Abstract

We present an ultrafast, large-field multiphoton excitation fluorescence microscope with high lateral and axial resolutions based on a two-dimensional (2-D) acousto-optical deflector (AOD) scanner and spatial light modulator (SLM). When a phase-only SLM is used to shape the near-infrared light from a mode-locked titanium:sapphire laser into a multifocus array including the 0-order beam, a 136μm×136μm field of view is achieved with a 60× objective using a 2-D AOD scanner without any mechanical scan element. The two-photon fluorescence image of a neuronal network that was obtained using this system demonstrates that our microscopy permits observation of dynamic biological events in a large field with high-temporal and -spatial resolution.

© 2012 Optical Society of America

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  1. W. R. Zipfel, R. M. Williams, and W. W. Webb, Nat. Biotechnol. 21, 1369 (2003).
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    [CrossRef]

2012 (1)

2010 (1)

B. E. Losavio, V. Iyer, S. Patel, and P. Saggau, J. Neural Eng. 7, 045002 (2010).
[CrossRef]

2009 (1)

M. Agour, E. Kolenovic, C. Falldorf, and C. von Kopylow, J. Opt. Pure Appl. Opt. 11, 105405 (2009).
[CrossRef]

2008 (2)

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

N. Ji, J. C. Magee, and E. Betzig, Nat. Methods 5, 197 (2008).
[CrossRef]

2006 (3)

R. Salomé, Y. Kremer, S. Dieudonné, J.-F. Léger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, J. Neurosci. Methods 154, 161 (2006).
[CrossRef]

S. Zeng, X. Lv, C. Zhan, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, Opt. Lett. 31, 1091 (2006).
[CrossRef]

M. J. Escuti and W. M. Jones, Proc. SPIE 6332, 63320M (2006).
[CrossRef]

2005 (1)

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

2004 (1)

R. D. Roorda, T. M. Hohl, R. Toledo-Crow, and G. Miesenböck, J. Neurophysiol. 92, 609 (2004).
[CrossRef]

2003 (2)

W. R. Zipfel, R. M. Williams, and W. W. Webb, Nat. Biotechnol. 21, 1369 (2003).
[CrossRef]

V. Iyer, Br. E. Losavio, and P. Saggau, J. Biomed. Opt. 8, 460 (2003).
[CrossRef]

2002 (1)

J. D. Lechleiter, D.-T. Lin, and I. Sieneart, Biophys. J. 83, 2292 (2002).
[CrossRef]

2001 (1)

1998 (1)

1990 (1)

S. R. Goldstein, T. Hubin, S. Rosenthal, and C. Washburn, J. Microsc. 157, 29 (1990).
[CrossRef]

Agour, M.

M. Agour, E. Kolenovic, C. Falldorf, and C. von Kopylow, J. Opt. Pure Appl. Opt. 11, 105405 (2009).
[CrossRef]

Andresen, V.

Araya, R.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Betzig, E.

N. Ji, J. C. Magee, and E. Betzig, Nat. Methods 5, 197 (2008).
[CrossRef]

Bewersdorf, J.

Bourdieu, L.

R. Salomé, Y. Kremer, S. Dieudonné, J.-F. Léger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, J. Neurosci. Methods 154, 161 (2006).
[CrossRef]

Chatenay, D.

R. Salomé, Y. Kremer, S. Dieudonné, J.-F. Léger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, J. Neurosci. Methods 154, 161 (2006).
[CrossRef]

Chen, W. R.

Dieudonné, S.

R. Salomé, Y. Kremer, S. Dieudonné, J.-F. Léger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, J. Neurosci. Methods 154, 161 (2006).
[CrossRef]

Egner, A.

Escuti, M. J.

M. J. Escuti and W. M. Jones, Proc. SPIE 6332, 63320M (2006).
[CrossRef]

Falldorf, C.

M. Agour, E. Kolenovic, C. Falldorf, and C. von Kopylow, J. Opt. Pure Appl. Opt. 11, 105405 (2009).
[CrossRef]

Gao, B.

Goldstein, S. R.

S. R. Goldstein, T. Hubin, S. Rosenthal, and C. Washburn, J. Microsc. 157, 29 (1990).
[CrossRef]

Hell, S. W.

Hohl, T. M.

R. D. Roorda, T. M. Hohl, R. Toledo-Crow, and G. Miesenböck, J. Neurophysiol. 92, 609 (2004).
[CrossRef]

Hubin, T.

