[3DEM] Gatan cryoplunge

[Bill Tivol]

On Feb 9, 2005, at 2:39 AM, Koning, R.I. (MCB_EM) wrote:

>
> We found that especially the lower part of the original vitrobot, 
> holding the ethane, is poorly designed. The grid has to go through air 
> when going from ethane to the grid container in the nitrogen, the 
> ethane freezes up quickly, and the grids are very difficult to place 
> inside their containers, which lie deep in the bubbling nitrogen. So 
> we build our own device mainly changing the lower part, adapting it a 
> bit to the design of the old Reichert KF80 plunger. We build in a 
> small heating device and a special chamber to hold the grid boxes, 
> which are shielded from air and do not suffer from any bubbling of 
> nitrogen. Now we can operate our machine a full day without refilling 
> any ethane (which is usually not necessary...). Our machine has also 
> been proven to be usefull for freezing cells (single layers). If you 
> want I can send you a picture of the lower part.
>  
> I have to add that I found that treating your grid right is important 
> for getting good samples. I have the impression that on all home made 
> holey grids (I never got around making usable ones myself) and also on 
> commercial grids, there is always some plastic resin left. So what 
> helped me A LOT is that I put thin layers of carbon ON BOTH SIDES of 
> the grids I buy. Critical seems to be that directly before use I glow 
> discharge the grid ON BOTH SIDES. It seems that this increases the 
> spreading of the sample on the grid. This also when using proteins 
> that prefer to stick to your grid and are not located in the holes, 
> and I had very good results using ribosomes and no problem at all in 
> getting them in the holes. I must say that this might ONLY be 
> advantageous when you put the sample on BOTH sides of your grid, which 
> I always do...

Dear Roman,
	We have two of the older style cryogen containers, and one freezes 
faster than the other.  We have not yet installed a heater, but that is 
a very good idea.  One of our postdocs solved the problem of placing 
the grids in the storage boxes; use a forceps to lift the aluminum box 
holder near the liquid nitrogen surface, then it is much easier to 
place the grid in the desired slot.  I agree with you that 
glow-discharging is important, and we have found that it is best if 
this is done very close to the plunging, so we glow discharge a few 
grids at a time.  We have not found any plastic residue on either 
Quantifoils or commercial lacy carbon grids, so we find no need to glow 
discharge both sides.  When we apply the specimen manually to one side, 
we always see liquid on both sides before blotting.  If not, then we 
use a longer glow discharge time for the next set of grids.  It can 
also happen that a particular specimen will distribute better in the 
holes for either longer or shorter glow discharge times, so we always 
investigate all the parameters before settling on a protocol for a 
particular specimen.
Yours,
Bill Tivol, PhD
EM Scientist and Manager
Cryo-Electron 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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