7. Use the Hamburger-Hamilton stage series [7] as a guide for
incubation times, but establish empirically the number of days/
hours required as this will vary with age of flock and season.
8. Only choose well-developed embryos for harvesting.
9. For silicone plates, ensure that there is a coating of at least
3 mm thick on the bottom of the dish. Alternative methods
could be used such as agarose.
10. Ensure that embryos are pinned down under tension using the
extraembryonic tissues. This aids the dissection and enhances
tissue integrity and survival.
11. It is important to perform the dissections of the desired tissue
“chunk” rapidly and effectively.
12. Use a fine pipette tip to transfer the tissue. Depending on tissue
size, it may be necessary to cut the tip to create a wider
opening. Part-fill the tip with agarose/medium mixture so as
not to dilute the agarose/medium mixture on the dish during
the tissue transfer.
13. Use fine plastic tip to position the tissue.
14. Clean glass on chamber with alcohol and lens wipe tissue to
ensure no debris or scratches.
15. We describe the techniques to prepare dissected somites for
imaging; however, the method can also be modified for imaging whole embryos at younger stages.
16. When setting up time-lapse imaging using multi-photon
microscopes, a trial run will help determine orientation,
depth, and duration of images needed to be captured to
achieve optimal image quality.
17. When imaging deeper into the tissue, the light will scatter.
Hence it is important to increase the laser power exponentially
when moving deeper into the tissue.
18. For the analysis, make sure images are well aligned and the
system does not drift. If drift does occur, use the registration
plugin (ImageJ) to correct or discard the drifting data.
19. When tracking the inverted images, first confirm by eye on a
small data set that the program is picking out individual cells
and that they are tracked well. If not, you may need to adjust
the threshold and tracking parameters.
References
1. Stern CD (2005) The chick; a great model system becomes even greater. Dev Cell 8(1):9–17.
https://doi.org/10.1016/j.devcel.2004.11.
018
2. McColl J, Mok GF, Lippert AH, Ponjavic A,
Muresan L, Munsterberg A (2018) 4D imaging
reveals stage dependent random and directed
cell motion during somite morphogenesis. Sci
180
Gi Fay Mok et al.
incubation times, but establish empirically the number of days/
hours required as this will vary with age of flock and season.
8. Only choose well-developed embryos for harvesting.
9. For silicone plates, ensure that there is a coating of at least
3 mm thick on the bottom of the dish. Alternative methods
could be used such as agarose.
10. Ensure that embryos are pinned down under tension using the
extraembryonic tissues. This aids the dissection and enhances
tissue integrity and survival.
11. It is important to perform the dissections of the desired tissue
“chunk” rapidly and effectively.
12. Use a fine pipette tip to transfer the tissue. Depending on tissue
size, it may be necessary to cut the tip to create a wider
opening. Part-fill the tip with agarose/medium mixture so as
not to dilute the agarose/medium mixture on the dish during
the tissue transfer.
13. Use fine plastic tip to position the tissue.
14. Clean glass on chamber with alcohol and lens wipe tissue to
ensure no debris or scratches.
15. We describe the techniques to prepare dissected somites for
imaging; however, the method can also be modified for imaging whole embryos at younger stages.
16. When setting up time-lapse imaging using multi-photon
microscopes, a trial run will help determine orientation,
depth, and duration of images needed to be captured to
achieve optimal image quality.
17. When imaging deeper into the tissue, the light will scatter.
Hence it is important to increase the laser power exponentially
when moving deeper into the tissue.
18. For the analysis, make sure images are well aligned and the
system does not drift. If drift does occur, use the registration
plugin (ImageJ) to correct or discard the drifting data.
19. When tracking the inverted images, first confirm by eye on a
small data set that the program is picking out individual cells
and that they are tracked well. If not, you may need to adjust
the threshold and tracking parameters.
References
1. Stern CD (2005) The chick; a great model system becomes even greater. Dev Cell 8(1):9–17.
https://doi.org/10.1016/j.devcel.2004.11.
018
2. McColl J, Mok GF, Lippert AH, Ponjavic A,
Muresan L, Munsterberg A (2018) 4D imaging
reveals stage dependent random and directed
cell motion during somite morphogenesis. Sci
180
Gi Fay Mok et al.