Cell Identification and Cell Lineage Analysis - Caenorhabditis Elegans: Molecular Genetics and Development - page 331

these goals Long et al. used DAPI stained worms to mark all cell nuclei, followed
by several image processing steps. First, the images of larvae are straightened to
arodshape( Peng et al., 2008 ). Next, nuclei are automatically identified by
adaptive thresholding and rule- and training-based segmentation. Nuclei can be
validated and curated using the volume-object image annotation system
( Peng et al.,2009 ). In this way, 357 out of the 558 L1 larval nuclei could be
faithfully identified.
Using fiduciary muscle nuclei GFP markers, Long et al. registered as many as 40
L1 larvae samples into a 3D standard representation. However, as few as 15 samples
were sufficient to infer correct nuclear positions along the body. A comparison of
cell positions in the atlas samples along the three axes of the body showed that
nucleus-to-nucleus spatial relationships are invariant, especially among cells
belonging to the same tissue. After building the standard representation, Long
et al. developed an automated procedure for mapping and annotating novel samples,
such as expression patterns, onto this reference ( Liu et al., 2009 ). Overall, their
automated segmentation can identify nuclei in certain tissues with > 80% accuracy.
Improving accuracy and the ability to segment all of the 558 nuclei of larvae will
entail using higher resolution microscopy methods like selective plane illumination
or stimulated emission depletion.
VI. Materials, Methods, and Protocols
A general protocol for mounting C. elegans on agar pads for live analysis is
provided in the chapter by Shaham in Worm Methods ( http://www.wormbook.
org/toc_wormmethods.html ) ; see also the methods appendix to the C. elegans I
book.
A. Protocol 1: Analysis of Embryonic Cell Lineages
1. Mounting
Detailed protocols for mounting C. elegans embryos are provided in the chapter by
Hardin. Traditionally C. elegans embryos have been mounted on agar pads with
buffer and a coverslip. Although embryos are completely viable under such condi-
tions, it is clear that this method compresses the egg and eggshell. An uncompressed
egg mounted in an aqueous medium is 50 m mlongand30 m m in diameter
( Deppe et al., 1978 ). Also Ref. Blanchoud et al., 2010; Dev Dyn 239: 3285-96.
Embryos mounted on agar pads are compressed to a thickness of 20 m m
( Schnabel et al.,1997 ). Mounting using spacer beads can also compress the embryo,
depending on the bead size used. The lateral compression is helpful in reducing the
number of optical sections needed for 4D lineage analysis and constraining the
embryo to a fixed orientation for observation. However as mentioned in the text,
compression may contribute to the variability in cell positions in early embryogenesis.