S. R. Goldstein, T. Hubin, S. Rosenthal, and C. Washburn, J. Microsc. 157, 29 (1990).
[CrossRef]

Hüttman, G.

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Iyer, V.

B. E. Losavio, V. Iyer, S. Patel, and P. Saggau, J. Neural Eng. 7, 045002 (2010).
[CrossRef]

V. Iyer, Br. E. Losavio, and P. Saggau, J. Biomed. Opt. 8, 460 (2003).
[CrossRef]

Jacques, S. L.

Ji, N.

N. Ji, J. C. Magee, and E. Betzig, Nat. Methods 5, 197 (2008).
[CrossRef]

Jones, W. M.

M. J. Escuti and W. M. Jones, Proc. SPIE 6332, 63320M (2006).
[CrossRef]

Kolenovic, E.

M. Agour, E. Kolenovic, C. Falldorf, and C. von Kopylow, J. Opt. Pure Appl. Opt. 11, 105405 (2009).
[CrossRef]

Kremer, Y.

R. Salomé, Y. Kremer, S. Dieudonné, J.-F. Léger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, J. Neurosci. Methods 154, 161 (2006).
[CrossRef]

Krichevsky, O.

R. Salomé, Y. Kremer, S. Dieudonné, J.-F. Léger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, J. Neurosci. Methods 154, 161 (2006).
[CrossRef]

Lechleiter, J. D.

J. D. Lechleiter, D.-T. Lin, and I. Sieneart, Biophys. J. 83, 2292 (2002).
[CrossRef]

Léger, J.-F.

R. Salomé, Y. Kremer, S. Dieudonné, J.-F. Léger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, J. Neurosci. Methods 154, 161 (2006).
[CrossRef]

Lin, D.-T.

J. D. Lechleiter, D.-T. Lin, and I. Sieneart, Biophys. J. 83, 2292 (2002).
[CrossRef]

Liu, H.

Losavio, B. E.

B. E. Losavio, V. Iyer, S. Patel, and P. Saggau, J. Neural Eng. 7, 045002 (2010).
[CrossRef]

Losavio, Br. E.

V. Iyer, Br. E. Losavio, and P. Saggau, J. Biomed. Opt. 8, 460 (2003).
[CrossRef]

Luo, Q.

Lv, X.

Magee, J. C.

N. Ji, J. C. Magee, and E. Betzig, Nat. Methods 5, 197 (2008).
[CrossRef]

Miesenböck, G.

R. D. Roorda, T. M. Hohl, R. Toledo-Crow, and G. Miesenböck, J. Neurophysiol. 92, 609 (2004).
[CrossRef]

Nikolenko, V.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Niu, H.

Noack, J.

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Paltauf, G.

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Patel, S.

B. E. Losavio, V. Iyer, S. Patel, and P. Saggau, J. Neural Eng. 7, 045002 (2010).
[CrossRef]

Peng, X.

Peterka, D. S.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Pick, R.

Qin, W.

Roorda, R. D.

R. D. Roorda, T. M. Hohl, R. Toledo-Crow, and G. Miesenböck, J. Neurophysiol. 92, 609 (2004).
[CrossRef]

Rosenthal, S.

S. R. Goldstein, T. Hubin, S. Rosenthal, and C. Washburn, J. Microsc. 157, 29 (1990).
[CrossRef]

Saggau, P.

B. E. Losavio, V. Iyer, S. Patel, and P. Saggau, J. Neural Eng. 7, 045002 (2010).
[CrossRef]

V. Iyer, Br. E. Losavio, and P. Saggau, J. Biomed. Opt. 8, 460 (2003).
[CrossRef]

Salomé, R.

R. Salomé, Y. Kremer, S. Dieudonné, J.-F. Léger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, J. Neurosci. Methods 154, 161 (2006).
[CrossRef]

Shao, Y.

Sieneart, I.

J. D. Lechleiter, D.-T. Lin, and I. Sieneart, Biophys. J. 83, 2292 (2002).
[CrossRef]

Toledo-Crow, R.

R. D. Roorda, T. M. Hohl, R. Toledo-Crow, and G. Miesenböck, J. Neurophysiol. 92, 609 (2004).
[CrossRef]

Vogel, A.

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

von Kopylow, C.

M. Agour, E. Kolenovic, C. Falldorf, and C. von Kopylow, J. Opt. Pure Appl. Opt. 11, 105405 (2009).
[CrossRef]

Washburn, C.

