Abstract

Multiphoton laser scanning microscopy (MPLSM) enables the production of long timelapse recordings from live fluorescent specimens. 1047- and 900-nm excitation were used to image both a vital fluorescent membrane probe, FM 4-64, and a modified green fluorescent protein (GFP) in live Caenorhabditis elegans embryos. Automated four-dimensional (4D) data collection yielded individual recordings comprising thousands of images, each allowing analysis of all of the cell divisions, contacts, migrations, and fusions that occur during a span of several hours of embryogenesis.

© 1998 Optical Society of America

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References

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  1. W. Denk, J. H. Strickler, and W. W. Webb, “2-photon laser scanning fluorescence microscopy.,” Science 24873–76 (1990).
    [Crossref] [PubMed]
  2. V. E. Centonze, D. L. Wokosin, and J. G. White, “Improved deep optical sectioning capabilities rendered by 2-photon excitation imaging,” Mol. Biol. of Cell 6113a (1995).
  3. M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, “Green fluorescent protein as a marker for gene expression,” Science 263802–805 (1994).
    [Crossref] [PubMed]
  4. J. E. Sulston, E. Schierenberg, J. G. White, and J. N. Thomson, “The embryonic cell lineage of the nematode Caenorhabditis elegans,” Dev. Biol. 10064–119 (1983).
    [Crossref] [PubMed]
  5. S. N. Hird and J. G. White, “Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans,” J. Cell Biol. 1211343–1355 (1993).
    [Crossref] [PubMed]
  6. A. Fire, “A four-dimensional digital image archiving system for cell lineage tracing and retrospective embryology,” Comput. Appl. Biosci. 10443–447 (1994).
    [PubMed]
  7. C. Thomas, P. DeVries, J. Hardin, and J. White, “Four-dimensional imaging: computer visualization of 3D movements in living specimens,” Science 273603–607 (1996). http://www.bocklabs.wisc.edu/imr/facility/4D/4d.htm
    [Crossref] [PubMed]
  8. D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multiple-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 267838–49 (1996).
  9. W. A. Mohler and J. G. White, “Stereo-4-D reconstruction and animation from living fluorescent specimens,” BioTechniques 241006–1012 (1998).http://www.bocklabs.wisc.edu/imr/stereo4d/stereo4d.html
    [PubMed]
  10. W. A. Mohler, J. S. Simske, E. M. Williams-Masson, J. D. Hardin, and J. G. White, “Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis,” Curr. Biol. 81087–1090 (1998). View supplementary movie files at: http://current-biology.com/supmat/cub/bb8s53s1.movhttp://current-biology.com/supmat/cub/bb8s53s2.mov
    [Crossref] [PubMed]
  11. R. H. Waterston, “Muscle” in The Nematode Caenorhabditis elegans, W. B. Wood, ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

1998 (2)

W. A. Mohler and J. G. White, “Stereo-4-D reconstruction and animation from living fluorescent specimens,” BioTechniques 241006–1012 (1998).http://www.bocklabs.wisc.edu/imr/stereo4d/stereo4d.html
[PubMed]

W. A. Mohler, J. S. Simske, E. M. Williams-Masson, J. D. Hardin, and J. G. White, “Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis,” Curr. Biol. 81087–1090 (1998). View supplementary movie files at: http://current-biology.com/supmat/cub/bb8s53s1.movhttp://current-biology.com/supmat/cub/bb8s53s2.mov
[Crossref] [PubMed]

1996 (2)

C. Thomas, P. DeVries, J. Hardin, and J. White, “Four-dimensional imaging: computer visualization of 3D movements in living specimens,” Science 273603–607 (1996). http://www.bocklabs.wisc.edu/imr/facility/4D/4d.htm
[Crossref] [PubMed]

D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multiple-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 267838–49 (1996).

1995 (1)

V. E. Centonze, D. L. Wokosin, and J. G. White, “Improved deep optical sectioning capabilities rendered by 2-photon excitation imaging,” Mol. Biol. of Cell 6113a (1995).

