[3dem] Strange ice behaviour - advice welcome from community!

[Ernesto Arias]

Hi Steinar,

Not sure if it's the same problem, but we had issues with cryo-grids
melting on the Gatan 910 multi-specimen cryo holder.

https://urldefense.com/v3/__https://x.com/EAriasPalomo/status/1309506212204081153__;!!Mih3wA!Fg6BgOGudDTs2gpnQXrl0Z5ynuBKSSr7zQ9MM-0nimpeLQf1c6wpBWK5y0ggOcANLwE5yh1w1B1NUkHXdmuxmPjh0JI$ 

Initially, the holder worked perfectly, but then we noticed that some grids
would melt either from the beginning of a session or randomly during use.
After troubleshooting, we identified the root cause as the splitrings. Over
time, these rings tend to constrict, with their C-shaped gap closing
slightly. This results in the splitrings sitting too loosely on the holder;
they can even rotate in place when clipped, which prevents proper contact
with the grid and leads to temperature loss.

To address this, we’ve started regularly opening up the splitrings slightly
using a pipette tip. This simple adjustment has completely resolved the
issue for us. I hope this solution is helpful to you as well and saves you
the frustration we initially faced.

Best wishes,

Ernesto.



On Tue, Dec 10, 2024 at 2:42 PM Halldorsson, Steinar <
steinar.halldorsson at roche.com> wrote:

> Dear 3DEM community
>
> Thanks for years of great forum discussions!
>
> The EM team at Roche in Basel has recently been observing some strange
> events in our JEM1400 microscope and we are curious if other users have
> seen this or if there have even been previous posts about this on the forum.
>
> Stochastically, some of our grids appear to "sublimate" when they are
> placed under the beam in the microscope. The observation is as such:
>
> After a minute or two of illumination of a grid in low mag or higher mag,
> it seems that the beam induces some sort of a cascade that seems to dry up
> the entire grid and leaves only strings or lumps of protein most likely. At
> the beginning the grid is full of vitreous buffer and within about 5-10
> seconds the entire buffer/water seems to have sublimated (see images
> attached of the dried up grids). One can even watch the grid live as it
> dries up.
>
> Some key facts:
> - Seems that frequency of this event has increased in the last couple of
> months
> - We have seen this only on our JEM1400 microscope and we use a Gatan 910
> multi specimen cryo holder
> - This has not been observed on our cryoARM (Jeol)
> - This happens independent of the slot used in the cryo holder
> - This only happens if a grid has been illuminated (not just stored in the
> holder)
> - Higher beam intensity appears to increase the speed of this happening
> but this is not as clear yet
> - We've monitored the holder temperature and it doesn't spike, seems to be
> stable below -170 deg C.
> - Happens with multiple samples and with multiple grid types (quantifoils,
> hexafoils, ultrafoils)
> - Happens with grids frozen from our Leica plunger and with the CryoWriter
> - We have high grade ethane and the bottle has not changed for over a year
> (we have a 10 L bottle)
>
> Our thoughts after a number of discussions:
> - Maybe we have a systematic contamination of something on the grids that
> causes this phenomena - unclear what it could be though
> - Is freezing problematic, due to temperature of ethan or other factors?
> Is ethan forming a layer outside the vitreous buffer? Does it get embedded
> even? (Would ethane even exhibit this kind of behaviour?)
> - Perhaps this is from the grids themselves, do other users observe this
> for more recent grid batches?
> - Is our storage dewar contaminated with something that attaches to our
> grids that reacts with the electron beam? Do some plastics degrade in
> liquid nitrogen?
>
>
> If anyone has observed similar issues we would like to hear from you, and
> even better if you have identified the source of those issues we would of
> course really like to hear from you.
>
> Hope to hear from some of you soon!
> Best wishes,
> Steinar
>
>
> --
> Steinar Halldórsson DPhil (he/him)
>
> F. Hoffmann-La Roche AG
> pRED/Lead Discovery/Structural Biology
> Grenzacherstrasse 124, 006/OG03, 4070 Basel, Switzerland
> Email: steinar.halldorsson at roche.com
> Tele: +41 61 68 82374
> _______________________________________________
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> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>
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