Abstract

We demonstrate the association of third-harmonic generation (THG) microscopy and particle image velocimetry (PIV) analysis as a novel functional imaging technique for automated micrometer-scale characterization of morphogenetic movements in developing embryos. Using a combined two-photon-excited fluorescence and THG microscope, we characterize the optical properties of Drosophila embryos and show that sustained THG imaging does not perturb sensitive developmental dynamics. Velocimetric THG imaging provides a quantitative description of the dynamics of internal structures in unstained wild-type and mutant embryos.

© 2004 Optical Society of America

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  1. L. Solnica-Krezel and S. Eaton, Development 130, 4229 (2003).
    [CrossRef] [PubMed]
  2. M. Bate and A. Martinez-Arias, The Development of Drosophila melanogaster (Cold Spring Harbor Laboratory Press, New York, 1993).
  3. Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
    [CrossRef]
  4. D. Yelin and Y. Silberberg, Opt. Express 5, 196 (1999), http://www.opticsexpress.org .
    [CrossRef]
  5. S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, H.-J. Tsai, and C.-K. Sun, Opt. Express 11, 3093 (2003), http://www.opticsexpress.org .
    [CrossRef] [PubMed]
  6. J.-X. Cheng and X. S. Xie, J. Opt. Soc. Am. B 19, 1604 (2002).
    [CrossRef]
  7. M. A. Welte, S. P. Gross, M. Postner, S. M. Block, and E. F. Wieschaus, Cell 92, 547 (1998).
    [CrossRef] [PubMed]
  8. M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
    [CrossRef]
  9. A. Royou, C. Field, J. C. Sisson, W. Sullivan, and R. Karess, Mol. Biol. Cell 15, 838 (2004).
    [CrossRef]
  10. M. Raffel, C. Willert, and J. Kompenhans, Particle Image Velocimetry: A Practical Guide (Springer-Verlag, Berlin, 1998).
    [CrossRef]
  11. J. K. Sveen, “An introduction to MatPIV v. 1.6.1,” eprint series (Department of Mathematics, University of Oslo, Oslo, Norway, 2004).

2004 (1)

A. Royou, C. Field, J. C. Sisson, W. Sullivan, and R. Karess, Mol. Biol. Cell 15, 838 (2004).
[CrossRef]

2003 (2)

2002 (1)

2001 (1)

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

1999 (1)

1998 (1)

M. A. Welte, S. P. Gross, M. Postner, S. M. Block, and E. F. Wieschaus, Cell 92, 547 (1998).
[CrossRef] [PubMed]

1997 (1)

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Barad, Y.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Bate, M.

M. Bate and A. Martinez-Arias, The Development of Drosophila melanogaster (Cold Spring Harbor Laboratory Press, New York, 1993).

Beaurepaire, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

Block, S. M.

M. A. Welte, S. P. Gross, M. Postner, S. M. Block, and E. F. Wieschaus, Cell 92, 547 (1998).
[CrossRef] [PubMed]

Chaigneau, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

Charpak, S.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

Chen, S.-Y.

Cheng, J.-X.

Chu, S.-W.

Eaton, S.

L. Solnica-Krezel and S. Eaton, Development 130, 4229 (2003).
[CrossRef] [PubMed]

Eisenberg, H.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Field, C.

A. Royou, C. Field, J. C. Sisson, W. Sullivan, and R. Karess, Mol. Biol. Cell 15, 838 (2004).
[CrossRef]

Gross, S. P.

M. A. Welte, S. P. Gross, M. Postner, S. M. Block, and E. F. Wieschaus, Cell 92, 547 (1998).
[CrossRef] [PubMed]

Horowitz, M.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Karess, R.

A. Royou, C. Field, J. C. Sisson, W. Sullivan, and R. Karess, Mol. Biol. Cell 15, 838 (2004).
[CrossRef]

Kompenhans, J.

M. Raffel, C. Willert, and J. Kompenhans, Particle Image Velocimetry: A Practical Guide (Springer-Verlag, Berlin, 1998).
[CrossRef]

Lin, C.-Y.

Liu, T.-M.

Martinez-Arias, A.

M. Bate and A. Martinez-Arias, The Development of Drosophila melanogaster (Cold Spring Harbor Laboratory Press, New York, 1993).

Mertz, J.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

Oheim, M.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

Postner, M.

M. A. Welte, S. P. Gross, M. Postner, S. M. Block, and E. F. Wieschaus, Cell 92, 547 (1998).
[CrossRef] [PubMed]

Raffel, M.

M. Raffel, C. Willert, and J. Kompenhans, Particle Image Velocimetry: A Practical Guide (Springer-Verlag, Berlin, 1998).
[CrossRef]

Royou, A.

