Abstract

We demonstrate a fast, direct wavefront-sensing method for dynamic in vivo adaptive optical two-photon microscopy. By using a Shack–Hartmann wavefront sensor and open-loop control, the system provides high-speed wavefront measurement and correction. To measure the wavefront in the middle of a Drosophila embryo at early stages, autofluorescence from endogenous fluorophores in the yolk were used as reference guide stars. The method was tested through live imaging of a Drosophila embryo. The aberration in the middle of the embryo was measured directly for the first time. After correction, the contrast and signal intensity of the structure in the middle of the embryo was improved.

© 2013 Optical Society of America

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2012 (1)

2011 (4)

2009 (2)

2008 (1)

M. Mavrakis, R. Rikhy, M. Lilly, and J. Lippincott-Schwartz, Curr. Protoc. Cell Biol. 4, 18 (2008).
[CrossRef]

2007 (1)

K. M. Morzinski, K. B. W. Harpsee, D. T. Gavel, and S. M. Ammons, Proc. SPIE 6467, 64670G (2007).
[CrossRef]

2006 (1)

2005 (1)

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef]

2003 (2)

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).

P. Marsh, D. Burns, and J. Girkin, Opt. Express 11, 1123 (2003).
[CrossRef]

2000 (1)

1999 (1)

Albert, O.

Ammons, S. M.

K. M. Morzinski, K. B. W. Harpsee, D. T. Gavel, and S. M. Ammons, Proc. SPIE 6467, 64670G (2007).
[CrossRef]

Andilla, J.

Artigas, D.

Ashburner, M.

M. Ashburner, K. G. Golic, and R. S. Hawley, Drosophila: A Laboratory Handbook, 2nd ed. (Cold Spring Harbor Laboratory, 2004).

Aviles-Espinosa, R.

Azucena, O.

Beaurepaire, E.

Betzig, E.

N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141 (2009).
[CrossRef]

Booth, M. J.

Botcherby, E. J.

Burns, D.

Chen, D. C.

Christie, R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).

Crest, J.

Dainty, J. C.

Debarre, D.

Denk, W.

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef]

Fernandez, B.

Fu, M.

Garcia, D.

Gavel, D.

Gavel, D. T.

K. M. Morzinski, K. B. W. Harpsee, D. T. Gavel, and S. M. Ammons, Proc. SPIE 6467, 64670G (2007).
[CrossRef]

Girkin, J.

Golic, K. G.

M. Ashburner, K. G. Golic, and R. S. Hawley, Drosophila: A Laboratory Handbook, 2nd ed. (Cold Spring Harbor Laboratory, 2004).

Haro, L. D. S.

Harpsee, K. B. W.

K. M. Morzinski, K. B. W. Harpsee, D. T. Gavel, and S. M. Ammons, Proc. SPIE 6467, 64670G (2007).
[CrossRef]

Hawley, R. S.

M. Ashburner, K. G. Golic, and R. S. Hawley, Drosophila: A Laboratory Handbook, 2nd ed. (Cold Spring Harbor Laboratory, 2004).

Helmchen, F.

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef]

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).

Ji, N.

N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141 (2009).
[CrossRef]

Joffre, M.

Kotadia, S.

Kubby, J.

Kubby, J. A.

J. A. Kubby, Adaptive Optics for Biological Imaging (CRC Press, 2013).

Levecq, X.

Lilly, M.

M. Mavrakis, R. Rikhy, M. Lilly, and J. Lippincott-Schwartz, Curr. Protoc. Cell Biol. 4, 18 (2008).
[CrossRef]

Lippincott-Schwartz, J.

M. Mavrakis, R. Rikhy, M. Lilly, and J. Lippincott-Schwartz, Curr. Protoc. Cell Biol. 4, 18 (2008).
[CrossRef]

Loza-Alvarez, P.

Marsh, P.

Martin, J.

Mavrakis, M.

M. Mavrakis, R. Rikhy, M. Lilly, and J. Lippincott-Schwartz, Curr. Protoc. Cell Biol. 4, 18 (2008).
[CrossRef]

Milkie, D. E.

N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141 (2009).
[CrossRef]

Morzinski, K. M.

K. M. Morzinski, K. B. W. Harpsee, D. T. Gavel, and S. M. Ammons, Proc. SPIE 6467, 64670G (2007).
[CrossRef]

Mourou, G.

Nieto, M.

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).

Norris, T. B.

Ogilvie, J. P.

Olarte, O. E.

Olivier, S.

Porcar-Guezenec, R.

Reinig, M.

Rikhy, R.

M. Mavrakis, R. Rikhy, M. Lilly, and J. Lippincott-Schwartz, Curr. Protoc. Cell Biol. 4, 18 (2008).
[CrossRef]

Sherman, L.

Solinas, X.

Srinivas, S.

Sullivan, W.

Tao, X.

Vdovin, G.

Watanabe, T.

Webb, W. W.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).

Williams, R. M.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).

Wilson, T.

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).

Zuo, Y.

Biomed. Opt. Express (1)

Curr. Protoc. Cell Biol. (1)

M. Mavrakis, R. Rikhy, M. Lilly, and J. Lippincott-Schwartz, Curr. Protoc. Cell Biol. 4, 18 (2008).
[CrossRef]

Nat. Methods (2)

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef]

N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141 (2009).
[CrossRef]

Opt. Express (3)

Opt. Lett. (6)

Proc. Natl. Acad. Sci. USA (1)

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).

Proc. SPIE (1)

K. M. Morzinski, K. B. W. Harpsee, D. T. Gavel, and S. M. Ammons, Proc. SPIE 6467, 64670G (2007).
[CrossRef]

Other (2)

M. Ashburner, K. G. Golic, and R. S. Hawley, Drosophila: A Laboratory Handbook, 2nd ed. (Cold Spring Harbor Laboratory, 2004).

J. A. Kubby, Adaptive Optics for Biological Imaging (CRC Press, 2013).

Supplementary Material (1)

» Media 1: AVI (545 KB)     

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Figures (4)

Fig. 1.
Fig. 1.

Schematic of AO two-photon microscope: L, lens; F, filter; M, mirror flipper; BE, beam expender; EOM, electro-optic modulator; DM, deformable mirror; DB, dichroic beam splitters; SHWS, Shack–Hartmann wavefront sensor; PMT, photomultiplier tube. See text for details.

Fig. 2.
Fig. 2.

Two-photon images of a Drosophila embryo at a depth of 51 μm with excitation wavelengths of (a) 1000 nm, (b) 920 nm, and (c) 800 nm. The wavefront (d) at the middle of embryo is measured by a SHWS in 30 ms using an autofluorescent guide star. During wavefront sensing, the laser illuminates the intrinsic fluorophores indicated by an arrow shown in (c). The scale bars are 10 μm.

Fig. 3.
Fig. 3.

Two-photon imaging of a live Drosophila embryo at a depth of 51 μm. The images of yolk autofluorescence before (a) and after correction. The RFP labeled nuclei structure before (a) and after (b) correction. The intensity profiles (e) along lines in (c) and (d) before (blue) and after (red) correction. The scale bars are 10 μm.

Fig. 4.
Fig. 4.

RMS wavefront change during 300 s with a time resolution of 10 s (Media 1). The phase maps of the wavefront change at 30, 50, and 150 s are shown at the top.

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4ω(r)=fP(r)/D,

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