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High-speed molecular spectral imaging of tissue with stimulated Raman scattering

Nature Photonics 6, 845–851 (2012) | Download Citation

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Abstract

To date, medical imaging of tissues has largely relied on time-consuming staining processes, and there is a need for rapid, label-free imaging techniques. Stimulated Raman scattering microscopy offers a three-dimensional, real-time imaging capability with chemical specificity. However, it can be difficult to differentiate between several constituents in tissues because their spectral characteristics can overlap. Furthermore, imaging speeds in previous multispectral stimulated Raman scattering imaging techniques were limited. Here, we demonstrate label-free imaging of tissues by 30 frames/s stimulated Raman scattering microscopy with frame-by-frame wavelength tunability. To produce multicolour images showing different constituents, spectral images were processed by modified independent component analysis, which can extract small differences in spectral features. We present various imaging modalities such as two-dimensional spectral imaging of rat liver, two-colour three-dimensional imaging of a vessel in rat liver, spectral imaging of several sections of intestinal villi in mouse, and in vivo spectral imaging of mouse ear skin.

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References

  1. 1.

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

  2. 2.

    , , , & Analysis and experimental assessment of the sensitivity of stimulated Raman scattering microscopy. Opt. Express 17, 3651–3658 (2009).

  3. 3.

    , & Vibrational imaging based on stimulated Raman scattering microscopy. New J. Phys. 11, 033026 (2009).

  4. 4.

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

  5. 5.

    , , & High-speed vibrational imaging and spectral analysis of lipid bodies by compound Raman microscopy. J. Phys. Chem. B 113, 7681–7686 (2009).

  6. 6.

    et al. Vibrational imaging of tablets by epi-detected stimulated Raman scattering microscopy. Analyst 135, 2613–2619 (2010).

  7. 7.

    et al. Label-free, real-time monitoring of biomass processing with stimulated Raman scattering microscopy. Angew. Chem. Int. Ed. 122, 5608–5611 (2010).

  8. 8.

    , , , & RNAi screening for fat regulatory genes with SRS microscopy. Nature Methods 8, 135–138 (2011).

  9. 9.

    , , & Imaging drug delivery to skin with stimulated Raman scattering microscopy. Mol. Pharmaceut. 8, 969–975 (2011).

  10. 10.

    et al. Label-free imaging of biomolecules in food products using stimulated Raman microscopy. J. Biomed. Opt. 16, 021118 (2011).

  11. 11.

    et al. Label-free live-cell imaging of nucleic acids using stimulated Raman scattering microscopy. ChemPhysChem 13, 1054–1059 (2012).

  12. 12.

    et al. Highly specific label-free molecular imaging with spectrally tailored excitation-stimulated Raman scattering (STE-SRS) microscopy. Nature Photon. 5, 103–109 (2011).

  13. 13.

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

  14. 14.

    et al. Multicolor stimulated Raman scattering microscopy. Mol. Phys. 110, 1927–1932 (2012).

  15. 15.

    , , , & Femtosecond stimulated Raman microscopy. Appl. Phys. B 87, 389–393 (2007).

  16. 16.

    , & Stimulated Raman scattering microscopy by spectral focusing and fiber-generated soliton as Stokes pulse. Opt. Lett. 36, 2387–2389 (2011).

  17. 17.

    , & Stimulated Raman scattering using a single femtosecond oscillator with flexibility for imaging and spectral applications. Opt. Express 19, 18885–18892 (2011).

  18. 18.

    et al. Stimulated Raman hyperspectral imaging based on spectral filtering of broadband fiber laser pulses. Opt. Lett. 37, 431–433 (2011).

  19. 19.

    et al. in Photonics West 2012, paper 8226-58 (SPIE, 2012).

  20. 20.

    et al. Stimulated Raman scattering microscope with shot noise limited sensitivity using subharmonically synchronized laser pulses. Opt. Express 18, 13708–13719 (2010).

  21. 21.

    et al. Subharmonic synchronization of picosecond Yb fiber laser to picosecond Ti:sapphire laser for stimulated Raman scattering microscopy. Jpn J. Appl. Phys. 51, 022702 (2012).

  22. 22.

    , & Independent Component Analysis (Wiley, 2001).

  23. 23.

    et al. Independent component analysis of Raman spectra: application on paraffin-embedded skin biopsies. Biomed. Signal Process. Control 2, 40–50 (2007).

  24. 24.

    et al. Double-clad hollow core photonic crystal fiber for coherent Raman endoscope. Opt. Express 19, 12562–12568 (2011).

  25. 25.

    , , , & Coherent Raman scanning fiber endoscopy. Opt. Lett. 36, 2396–2398 (2011).

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Acknowledgements

The authors thank H. Watanabe for his kind support with the experiment on mouse skin imaging. They also thank N. Smith for his careful editing of this manuscript. This research was supported by Japan Science and Technology (JST)–Precursory Research for Embryonic Science and Technology (PRESTO) (Y. Ozeki) and partially supported by KAKENHI (21248040).

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Affiliations

  1. Department of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

    • Yasuyuki Ozeki
    • , Wataru Umemura
    • , Kazuhiko Sumimura
    • , Kiichi Fukui
    •  & Kazuyoshi Itoh

  2. Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

    • Yasuyuki Ozeki

  3. Canon Inc., Frontier Research Center, 30-2 Shimomaruko 3-chome, Ota-ku, Tokyo 146-8501, Japan

    • Yoichi Otsuka
    • , Shuya Satoh
    •  & Hiroyuki Hashimoto

  4. Department of Electrical Engineering and Computer Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8603, Japan

    • Norihiko Nishizawa

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Contributions

Y. Ozeki conceived the idea and drafted the manuscript. Y. Ozeki and W.U. built the instrument and coded data analysis programs. K.S. and N.N. built and modified the fibre laser. Y. Otsuka and S.S. prepared biological samples. Y. Ozeki, W.U. and Y. Otsuka conducted the experiments. H.H., K.F. and K.I. supervised the research.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Yasuyuki Ozeki.

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DOI

https://doi.org/10.1038/nphoton.2012.263