A. Royou, C. Field, J. C. Sisson, W. Sullivan, and R. Karess, Mol. Biol. Cell 15, 838 (2004).
[CrossRef]

Silberberg, Y.

D. Yelin and Y. Silberberg, Opt. Express 5, 196 (1999), http://www.opticsexpress.org .
[CrossRef]

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Sisson, J. C.

A. Royou, C. Field, J. C. Sisson, W. Sullivan, and R. Karess, Mol. Biol. Cell 15, 838 (2004).
[CrossRef]

Solnica-Krezel, L.

L. Solnica-Krezel and S. Eaton, Development 130, 4229 (2003).
[CrossRef] [PubMed]

Sullivan, W.

A. Royou, C. Field, J. C. Sisson, W. Sullivan, and R. Karess, Mol. Biol. Cell 15, 838 (2004).
[CrossRef]

Sun, C.-K.

Sveen, J. K.

J. K. Sveen, “An introduction to MatPIV v. 1.6.1,” eprint series (Department of Mathematics, University of Oslo, Oslo, Norway, 2004).

Tsai, H.-J.

Tsai, T.-H.

Welte, M. A.

M. A. Welte, S. P. Gross, M. Postner, S. M. Block, and E. F. Wieschaus, Cell 92, 547 (1998).
[CrossRef] [PubMed]

Wieschaus, E. F.

M. A. Welte, S. P. Gross, M. Postner, S. M. Block, and E. F. Wieschaus, Cell 92, 547 (1998).
[CrossRef] [PubMed]

Willert, C.

M. Raffel, C. Willert, and J. Kompenhans, Particle Image Velocimetry: A Practical Guide (Springer-Verlag, Berlin, 1998).
[CrossRef]

Xie, X. S.

Yelin, D.

Appl. Phys. Lett. (1)

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Cell (1)

M. A. Welte, S. P. Gross, M. Postner, S. M. Block, and E. F. Wieschaus, Cell 92, 547 (1998).
[CrossRef] [PubMed]

Development (1)

L. Solnica-Krezel and S. Eaton, Development 130, 4229 (2003).
[CrossRef] [PubMed]

J. Neurosci. Methods (1)

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

J. Opt. Soc. Am. B (1)

Mol. Biol. Cell (1)

A. Royou, C. Field, J. C. Sisson, W. Sullivan, and R. Karess, Mol. Biol. Cell 15, 838 (2004).
[CrossRef]

Opt. Express (2)

Other (3)

M. Bate and A. Martinez-Arias, The Development of Drosophila melanogaster (Cold Spring Harbor Laboratory Press, New York, 1993).

M. Raffel, C. Willert, and J. Kompenhans, Particle Image Velocimetry: A Practical Guide (Springer-Verlag, Berlin, 1998).
[CrossRef]

J. K. Sveen, “An introduction to MatPIV v. 1.6.1,” eprint series (Department of Mathematics, University of Oslo, Oslo, Norway, 2004).

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Figures (4)

Fig. 1
Fig. 1

Optical properties of early Drosophila embryos investigated by 2PEF–THG microscopy. 2PEF imaging (820-nm excitation) reveals GFP-labeled nuclei and endogenous yolk fluorescence. Lipid droplets are a major source of contrast in THG images (1180-nm excitation). ls, scattering mean free path (see text); scale bar, 15 µm.

Fig. 2
Fig. 2

Cellularization dynamics were followed at 19 °C in control sGMCA embryos by 2PEF microscopy (920-nm excitation) and in wild-type embryos by THG microscopy (1180-nm excitation). Image sequences were recorded, and space–time (YT) projections of the signal in the white rectangle areas were extracted. In the YT representation, slopes reflect the rate of CFI; see text. Similar speeds (indicated in micrometers per minute) are found in both experiments, which show that sustained THG imaging does not perturb the dynamics of this sensitive, temperature-dependent process.

Fig. 3
Fig. 3

A, Velocimetric THG microscopy provides in vivo a micrometer-scale description of morphogenetic movements through entire unstained wild-type embryos. B, Velocimetric analysis of 2PEF images of a nuclei-labeled embryo. C, Velocimetric analysis of transmitted-light (TL) images of an unstained embryo does not provide three-dimensional sectioning. Scale arrow, 5 µm/min. One image was recorded every 20 s, with 0.6µm pixel size.

Fig. 4
Fig. 4

THG microscopy provides micrometer-scale quantification of disrupted morphogenetic movements in unstained mutant embryos. The mean velocity in the area defined by a black rectangle, as determined by velocimetric THG in a wild-type embryo, is shown as a function of time. The same measurements in a Dorsal embryo with disrupted movements and in a wild-type embryo from transmitted-light (TL) microscopy are also shown. Velocities are projected in the vertical direction, positive up.

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