Abstract

We present a real-time, direct-view multiphoton excitation fluorescence microscope that provides three-dimensional imaging at high resolution. Using a rotating microlens disk, we split the near-infrared light of a mode-locked titanium:sapphire laser into an array of beams that are transformed into an array of high-aperture foci at the object. We typically scan at 225 frames per second and image the fluorescence with a camera that reads out the images at video rate. For 1.4 aperture oil and 1.2 water immersion lenses at 780-nm excitation we obtained axial resolutions of 0.84 and 1.4 µm, respectively, which are similar to that of a single-beam two-photon microscope. Compared with the latter setup, our system represents a 40–100-fold increase in efficiency, or imaging speed. Moreover, it permits the observation with the eye of high-resolution two-photon images of (live) samples.

© 1998 Optical Society of America

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  1. W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]
  2. K. König, P. C. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).
  3. G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. 181, 253 (1996).
    [CrossRef] [PubMed]
  4. H. J. Tiziani, R. Achi, R. N. Krämer, and L. Wiegers, Appl. Opt. 35, 120 (1996).
    [CrossRef] [PubMed]
  5. A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 52 (1996).
    [CrossRef]
  6. M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. (to be published).
  7. S. W. Hell, P. E. Hänninen, A. Kuusisto, M. Schrader, and E. Soini, Opt. Commun. 117, 20 (1995).
    [CrossRef]
  8. S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
    [CrossRef] [PubMed]
  9. D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
    [CrossRef]

1996 (6)

K. König, P. C. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. 181, 253 (1996).
[CrossRef] [PubMed]

H. J. Tiziani, R. Achi, R. N. Krämer, and L. Wiegers, Appl. Opt. 35, 120 (1996).
[CrossRef] [PubMed]

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 52 (1996).
[CrossRef]

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

1995 (1)

S. W. Hell, P. E. Hänninen, A. Kuusisto, M. Schrader, and E. Soini, Opt. Commun. 117, 20 (1995).
[CrossRef]

1990 (1)

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

Abe, M.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 52 (1996).
[CrossRef]

Achi, R.

Athey, B.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. 181, 253 (1996).
[CrossRef] [PubMed]

Bahlmann, K.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Bliton, A. C.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. 181, 253 (1996).
[CrossRef] [PubMed]

Brakenhoff, G. J.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. 181, 253 (1996).
[CrossRef] [PubMed]

Centonze, V. E.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

Crittenden, S.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

Denk, W.

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

Gratton, E.

K. König, P. C. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

Gryczynski, I.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Hänninen, P. E.

S. W. Hell, P. E. Hänninen, A. Kuusisto, M. Schrader, and E. Soini, Opt. Commun. 117, 20 (1995).
[CrossRef]

Hell, S. W.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

S. W. Hell, P. E. Hänninen, A. Kuusisto, M. Schrader, and E. Soini, Opt. Commun. 117, 20 (1995).
[CrossRef]

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. (to be published).

Hofmann, U. G.

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. (to be published).

Ichihara, A.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 52 (1996).
[CrossRef]

Isozaki, K.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 52 (1996).
[CrossRef]

König, K.

K. König, P. C. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

Kosugi, Y.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 52 (1996).
[CrossRef]

Krämer, R. N.

Kuusisto, A.

S. W. Hell, P. E. Hänninen, A. Kuusisto, M. Schrader, and E. Soini, Opt. Commun. 117, 20 (1995).
[CrossRef]

Lakowicz, J. R.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Malak, H.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Mantulin, W. W.

K. König, P. C. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

Mikuriya, K.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 52 (1996).
[CrossRef]

Norris, T.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. 181, 253 (1996).
[CrossRef] [PubMed]

Schrader, M.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

S. W. Hell, P. E. Hänninen, A. Kuusisto, M. Schrader, and E. Soini, Opt. Commun. 117, 20 (1995).
[CrossRef]

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. (to be published).

So, P. C. T.

K. König, P. C. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

Soini, A.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Soini, E.

S. W. Hell, P. E. Hänninen, A. Kuusisto, M. Schrader, and E. Soini, Opt. Commun. 117, 20 (1995).
[CrossRef]

Squier, J.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. 181, 253 (1996).
[CrossRef] [PubMed]

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, 52 (1996).
[CrossRef]

Tanaami, T.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 52 (1996).
[CrossRef]

Tiziani, H. J.

Tromberg, B. J.

K. König, P. C. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

Uemura, I.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 52 (1996).
[CrossRef]

Wade, M. H.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. 181, 253 (1996).
[CrossRef] [PubMed]

Webb, W. W.

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

White, J.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

Wiegers, L.

Wokosin, D. L.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

Appl. Opt. (1)

Bioimages (1)

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, Bioimages 4, 52 (1996).
[CrossRef]

Bioimaging (1)

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

J. Biomed. Opt. (1)

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

J. Microsc. (2)

K. König, P. C. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. 181, 253 (1996).
[CrossRef] [PubMed]

Opt. Commun. (1)

S. W. Hell, P. E. Hänninen, A. Kuusisto, M. Schrader, and E. Soini, Opt. Commun. 117, 20 (1995).
[CrossRef]

Science (1)

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

Other (1)

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. (to be published).

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Figures (4)

Fig. 1
Fig. 1

Schematic of the MMM for real-time, direct-view nonlinear microscopy: L’s, lenses; ML, microlens disk; M, optional mirror; F, short-pass filter; DM, dichroic mirror. Inset, the rotating microlens array.

Fig. 2
Fig. 2

Axial response to an ultrathin fluorescent plane, revealing the axial resolution of the MMM for high-N.A. water and oil immersion lenses.

Fig. 3
Fig. 3

Two-photon image stack of a pollen grain consisting of 12 xy layers at 4µm axial distance. The imaging time for each layer was 100  ms; the stack was taken within 1.2  s. The pixel size is 62.5 nm×62.5 nm; the data are not processed. Alternatively, the images can be viewed with the eye.

Fig. 4
Fig. 4

xy images of live boar-sperm cells taken within 33  ms (30  images/s). Four typical images of a movie of 191 images are displayed. Note the movement of the sperm tail within the first 33  ms. The movie data are available on request.

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