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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 5, Iss. 11 — Aug. 25, 2010
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Infrared imaging of an A549 cultured cell by a vibrational sum-frequency generation detected infrared super-resolution microscope

Satoshi Kogure, Keiichi Inoue, Tsutomu Ohmori, Miya Ishihara, Makoto Kikuchi, Masaaki Fujii, and Makoto Sakai  »View Author Affiliations


Optics Express, Vol. 18, Issue 13, pp. 13402-13406 (2010)
http://dx.doi.org/10.1364/OE.18.013402


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Abstract

We performed infrared (IR) spectroscopic imaging of molecular species in cultured cell interiors of A549 cells using in-house developed vibrational sum-frequency generation detected IR super-resolution microscope. The spatial resolution of this IR microscope was approximately 1.1 µm, which exceeds the diffraction limit of IR light. Therefore, we clearly observed differences in the signal intensity at various IR wavelengths which appear to originate from the differing IR absorptions of specific vibrational modes, and reveal the distribution of molecular species in the single cell. These results were never imaged with the conventional IR microscope.

© 2010 OSA

1. Introduction

Biological and medical disciplines have seen a rapid recent increase in the importance of vibrational imaging of living samples such as tissues and cells, to study the distribution of molecular species in cells and to obtain information about their structure and environment. For this purpose, infrared (IR) absorption and Raman scattering microscopes enable in situ measurement of molecular vibrations without labeling and/or physiological damage to targets [1

1. P. Heraud, S. Caine, N. Campanale, T. Karnezis, D. McNaughton, B. R. Wood, M. J. Tobin, and C. C. A. Bernard, “Early detection of the chemical changes occurring during the induction and prevention of autoimmune-mediated demyelination detected by FT-IR imaging,” Neuroimage 49(2), 1180–1189 (2010). [CrossRef]

5

5. H.-W. Wang, T. T. Le, and J.-X. Cheng, “Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope,” Opt. Commun. 281(7), 1813–1822 (2008). [CrossRef]

]. Furthermore, Raman and CARS microscopes posses the ability to obtain the vibrational image of a single cell through sub-micrometer imaging [2

2. Y. Naito, A. Toh-e, and H. Hamaguchi, “In vivo time-resolved Raman Imaging of a spontaneous death process of a single budding yeast cell,” J. Raman Spectrosc. 36(9), 837–839 (2005). [CrossRef]

4

4. H. Kano and H. O. Hamaguchi, “In-vivo multi-nonlinear optical imaging of a living cell using a supercontinuum light source generated from a photonic crystal fiber,” Opt. Express 14(7), 2798–2804 (2006). [CrossRef] [PubMed]

]. On the other hand, due to the longer wavelength and larger diffraction limit of IR light, the application of IR microscopes for the vibrational imaging of minute samples, such as single cells, is rather underdeveloped compared to Raman microscopes, which employ shorter wavelength visible light [6

6. P. Lasch and D. Naumann, “Spatial resolution in infrared microspectroscopic imaging of tissues,” Biochim. Biophys. Acta 1758(7), 814–829 (2006). [CrossRef] [PubMed]

,7

7. H. Fabian, N. A. N. Thi, M. Eiden, P. Lasch, J. Schmitt, and D. Naumann, “Diagnosing benign and malignant lesions in breast tissue sections by using IR-microspectroscopy,” Biochim. Biophys. Acta 1758(7), 874–882 (2006). [CrossRef] [PubMed]

]. Importantly though, IR spectroscopy is a complementary technique to Raman spectroscopy, and both of them are indispensible for the complete understanding of molecular vibrations of biological molecules in the cell

To overcome the shortcoming of existing IR-based techniques, we have developed a new vibrational sum-frequency generation (VSFG) detected IR super-resolution microscope. VSFG is a nonlinear optical process in which a visible and an IR photon are incident with a molecule, and a new photon, whose energy is equal to the sum-frequency of first two photons (νvis + νIR) [8

