Abstract

We describe a simple setup that allows depth of field switching at kilohertz rates in a nonlinear microscope. Beam profile and/or divergence are modulated using a tunable, acoustically driven gradient-index fluid lens. We demonstrate two modulation strategies, one based on fast varifocus scanning during each pixel and the other based on pseudo-Bessel beam excitation. Average beam shape is switched every line during scanning, resulting in the interlaced acquisition of two different images. We apply this approach to the simultaneous standard and 4.5×-extended depth-of-field imaging of developing embryos.

© 2009 Optical Society of America

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2008

2007

2006

P. Dufour, M. Piché, Y. De Koninck, and N. McCarthy, Appl. Opt. 45, 9246 (2006).
[CrossRef] [PubMed]

E. J. Botcherby, R. Juskaitis, and T. Wilson, Opt. Commun. 268, 253 (2006).
[CrossRef]

2005

D. McGloin and K. Dholakia, Contemp. Phys. 46, 15 (2005).
[CrossRef]

2004

K. A. Higginson, M. A. Costolo, and E. A. Rietman, Appl. Phys. Lett. 84, 843 (2004).
[CrossRef]

2002

1990

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

1987

J. Durnin, J. J. Miceli, and J. H. Eberly, Phys. Rev. Lett. 58, 1499 (1987).
[CrossRef] [PubMed]

Arnold, C. B.

Botcherby, E. J.

E. J. Botcherby, R. Juskaitis, and T. Wilson, Opt. Commun. 268, 253 (2006).
[CrossRef]

Campos, J.

Chen, Z.

Costolo, M. A.

K. A. Higginson, M. A. Costolo, and E. A. Rietman, Appl. Phys. Lett. 84, 843 (2004).
[CrossRef]

Davis, J. A.

De Koninck, Y.

Denk, W.

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

Dholakia, K.

D. McGloin and K. Dholakia, Contemp. Phys. 46, 15 (2005).
[CrossRef]

Ding, Z.

Dufour, P.

Durnin, J.

J. Durnin, J. J. Miceli, and J. H. Eberly, Phys. Rev. Lett. 58, 1499 (1987).
[CrossRef] [PubMed]

Eberly, J. H.

J. Durnin, J. J. Miceli, and J. H. Eberly, Phys. Rev. Lett. 58, 1499 (1987).
[CrossRef] [PubMed]

Higginson, K. A.

K. A. Higginson, M. A. Costolo, and E. A. Rietman, Appl. Phys. Lett. 84, 843 (2004).
[CrossRef]

Iemmi, C.

Juskaitis, R.

E. J. Botcherby, R. Juskaitis, and T. Wilson, Opt. Commun. 268, 253 (2006).
[CrossRef]

Lopez-Coronado, O.

McCarthy, N.

McGloin, D.

D. McGloin and K. Dholakia, Contemp. Phys. 46, 15 (2005).
[CrossRef]

McLeod, E.

Mermillod-Blondin, A.

Miceli, J. J.

J. Durnin, J. J. Miceli, and J. H. Eberly, Phys. Rev. Lett. 58, 1499 (1987).
[CrossRef] [PubMed]

Nelson, J. S.

Piché, M.

Ren, H.

Rietman, E. A.

K. A. Higginson, M. A. Costolo, and E. A. Rietman, Appl. Phys. Lett. 84, 843 (2004).
[CrossRef]

Strickler, J. H.

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

Tuvey, C. S.

Webb, W. W.

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

Wilson, T.

E. J. Botcherby, R. Juskaitis, and T. Wilson, Opt. Commun. 268, 253 (2006).
[CrossRef]

Yzuel, M. J.

Zhao, Y.

Appl. Opt.

Appl. Phys. Lett.

K. A. Higginson, M. A. Costolo, and E. A. Rietman, Appl. Phys. Lett. 84, 843 (2004).
[CrossRef]

Contemp. Phys.

D. McGloin and K. Dholakia, Contemp. Phys. 46, 15 (2005).
[CrossRef]

J. Appl. Phys.

E. McLeod and C. B. Arnold, J. Appl. Phys. 102, 033104 (2007).
[CrossRef]

Opt. Commun.

E. J. Botcherby, R. Juskaitis, and T. Wilson, Opt. Commun. 268, 253 (2006).
[CrossRef]

Opt. Lett.

Phys. Rev. Lett.

J. Durnin, J. J. Miceli, and J. H. Eberly, Phys. Rev. Lett. 58, 1499 (1987).
[CrossRef] [PubMed]

Science

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

Supplementary Material (1)

» Media 1: AVI (4311 KB)     

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Figures (4)

Fig. 1
Fig. 1

(a) Microscopy with acoustically modulated beams. TAG lens, tunable acoustic gradient-index lens (inset). XY, lateral scanning; dic, dichroic; PMT, photomultiplier; obj, 20 × 0.95     NA objective with underfilled pupil. (b) Principle of the interlaced acquisition with standard (off) and modulated (on) beams.

Fig. 2
Fig. 2

Point-spread functions (PSFs) obtained when using the TAG lens as a fast varifocus device. (a) Principle: beam divergence at the objective pupil is rapidly modulated during one pixel acquisition time. (b), (c) Numerical simulations of the instantaneous (b) and time-averaged (c) on-axis intensity distribution. (d) Experimental axial 2PEF PSFs recorded from 330 nm beads switches from 2.5 μ m (lens off) to 6 μ m (on) FWHM and exhibits a two-lobe distribution when applying large modulation amplitudes (see text).

Fig. 3
Fig. 3

TAG lens used as a pseudo-Bessel beam generator. (a), (b) Average intensity distribution at the back aperture of the objective with the lens off and on. (c), (d) ZX, 2PEF images of 330 nm fluorescent beads recorded with standard [(c), off)] and extended [(d), on] depth of field. (e) z profiles through several bead images. FWHM are 4.0 ± 0.2 μ m (off) and 18.3 ± 0.4 μ m (on). (f), (g) Beads in a 3D gel imaged with standard and extended depth of field. Scale bars, 3 μ m .

Fig. 4
Fig. 4

Two-photon imaging of a developing Drosophila embryo and of a pollen grain with simultaneous standard and extended depth of field. (a1), (a2) Images of a fixed pollen grain recorded at axial locations separated by 12 μ m . The standard (off) images provide optical slices, whereas the simultaneously recorded extended-field images (on) provide a more global view of the sample. (b1), (b2) (Media 1) Time-lapse imaging of a developing Drosophila embryo with GFP-labeled cell nuclei, during ventral furrow formation. The beam focus was kept just below the outer cells of the ventral side, as illustrated in (b1). The standard movie shows the invaginating ventral cells passing through the focal plane, whereas the simultaneous extended-depth movie provides a global view of lateral cell movements during furrow formation. See Media 1. Scale bars, 50 μ m .

Equations (2)

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n ( ρ , t ) = n 0 + ( n a n a ω 2 ρ 2 4 v 2 ) sin ( ω t ) ,
n ( ρ , t ) = n 0 + n a J 0 ( ω ρ v ) sin ( ω t ) .

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