Hi,
I am working with an osmotically-sensitive strain and prefer to use embryonic growth medium (EGM) for
4D imaging. However, I have noticed that EGM appears to be toxic when I image GFP/ mCherry with
a spinning disk confocal microscope. After about 10 minutes, the embryos arrest and I have observed this
with wild-type strains expressing GFP-tubulin/mCherry-histone as well.
I was wondering if anyone else has encountered this problem and can recommend a different media
in which to image my embryos.
Thanks!
Nick
Nick,
I spent many a wasted experiment having precisely the same problem. After extensive troubleshooting, I found that removing the mother carcass and trying to reduce the amount of bacteria in the mount is essential. In fact, you can titrate this killing effect, what I call SuperDeath (due to the arrest phenotype that looks as though the embryos were put in a fixative as you described), by leaving more mother carcasses in the mount. So, I do not dissect more than 3 animals when making a mount to limit “superdeath.” It was very frustrating to troubleshoot because I found the killing effect variable, probably due to the proximity of the embryo to the mother carcass and the diffusion of the “superdeath” agent. Permeable embryos are likely more sensitive to this effect. Additionally, something about agar pad mounts limits this effect so that you rarely see embryos freeze like this- people in the lab couldn’t figure out why I was having this problem, which in hindsight makes more sense.
A troubleshooting tip: mount embryos in hanging drop and let them sit on the benchtop overnight. Whatever condition you use, all embryos should hatch overnight, even early meiosis I embryos. This will also eliminate any potential toxicity due to your particular imaging method, I saw superdeath using DIC as well. Make a mount +/- mother carcass and superdeath should be obvious.
After eliminating this problem, I found embryos behave well in 70% egg buffer (this depends on the osmolality of your prep of egg buffer which can be variable, and early meiotic embryos tend to be rather finicky so I don’t routinely use this) and are perfectly happy in blastomere culture media (early meiotic embryos survive to hatching, see: Shelton, C. A. and Bowerman, B. (1996). Time-dependent responses to glp-1-mediated inductions in early C. elegans embryos. Development 122,2043 -2050.) This media is much easier to make than EGM as well.
Hope this solves your problem.
Josh