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Raman and SERS microscopy for molecular imaging of live cells

Nature Protocols volume 8, pages 677–692 (2013)Cite this article

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Abstract

Raman microscopy is a promising technology for visualizing the distribution of molecules in cells. A challenge for live-cell imaging using Raman microscopy has been long imaging times owing to the weak Raman signal. Here we present a protocol for constructing and using a Raman microscope equipped with both a slit-scanning excitation and detection system and a laser steering and nanoparticle-tracking system. Slit scanning allows Raman imaging with high temporal and spatial resolution, whereas the laser beam steering system enables dynamic surface-enhanced Raman imaging using gold nanoparticles. Both features enable mapping of the distributions of molecules in live cells and visualization of cellular transport pathways. Furthermore, its utility can be expanded to small-molecule imaging by using tiny Raman-active tags such as alkyne. For example, DNA synthesis in a cell can be visualized by detecting 5-ethynyl-2′-deoxyuridine (EdU), a deoxyuridine derivative with an alkyne moiety. We describe the optics, hardware and software to construct the Raman microscope, and discuss the conditions and parameters involved in live-cell imaging. The whole system can be built in ∼8 h.

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Figure 1: Schematic diagram of the experimental setup for Raman imaging.
Figure 2: Raman spectra obtained from glass, quartz and CaF2 substrate.
Figure 3: Schematic diagram for analysis with a Raman tag.
Figure 4: Optics for the laser beam steering and tracking system.
Figure 5: Principle of the laser steering and nanoparticle-tracking system.
Figure 6: Raman scattering images of unstained living HeLa cells.
Figure 7: Raman images of alkyne-tagged cell proliferation probe, EdU, in HeLa cells.
Figure 8: Time-resolved observation of SERS from gold nanoparticles in cells.
Figure 9: Observation of cellular transport pathways by dynamic SERS imaging.

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References

  1. Huang, Y.-S., Karashima, T., Yamamoto, M. & Hamaguchi, H. Molecular-level pursuit of yeast mitosis by time- and space-resolved Raman spectroscopy. J. Raman Spectrosc. 34, 1–3 (2003).

    Article  CAS  Google Scholar 

  2. Hamada, K. et al. Raman microscopy for dynamic molecular imaging of living cells. J. Biomed. Opt. 13, 044027 (2008).

    Article  Google Scholar 

  3. Puppels, G.J. et al. Studying single living cells and chromosomes by confocal Raman microspectroscopy. Nature 347, 301–303 (1990).

    Article  CAS  Google Scholar 

  4. Puppels, G.J., Grond, M. & Greve, J. Direct imaging Raman microscope based on tunable wavelength excitation and narrow-band emission detection. Appl. Spectrosc. 47, 1256–1267 (1993).

    Article  CAS  Google Scholar 

  5. Manen, H.-J., Kraan, Y.M., Roos, D. & Otto, C. Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes. Proc. Natl. Acad. Sci. USA 102, 10159–10164 (2005).

    Article  Google Scholar 

  6. Matthaus, C., Chernenko, T., Newmark, J.A., Warner, C.M. & Diex, M. Label-free detection of mitochondrial distribution in cells by nonresonant Raman microspectroscopy. Biophys. J. 93, 668–673 (2007).

    Article  CAS  Google Scholar 

  7. Manen, H.-J., Kraan, Y.M., Roos, D. & Otto, C. Intracellular chemical imaging of heme containing enzymes involved in innate immunity using resonance Raman microscopy. J. Phys. Chem. B 108, 18762–18771 (2004).

    Article  Google Scholar 

  8. Puppels, G.J. et al. Description and performance of a highly sensitive confocal Raman microspectrometer. J. Raman Spectrosc. 22, 217–225 (1991).

    Article  CAS  Google Scholar 

  9. Veirs, D.K., Ager, J.W. III, Loucks, E.T. & Rosenblatt, G.M. Mapping materials properties with Raman spectroscopy utilizing a 2D detector. Appl. Opt. 29, 4969–4980 (1990).

    Article  CAS  Google Scholar 

  10. Okuno, M. & Hamaguchi, H. Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells. Opt. Lett. 35, 4096–4098 (2010).

    Article  Google Scholar 

  11. Kong, L.B., Zhang, P.F., Yu, J., Setlow, P. & Li, Y.Q. Rapid confocal Raman imaging using a synchro multifoci-scan scheme for dynamic monitoring of single living cells. Appl. Phys. Lett. 98, 213803 (2011).

    Google Scholar 

  12. Ling, J., Weitman, S.D., Miller, M.A., Moore, R.V. & Bovik, A.C. Direct Raman imaging techniques for study of the subcellular distribution of a drug. Appl. Opt. 41, 6006–6017 (2002).

    Article  CAS  Google Scholar 

  13. Okada, M. et al. Label-free Raman observation of cytochrome cdynamics during apoptosis. Proc. Natl. Acad. Sci. USA 109, 28–32 (2012).

    Article  CAS  Google Scholar 

  14. Duncan, M., Reintjes, J. & Manuccia, T.J. Scanning coherent anti-Stokes Raman microscope. Opt. Lett. 7, 350–352 (1982).

