Biology Reference
In-Depth Information
(
240 min post first cleavage) cellular differentiation begins. Cells can be iden-
tified based on position relative to landmarks such as the first apoptotic cell
deaths; maps of cell nuclei at 270 nuclei are in Figure 7 of
Sulston et al. (1983)
.
Cell and nuclear identification is more or less straightforward using the available
maps up to the 1.75-fold stage when body wall muscle contractions begin. By the
twofold stage the rolling activity of the embryo severely hampers attempts to identify
cells in live specimens. To prevent this rapid movement the microscope objective can
be transiently and reversibly cooled (
Sulston et al., 1983
). Overall the last 3 h of
embryonic development remain relatively little studied.
The cell lineage reported in
Sulston et al. (1983)
was a composite built up from
partial lineages of hundreds of embryos. With the improvement of imaging and
storage technology by the late 1980s it became possible to record the complete
development of single embryos using timelapse imaging in multiple focal planes.
Such ''four dimensional'' (4D) microscopy was developed by J. G. White and used
for studies of early C. elegans embryos (
Hird and White, 1993
). Several groups
developed software for 4D acquisition and playback (
Fire, 1994; Thomas et al.,
1996
). Commercial imaging software suites such as Improvision or Amira include
options for automated 4D capture, as do the software suites for most commercial
compound microscopes. Finally the Open Source software suite Micro-Manager
automated 4D acquisition.
Cell-lineage tracing from DIC 4D movies requires manual identification of nuclei
at each time point. Software such as SIMI Biocell (
Schnabel et al., 1997
)(
Table III
)
Table III
Comparison of some methods for manual and semiautomatic embryonic lineage recording
Software name
Data
Maximum nuclei
tracked
Method
Time required
Error rate
Reference
SIMI Biocell
DIC
385 cells full/114
cells up to 1.5 fold
Manual
N/A
N/A
(
Schnabel et al.,
1997
)
Angler
DIC
end of gastrulation/
300 cells
Manual
N/A
N/A
(
Martinelli et al.,
1997
)
N/A
DIC
24 cells
Automated
N/A
N/A
(
Hamahashi et al.,
2005
)
StarryNite
GFP
350 cells
Automated
8 h up to 350
cells
1-3% up to 194
cells, larger
up to 350 cells
(
Bao et al., 2006
)
Endrov
DIC/GFP 150 cells full/partial
later stage
Manual
N/A
N/A
(
Hench et al.,
2009
)
NucleiTracker4D GFP
566 live/ 669 total
cells
Semiautomated For GFP
confocal = 4 h
up to 350 cells
8-16 h to
525 cells
Up to 3% to 350
cells, up to
15% to 525
cells (at each
time-point).
CAG & ADC,
unpublished
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