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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 15 — Jul. 16, 2012
  • pp: 16195–16204

Light sheet-excited spontaneous Raman imaging of a living fish by optical sectioning in a wide field Raman microscope

Yusuke Oshima, Hidetoshi Sato, Hiroko Kajiura-Kobayashi, Tetsuaki Kimura, Kiyoshi Naruse, and Shigenori Nonaka  »View Author Affiliations

Optics Express, Vol. 20, Issue 15, pp. 16195-16204 (2012)

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Spontaneous Raman microscopy is a potentially useful technique for imaging living cells, tissue and small animals without any probe or dye labeling. We have developed a spontaneous Raman imaging system in wide-field view, which we term ‘light sheet-excited direct Raman spectroscopy’ (LSDRS). This system, which we reported previously, consists of a background-free electrically tunable Ti:Sapphire laser (BF-ETL), a cylindrical lens, a CCD camera, and a narrow bandpass filter. Here, we have adapted the LSDRS system for microscopy systems, such as single-plane illumination microscopy (SPIM) for biomedical applications, and demonstrated spontaneous Raman imaging of a living fish. The results suggest that our Raman microscopy system enables investigation of the differentiation process and mechanism of iridocytes during development. This is the first report in which Raman imaging of a living animal was successfully demonstrated by spontaneous Raman scattering signals, but not nonlinear Raman effects such as CARS and SRS.

© 2012 OSA

OCIS Codes
(110.0110) Imaging systems : Imaging systems
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(300.6450) Spectroscopy : Spectroscopy, Raman
(110.4234) Imaging systems : Multispectral and hyperspectral imaging
(180.5655) Microscopy : Raman microscopy

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: April 11, 2012
Revised Manuscript: June 15, 2012
Manuscript Accepted: June 19, 2012
Published: July 2, 2012

Virtual Issues
Vol. 7, Iss. 9 Virtual Journal for Biomedical Optics

Yusuke Oshima, Hidetoshi Sato, Hiroko Kajiura-Kobayashi, Tetsuaki Kimura, Kiyoshi Naruse, and Shigenori Nonaka, "Light sheet-excited spontaneous Raman imaging of a living fish by optical sectioning in a wide field Raman microscope," Opt. Express 20, 16195-16204 (2012)

