Abstract

We propose a multifocus scanning technique in second-harmonic-generation (SHG) microscopy in which the SHG detection efficiency and the image acquisition rate are improved by several tens of times compared with typical single-focus scanning techniques. Because of a microlens array scanner, the laser beam of a mode-locked Ti:sapphire laser is split into 100 beamlets, and the same number of foci are formed in the specimen. Parallel scanning of the foci enables us to observe real-time images of the specimen. The imaging properties and the usefulness of the microscope are demonstrated by SHG images of living HeLa cells and rat cardiac myocytes.

© 2002 Optical Society of America

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  1. I. Freund, M. Deutsch, and A. Sprecher, Biophys. J. 50, 693 (1986).
  2. L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, Opt. Lett. 25, 320 (2000).
    [CrossRef]
  3. W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]
  4. I. Ben-Oren, G. Peleg, A. Lewis, B. Minke, and L. M. Loew, Biophys. J. 71, 1616 (1996).
  5. L. Moreaux, O. Sandre, Serge Charpak, M. Blanchard-Desce, and J. Mertz, Biophys. J. 80, 1568 (2001).
  6. P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).
  7. A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 57 (1996).
  8. K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, Opt. Commun. 174, 7 (2000).
    [CrossRef]
  9. J. Bewersdorf, R. Pick, and S. W. Hell, Opt. Lett. 23, 655 (1998).
    [CrossRef]
  10. V. Andresen, A. Egner, and S. W. Hell, J. Opt. Soc. Am. A 17, 1192 (2000).
    [CrossRef]
  11. K. König, P. T. C. So, W. W. Mantulin, and E. Gratton, Opt. Lett. 22, 135 (1997).
    [CrossRef]

2001 (1)

L. Moreaux, O. Sandre, Serge Charpak, M. Blanchard-Desce, and J. Mertz, Biophys. J. 80, 1568 (2001).

2000 (3)

1999 (1)

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).

1998 (1)

1997 (1)

1996 (2)

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 57 (1996).

I. Ben-Oren, G. Peleg, A. Lewis, B. Minke, and L. M. Loew, Biophys. J. 71, 1616 (1996).

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

1986 (1)

I. Freund, M. Deutsch, and A. Sprecher, Biophys. J. 50, 693 (1986).

Abe, M.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 57 (1996).

Andresen, V.

Ben-Oren, I.

I. Ben-Oren, G. Peleg, A. Lewis, B. Minke, and L. M. Loew, Biophys. J. 71, 1616 (1996).

Bewersdorf, J.

Blanchard-Desce, M.

L. Moreaux, O. Sandre, Serge Charpak, M. Blanchard-Desce, and J. Mertz, Biophys. J. 80, 1568 (2001).

L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, Opt. Lett. 25, 320 (2000).
[CrossRef]

Campagnola, P. J.

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).

Charpak, Serge

L. Moreaux, O. Sandre, Serge Charpak, M. Blanchard-Desce, and J. Mertz, Biophys. J. 80, 1568 (2001).

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Deutsch, M.

I. Freund, M. Deutsch, and A. Sprecher, Biophys. J. 50, 693 (1986).

Egner, A.

Freund, I.

I. Freund, M. Deutsch, and A. Sprecher, Biophys. J. 50, 693 (1986).

Fujita, K.

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, Opt. Commun. 174, 7 (2000).
[CrossRef]

Gratton, E.

Hell, S. W.

Ichihara, A.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 57 (1996).

Isozaki, K.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 57 (1996).

Kaneko, T.

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, Opt. Commun. 174, 7 (2000).
[CrossRef]

Kawata, S.

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, Opt. Commun. 174, 7 (2000).
[CrossRef]

König, K.

Kosugi, Y.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 57 (1996).

Lewis, A.

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).

I. Ben-Oren, G. Peleg, A. Lewis, B. Minke, and L. M. Loew, Biophys. J. 71, 1616 (1996).

Loew, L. M.

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).

I. Ben-Oren, G. Peleg, A. Lewis, B. Minke, and L. M. Loew, Biophys. J. 71, 1616 (1996).

Mantulin, W. W.

Mertz, J.

L. Moreaux, O. Sandre, Serge Charpak, M. Blanchard-Desce, and J. Mertz, Biophys. J. 80, 1568 (2001).

L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, Opt. Lett. 25, 320 (2000).
[CrossRef]

Mikuriya, K.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 57 (1996).

Minke, B.

I. Ben-Oren, G. Peleg, A. Lewis, B. Minke, and L. M. Loew, Biophys. J. 71, 1616 (1996).

Moreaux, L.

L. Moreaux, O. Sandre, Serge Charpak, M. Blanchard-Desce, and J. Mertz, Biophys. J. 80, 1568 (2001).

L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, Opt. Lett. 25, 320 (2000).
[CrossRef]

Nakamura, O.

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, Opt. Commun. 174, 7 (2000).
[CrossRef]

Oyamada, M.

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, Opt. Commun. 174, 7 (2000).
[CrossRef]

Peleg, G.

I. Ben-Oren, G. Peleg, A. Lewis, B. Minke, and L. M. Loew, Biophys. J. 71, 1616 (1996).

Pick, R.

Sandre, O.

L. Moreaux, O. Sandre, Serge Charpak, M. Blanchard-Desce, and J. Mertz, Biophys. J. 80, 1568 (2001).

L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, Opt. Lett. 25, 320 (2000).
[CrossRef]

So, P. T. C.

Sprecher, A.

I. Freund, M. Deutsch, and A. Sprecher, Biophys. J. 50, 693 (1986).

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Sugiyama, Y.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 57 (1996).

Takamatsu, T.

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, Opt. Commun. 174, 7 (2000).
[CrossRef]

Tanaami, T.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 57 (1996).

Uemura, I.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 57 (1996).

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Wei, M.

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).

Bioimages (1)

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 57 (1996).

Biophys. J. (4)

I. Ben-Oren, G. Peleg, A. Lewis, B. Minke, and L. M. Loew, Biophys. J. 71, 1616 (1996).

L. Moreaux, O. Sandre, Serge Charpak, M. Blanchard-Desce, and J. Mertz, Biophys. J. 80, 1568 (2001).

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).

I. Freund, M. Deutsch, and A. Sprecher, Biophys. J. 50, 693 (1986).

J. Opt. Soc. Am. A (1)

Opt. Commun. (1)

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, Opt. Commun. 174, 7 (2000).
[CrossRef]

Opt. Lett. (3)

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

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Figures (4)

Fig. 1
Fig. 1

Optical setup of a SHG microscope with a microlens array scanner.

Fig. 2
Fig. 2

(a), (b) SH images of HeLa cells labeled with RH237. (c), (d) Two-photon fluorescence images of the same sample. Each image was averaged from 16 images obtained with an exposure of 33 ms. The total incident power was 150 mW at the sample. The scale bar is 10 µm.

Fig. 3
Fig. 3

(a) Spectrum of SHG emission from RH237 on the plasma membrane of the HeLa cells. (b) The spectrum of fluorescence emission was also observed by removal of Filter 2 (b).

Fig. 4
Fig. 4

SH images of rat cardiac myocytes under contraction. Each image shows the cells (a) before contraction, (b) after contraction has started, (c) contracted the most, (d) after relaxation, (e) during the next contraction, and (f) when relaxed. Six images were chosen from a time series of SH images recorded at video rate. The exposure time for each image was 33 ms. The cells were labeled with RH237. The total incident power was 170 mW at the sample. The scale bar is 2 µm.

Equations (1)

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P2ω=ΓSHGPω/sτf2τf,

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