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

[Halldorsson, Steinar]

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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