[3DEM] Gatan cryoplunge

[Paul Chipman]

Dear All,

We regularly use the Vitrobot and as Bob and others have already 
mentioned, we routinely see a gradient of density.  Since we work 
with viral particles of varying diameter the gradient is useful as 
most grids have at least some ice of proper thickness.  It would 
however, be detrimental to those wishing to use Leginon or some other 
automated package.  In that case I think more a more rigorous testing 
of parameters might be needed to yield the uniform ice that Bill 
Tivol is able to achieve.

As Norm already mentioned, the Vitrobot is extremely useful for 
teaching purposes as it eliminates the need to acquire this skill 
while the student is still learning how to properly transfer the grid 
to holder and microscope.

One other thing we have noticed is that occasionally we get a batch 
of Quantifoil grids that continuously have most grid squares ruptured 
while using the Vitrobot (using the same parameters that usually 
work).  These same Quantifoil grids work fine with manual, plunge 
freezing.  We're still not sure if the Vitrobot is less gentle than 
freezing by hand or if some other factor is involved.

Cheers,
paul



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>Dear All,
>         Our experience with the Vitrobot is this.  As you blot the 
>grid initially you get this gradient.  Depending on how much longer 
>and the absorbancy of the filter paper you use this gradient moves 
>up the grid.  This comes from the design of the blotting pads which 
>are at an angle.  Depending on how hydrophylic your grids are will 
>determine if the ice remains over the holes or they become open as 
>the thin part of the gradient traverses the grid.  There is a slight 
>window of time where you will have the proper ice thickness on the 
>grid.  You must also realize that different filter paper will absorb 
>differing amounts of buffer before it is saturated, which comes into 
>play as well.  Once you have found the right parameters stick with 
>it and you will get consistent results.  Good luck.
>         Bob
>
>At 11:39 AM 2/9/2005 +0100, Koning, R.I. \(MCB_EM\) wrote:
>
>********************************
>Robert Grassucci
>Howard Hughes Medical Institute
>Wadsworth Center
>Empire State Plaza
>Albany, NY 12201-0509
>
>bobg at wadsworth.org
>Phone: (518)474-5821
>Fax:	(518)486-2191
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-- 
Paul Chipman
Electron Microscopy Facility Manager
Dept. of Biology, Purdue University
Lilly Hall, Rm. B216
Phone: 765-494-1487
Fax:765-496-1189