Biology Reference
In-Depth Information
2. DIC Cell-Lineage Analysis and 4D Recording
Procedures for manual lineaging of embryos are described by Sulston et al.
(1983) . A number of software tools have been described over the years to allow
time-lapse recording in multiple focal planes (4D recording) (see above). At present
most commercial microscope vendors include 4D acquisition as an option. The
minimal requirements are microscope equipped for Nomarski DIC optics, a motor-
ized z-drive, a camera, and a computer workstation that controls the z-drive. A high-
N.A. DIC objective (e.g., Zeiss Plan-Neofluar 100 ) is essential for any lineage
studies. Cell lineages can be traced manually from DIC 4D data sets. The software
package SIMI Biocell is specifically designed to facilitate lineage construction from
DIC 4D movies.
3. 4D Lineaging Using Histone-GFP
Procedures for 4D imaging of histone-GFP marked embryos are defined in
Murray et al. (2006) . Briefly, single transgenic HIS-72::GFP(zuIs178) embryos
are mounted between coverslips in 8 m L of a mixture of 20 m m polystyrene beads
(Polysciences Inc., Warrington, PA) in 1% methlycellulose in M9 (15% v/v beads,
85% v/v 1% methylcellulose in M9). The coverslip sandwich can be flipped to
display the desired late development dorsal or ventral aspect and then attached to
a slide and sealed.
We use a Zeiss LSM510 confocal with a 30 mWArgon laser. We acquire confocal
z-stacks of size 64 35 30 m m 3 with resolution of 0.125 0.125 0.85 m m 3 /
voxel every minute for the first 300 min of development then every 2 min for the next
180 min. Two-color (GFP/mCherry) movies can be acquired to correlate cell-spe-
cific expression patterns with the ubiquitously expressed histone-GFP. Laser power,
detector, and acquisition configurations are loaded through the MultiTime macro in
the Zeiss LSM software.
Precise temperature control is extremely important to maintain embryo viability
over prolonged periods of confocal imaging. Although embryos will survive 4D DIC
imaging throughout embryogenesis at 25 C, we find the upper limit for confocal
imaging is 24 C; the viability of imaged embryos should be checked whenever 4D
imaging is being set up for the first time. There are several options for control of
specimen temperature; we have used a custom-designed aluminum casing for the
objective. The casing is cooled or heated by a small Peltier element and a liquid
cooling system designed for computer chips.
The analysis of 4D LSM data sets by Starrynite and Acetree is described in
detail by ( Murray et al., 2006 ). We provide here a brief overview of our nuclear
tracking approach (Giurumescu et al., in preparation). We analyze 4D LSM data
sets with a user interface that combines the automated tracking and user-selected
curation. At the first time point (usually 4-6 nuclei) the user manually identifies
nuclei and names them according to the canonical wild-type lineage. For subse-
quent time z-stacks, the software first performs an automatic segmentation and
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