Abstract

We have developed a multifocus confocal Raman microspectroscopic system for the fast multimode vibrational imaging of living cells. It consists of an inverted microscope equipped with a microlens array, a pinhole array, a fiber bundle, and a multichannel Raman spectrometer. Forty-eight Raman spectra from 48 foci under the microscope are simultaneously obtained by using multifocus excitation and image-compression techniques. The multifocus confocal configuration suppresses the background generated from the cover glass and the cell culturing medium so that high-contrast images are obtainable with a short accumulation time. The system enables us to obtain multimode (10 different vibrational modes) vibrational images of living cells in tens of seconds with only 1mW laser power at one focal point. This image acquisition time is more than 10 times faster than that in conventional single-focus Raman microspectroscopy.

© 2010 Optical Society of America

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2009 (2)

2005 (1)

Y. S. Huang, T. Karashima, M. Yamamoto, and H. Hamaguchi, Biochemistry 44, 10009 (2005).
[CrossRef] [PubMed]

2004 (2)

Y. S. Huang, T. Karashima, M. Yamamoto, T. Ogura, and H. Hamaguchi, J. Raman Spectrosc. 35, 525 (2004).
[CrossRef]

T. W. Kee and M. T. Cicerone, Opt. Lett. 29, 2701 (2004).
[CrossRef] [PubMed]

2002 (2)

1999 (1)

A. D. Gift, J. Y. Ma, K. S. Haber, B. L. McClain, and D. Ben-Amotz, J. Raman Spectrosc. 30, 757 (1999).
[CrossRef]

1998 (1)

1997 (1)

1993 (1)

S. Z. Hu, I. K. Morris, J. P. Singh, K. M. Smith, and T. G. Spiro, J. Am. Chem. Soc. 115, 12446 (1993).
[CrossRef]

1991 (1)

G. J. Puppels, W. Colier, J. H. F. Olminkhof, C. Otto, F. F. M. Demul, and J. Greve, J. Raman Spectrosc. 22, 217 (1991).
[CrossRef]

1990 (1)

G. J. Puppels, F. F. M. Demul, C. Otto, J. Greve, M. Robertnicoud, D. J. Arndtjovin, and T. M. Jovin, Nature 347, 301 (1990).
[CrossRef] [PubMed]

Araki, T.

Arndtjovin, D. J.

G. J. Puppels, F. F. M. Demul, C. Otto, J. Greve, M. Robertnicoud, D. J. Arndtjovin, and T. M. Jovin, Nature 347, 301 (1990).
[CrossRef] [PubMed]

Bachler, B. R.

Ben-Amotz, D.

A. D. Gift, J. Y. Ma, K. S. Haber, B. L. McClain, and D. Ben-Amotz, J. Raman Spectrosc. 30, 757 (1999).
[CrossRef]

J. Y. Ma and D. Ben-Amotz, Appl. Spectrosc. 51, 1845(1997).
[CrossRef]

Bewersdorf, J.

Chen, B. C.

J. Sung, B. C. Chen, and S. H. Lim, “Fast three-dimensional chemical imaging by interferometric multiplex coherent anti-Stokes Raman scattering microscopy,” J. Raman Spectrosc. (to be published).

Cicerone, M. T.

Colier, W.

G. J. Puppels, W. Colier, J. H. F. Olminkhof, C. Otto, F. F. M. Demul, and J. Greve, J. Raman Spectrosc. 22, 217 (1991).
[CrossRef]

Cui, M.

Demul, F. F. M.

G. J. Puppels, W. Colier, J. H. F. Olminkhof, C. Otto, F. F. M. Demul, and J. Greve, J. Raman Spectrosc. 22, 217 (1991).
[CrossRef]

G. J. Puppels, F. F. M. Demul, C. Otto, J. Greve, M. Robertnicoud, D. J. Arndtjovin, and T. M. Jovin, Nature 347, 301 (1990).
[CrossRef] [PubMed]

Fujita, K.

Gift, A. D.

A. D. Gift, J. Y. Ma, K. S. Haber, B. L. McClain, and D. Ben-Amotz, J. Raman Spectrosc. 30, 757 (1999).
[CrossRef]

Greve, J.

G. J. Puppels, W. Colier, J. H. F. Olminkhof, C. Otto, F. F. M. Demul, and J. Greve, J. Raman Spectrosc. 22, 217 (1991).
[CrossRef]

G. J. Puppels, F. F. M. Demul, C. Otto, J. Greve, M. Robertnicoud, D. J. Arndtjovin, and T. M. Jovin, Nature 347, 301 (1990).
[CrossRef] [PubMed]

Haber, K. S.

