Hello everybody,
The meniscus in the wells of the 96-well plate really affects the image quality in transmission imaging (it’s like adding a lens before the sample…). Do you know of anything that could be used to reduce this effect? Is there a surfactant compatible with live imaging that could be used to reduce curvature? Maybe some oil above the wells?
Thank you for your ideas!
Nicolas
Welcome @NicoKiaru!
Definitely curious to hear what others have to suggest, but if your media is not too precious, one option is to fill the wells to the top.
Flooding with media works, as Talley suggested. If you don’t need all 96 wells, switching to less/larger wells will help.
Thanks for your answers, filling the well looks like the easiest solution I agree.
I was also wondering whether oil could be a good choice. IVF is done under silicon oil, so it should be pretty unharmful for cells I guess… Is it ? Also is silicon oil permeable to O2 ?
I haven’t tried to use oil for this purpose, but we use mineral oil layered on top of media in multi-well plates to avoid evaporation. Mineral oil allows gas exchange. You can purchase mineral oil from a pharmacy or (for a lot more $) from Sigma - both have worked fine for us. If you try it, let us know if it helps!
As Jennifer indicates, mineral oil can be used. Do you absolutely need 96 wells? As the wells get smaller, this is more of an issue. On 24 and 48 well plates, there is typically enough area within the well center, where you can avoid the edges. From a practical sense, do you need to image out to the edges of the wells? There are special imaging plates you can buy (ibidi typically has these products), that are quite a bit more expensive, where the material used for the wall of the wells is different or has been treated, so that the media stays flat, there is no ‘smiling’ or meniscus issue. I can’t remember off hand a pretreat that you could do. For dead cells, leaving in 0.1% triton X100 does a nice job of breaking the surface tension. Good luck.
How do you flood the wells without making a mess? Any tricks or tools? Are there any brands of 96 well plates that are easier to flood (maybe they have a lip around the outer wells)?
Some plates (just looked at a Greiner 96 well Sensoplate and a CellVis) do have a very small ‘lip’ around the entire perimeter of the plate, this could be enough, be careful though. If I did that I would consider putting the lid on (if its phase contrast or brightfield) and using parafilm or something around the edge to prevent spilling of medium or buffer into the stage or nosepiece.
I have a behind-the-desk suggestion, heh. Since the meniscus is caused by wetting, if you changed the hydrophobicity of the upper part of the well it should make it easier to create a flat liquid interface. For example, by dipping in silane then filling the well with media just to the level of the silane. Seems optimizable, but of course no promises since I haven’t tried it!
There are actually a patent regarding material for preventing meniscus effect: https://worldwide.espacenet.com/patent/search/family/039720811/publication/US2010047845A1?q=US9062283B2.
You can try to find this patented material or one of mentioned in patent competitors.
I personally switched to lower throughput with ibidi Ph+ slides. (they have up to 8-well slides).