1994 (2)

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, “Green fluorescent protein as a marker for gene expression,” Science 263802–805 (1994).
[Crossref] [PubMed]

A. Fire, “A four-dimensional digital image archiving system for cell lineage tracing and retrospective embryology,” Comput. Appl. Biosci. 10443–447 (1994).
[PubMed]

1993 (1)

S. N. Hird and J. G. White, “Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans,” J. Cell Biol. 1211343–1355 (1993).
[Crossref] [PubMed]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “2-photon laser scanning fluorescence microscopy.,” Science 24873–76 (1990).
[Crossref] [PubMed]

1983 (1)

J. E. Sulston, E. Schierenberg, J. G. White, and J. N. Thomson, “The embryonic cell lineage of the nematode Caenorhabditis elegans,” Dev. Biol. 10064–119 (1983).
[Crossref] [PubMed]

Centonze, V. E.

D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multiple-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 267838–49 (1996).

V. E. Centonze, D. L. Wokosin, and J. G. White, “Improved deep optical sectioning capabilities rendered by 2-photon excitation imaging,” Mol. Biol. of Cell 6113a (1995).

Chalfie, M.

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, “Green fluorescent protein as a marker for gene expression,” Science 263802–805 (1994).
[Crossref] [PubMed]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “2-photon laser scanning fluorescence microscopy.,” Science 24873–76 (1990).
[Crossref] [PubMed]

DeVries, P.

C. Thomas, P. DeVries, J. Hardin, and J. White, “Four-dimensional imaging: computer visualization of 3D movements in living specimens,” Science 273603–607 (1996). http://www.bocklabs.wisc.edu/imr/facility/4D/4d.htm
[Crossref] [PubMed]

Euskirchen, G.

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, “Green fluorescent protein as a marker for gene expression,” Science 263802–805 (1994).
[Crossref] [PubMed]

Ferguson, A. I.

D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multiple-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 267838–49 (1996).

Fire, A.

A. Fire, “A four-dimensional digital image archiving system for cell lineage tracing and retrospective embryology,” Comput. Appl. Biosci. 10443–447 (1994).
[PubMed]

Hardin, J.

C. Thomas, P. DeVries, J. Hardin, and J. White, “Four-dimensional imaging: computer visualization of 3D movements in living specimens,” Science 273603–607 (1996). http://www.bocklabs.wisc.edu/imr/facility/4D/4d.htm
[Crossref] [PubMed]

Hardin, J. D.

W. A. Mohler, J. S. Simske, E. M. Williams-Masson, J. D. Hardin, and J. G. White, “Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis,” Curr. Biol. 81087–1090 (1998). View supplementary movie files at: http://current-biology.com/supmat/cub/bb8s53s1.movhttp://current-biology.com/supmat/cub/bb8s53s2.mov
[Crossref] [PubMed]

Hird, S. N.

D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multiple-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 267838–49 (1996).

S. N. Hird and J. G. White, “Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans,” J. Cell Biol. 1211343–1355 (1993).
[Crossref] [PubMed]

Maker, G. T.

D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multiple-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 267838–49 (1996).

Malcolm, G. P. A.

D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multiple-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 267838–49 (1996).

Mohler, W. A.

W. A. Mohler and J. G. White, “Stereo-4-D reconstruction and animation from living fluorescent specimens,” BioTechniques 241006–1012 (1998).http://www.bocklabs.wisc.edu/imr/stereo4d/stereo4d.html
[PubMed]

W. A. Mohler, J. S. Simske, E. M. Williams-Masson, J. D. Hardin, and J. G. White, “Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis,” Curr. Biol. 81087–1090 (1998). View supplementary movie files at: http://current-biology.com/supmat/cub/bb8s53s1.movhttp://current-biology.com/supmat/cub/bb8s53s2.mov
[Crossref] [PubMed]

Prasher, D. C.

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, “Green fluorescent protein as a marker for gene expression,” Science 263802–805 (1994).
[Crossref] [PubMed]

Schierenberg, E.

J. E. Sulston, E. Schierenberg, J. G. White, and J. N. Thomson, “The embryonic cell lineage of the nematode Caenorhabditis elegans,” Dev. Biol. 10064–119 (1983).
[Crossref] [PubMed]

Sepsenwol, S.

D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multiple-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 267838–49 (1996).

Simske, J. S.