A. D. Gift, J. Y. Ma, K. S. Haber, B. L. McClain, and D. Ben-Amotz, J. Raman Spectrosc. 30, 757 (1999).
[CrossRef]

Hamaguchi, H.

Y. S. Huang, T. Karashima, M. Yamamoto, and H. Hamaguchi, Biochemistry 44, 10009 (2005).
[CrossRef] [PubMed]

Y. S. Huang, T. Karashima, M. Yamamoto, T. Ogura, and H. Hamaguchi, J. Raman Spectrosc. 35, 525 (2004).
[CrossRef]

M. Okuno, H. Kano, and H. Hamaguchi are preparing a manuscript to be called “Raman and CARS: which wins in living cell spectroscopy and imaging?”

Hashimoto, M.

Hell, S. W.

Hu, S. Z.

S. Z. Hu, I. K. Morris, J. P. Singh, K. M. Smith, and T. G. Spiro, J. Am. Chem. Soc. 115, 12446 (1993).
[CrossRef]

Huang, Y. S.

Y. S. Huang, T. Karashima, M. Yamamoto, and H. Hamaguchi, Biochemistry 44, 10009 (2005).
[CrossRef] [PubMed]

Y. S. Huang, T. Karashima, M. Yamamoto, T. Ogura, and H. Hamaguchi, J. Raman Spectrosc. 35, 525 (2004).
[CrossRef]

Ishida, H.

Jovin, T. M.

G. J. Puppels, F. F. M. Demul, C. Otto, J. Greve, M. Robertnicoud, D. J. Arndtjovin, and T. M. Jovin, Nature 347, 301 (1990).
[CrossRef] [PubMed]

Kaneko, T.

Kano, H.

M. Okuno, H. Kano, and H. Hamaguchi are preparing a manuscript to be called “Raman and CARS: which wins in living cell spectroscopy and imaging?”

Karashima, T.

Y. S. Huang, T. Karashima, M. Yamamoto, and H. Hamaguchi, Biochemistry 44, 10009 (2005).
[CrossRef] [PubMed]

Y. S. Huang, T. Karashima, M. Yamamoto, T. Ogura, and H. Hamaguchi, J. Raman Spectrosc. 35, 525 (2004).
[CrossRef]

Kawata, S.

Kee, T. W.

Kobayashi, M.

Kosugi, Y.

Lim, S. H.

J. Sung, B. C. Chen, and S. H. Lim, “Fast three-dimensional chemical imaging by interferometric multiplex coherent anti-Stokes Raman scattering microscopy,” J. Raman Spectrosc. (to be published).

Ma, J. Y.

A. D. Gift, J. Y. Ma, K. S. Haber, B. L. McClain, and D. Ben-Amotz, J. Raman Spectrosc. 30, 757 (1999).
[CrossRef]

J. Y. Ma and D. Ben-Amotz, Appl. Spectrosc. 51, 1845(1997).
[CrossRef]

McClain, B. L.

A. D. Gift, J. Y. Ma, K. S. Haber, B. L. McClain, and D. Ben-Amotz, J. Raman Spectrosc. 30, 757 (1999).
[CrossRef]

Minamikawa, T.

Morris, I. K.

S. Z. Hu, I. K. Morris, J. P. Singh, K. M. Smith, and T. G. Spiro, J. Am. Chem. Soc. 115, 12446 (1993).
[CrossRef]

Nakamura, O.

Ogilvie, J. P.

Ogura, T.

Y. S. Huang, T. Karashima, M. Yamamoto, T. Ogura, and H. Hamaguchi, J. Raman Spectrosc. 35, 525 (2004).
[CrossRef]

Okuno, M.

M. Okuno, H. Kano, and H. Hamaguchi are preparing a manuscript to be called “Raman and CARS: which wins in living cell spectroscopy and imaging?”

Olminkhof, J. H. F.

G. J. Puppels, W. Colier, J. H. F. Olminkhof, C. Otto, F. F. M. Demul, and J. Greve, J. Raman Spectrosc. 22, 217 (1991).
[CrossRef]

Otsuki, S.

Otto, C.

G. J. Puppels, W. Colier, J. H. F. Olminkhof, C. Otto, F. F. M. Demul, and J. Greve, J. Raman Spectrosc. 22, 217 (1991).
[CrossRef]

G. J. Puppels, F. F. M. Demul, C. Otto, J. Greve, M. Robertnicoud, D. J. Arndtjovin, and T. M. Jovin, Nature 347, 301 (1990).
[CrossRef] [PubMed]

Pick, R.

Puppels, G. J.

