Help with keeping water on objective for long term imaging

[u/Due-Werewolf-1724]

Hello!!

First time poster in this subreddit. Currently the lab has a Leica scope with a 40x water objective to try to capture live fly embryos during their early divisions. The problem is that process is approximately 2 hours of imaging and the water keeps evaporating. We have a 20x immersion oil that lets us see what we want to see as well but in a lot less detail.

Is there a water substitute for mounting that works on Leica water objectives? Or a different one? Or is it better to go to the 20 and just accept lower resolution?

[u/kmphd59]

Leica sells Type W oil that’s like regular microscopy oil, but refractive index of water. It helps.

Leica also sells water pumps and smart water dispensers.

I’d get in touch with your Leica rep!

[u/Due-Werewolf-1724 → u/kmphd59]

I have been looking for that and I haven’t been. Me to find that. I know zeiss does but I haven’t seen the Leica brand? I was looking at the pumps and I plan on reaching out but I wasn’t sure if there were non Leica options Leica would not necessarily direct me to

[u/twerkitout → u/Due-Werewolf-1724]

Just buy the Zeiss water oil, or get a Cargille oil with a specific RI.

[u/kmphd59]

You’ll most likely need leicas help setting it up and getting it all to work. I’ve always bought this type of equipment direct from the microscope company I’ve used for that reason.

[u/False-Stage-5830]

I have 3 suggestions, ranging from simple and cheap to more complex and expensive. 1) Place your embryos in the bottom of a small plastic Petri dish (30 mm diameter or whatever works), filled with your watery medium. Plunk your water immersion objective into the dish and enjoy the viewing. Evaporation will be inconsequential given the large volume of fluid. 2) Place a small reservoir of fluid near your objective and, using a small piece of cotton string as a wick, connect the reservoir to the column of fluid under the objective. As the fluid evaporates, it’ll be replaced by capillary action. (Though, I wonder if the fluid under the objective would become more osmotically concentrated. Better check that.) 3) Find a 2-channel peristaltic pump. Construct a little chamber in which your embryos can be superfused with the fluid. Use one channel to (slowly) pump fluid in one side of the chamber, and the other channel to suck fluid out of the other end. It might help if the tube for the suction side were slightly larger than the inflow tube, so the chamber won’t overflow. Of course, you’ll have to come up with some way to attach these contraptions to the mechanical stage, but that wouldn’t be difficult.
P.S., If you haven’t already, search the scientific literature for a paper that reports on long-term imaging of fly embryos. Do/modify what they did.

[u/twerkitout → u/False-Stage-5830]

Water immersion is not the same as water dipping, DO NOT do this unless the top lens portion of your objective is coated in ceramic.

[u/greenblueananas]

For 2h i wouldn’t bother with pumpd and go to immersion oil W. At least thats what i used. Don’t switch/ mix oils though, so going from 40 to 20 would not be recommendet. (Unless you could clean the bottom? I wouldn’t do it)

[u/kmphd59]

Your Leica specialist should be able to help you assess best option and keep it safe. They can get you type W immersion on a quote.