W. A. Mohler, J. S. Simske, E. M. Williams-Masson, J. D. Hardin, and J. G. White, “Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis,” Curr. Biol. 81087–1090 (1998). View supplementary movie files at: http://current-biology.com/supmat/cub/bb8s53s1.movhttp://current-biology.com/supmat/cub/bb8s53s2.mov
[Crossref] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “2-photon laser scanning fluorescence microscopy.,” Science 24873–76 (1990).
[Crossref] [PubMed]

Sulston, J. E.

J. E. Sulston, E. Schierenberg, J. G. White, and J. N. Thomson, “The embryonic cell lineage of the nematode Caenorhabditis elegans,” Dev. Biol. 10064–119 (1983).
[Crossref] [PubMed]

Thomas, C.

C. Thomas, P. DeVries, J. Hardin, and J. White, “Four-dimensional imaging: computer visualization of 3D movements in living specimens,” Science 273603–607 (1996). http://www.bocklabs.wisc.edu/imr/facility/4D/4d.htm
[Crossref] [PubMed]

Thomson, J. N.

J. E. Sulston, E. Schierenberg, J. G. White, and J. N. Thomson, “The embryonic cell lineage of the nematode Caenorhabditis elegans,” Dev. Biol. 10064–119 (1983).
[Crossref] [PubMed]

Tu, Y.

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, “Green fluorescent protein as a marker for gene expression,” Science 263802–805 (1994).
[Crossref] [PubMed]

Ward, W. W.

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, “Green fluorescent protein as a marker for gene expression,” Science 263802–805 (1994).
[Crossref] [PubMed]

Waterston, R. H.

R. H. Waterston, “Muscle” in The Nematode Caenorhabditis elegans, W. B. Wood, ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “2-photon laser scanning fluorescence microscopy.,” Science 24873–76 (1990).
[Crossref] [PubMed]

White, J.

C. Thomas, P. DeVries, J. Hardin, and J. White, “Four-dimensional imaging: computer visualization of 3D movements in living specimens,” Science 273603–607 (1996). http://www.bocklabs.wisc.edu/imr/facility/4D/4d.htm
[Crossref] [PubMed]

White, J. G.

W. A. Mohler and J. G. White, “Stereo-4-D reconstruction and animation from living fluorescent specimens,” BioTechniques 241006–1012 (1998).http://www.bocklabs.wisc.edu/imr/stereo4d/stereo4d.html
[PubMed]

W. A. Mohler, J. S. Simske, E. M. Williams-Masson, J. D. Hardin, and J. G. White, “Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis,” Curr. Biol. 81087–1090 (1998). View supplementary movie files at: http://current-biology.com/supmat/cub/bb8s53s1.movhttp://current-biology.com/supmat/cub/bb8s53s2.mov
[Crossref] [PubMed]

D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multiple-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 267838–49 (1996).

V. E. Centonze, D. L. Wokosin, and J. G. White, “Improved deep optical sectioning capabilities rendered by 2-photon excitation imaging,” Mol. Biol. of Cell 6113a (1995).

S. N. Hird and J. G. White, “Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans,” J. Cell Biol. 1211343–1355 (1993).
[Crossref] [PubMed]

J. E. Sulston, E. Schierenberg, J. G. White, and J. N. Thomson, “The embryonic cell lineage of the nematode Caenorhabditis elegans,” Dev. Biol. 10064–119 (1983).
[Crossref] [PubMed]

Williams-Masson, E. M.

W. A. Mohler, J. S. Simske, E. M. Williams-Masson, J. D. Hardin, and J. G. White, “Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis,” Curr. Biol. 81087–1090 (1998). View supplementary movie files at: http://current-biology.com/supmat/cub/bb8s53s1.movhttp://current-biology.com/supmat/cub/bb8s53s2.mov
[Crossref] [PubMed]

Wokosin, D. L.

D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multiple-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 267838–49 (1996).

V. E. Centonze, D. L. Wokosin, and J. G. White, “Improved deep optical sectioning capabilities rendered by 2-photon excitation imaging,” Mol. Biol. of Cell 6113a (1995).