G. J. Puppels, W. Colier, J. H. F. Olminkhof, C. Otto, F. F. M. Demul, and J. Greve, J. Raman Spectrosc. 22, 217 (1991).
[CrossRef]

G. J. Puppels, F. F. M. Demul, C. Otto, J. Greve, M. Robertnicoud, D. J. Arndtjovin, and T. M. Jovin, Nature 347, 301 (1990).
[CrossRef] [PubMed]

Robertnicoud, M.

G. J. Puppels, F. F. M. Demul, C. Otto, J. Greve, M. Robertnicoud, D. J. Arndtjovin, and T. M. Jovin, Nature 347, 301 (1990).
[CrossRef] [PubMed]

Shimizu, M.

Singh, J. P.

S. Z. Hu, I. K. Morris, J. P. Singh, K. M. Smith, and T. G. Spiro, J. Am. Chem. Soc. 115, 12446 (1993).
[CrossRef]

Smith, K. M.

S. Z. Hu, I. K. Morris, J. P. Singh, K. M. Smith, and T. G. Spiro, J. Am. Chem. Soc. 115, 12446 (1993).
[CrossRef]

Spiro, T. G.

S. Z. Hu, I. K. Morris, J. P. Singh, K. M. Smith, and T. G. Spiro, J. Am. Chem. Soc. 115, 12446 (1993).
[CrossRef]

Sung, J.

J. Sung, B. C. Chen, and S. H. Lim, “Fast three-dimensional chemical imaging by interferometric multiplex coherent anti-Stokes Raman scattering microscopy,” J. Raman Spectrosc. (to be published).

Takamatsu, T.

Tanaami, T.

Tomosada, N.

Yamamoto, M.

Y. S. Huang, T. Karashima, M. Yamamoto, and H. Hamaguchi, Biochemistry 44, 10009 (2005).
[CrossRef] [PubMed]

Y. S. Huang, T. Karashima, M. Yamamoto, T. Ogura, and H. Hamaguchi, J. Raman Spectrosc. 35, 525 (2004).
[CrossRef]

Appl. Opt. (1)

Appl. Spectrosc. (1)

Biochemistry (1)

Y. S. Huang, T. Karashima, M. Yamamoto, and H. Hamaguchi, Biochemistry 44, 10009 (2005).
[CrossRef] [PubMed]

J. Am. Chem. Soc. (1)

S. Z. Hu, I. K. Morris, J. P. Singh, K. M. Smith, and T. G. Spiro, J. Am. Chem. Soc. 115, 12446 (1993).
[CrossRef]

J. Raman Spectrosc. (3)

Y. S. Huang, T. Karashima, M. Yamamoto, T. Ogura, and H. Hamaguchi, J. Raman Spectrosc. 35, 525 (2004).
[CrossRef]

G. J. Puppels, W. Colier, J. H. F. Olminkhof, C. Otto, F. F. M. Demul, and J. Greve, J. Raman Spectrosc. 22, 217 (1991).
[CrossRef]

A. D. Gift, J. Y. Ma, K. S. Haber, B. L. McClain, and D. Ben-Amotz, J. Raman Spectrosc. 30, 757 (1999).
[CrossRef]

Nature (1)

G. J. Puppels, F. F. M. Demul, C. Otto, J. Greve, M. Robertnicoud, D. J. Arndtjovin, and T. M. Jovin, Nature 347, 301 (1990).
[CrossRef] [PubMed]

Opt. Express (1)

Opt. Lett. (4)

Other (2)

J. Sung, B. C. Chen, and S. H. Lim, “Fast three-dimensional chemical imaging by interferometric multiplex coherent anti-Stokes Raman scattering microscopy,” J. Raman Spectrosc. (to be published).

M. Okuno, H. Kano, and H. Hamaguchi are preparing a manuscript to be called “Raman and CARS: which wins in living cell spectroscopy and imaging?”

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Figures (4)

Fig. 1
Fig. 1

Schematic (top and side views) of the multifocus confocal Raman microspectroscopic system.

Fig. 2
Fig. 2

(a) Lateral and (b) depth profiles of a single polystyrene bead with 356 nm diameter.

Fig. 3
Fig. 3

(a) CCD image consisting of 48 Raman spectra of budding yeast cells. (b) Correspondence between the spectrum number in (a) and the position of the focus under the microscope. Extracted Raman spectra at (c) 27 and (d) 30 in (a). Spectra are intensity-corrected by the fluorescence spectra of a Rhodamine 6G ethanol solution.

Fig. 4
Fig. 4

Raman images of budding yeast cells of the Raman bands at (a) 1446, (b) 1583, (c) 1602, and (d) 1655 cm 1 . The total acquisition time is approximately 20 s . The scale bar in the image is 4 μm .

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