[3dem] cryoEM and humidity control

[Lisa Craig]

Hi,

We have recently started doing cryoEM on a Tecnai F20 that has until 
now been used mostly for material sciences. Our first few sessions went 
well but lately we find that the vacuum crashes almost every time we 
insert the Gatan 626 cold stage. We also noticed that the tip of the 
stage, where the grid is held, gets frosted up immediately after 
pulling it out of the workstation prior to insertion into the scope. 
Inserting the cold stage at room temp doesn't cause the crash. Its been 
suggested to us that this happens because the humidity in the room is 
too high and this is what's been causing the crash. Our humidity 
fluctuates a lot and is lately hovering at 35%. This does make sense to 
me that putting a wet tip into the column would affect the vacuum, but 
(i) there are times when we see the frosting at the tip but don't get a 
crash; (ii) others have told us they do cryo successfully in even 
higher humidity; and (iii) preliminary tests suggest that if we turn 
the stage counterclockwise very slowly into position for final 
insertion into the column after the initial evacuation of the airlock, 
we seem to avoid a crash.

Regarding (iii), normally we rotate the goniometer to -55 degrees, 
insert the stage with the LN2 opening on the dewar pointing at 3:00 for 
pumping of the airlock, then bring the goniometer back to 0 degrees and 
simultaneously rotate the stage in the opposite direction, 
counterclockwise into position and guide it in. This is the way I've 
always inserted the cryoholder and never had problems. But on this 
microscope, during this simultaneous counter-rotation of the goniometer 
and coldstage we usually see a big jump in column pressure and often a 
crash. However, if instead we allow the stage to rotate clockwise with 
the goniometer back to 0 degrees, then very slowly rotate the stage 
counterclockwise into its final position this jump and vacuum crash 
don't occur, although we've only tried this a few times.

So I was hoping to get some input from the community into how critical 
the humidity is, whether others have observed the frosting of the stage 
tip, and whether this sounds like a humidity problem or something 
deeper, like the cold stage not cooling properly or the seal between 
the coldstage rod and the goniometer failing. I actually think the cold 
stage is working fine because the temp stays at ~-175 degrees C and we 
don't have a problem with contamination as long as the vacuum doesn't 
crash. We've also cleaned the o-ring on the goniometer and checked it 
for flaws.

Thanks for your time,
Lisa.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Lisa Craig, Assistant Professor
Dept. of Molecular Biology & Biochemistry
Simon Fraser University, Rm. SSB 7151
8888 University Dr., Burnaby, BC Canada V5A 1S6
http://www.sfu.ca/mbb/mbb/faculty/craig

Office phone: 778-782-7140
Lab phone: 778-782-7141
Fax: 778-782-5583