Imaging one cell embryo

Model Organism: Worms Methods & Reagents
1

Hi All,

I’m looking for tips/tricks to imaging the one cell zygote. Currently I am cutting open worms in a watch glass. gathering the embryos and moving them to a slide with an agar pad. Then sealing and finally imaging. After all this time has elapsed when I view my embryos under the scope they’re at the 2 cell or older stage.
I’d really like to get pseudocleavage and watch the first cell division.

Any suggestions to help me make mounts faster/get earlier embryos?
Thanks in advance.

2

I need more information. How do you gather the embryos and transfer to an agar pad? How long does it take you to dissect the mothers?

Back when I was doing this, we used a pulled capillary (I think from a 50 or 100ul capillary) and mouth pipetted using aspirators (VWR# 53507-278). Before dissed ecting I had my agar pad between two microscope slides. I quickly dissected 10-15 mothers, and mouth pipetted all the young embryos (It was easy for me to specifically pick 1 and 2-cell embryos under the stereoscope) onto the agar pad. I would remove excess liquid with the mouth pipet, then using an eyelash glued to a toothpick using nail polish I would push the embryos closer together, then I popped a cover slip on. I did all of this in a couple of minutes, then rushed to the microscope.

Hope this helps!

Steve V

3

I would skip the sealing step if you are only imaging 1-2 cell embryos. Just make sure there is plenty of liquid to keep the agar pad drying/shrinking for the short time you are imaging.
Cut open fewer worms (don’t be greedy), and make sure they are young adults so you don’t spend time hunting for 1 cell embryos. Also, have your agar pad ready to go before you cut open.
Can you move your dissecting scope next to the imaging scope as well?
Good luck!

4

Thanks for the advice! To be more specific.
The first thing I do is make my agar pad on the microscope slide. I then cut open 5 or 6 moms in a watch glass of Egg Salts. I then gather up the embryos with an eyelash brush. After this, I get my agar pad and transfer the embryos with my mouth pipette to the pad. I remove all the excess liquid with my mouth pipette (so the embryos will stick). I then add 10uLs of Egg salts to the embryo pile, and put 10uLs on the cover slip. I gently lay down the coverslip (with a razor), then add more egg salt to the slide so there is enough liquid. Finally I seal my slide with velap.
I would say in total this takes 10mins or less. The longest part for me is the gathering of the embryos.

I will cut open young adults, and use our stereoscope by the our imaging scope.

5

I did some of these several years ago, hosted by a nearby lab. They used poly-Lysine slide coating (added from an aliquot then cooked briefly on a 95C temp block). Embryos in egg salts were squirted onto this slide with mouth pipette/capillary, and cover slip gently dropped. NOT an agar pad.

They also had one stereo scope in the lab with a high mag objective: in this, one could see the # of cells in embryos that had been spilled out when the mother was cut. This was a very nice feature when selecting embryos for imaging, because you could select your stage.

6

Hi,
Few pointers …

  1. If you are imaging at 60x or 100x, all you need is one or two embryos. I always dissect 1 worm. You are sure to get 1-celled embryo.
  2. Pick young adult ( if your expt allows you). There are fewer embryos to screen.
  3. Since its dissection of one worm, I prefer to dissect it on a drop of egg buffer placed on a coverslip. That makes it easier to visualize and aspirate it using mouth pipette.
  4. After placing the embryo on agarose pad, mark the position on glass slide (do not mark on cover slip; if you are using a DIC condenser, skip this).
  5. Seal only the corners, enough to hold the coverslip in place and prevent it from sticking on to the objective glass.

All the best!

Anup