[3dem] cryoEM and humidity control

Bob Grassucci bob.grassucci at wadsworth.org
Tue Nov 6 11:27:17 PST 2007

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.

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

IMPORTANT NOTICE: This e-mail and any attachments may contain
confidential or sensitive information which is, or may be, legally
privileged or otherwise protected by law from further disclosure.  It
is intended only for the addressee.  If you received this in error or
from someone who was not authorized to send it to you, please do not
distribute, copy or use it or any attachments.  Please notify the
sender immediately by reply e-mail and delete this from your
system. Thank you for your cooperation.

More information about the 3dem mailing list