[3DEM] Gatan cryopluge

[Bill Tivol]

On Feb 9, 2005, at 1:24 AM, Ishikawa, Takashi wrote:

> We are also happy with the FEI Vitrobot, basically. Students without 
> any
>  experience of cryo can make a good freezing on the first day. 
> However, we
>  realized one problem: Frozen grids have gradient of thickness quite 
> often. In
>  other words, ice is too thick at one end and the grid is totally 
> dried at the
>  other end, while the area (a few to 10 meshes) in between has optimum
>  thickness. I guess this is because the grid is blotted by tilted 
> filter paper
>  attached on pads. Undoubtedly, It would be nicer if we have even 
> thickness
>  of ice.
>
> Did anybody have the same problem as we have and solve this issue ? Or
> anybody has the idea how this problem happened ?
>
Dear Takashi,
	The Vitrobot is, indeed, designed to blot the bottom of the grid more 
firmly than the top.  We have not seen as bad a problem with ice 
thickness gradients as you have, but this may be due either to the 
types of specimen or blotting parameters.  We usually freeze in dilute 
aqueous buffer, but also in the medium that the particular organism is 
grown in--in one case very high [NaCl].  We use 200 mesh grids with 
Quantifoil substrate, blot times of 2-3 sec, and an offset of -2 or -3 
for typical specimens.  Perhaps the Quantifoil substrate prevents the 
grid from drying, but we usually get a very large fraction of usable 
area.  (I have no connection with Quantifoil, except as a satisfied 
user.)
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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