[3dem] cryoEM and humidity control

[Bill Tivol]

On Nov 6, 2007, at 10:34 AM, Lisa Craig wrote:

> 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.

Dear Lisa,
	We have success on our T12 pumping for 50 sec, but YMMV, so Bob's  
suggestion is a good one.  I'd remove the o-ring and check the groove  
for small score marks or fibers, replace the o-ring, and try again.   
It seems like the crash occurs when the o-ring is traveling along the  
tube in the stage, and moving slowly prevents a crash, so if the o- 
ring is not the problem, I'd look into the surface in the stage that  
it moves on.
Yours,
Bill Tivol, PhD
EM Scientist
Electron Cryo-Microscopy Facility
Broad Center, Mail Code 114-96
California Institute of Technology
Pasadena CA 91125
(626) 395-8833
tivol at caltech.edu

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