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  1. S. Wachsmann-Hogiu, T. Weeks, and T. Huser, “Chemical analysis in vivo and in vitro by Raman spectroscopy--from single cells to humans,” Curr. Opin. Biotechnol.20(1), 63–73 (2009). [CrossRef] [PubMed]
  2. Y. Zhang, H. Hong, and W. Cai, “Imaging with Raman spectroscopy,” Curr. Pharm. Biotechnol.11(6), 654–661 (2010). [CrossRef] [PubMed]
  3. K. Fujita and N. I. Smith, “Label-free molecular imaging of living cells,” Mol. Cells26(6), 530–535 (2008). [PubMed]
  4. M. D. Morris and G. S. Mandair, “Raman assessment of bone quality,” Clin. Orthop. Relat. Res.469(8), 2160–2169 (2011). [CrossRef] [PubMed]
  5. C. Krafft, B. Dietzek, and J. Popp, “Raman and CARS microspectroscopy of cells and tissues,” Analyst (Lond.)134(6), 1046–1057 (2009). [CrossRef] [PubMed]
  6. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004). [CrossRef] [PubMed]
  7. C. J. Engelbrecht and E. H. Stelzer, “Resolution enhancement in a light-sheet-based microscope (SPIM),” Opt. Lett.31(10), 1477–1479 (2006). [CrossRef] [PubMed]
  8. C. J. Engelbrecht, K. Greger, E. G. Reynaud, U. Krzic, J. Colombelli, and E. H. Stelzer, “Three-dimensional laser microsurgery in light-sheet based microscopy (SPIM),” Opt. Express15(10), 6420–6430 (2007). [CrossRef] [PubMed]
  9. P. J. Keller and E. H. Stelzer, “Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy,” Curr. Opin. Neurobiol.18(6), 624–632 (2008). [CrossRef] [PubMed]
  10. P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008). [CrossRef] [PubMed]
  11. J. Huisken, “Slicing embryos gently with laser light sheets,” Bioessays34(5), 406–411 (2012). [CrossRef] [PubMed]
  12. B. G. Saar, C. W. Freudiger, J. Reichman, C. M. Stanley, G. R. Holtom, and X. S. Xie, “Video-rate molecular imaging in vivo with stimulated Raman scattering,” Science330(6009), 1368–1370 (2010). [CrossRef] [PubMed]
  13. B. G. Saar, L. R. Contreras-Rojas, X. S. Xie, and R. H. Guy, “Imaging drug delivery to skin with stimulated Raman scattering microscopy,” Mol. Pharm.8(3), 969–975 (2011). [CrossRef] [PubMed]
  14. J. P. Pezacki, J. A. Blake, D. C. Danielson, D. C. Kennedy, R. K. Lyn, and R. Singaravelu, “Chemical contrast for imaging living systems: molecular vibrations drive CARS microscopy,” Nat. Chem. Biol.7(3), 137–145 (2011). [CrossRef] [PubMed]
  15. T. Minamikawa, M. Hashimoto, K. Fujita, S. Kawata, and T. Araki, “Multi-focus excitation coherent anti-Stokes Raman scattering (CARS) microscopy and its applications for real-time imaging,” Opt. Express17(12), 9526–9536 (2009). [CrossRef] [PubMed]
  16. J. Y. Lee, S. H. Kim, D. W. Moon, and E. S. Lee, “Three-color multiplex CARS for fast imaging and microspectroscopy in the entire CHn stretching vibrational region,” Opt. Express17(25), 22281–22295 (2009). [CrossRef] [PubMed]
  17. K. Hamada, K. Fujita, N. I. Smith, M. Kobayashi, Y. Inouye, and S. Kawata, “Raman microscopy for dynamic molecular imaging of living cells,” J. Biomed. Opt.13(4), 044027 (2008). [CrossRef] [PubMed]
  18. I. Barman, K. M. Tan, and G. P. Singh, “Optical sectioning using single-plane-illumination Raman imaging,” J. Raman Spectrosc.41(10), 1099–1101 (2010). [CrossRef]
  19. Y. Oshima, C. Furihata, and H. Sato, “Light sheet direct Raman imaging technique for observation of mixing of solvents,” Appl. Spectrosc.63(10), 1115–1120 (2009). [CrossRef] [PubMed]
  20. H. Sato, S. Wada, M. Y. Ling, and H. Tashiro, “Noninvasive measurement of oxygenation of hemoglobin by direct transmission of near-infrared energy (700-1000 nm) from an electronically tuned Ti: sapphire laser driven by a dual radio-frequency driving method,” Appl. Spectrosc.54(8), 1163–1167 (2000). [CrossRef]
  21. H. Sato, S. Wada, and H. Tashiro, “Fluorescence backgroundless Ti: Sapphire laser using acousto-optical tunable filter for Raman spectroscopic measurements,” Appl. Spectrosc.56(10), 1303–1307 (2002). [CrossRef]
  22. M. Lynn Lamoreux, R. N. Kelsh, Y. Wakamatsu, and K. Ozato, “Pigment pattern formation in the medaka embryo,” Pigment Cell Res.18(2), 64–73 (2005). [CrossRef] [PubMed]
  23. T. Yamamoto, “Chromatophores and Iridocytes,” MEDAKA(killifish): Biology and Strains, http://www.bio.nagoya-u.ac.jp:8000/iridocytes.html
  24. G. N. Ten, T. G. Bourova, and V. I. Baranov, “Calculation of Raman spectra and structure elucidation of guanine in the polycrystalline state and in aqueous solution,” J. Struct. Chem.46(6), 998–1005 (2005). [CrossRef]
  25. S. Thawornchinsombut, J. W. Park, G. Meng, and E. C. Li-Chan, “Raman spectroscopy determines structural changes associated with gelation properties of fish proteins recovered at alkaline pH,” J. Agric. Food Chem.54(6), 2178–2187 (2006). [CrossRef] [PubMed]
  26. Z. Movasaghi, S. Rehman, and I. U. Rehman, “Raman spectroscopy of biological tissues,” Appl. Spectrosc. Rev.42(5), 493–541 (2007). [CrossRef]
  27. Y. Kamei, M. Suzuki, K. Watanabe, K. Fujimori, T. Kawasaki, T. Deguchi, Y. Yoneda, T. Todo, S. Takagi, T. Funatsu, and S. Yuba, “Infrared laser-mediated gene induction in targeted single cells in vivo,” Nat. Methods6(1), 79–81 (2009). [CrossRef] [PubMed]
  28. L. Gao, H. Zhou, M. J. Thrall, F. Li, Y. Yang, Z. Wang, P. Luo, K. K. Wong, G. S. Palapattu, and S. T. Wong, “Label-free high-resolution imaging of prostate glands and cavernous nerves using coherent anti-Stokes Raman scattering microscopy,” Biomed. Opt. Express2(4), 915–926 (2011). [CrossRef] [PubMed]
  29. J. Horsnell, P. Stonelake, J. Christie-Brown, G. Shetty, J. Hutchings, C. Kendall, and N. Stone, “Raman spectroscopy--a new method for the intra-operative assessment of axillary lymph nodes,” Analyst (Lond.)135(12), 3042–3047 (2010). [CrossRef] [PubMed]

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