[3dem] weird ice?

[Shi, Dan (NIH/NCI) [C]]

Hi Peter,

As people pointed out the crystalized ice should be caused by the grid's temperature was raised before recording the images. Loosen clip ring usually associated longer settle time and severe drifting. Extreme thing ice could be devitrified within a second in RT air as Tommi suggested, you could take an image from the thicker area of the grid to confirm it.

Beside slightly increasing the ice thickness, shortening the grids travel distance/time between liquid ethane to LN2 during freezing may help too. I observed very similar incidents of grids with relative thin ice on our Krios.

Good luck,

Dan Shi
Frederick National Lab for Cancer research
CCR, NCI

From: White, Tommi A. <whiteto at missouri.edu>
Sent: Wednesday, October 17, 2018 1:03 PM
To: Peter Shen <peter.shen at biochem.utah.edu>
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] weird ice?

Hi Peter.

When I see this, I assumed I got my sample too dry, and what remains is protein film.  I usually load a grid that should have "thicker ice".  The way I assess which ones should have thicker ice is by recording both humidity in my vitrobot chamber and assessing blot size on my vitrobot filter paper immediately after the blot has happened (somewhat subjective, but with a single sample, side-entry cryoholder - I've gotten pretty good at picking out which grids will have suitable ice).

For example...(assuming everything else is the same)
lower humidity with a big blot on filter paper = thinner ice (maybe dried, as in your case)
lower humidity with smaller blot = thicker grid
higher humidity with small blot = thickest grid

My two cents, for what it's worth! :)

HTH,
Tommi
Tommi A. White, Ph.D.
Assistant Professor of Biochemistry
Director, Electron Microscopy Core Facility
University of Missouri
W117 Veterinary Medicine Building
1600 East Rollins Street
Columbia, MO 65211
573-882-8304
WhiteTo at missouri.edu<mailto:WhiteTo at missouri.edu>
http://emc.missouri.edu<http://emc.missouri.edu/>



From: 3dem [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of Peter Shen
Sent: Tuesday, October 16, 2018 6:39 PM
To: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
Subject: [3dem] weird ice?

Hi everyone,

We've recently started to see some funky ice on our TF20. Here are a couple of screenshots:

https://www.dropbox.com/sh/mlfado5rf9o447c/AAAt9VJM28KG6sCaNnCXIXQga?dl=0

Has anyone seen this before? We see this regardless of sample and grid type (a few different protein preps and UltrAuFoil/Quantifoil tested). Any insight would be greatly appreciated! We're pulling our hair out here...

Peter
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