[3dem] cryoEM and humidity control

[Bob Grassucci]

Hi Lisa,
    How long are you pumping the tip while it is in the airlock?  You 
may want to increase it to at least 120 seconds to remove any loose 
ice.  35% Rh is pretty low so I do not think that is a problem.  We load 
our cryo holder as you describe a -55 degrees, then once the tip is 
pumped start the rotation manually until the holder top is level and 
then reset the tilt to zero.  There have been instances where a user has 
banged the cold holder inside the guide tube and caused a pole touch but 
that  is as bad as it got.  Is your guide pin straight?  This can cause 
crashes as well.  I hope these suggestions help.  Good luck.
    Regards,
    Bob

Lisa Craig wrote:
> 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
>
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>


-- 
********************************

-Robert Grassucci-
Howard Hughes Medical Institute
Wadsworth Center Empire State Plaza
Albany, NY 12201-0509
bobg at wadsworth.org
Phone: (518)474-5821
Fax: (518)486-2191




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