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.

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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 248 73-76 (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 6 113a (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 263 802-805 (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. 100 64-119 (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. 121 1343-1355 (1993).
    [CrossRef] [PubMed]
  6. A. Fire, "A four-dimensional digital image archiving system for cell lineage tracing and retrospective embryology," Comput. Appl. Biosci. 10 443-447 (1994).
    [PubMed]
  7. C. Thomas, P. DeVries, J. Hardin and J. White, "Four-dimensional imaging: computer visualization of 3D movements in living specimens," Science 273 603-607 (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 2678 38-49 (1996).
  9. W. A. Mohler and J. G. White, "Stereo-4-D reconstruction and animation from living fluorescent specimens," BioTechniques 24 1006-1012 (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. 8 1087-1090 (1998). View supplementary movie files at: http://current-biology.com/supmat/cub/bb8s53s1.mov http://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).

Other (11)

W. Denk, J. H. Strickler, and W. W. Webb, "2-photon laser scanning fluorescence microscopy," Science 248 73-76 (1990).
[CrossRef] [PubMed]

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 6 113a (1995).

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward and D. C. Prasher, "Green fluorescent protein as a marker for gene expression," Science 263 802-805 (1994).
[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. 100 64-119 (1983).
[CrossRef] [PubMed]

S. N. Hird and J. G. White, "Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans," J. Cell Biol. 121 1343-1355 (1993).
[CrossRef] [PubMed]

A. Fire, "A four-dimensional digital image archiving system for cell lineage tracing and retrospective embryology," Comput. Appl. Biosci. 10 443-447 (1994).
[PubMed]

C. Thomas, P. DeVries, J. Hardin and J. White, "Four-dimensional imaging: computer visualization of 3D movements in living specimens," Science 273 603-607 (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 2678 38-49 (1996).

W. A. Mohler and J. G. White, "Stereo-4-D reconstruction and animation from living fluorescent specimens," BioTechniques 24 1006-1012 (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. 8 1087-1090 (1998). View supplementary movie files at: http://current-biology.com/supmat/cub/bb8s53s1.mov http://current-biology.com/supmat/cub/bb8s53s2.mov
[CrossRef] [PubMed]

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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