Abstract

We resolve the classical conflict between parallelization and axial resolution in three-dimensional fluorescence microscopy through time-multiplexed multifocal multiphoton excitation. A rotating array of microlenses on a disk splits ultrafast laser pulses in such a way that an array of high-aperture foci are created in the sample. Two rigidly mounted corotating glass disks with suitable arrays of holes ensure that adjacent foci illuminate the sample at different time points. Recordings of biological specimens demonstrate elimination of out-of-focus haze for densely packed foci and concomitant substantial improvement of contrast and resolution.

© 2001 Optical Society of America

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2000

1998

J. Bewersdorf, R. Pick, and S. W. Hell, Opt. Lett. 23, 655 (1998).
[CrossRef]

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford) 192, 217 (1998).
[CrossRef]

M. Straub and S. W. Hell, Appl. Phys. Lett. 73, 1769 (1998).
[CrossRef]

Q. S. Hanley, P. J. Verveer, and T. M. Jovin, Appl. Spectrosc. 52, 783 (1998).
[CrossRef]

1996

1990

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

1988

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (1988).
[CrossRef]

1968

Achi, R.

Bewersdorf, J.

Brakenhoff, G. J.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford) 192, 217 (1998).
[CrossRef]

Buist, A. H.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford) 192, 217 (1998).
[CrossRef]

Corle, T. R.

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (1988).
[CrossRef]

Denk, W.

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

Egger, M. D.

Egner, A.

Fewer, D. T.

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, and J. Hegarty, J. Microsc. (Oxford) 184, 95 (1996).
[CrossRef]

Galambos, R.

Hadravsky, M.

Hanley, Q. S.

Hegarty, J.

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, and J. Hegarty, J. Microsc. (Oxford) 184, 95 (1996).
[CrossRef]

Hell, S. W.

Hewlett, S. J.

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, and J. Hegarty, J. Microsc. (Oxford) 184, 95 (1996).
[CrossRef]

Jovin, T. M.

Juskaitis, R.

Kino, G. S.

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (1988).
[CrossRef]

Kozubek, M.

Krämer, R. N.

McCabe, E. M.

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, and J. Hegarty, J. Microsc. (Oxford) 184, 95 (1996).
[CrossRef]

Müller, M.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford) 192, 217 (1998).
[CrossRef]

Neil, M.

Ottewill, A. C.

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, and J. Hegarty, J. Microsc. (Oxford) 184, 95 (1996).
[CrossRef]

Petran, M.

Pick, R.

Squier, J.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford) 192, 217 (1998).
[CrossRef]

Straub, M.

M. Straub and S. W. Hell, Appl. Phys. Lett. 73, 1769 (1998).
[CrossRef]

Strickler, J. H.

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

Tiziani, H. J.

Verveer, P. J.

Webb, W. W.

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

Wiegers, L.

Wilson, T.

Xiao, G. Q.

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (1988).
[CrossRef]

Appl. Opt.

Appl. Phys. Lett.

M. Straub and S. W. Hell, Appl. Phys. Lett. 73, 1769 (1998).
[CrossRef]

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (1988).
[CrossRef]

Appl. Spectrosc.

J. Microsc. (Oxford)

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford) 192, 217 (1998).
[CrossRef]

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, and J. Hegarty, J. Microsc. (Oxford) 184, 95 (1996).
[CrossRef]

J. Opt. Soc. Am.

J. Opt. Soc. Am. A

Opt. Lett.

Science

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

Time-multiplexed MMM. An array of microlenses (ML) on a rotating disk splits the beam of a mode-locked laser into beamlets, producing an array of diffraction-limited foci in the focal plane inside the specimen. Inset, temporal delay mask (TMX) placed behind the microlenses, ensuring that neighboring foci pass the focal plane at different time points. L1,L2, lenses; M, flip mirror; DM, dichroic mirror.

Fig. 2
Fig. 2

Disk (left) and photograph (right) of a fraction of the microlens array. The disk is rigidly attached to two glass disks featuring suitable arrays of holes. Three classes of temporal delay, A, B, and C, are produced, as indicated by the different gray values. The amplitude transmission is nearly the same for all classes. The effective interfocal distance d is increased by 3 to dTMX.

Fig. 3
Fig. 3

Fluorescence sea response Iseaz obtained with three different microlens arrays: (a) comparison of disk MMM-6.4, MMM-TMX-6.4, and MMM-TMX-4.7. (b) derivative Vz of the curves shown in (a).

Fig. 4
Fig. 4

Effect of TMX studied by 3D imaging of a spiky pollen grain. (a)–(c) xy images recorded at the same axial position with disk (a) MMM-6.4, (b) MMM-TMX-6.4, and (c) MMM-TMX-4.7. The respective volume-rendered 3D data are shown in (d)–(f). The color table has been chosen to highlight the haze around the spikes. TMX removes the haze, as demonstrated by the comparison between (a) and (b), as well as between (d) and (e). In (c) and (f) TMX permitted sectioning with a higher focal density, at a resolution similar to that of (a) and (d) but at higher speed.

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