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

[Zongli Li]

Hi Steinar,

If the holder vacuum is good, this behavior is most likely caused by grid
clamping issue. We have similar experience in our Tecnai F20 with Gatan 626
cryo holder. If the clamp ring does not make good contact with your grid
(grid can freely move after clamping). When you move the stage, the
mechanical vibration can make the grid jump up and down and if the grid
loses contact with the cryo holder at any moment, it will warm up
immediately, as the cryo holder is the only thing that keeps the grid cold.
In good vacuum, the warmed grid will show as "sublimated". This
phenomenon has nothing to do with the beam.

Best,

Zongli



On Tue, Dec 10, 2024 at 8:42 AM 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
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20241210/35d98d68/attachment.html>