8. Y. R. Shen, The principles of nonlinear optics (John Wiley & Sons, New York, 1984).

], is emitted at interfaces such as cell walls and cytomembranes and/or from chiral molecules through vibrational resonance of the molecule with the IR light [9

9. C. Hirose, H. Yamamoto, N. Akamatsu, and K. Domen, “Orientation analysis by simulation of vibrational sum frequency generation spectrum: CH stretching bands of the methyl group,” J. Phys. Chem. 97(39), 10064–10069 (1993). [CrossRef]

13

13. N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006). [CrossRef] [PubMed]

]. The largest benefit of the VSFG method for IR microscopy is the conversion of IR absorption to visible emission. Because the VSFG signal has a visible wavelength, the image is observed at the resolution of visible light, which is about 10 times smaller than that of IR light. Furthermore, it is possible to apply this method to the majority of non-fluorescencing biological molecules.

To date, by using the VSFG detected IR super-resolution microscope, we have succeeded in IR imaging onion root cells at a spatial resolution of 1.1 μm, which exceeds the diffraction limit of IR light. In addition, we have also successfully observed the difference of the distribution of specific molecular species in the cell interior by changing the IR wavelength [14

14. K. Inoue, M. Fujii, and M. Sakai, “Development of a non-scanning vibrational sum-frequency generation detected infrared super-resolution microscope and its application to biological cells,” Appl. Spectrosc. 64(3), 275–281 (2010). [CrossRef] [PubMed]

]. The present work aims at the IR imaging of molecular species in cultured cancer cell interiors using this microscope. This ability is indispensible to cell biological and/or medical applications of the VSFG microscope.

2. Experimental methods

The laser setup for our two-color IR super-resolution microscope has been described previously [14

14. K. Inoue, M. Fujii, and M. Sakai, “Development of a non-scanning vibrational sum-frequency generation detected infrared super-resolution microscope and its application to biological cells,” Appl. Spectrosc. 64(3), 275–281 (2010). [CrossRef] [PubMed]

,15

15. K. Inoue, N. Bokor, S. Kogure, M. Fujii, and M. Sakai, “Two-point-separation in a sub-micron nonscanning IR super-resolution microscope based on transient fluorescence detected IR spectroscopy,” Opt. Express 17(14), 12013–12018 (2009). [CrossRef] [PubMed]

]. Figure 1
Fig. 1 Schematic diagram of the experimental setup of the VSFG microscope.
shows optical layout used for IR imaging detection. Both IR and visible light with a spectral bandwidth of 20 cm−1 and a pulse width of 2 ps, generated by a picosecond laser system, were introduced into a home-made laser fluorescence microscope. IR and visible light beams were superposed on a colinear path by a beamcombiner and focused into the sample using a CaF2 lens (f = 100 mm). The focal spot sizes of IR and visible light beams were adjusted to about 100 μm diameter at the sample position. During the experiment, IR light of < 10 μJ / pulse and visible light of < 10 nJ / pulse were used to irradiate the sample at a repetition rate of 1 kHz. The VSFG light from samples was collected from the opposite side of the sample from the incident light, by a NA = 0.5 objective lens (Newport, M-50X), projected onto a CCD camera with an image-intensifier (Princeton Instruments; PI-MAX-1K filmless) and recorded by a personal computer as a VSFG image. Here, we used the 610 nm wavelength of the visible light. This is because the wavelength of VSFG signal is fixed to the photon detection maximum (~500 - 600 nm) of ICCD detector. To remove the excitation lasers, notch and IR-cut (Y48, HOYA, Tokyo, Japan) filters were placed behind the objective. In addition, we used short (Y48, HOYA, Tokyo, Japan) and long cut (Asahi Spectra, Tokyo, Japan) filters, which acted together as a band-pass filter to eliminate background light and to allow selective detection of the VSFG signal.

A549 cells, a lung carcinoma cells were used as samples. The cells were cultured in Dulbecco modified Eagle medium (GIBCO) with 10% fetal bovine serum on a cover glass. Just before VSFG imaging, the sample was covered with another cover glass and sealed using nail varnish to prevent it from drying.