    Article  CAS  Google Scholar 

  15. Zumbusch, A., Holtom, G. & Xie, X.S. Vibrational microscopy using coherent anti-Stokes Raman scattering. Phys. Rev. Lett. 82, 4142–4145 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. 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  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Kneipp, K. et al. Surface-enhanced Raman spectroscopy in single living cells using gold nano particles. Appl. Spectrosc. 56, 150–154 (2002).

    Article  CAS  Google Scholar 

  20. Bell, S.E.J. & Sirimuthu, N.M.S. Surface-enhanced Raman spesctroscopy (SERS) for sub-micromolar detection of DNA/RNA mononucleotides. J. Am. Chem. Soc. 128, 15580–15581 (2006).

    Article  CAS  Google Scholar 

  21. Fujita, K. et al. Time-resolved observation of surface-enhanced Raman scattering from gold nano particles during transport through a living cell. J. Biomed. Opt. 14, 024038 (2009).

    Article  Google Scholar 

  22. Ando, J., Fujita, K., Smith, N.I. & Kawata, S. Dynamic SERS imaging of cellular transport pathways with endocytosed gold nanoparticles. Nano Lett. 11, 5344–5348 (2011).

    Article  CAS  Google Scholar 

  23. Watanabe, T.M., Sato, T., Gonda, K. & Higuchi, H. Three-dimensional nanometry of vesicle transport in living cells using dual-focus imaging optics. Biochem. Biophys. Res. Commun. 359, 1–7 (2007).

    Article  CAS  Google Scholar 

  24. Toprak, E., Balci, H., Blehm, B.H. & Selvin, P.R. Three-dimensional particle tracking via bifocal imaging. Nano Lett. 7, 2043–2045 (2007).

    Article  CAS  Google Scholar 

  25. Yamakoshi, H. et al. Imaging of EdU, an alkyne-tagged cell proliferation probe, by Raman microscopy. J. Am. Chem. Soc. 133, 6102–6105 (2011).

    Article  CAS  Google Scholar 

  26. Yamakoshi, H. et al. Alkyne-tag Raman imaging for visualization of mobile small molecules in live cells. J. Am. Chem. Soc. 134, 20681–20689 (2012).

    Article  CAS  Google Scholar 

  27. Kawata, S., Aritomoto, R. & Nakamura, O. Three-dimensional optical-transfer-function analysis for a laser-scan fluorescence microscope with an extended detector. J. Opt. Soc. Am. A 8, 171–175 (1991).

    Article  Google Scholar 

  28. Puppels, G.J. et al. Laser irradiation and Raman spectroscopy of single living cells and chromosomes: sample degradation occurs with 514.5-nm but not with 660-nm laser light. Exp. Cell Res. 195, 361–367 (1991).

    Article  CAS  Google Scholar 

  29. Carey, P.R. Biochemical Applications of Raman and Resonance Raman Spectroscopies (Molecular Biology) (Academic Press, 1982).

  30. Lieber, C.A. & Mahadevan-Jansen, A. Automated method for subtraction of fluorescence from biological Raman spectra. Appl. Spectrosc. 57, 1363–1367 (2003).

    Article  CAS  Google Scholar 

  31. Tu, A.T. Raman Spectroscopy in Biology: Principles and Applications (John Wiley & Sons, 1982).

  32. Notingher, I. & Hench, L. Raman microspectroscopy: a noninvasive tool for studies of individual living cells in vitro. Expet. Rev. Med. Dev. 3, 215–234 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Okada and N.I. Smith for technical support and helpful discussions. This work was partly supported by JST-ERATO, JST-CREST and JSPS KAKENHI grant nos. 24710267 and 23710276.

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Authors and Affiliations

  1. Department of Applied Physics, Osaka University, Suita, Osaka, Japan

    Almar F Palonpon, Satoshi Kawata & Katsumasa Fujita

  2. Sodeoka Live Cell Chemistry Project, Exploratory Research for Advanced Technology (ERATO) Program, Japan Science and Technology Agency (JST), Wako, Saitama, Japan

    Almar F Palonpon, Jun Ando, Hiroyuki Yamakoshi, Kosuke Dodo, Mikiko Sodeoka & Katsumasa Fujita

  3. RIKEN Advanced Science Institute, Wako, Saitama, Japan

    Jun Ando, Hiroyuki Yamakoshi, Kosuke Dodo, Mikiko Sodeoka & Satoshi Kawata

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  1. Almar F Palonpon
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Contributions

A.F.P., J.A. and K.F. developed and constructed the Raman microscope system. A.F.P., H.Y. and J.A. conducted the experiments presented here. K.D., M.S., S.K. and K.F. provided conceptual input and supervised the research. A.F.P., J.A. and K.F. wrote the manuscript with contributions from all other coauthors.

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Correspondence to Katsumasa Fujita.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Time-resolved observation of SERS from gold nanoparticles in cells. Left: SERS images. Color channels: red, 501 cm−1; blue, 807 cm−1; green, 1143 cm−1. Right: Darkfield images. (MOV 544 kb)

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Palonpon, A., Ando, J., Yamakoshi, H. et al. Raman and SERS microscopy for molecular imaging of live cells. Nat Protoc 8, 677–692 (2013). https://doi.org/10.1038/nprot.2013.030

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