen, E. M. Phase retrieval approach for coherent anti-Stokes Raman scattering spectrum analysis. J. Opt. Soc. Am. B 9, 1209–1214 (1992).

    Article  ADS  Google Scholar 

  27. Cicerone, M. T., Aamer, K. A., Lee, Y. J. & Vartiainen, E. Maximum entropy and time-domain Kramers–Kronig phase retrieval approaches are functionally equivalent for CARS microspectroscopy. J. Raman Spectrosc. 43, 637–643 (2012).

    Article  ADS  Google Scholar 

  28. Pohling, C., Buckup, T., Pagenstecher, A. & Motzkus, M. Chemoselective imaging of mouse brain tissue via multiplex CARS microscopy. Biomed. Opt. Express 2, 2110–2116 (2011).

    Article  Google Scholar 

  29. Parekh, S. H., Lee, Y. J., Aamer, K. A. & Cicerone, M. T. Label-free cellular imaging by broadband coherent anti-Stokes Raman scattering microscopy. Biophys. J. 99, 2695–2704 (2010).

    Article  ADS  Google Scholar 

  30. Lim, S.-H., Caster, A. G., Nicolet, O. & Leone, S. R. Chemical imaging by single pulse interferometric coherent anti-stokes Raman scattering microscopy. J. Phys. Chem. B 110, 5196–5204 (2006).

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

  32. Selm, R. et al. Ultrabroadband background-free coherent anti-Stokes Raman scattering microscopy based on a compact Er:fiber laser system. Opt. Lett. 35, 3282–3284 (2010).

    Article  ADS  Google Scholar 

  33. Ozeki, Y. et al. High-speed molecular spectral imaging of tissue with stimulated Raman scattering. Nature Photon. 6, 845–851 (2012).

    Article  ADS  Google Scholar 

  34. Deng, H., Bloomfield, V. A., Benevides, J. M. & Thomas, G. J. Jr Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence. Biopolymers 50, 656–666 (1999).

    Article  Google Scholar 

  35. Frushour, B. G. & Koenig, J. L. Raman scattering of collagen, gelatin, and elastin. Biopolymers 14, 379–391 (1975).

    Article  Google Scholar 

  36. Le, T. T., Langohr, I. M., Locker, M. J., Sturek, M. & Cheng, J.-X. Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy. J. Biomed. Opt. 12, 054007 (2007).

    Article  ADS  Google Scholar 

  37. Zipfel, W. R. et al. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl Acad. Sci. USA 100, 7075–7080 (2003).

    Article  ADS  Google Scholar 

  38. Dong, R., Yan, X., Pang, X. & Liu, S. Temperature-dependent Raman spectra of collagen and DNA. Spectrochim. Acta A 60, 557–561 (2004).

    Article  ADS  Google Scholar 

  39. Sun, Y. et al. Investigating mechanisms of collagen thermal denaturation by high resolution second-harmonic generation imaging. Biophys. J. 91, 2620–2625 (2006).

    Article  ADS  Google Scholar 

  40. Fu, Y., Wang, H., Shi, R. & Cheng, J.-X. Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues. Biophys. J. 92, 3251–3259 (2007).

    Article  ADS  Google Scholar 

  41. Theodossiou, T. et al. Thermally induced irreversible conformational changes in collagen probed by optical second harmonic generation and laser-induced fluorescence. Lasers Med. Sci. 17, 34–41 (2002).

    Article  Google Scholar 

  42. Krafft, C. et al. FTIR, Raman, and CARS microscopic imaging for histopathologic assessment of brain tumors. Proc. SPIE 7560, 756007 10.1117/12.851080(2010).

    Article  Google Scholar 

  43. Wood, B. R. & McNaughton, D. Raman excitation wavelength investigation of single red blood cells in vivo. J. Raman Spectrosc. 33, 517–523 (2002).

    Article  ADS  Google Scholar 

  44. Yates, A. J., Thompson, D. K., Boesel, C. P., Albrightson, C. & Hart, R. W. Lipid composition of human neural tumors. J. Lipid Res. 20, 428–436 (1979).

    Google Scholar 

  45. Krafft, C., Sobottka, S. B., Schackert, G. & Salzer, R. Analysis of human brain tissue, brain tumors and tumor cells by infrared spectroscopic mapping. Analyst 129, 921–925 (2004).

    Article  ADS  Google Scholar 

  46. Hartshorn, C. M. et al. Multicomponent chemical imaging of pharmaceutical solid dosage forms with broadband CARS microscopy. Anal. Chem. 85, 8102–8111 (2013).

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank Q. Wu, J. Hale and M. Sinyuk for preparing the pathological tissue specimens and S. Miller for preparation of neat chemical specimens. C.H.C., J.M.H. and C.M.H. also thank the National Research Council for support through the Research Associate Program (RAP). This work was supported in part by NIH/NIBIB grant 2P41EB001046-11.

Author information

Authors and Affiliations

Authors

Contributions

C.H.C. performed all experiments, analysed all data and drafted the manuscript. M.T.C. and C.H.C. designed all experiments and constructed the final manuscript. M.T.C. and Y.J.L. conceptualized the complementary two/three-colour excitation scheme. C.H.C. constructed the instrument, modified the laser system and developed the high-speed acquisition and processing software. C.H.C., Y.J.L., C.M.H. and M.T.C. developed the signal-processing methodology and protocols. M.T.C. developed the Kramers–Kronig transform and C.H.C. developed the parallelized, high-speed implementation. A.R.H.W., J.M.H., J.N.R. and J.D.L. provided materials and/or the tumour sections and provided histopathology insights and direction. J.M.H. assisted in performing the tumour section study, as well as contributing to the text of the manuscript. A.R.H.W., J.M.H. and C.H.C. collected the spontaneous Raman spectra of glycerol and C.H.C. performed the analysis. C.H.C. developed the presented mathematical framework of CARS generation and associated efficiencies with two/three-colour stimulation. M.T.C. supervised the study.

Corresponding author

Correspondence to Marcus T. Cicerone.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 1586 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Camp Jr, C., Lee, Y., Heddleston, J. et al. High-speed coherent Raman fingerprint imaging of biological tissues. Nature Photon 8, 627–634 (2014). https://doi.org/10.1038/nphoton.2014.145

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nphoton.2014.145

This article is cited by

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