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A 3D digital atlas of C. elegans and its application to single-cell analyses


We built a digital nuclear atlas of the newly hatched, first larval stage (L1) of the wild-type hermaphrodite of Caenorhabditis elegans at single-cell resolution from confocal image stacks of 15 individual worms. The atlas quantifies the stereotypy of nuclear locations and provides other statistics on the spatial patterns of the 357 nuclei that could be faithfully segmented and annotated out of the 558 present at this developmental stage. We then developed an automated approach to assign cell names to each nucleus in a three-dimensional image of an L1 worm. We achieved 86% accuracy in identifying the 357 nuclei automatically. This computational method will allow high-throughput single-cell analyses of the post-embryonic worm, such as gene expression analysis, or ablation or stimulation of cells under computer control in a high-throughput functional screen.

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Figure 1: Automatic processing of a 3D image of C. elegans.
Figure 2: Statistics of nuclear positions.
Figure 3: The nuclear spatial location patterns for different cell types.
Figure 4: The AP axis graphs for different cell types derived from the atlas.
Figure 5: Nuclear sizes for different cell types.
Figure 6: Accuracies of automated segmentation and annotation of 55 image stacks.


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We thank A. Fire for providing reagents and advice. The work of X.L. and S.K.K. was funded by the Ellison Medical Foundation and the US National Institutes of Health. X.L. was also funded by the Larry L. Hillblom Foundation. The work of F.L., H.P. and E.M. was funded by Howard Hughes Medical Institute.

Author information




F.L., H.P. and E.M. developed the pipeline and related algorithms, built the atlas, performed the statistical analysis and wrote the manuscript. X.L. and S.K.K. prepared the worm assays and images, manually annotated the nuclei and helped prepare the manuscript.

Corresponding author

Correspondence to Eugene Myers.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note (PDF 4181 kb)

Supplementary Video 1

Three-dimensional images before and after worm body straightening, and its segmentation result. (MOV 3204 kb)

Supplementary Video 2

Three-dimensional point-cloud rendering of the nuclei of 357 cells in L1 hermaphrodites larvae. (MOV 6041 kb)

Supplementary Video 3

A tutorial movie showing how to use V3D software to view the 3D digital atlas and interactively access/edit the content of individual nuclei annotation. (MOV 8675 kb)

Supplementary Data 1

3D digital atlas of L1 represented as a point cloud. (ZIP 11 kb)

Supplementary Data 2

CellExplorer software package, which also contains the sample data set. This software package can straighten worm stacks, segment nuclei, automatically annotate nuclei names and perform related analysis. Read the README file inside this package for further instructions. (ZIP 13487 kb)

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Long, F., Peng, H., Liu, X. et al. A 3D digital atlas of C. elegans and its application to single-cell analyses. Nat Methods 6, 667–672 (2009).

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