3. Results and Discussions

Figure 2a
Fig. 2 VSFG images of an A549 cultured cell. (a) Transmission image of the cell. Image obtained by (b) only visible (λ VIS = 610 nm) and (c) only IR (λ IR = 3273 nm (ν IR = 3055 cm−1)) beams, (d) simultaneous introduction of visible and IR beams (λ VIS = 610 nm, λ IR = 3273 nm (ν IR = 3055 cm−1)). (e) As for (d), but with the IR wavelength increased to 3666 nm (ν IR = 2728 cm−1). The white scale bars in each figure indicate 5 μm.
shows a transmission image of an A549 cultured cell. We applied only visible (Fig. 2b, wavelength = 610 nm), only IR (Fig. 2c, wavelength = 3300 nm), and (d) both IR and visible beams to the cell. No VSFG signal was detected for cases (b) (only visible) and (c) (only IR). However, a VSFG image was clearly observed by simultaneously introducing both IR and visible light (Fig. 2d). VSFG images almost disappeared when the IR wavelength was changed to 2728 cm−1, where the majority of biological molecules do not absorb IR light (Fig. 2e). The difference between Figs. 2d and 2e clearly indicates that stronger intensity VSFG signals correspond to stronger absorption of IR light. This relationship is similar to the previously reported results for onion root cells, and therefore we conclude that the images in Figs. 2c and 2d reflect the IR absorption of the molecule [14

14. K. Inoue, M. Fujii, and M. Sakai, “Development of a non-scanning vibrational sum-frequency generation detected infrared super-resolution microscope and its application to biological cells,” Appl. Spectrosc. 64(3), 275–281 (2010). [CrossRef] [PubMed]

]. Furthermore, we measured of the spectrum of the VSFG signal, observing a strong and very sharp peak at a wavelength of 514 nm, which is 3030 cm−1 greater than the visible excitation beam (data not shown). This strongly confirms that the image in Fig. 2(d) originates from VSFG. Therefore, we conclude that IR imaging of cultured A549 cell was successfully demonstrated by the VSFG detected IR microscope.

4. Conclusion

We successfully imaged an A549 cultured cell (from lung cancer) using a VSFG detected IR super-resolution microscope. In addition, we observed differences in the signal intensity at various IR wavelengths which appear to originate from the differing IR absorptions of specific vibrational modes, and reveal the distribution of molecular species for the first time. Finally, it is expected that alongside the Raman microscope, this kind of microscope will become a powerful technique for the vibrational spectroscopy of living cells.

Acknowledgments

We thank Dr. Nándor Bokor and Dr. Jonathan R. Woodward for stimulating discussions and kind help on the preparation of the manuscript. The authors are also grateful to Ms. Machiko Tanigawa and Ms. Yoshine Mayumi for preparing the cellular samples. The present work was financially supported in part by a Grants-in-Aid for Scientific Research (KAKENHI) on Priority Areas (Area No. [477]).

References and links

1.

P. Heraud, S. Caine, N. Campanale, T. Karnezis, D. McNaughton, B. R. Wood, M. J. Tobin, and C. C. A. Bernard, “Early detection of the chemical changes occurring during the induction and prevention of autoimmune-mediated demyelination detected by FT-IR imaging,” Neuroimage 49(2), 1180–1189 (2010). [CrossRef]

2.

Y. Naito, A. Toh-e, and H. Hamaguchi, “In vivo time-resolved Raman Imaging of a spontaneous death process of a single budding yeast cell,” J. Raman Spectrosc. 36(9), 837–839 (2005). [CrossRef]

3.

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An Epi-detected coherent anti-stokes Raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001). [CrossRef]

4.

H. Kano and H. O. Hamaguchi, “In-vivo multi-nonlinear optical imaging of a living cell using a supercontinuum light source generated from a photonic crystal fiber,” Opt. Express 14(7), 2798–2804 (2006). [CrossRef] [PubMed]

5.

H.-W. Wang, T. T. Le, and J.-X. Cheng, “Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope,” Opt. Commun. 281(7), 1813–1822 (2008). [CrossRef]

6.

