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Multiphoton laser scanning microscopy for four-dimensional analysis of Caenorhabditis elegans embryonic development.

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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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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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