S. R. Goldstein, T. Hubin, S. Rosenthal, and C. Washburn, J. Microsc. 157, 29 (1990).
[CrossRef]

Watson, B. O.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, Nat. Biotechnol. 21, 1369 (2003).
[CrossRef]

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, Nat. Biotechnol. 21, 1369 (2003).
[CrossRef]

Woodruff, A.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Wyart, C.

R. Salomé, Y. Kremer, S. Dieudonné, J.-F. Léger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, J. Neurosci. Methods 154, 161 (2006).
[CrossRef]

Xiong, W.

Yuste, R.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Zeng, S.

Zhan, C.

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, Nat. Biotechnol. 21, 1369 (2003).
[CrossRef]

Appl. Phys. B (1)

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Biophys. J. (1)

J. D. Lechleiter, D.-T. Lin, and I. Sieneart, Biophys. J. 83, 2292 (2002).
[CrossRef]

Front. Neural Circuits (1)

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

J. Biomed. Opt. (1)

V. Iyer, Br. E. Losavio, and P. Saggau, J. Biomed. Opt. 8, 460 (2003).
[CrossRef]

J. Microsc. (1)

S. R. Goldstein, T. Hubin, S. Rosenthal, and C. Washburn, J. Microsc. 157, 29 (1990).
[CrossRef]

J. Neural Eng. (1)

B. E. Losavio, V. Iyer, S. Patel, and P. Saggau, J. Neural Eng. 7, 045002 (2010).
[CrossRef]

J. Neurophysiol. (1)

R. D. Roorda, T. M. Hohl, R. Toledo-Crow, and G. Miesenböck, J. Neurophysiol. 92, 609 (2004).
[CrossRef]

J. Neurosci. Methods (1)

R. Salomé, Y. Kremer, S. Dieudonné, J.-F. Léger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, J. Neurosci. Methods 154, 161 (2006).
[CrossRef]

J. Opt. Pure Appl. Opt. (1)

M. Agour, E. Kolenovic, C. Falldorf, and C. von Kopylow, J. Opt. Pure Appl. Opt. 11, 105405 (2009).
[CrossRef]

Nat. Biotechnol. (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, Nat. Biotechnol. 21, 1369 (2003).
[CrossRef]

Nat. Methods (1)

N. Ji, J. C. Magee, and E. Betzig, Nat. Methods 5, 197 (2008).
[CrossRef]

Opt. Lett. (4)

Proc. SPIE (1)

M. J. Escuti and W. M. Jones, Proc. SPIE 6332, 63320M (2006).
[CrossRef]

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Figures (4)

Fig. 1.
Fig. 1.

Schematic of the fast, large FOV multiphoton microscope with an SLM and a 2-D AOD scanner. The prism is for compensating the dispersion introduced by AODs. An uncoated, 45 deg-tilted SF11 prism with an apex angle of 60 deg is used to compensate for the temporal and spatial dispersion of the 2-D AODs. The total path length between the prism and the 2-D AODs is 18.5 cm. The pulse width is 125 fs after AODs. M1 and M2 are beam-steering mirrors. The focal lengths of L1 and L2 are 30 and 100 mm, respectively. The SLM on the focal plane of the Fourier lens modulates the incidence beam to generate a multifocus array including the 0-order beam on the focal plane of the tube lens. The focal length of the tube lens is 180 mm. The distance from the objective back focal plane (BFP) to the SLM is 760 mm. The polarization of light can be tuned by the λ/2 plate. (Laser, an fs laser; M1, M2, and M3: gold mirror; Camera, EMCCD).

Fig. 2.
Fig. 2.

Design of a 3×3 multifocus array, (a) multifocus array of the high-order diffraction beams, (b) focal point of the 0-order diffraction beam, and (c) compound multifocus array.

Fig. 3.
Fig. 3.

Two-photon fluorescence intensity image of fresh pollen grains from the flower of the Yellow Leaf Oleander, obtained by recording the autofluorescence with an exposure time of 3 ms. Scale bar is 20 μm.

Fig. 4.
Fig. 4.

Two-photon fluorescence intensity image of a cultured neuronal network (3-day cultured chick forebrain neurons harvested at embryonic day 7) live-cell stained with DiO. Cell A, Cell B, and Cell C form a linear neuronal circuit connected by axons (yellow arrows). Scale bar is 20 μm.

Equations (2)

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FOV=fsΔθmaxM,
η+1=12[1S]sin2(πΔndλ),

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