[3DEM] Gatan cryoplunge

[Koning, R.I. (MCB_EM)]

Dear Oscar and all,
 
Automated plunging is very desirable, especially to get reproducable results, for students and people with little experience and for certain special cases (temperature and moist critical samples: liposomes, microtubules, cells and others). I have no experience with the GATAN plunger and little with the Vitrobot.
 
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 had no experience with the water layer thickness ranging in thickness over the grid, but I can imagine that if the blotting time is short, the rubbers on the upper side get no time to not touch the grid properly or are shielded by the tweezers. Also the grid might be located a bit too high between the filter papers, so that only part of the grid is blotted properly. You might wanne try different blotting paper (???). 
 
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...

Roman 

R.I.Koning, Ph.D. 
Molecular Cell Biology 
Leiden University Medical Center 
Wassenaarseweg 72, 2333 AL 
Leiden, The Netherlands 
(+31) 71 527 6463 (tel) 
(+31) 71 527 6440 (fax) 

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