Hi all,
I have grids that all have a confusing ice-quality. All grids (UAUFT and AU-Flat, 1.2/1.3) were prepared using the vitrobot with following settings: 100% humidity, 4°C, 4ul sample, 10sec wait time, 5 sec blot time, 1sec drain time.
The ice gradient in general looks promising, but when I make images, the ice breaks immediately, even in squares that look like they have thicker ice in holes. Also, there are some kind of patches with varying thickness, where in one particles have great contrast and in adjacient areas the ice is way too thick to see particles.
Anyone has seen similar behaviours?
It’s probably just really thin everywhere, and too thin to hold the particles in the empty areas. For gold foil, I recommend glow discharging (can be long/intense, even 2 minutes) to clean the foil and then when you apply the droplet of sample, use the surface tension at the pipette tip to drag or rub the drop around the grid. I use a circular motion. The tip shouldn’t contact the grid directly. You can also try reducing blot force instead of the time, I usually use -1 or -2. Hopefully you can get a better ice thickness, and also get rid of the ice domes in the squares.
Is there anything unusual in your buffer? E.g. some kind of sugar?
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perhaps eutectic ice? need the post-blot to plunged in ethane time to be absolutely minimal. when you fire a beam at the hole, does it snap or does it melt? snap and peel is eutectic (aka freeze-dried) ice. the “egg yolk” dark circles in the center of some squares is also common, arguably from ethane not being cooled enough.
agreed sugars/detergents/glycerol could be culprit, and your high-mag image does suggest ice is so thin it’s exclusionary.
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By the look of your highest mag. I can see a layer of crystalline ice there. As above, buffer composition could change the way ice forms and having this issue.
But also worth look out if your ethane is cold enough or not.
Good luck!
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Thank you all. I am pretty sure now it was the drain time. 1sec drain time and all grids looked like that. I made grids of the very same sample without drain time and ice / contrast / particle density was good.