BioTechniques (1)

W. A. Mohler and J. G. White, “Stereo-4-D reconstruction and animation from living fluorescent specimens,” BioTechniques 241006–1012 (1998).http://www.bocklabs.wisc.edu/imr/stereo4d/stereo4d.html
[PubMed]

Comput. Appl. Biosci. (1)

A. Fire, “A four-dimensional digital image archiving system for cell lineage tracing and retrospective embryology,” Comput. Appl. Biosci. 10443–447 (1994).
[PubMed]

Curr. Biol. (1)

W. A. Mohler, J. S. Simske, E. M. Williams-Masson, J. D. Hardin, and J. G. White, “Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis,” Curr. Biol. 81087–1090 (1998). View supplementary movie files at: http://current-biology.com/supmat/cub/bb8s53s1.movhttp://current-biology.com/supmat/cub/bb8s53s2.mov
[Crossref] [PubMed]

Dev. Biol. (1)

J. E. Sulston, E. Schierenberg, J. G. White, and J. N. Thomson, “The embryonic cell lineage of the nematode Caenorhabditis elegans,” Dev. Biol. 10064–119 (1983).
[Crossref] [PubMed]

J. Cell Biol. (1)

S. N. Hird and J. G. White, “Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans,” J. Cell Biol. 1211343–1355 (1993).
[Crossref] [PubMed]

Mol. Biol. of Cell (1)

V. E. Centonze, D. L. Wokosin, and J. G. White, “Improved deep optical sectioning capabilities rendered by 2-photon excitation imaging,” Mol. Biol. of Cell 6113a (1995).

Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE (1)

D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multiple-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 267838–49 (1996).

Science (3)

W. Denk, J. H. Strickler, and W. W. Webb, “2-photon laser scanning fluorescence microscopy.,” Science 24873–76 (1990).
[Crossref] [PubMed]

C. Thomas, P. DeVries, J. Hardin, and J. White, “Four-dimensional imaging: computer visualization of 3D movements in living specimens,” Science 273603–607 (1996). http://www.bocklabs.wisc.edu/imr/facility/4D/4d.htm
[Crossref] [PubMed]

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, “Green fluorescent protein as a marker for gene expression,” Science 263802–805 (1994).
[Crossref] [PubMed]

Other (1)

R. H. Waterston, “Muscle” in The Nematode Caenorhabditis elegans, W. B. Wood, ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

Supplementary Material (6)

» Media 1: MOV (953 KB)     
» Media 2: MOV (760 KB)     
» Media 3: MOV (1436 KB)     
» Media 4: MOV (1668 KB)     
» Media 5: MOV (1654 KB)     
» Media 6: MOV (2070 KB)     

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Figures (6)

Fig. 1.
Fig. 1.

(QuickTime movie) Timelapse animation of a single optical section through an FM 4–64-labeled embryo. Indicated by arrowheads are a cytokinesis event (pink) and the site of the gastrulation pore (yellow) through which ingressing cells move to the interior of the embryo. [Media 1]

Fig. 2.
Fig. 2.

(QuickTime movie) A programmed cell death occurring within the same embryo from Fig. 1. Timelapse sequence following cell from birth (arrowhead) to death and engulfment by a neighboring cell. Fluorescence intensity is plotted using a pseudocolor scale (black-red-yellow-white) to emphasize the change in labeling of the dying cell. [Media 2]

Fig. 3
Fig. 3

(QuickTime movie) Standard 4D and time-animated volume (stereo-4D) reconstruction of an embryo undergoing cleavage, gastrulation, and the preliminary phase of gut differentiation. Intestinal precursors acquire bright cytoplasmic spots. [Media 3]

Fig. 4.
Fig. 4.

(QuickTime movie) Stereo-4D reconstruction of an elongating embryo showing prominent “omega”-shaped arrangement of axon bundles around the nerve ring. [Media 4]

Fig. 5.
Fig. 5.

(QuickTime movie) Standard 4D, stereo-4D, and “cored” stereo-4D reconstruction of an embryo undergoing early morphogenesis. Arrowhead indicates the fusion of two cells within the dorsal hypodermis. [Media 5]

Fig. 6.
Fig. 6.

(QuickTime movie) Stereo-4D reconstruction of a transgenic animal expressing MH27-GFP. Timecourse follows the embryo from the onset of fluorescent transgene expression through hypodermal differentiation, migration, enclosure, and cell fusion. [Media 6]

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