P. Lasch and D. Naumann, “Spatial resolution in infrared microspectroscopic imaging of tissues,” Biochim. Biophys. Acta 1758(7), 814–829 (2006). [CrossRef] [PubMed]

7.

H. Fabian, N. A. N. Thi, M. Eiden, P. Lasch, J. Schmitt, and D. Naumann, “Diagnosing benign and malignant lesions in breast tissue sections by using IR-microspectroscopy,” Biochim. Biophys. Acta 1758(7), 874–882 (2006). [CrossRef] [PubMed]

8.

Y. R. Shen, The principles of nonlinear optics (John Wiley & Sons, New York, 1984).

9.

C. Hirose, H. Yamamoto, N. Akamatsu, and K. Domen, “Orientation analysis by simulation of vibrational sum frequency generation spectrum: CH stretching bands of the methyl group,” J. Phys. Chem. 97(39), 10064–10069 (1993). [CrossRef]

10.

Y. Goto, N. Akamatsu, K. Domen, and C. Hirose, “Vibration-induced order-disorder transitions in a Langmuir-Blodgett film as investigated by vibrational sum-frequency generation spectroscopy,” J. Phys. Chem. 99(12), 4086–4090 (1995). [CrossRef]

11.

N. Akamatsu, K. Domen, C. Hirose, T. Onishi, H. Shimizu, and K. Masutani, “SFG study of rotational anisotropy of cadmium arachidate Langmuir-Blodgett films,” Chem. Phys. Lett. 181(2-3), 175–178 (1991). [CrossRef]

12.

R. W. Boyd, Nonlinear optics, 2nd ed. (Academic Press, San Diego 2003)

13.

N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006). [CrossRef] [PubMed]

14.

K. Inoue, M. Fujii, and M. Sakai, “Development of a non-scanning vibrational sum-frequency generation detected infrared super-resolution microscope and its application to biological cells,” Appl. Spectrosc. 64(3), 275–281 (2010). [CrossRef] [PubMed]

15.

K. Inoue, N. Bokor, S. Kogure, M. Fujii, and M. Sakai, “Two-point-separation in a sub-micron nonscanning IR super-resolution microscope based on transient fluorescence detected IR spectroscopy,” Opt. Express 17(14), 12013–12018 (2009). [CrossRef] [PubMed]

16.

C. J. Pouchert, The Aldrich library of infrared spectra (Aldrich Chemical Co., Milwaukee, 1970)

17.

G. Mizutani, T. Koyama, S. Tomizawa, and H. Sano, “Distinction between some saccharides in scattered optical sum frequency intensity images,” Spectrochim. Acta [A] 62(4-5), 845–849 (2005). [CrossRef]

18.

Y. Miyauchi, H. Sano, and G. Mizutani, “Selective observation of starch in a water plant using optical sum-frequency microscopy,” J. Opt. Soc. Am. A 23(7), 1687–1690 (2006). [CrossRef]

OCIS Codes
(100.6640) Image processing : Superresolution
(110.3080) Imaging systems : Infrared imaging
(170.0110) Medical optics and biotechnology : Imaging systems
(170.1530) Medical optics and biotechnology : Cell analysis
(320.5390) Ultrafast optics : Picosecond phenomena
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Imaging Systems

History
Original Manuscript: April 27, 2010
Revised Manuscript: May 27, 2010
Manuscript Accepted: May 31, 2010
Published: June 7, 2010

Virtual Issues
Vol. 5, Iss. 11 Virtual Journal for Biomedical Optics

Citation
Satoshi Kogure, Keiichi Inoue, Tsutomu Ohmori, Miya Ishihara, Makoto Kikuchi, Masaaki Fujii, and Makoto Sakai, "Infrared imaging of an A549 cultured cell by a vibrational sum-frequency generation detected infrared super-resolution microscope," Opt. Express 18, 13402-13406 (2010)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-18-13-13402


