[3DEM] Gatan cryoplunge

[Bob Grassucci]

Dear Paul,
         We have found that Quantifoil grids vary from batch to 
batch.  Some are better than others with respect to strength as well as 
hydrophilic properties.  We routinely treat them with a chloroform 
atmosphere to get rid of any residual plastic and re-coat them with carbon 
just prior to putting our samples on them.  To get good cryo grids one 
needs to take into account not only the blotting parameters but also the 
substrate you are putting your sample on.  Another factor affecting quality 
is the buffer that your sample is in.  There are many components that can 
change the surface properties of water like detergents for example.  With 
good grids and the right buffer it is much easier to make the perfect 
grid.  My guess as to why you may get ruptured carbon is a combination of 
the carbon strength and what displacement you are setting the Vitrobot 
to.  A positive displacement causes the grid to be blotted harder since it 
is in the narrower space between the blotting pad "wedge".  Every Vitrobot 
has a sweet spot which varies from one to another.  You just need to find 
what it is on your particular instrument.
         Regards,
         Bob

At 09:54 AM 2/9/2005 -0500, Paul Chipman wrote:
>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