[3dem] cryoEM and humidity control
Lisa Craig
licraig at sfu.ca
Tue Nov 6 10:34:59 PST 2007
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
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