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References

  1. P. Heraud, S. Caine, N. Campanale, T. Karnezis, D. McNaughton, B. R. Wood, M. J. Tobin, and C. C. A. Bernard, “Early detection of the chemical changes occurring during the induction and prevention of autoimmune-mediated demyelination detected by FT-IR imaging,” Neuroimage 49(2), 1180–1189 (2010). [CrossRef]
  2. Y. Naito, A. Toh-e, and H. Hamaguchi, “In vivo time-resolved Raman Imaging of a spontaneous death process of a single budding yeast cell,” J. Raman Spectrosc. 36(9), 837–839 (2005). [CrossRef]
  3. J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An Epi-detected coherent anti-stokes Raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001). [CrossRef]
  4. H. Kano and H. O. Hamaguchi, “In-vivo multi-nonlinear optical imaging of a living cell using a supercontinuum light source generated from a photonic crystal fiber,” Opt. Express 14(7), 2798–2804 (2006). [CrossRef] [PubMed]
  5. H.-W. Wang, T. T. Le, and J.-X. Cheng, “Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope,” Opt. Commun. 281(7), 1813–1822 (2008). [CrossRef]
  6. P. Lasch and D. Naumann, “Spatial resolution in infrared microspectroscopic imaging of tissues,” Biochim. Biophys. Acta 1758(7), 814–829 (2006). [CrossRef] [PubMed]
  7. H. Fabian, N. A. N. Thi, M. Eiden, P. Lasch, J. Schmitt, and D. Naumann, “Diagnosing benign and malignant lesions in breast tissue sections by using IR-microspectroscopy,” Biochim. Biophys. Acta 1758(7), 874–882 (2006). [CrossRef] [PubMed]
  8. Y. R. Shen, The principles of nonlinear optics (John Wiley & Sons, New York, 1984).
  9. C. Hirose, H. Yamamoto, N. Akamatsu, and K. Domen, “Orientation analysis by simulation of vibrational sum frequency generation spectrum: CH stretching bands of the methyl group,” J. Phys. Chem. 97(39), 10064–10069 (1993). [CrossRef]
  10. Y. Goto, N. Akamatsu, K. Domen, and C. Hirose, “Vibration-induced order-disorder transitions in a Langmuir-Blodgett film as investigated by vibrational sum-frequency generation spectroscopy,” J. Phys. Chem. 99(12), 4086–4090 (1995). [CrossRef]
  11. N. Akamatsu, K. Domen, C. Hirose, T. Onishi, H. Shimizu, and K. Masutani, “SFG study of rotational anisotropy of cadmium arachidate Langmuir-Blodgett films,” Chem. Phys. Lett. 181(2-3), 175–178 (1991). [CrossRef]
  12. R. W. Boyd, Nonlinear optics, 2nd ed. (Academic Press, San Diego 2003)
  13. N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006). [CrossRef] [PubMed]
  14. K. Inoue, M. Fujii, and M. Sakai, “Development of a non-scanning vibrational sum-frequency generation detected infrared super-resolution microscope and its application to biological cells,” Appl. Spectrosc. 64(3), 275–281 (2010). [CrossRef] [PubMed]
  15. K. Inoue, N. Bokor, S. Kogure, M. Fujii, and M. Sakai, “Two-point-separation in a sub-micron nonscanning IR super-resolution microscope based on transient fluorescence detected IR spectroscopy,” Opt. Express 17(14), 12013–12018 (2009). [CrossRef] [PubMed]
  16. C. J. Pouchert, The Aldrich library of infrared spectra (Aldrich Chemical Co., Milwaukee, 1970)
  17. G. Mizutani, T. Koyama, S. Tomizawa, and H. Sano, “Distinction between some saccharides in scattered optical sum frequency intensity images,” Spectrochim. Acta [A] 62(4-5), 845–849 (2005). [CrossRef]
  18. Y. Miyauchi, H. Sano, and G. Mizutani, “Selective observation of starch in a water plant using optical sum-frequency microscopy,” J. Opt. Soc. Am. A 23(7), 1687–1690 (2006). [CrossRef]

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