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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  1. W. Denk, J. H. Strickler, and W. W. Webb, "2-photon laser scanning fluorescence microscopy," Science 248 73-76 (1990).
  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).
  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).
  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).
  6. A. Fire, "A four-dimensional digital image archiving system for cell lineage tracing and retrospective embryology," Comput. Appl. Biosci. 10 443-447 (1994).
  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
  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
  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
  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).

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).

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).

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).

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

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

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

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

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)     
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» Media 6: MOV (2070 KB)     

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