[3dem] Re: 3dem Digest, Vol 44, Issue 3

[DocDave50 at aol.com]

Hi Jim,
 
I have not had the opportunity to  exercise to use the Joel scope but the 
following procedure works excellent on  the Tecnai scopes.  Of course, the 
major issue is the holder type being  used.  It's a bit different using an 
Oxford Holder than the Gatan Holder  but the principle is still the same. 
 
When using any of these holders, it is  best if you can have some reserve 
left in the dewar after insertion.  One  possible issue to be concerned with 
when dumping all of the Liquid N2 is that it  is possible to get some on the 
viewing screen if not covered, a second is  that it is the cause for the 
cracks that appear on the scope desk  top.
 
On the Tecnai series scopes, using  Gatan holders, you rotate the 
Goniometer to -55o, start the TMP to  evacuate the airlock and remove most of the N2 
in the dewar leaving residual  liquid N2.  Upon removal from the Transfer 
Station, the holder is rotated  90o such that the mouth of the Holder would be 
facing away from the  front of the microscope and the dewar/alignment pin 
at 90o and oriented with the  airlock assembly.  Since the TMP is up to speed 
and the airlock  pumped, you fully insert the holder with no loss of 
remaining N2.  Since  the TMP is already at speed, it starts pumping the airlock 
immediately limiting  the potential for ice contamination on the cryo-shield. 
 Once the Airlock  is fully pumped, rotate the Holder (without pulling or 
pushing) to the 0o  position (opening to the top), and then, while holding 
the dewar  stable, rock the Goniometer back to zero.  At this point, the  
Holder will pull into the microscope and the Holder Dewar is refilled with  
Liquid N2.  
 
Under these conditions, the dewar  always has residual N2, there is no need 
to dump all that N2 out...on  whatever...the desktop (causing 
cracks)...potentially the front screen  (potentially imploding the Camera 
Chamber)...styrofoam box (throwing away all  that N2)...whatever.  With the dewar topped 
off and some time allotted for  sample stabilization, away you go to collect 
data.  Under these conditions,  I have found that the sample stabilizes to 
drift rates less than 1A/sec  reasonably quickly and I rarely have ice 
contamination.  I can't say that  this is related to the maintenance of liquid N2 
in the dewar but it works  quite well.
 
My only concern regarding the approach  you are outlining below, Jim, is 
that it would appear that you are inserting the  holder into the airlock and 
then starting to pump the airlock.  I'm not  sure what the JOEL designation 
for the airlock, which opened, pumps the inner  airlock (it is N2 on the 
Tecnai), but you will find it much easier to pre-pump  the airlock prior to 
insertion of the holder.  It is, then only moments,  before the airlock is fully 
pumped such that holder insertion can commence  rather than 5 minutes.  
Another benefit to the -55 counter clockwise  compustage rotation is that the 
holder insertion is easier because it's easier  to hold the dewar at that 
angle and you have obvious points for aligning the  holder as you are moving it 
from the transfer station to the scope.
 
Best Wishes
 
David W. Chester, Ph.D.
 
In a message dated 4/3/2011 3:00:06 P.M. Eastern Daylight Time,  
3dem-request at ncmir.ucsd.edu writes:

Hello,
Before cryoEM sample holder insertion, it is rotated 90  degree, so most
liquid N2 is poured toward user from dewar. I heard that  new TEM goniometer
could be tilted before holder insertion. Anyone try it  with older TEM?

I'm thinking two ways for our JEOL2010F.
1) tilt  gonimeter +60 degree, insert sample holder (dewar is -30 degree),
flip  airlock switch to "PUMP", after green light is on and wait for another
~5  min, turn the holder clockwise ~30 degree (now dewar is straight up),
tilt  gonimeter back to +30 degree, then turn the holder clockwise another
~60  degree (now dewar is +30 degree right), and finally tilt gonimeter back
to  0 degree (now dewar is straight up).

2) tilt gonimeter +45 degree,  insert sample (now dewar is -45 degreet), 
flip
airlock to "PUMP", after  greenlight is on and wait for ~5 min, turn the
holder ~30 degree (now dewar  is -15 degree left) and another ~60 degree 
(now
dewar is -45 degree),  finally tilt gonimeter back to 0 degree.

The first method need tilt  gonimeter two times at high angle, but the
holder's dewar is only rotated  within 30 degree.

Anyone try it on JEOL2010F? If it works, it reduce  one hazardous operation
in  cryoEM.
Thanks!
Jim

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