[3DEM] Vitrobot and a Question?

[Bob Grassucci]

Dear Jessica,
         I think the answer lies in the ice thickness.  I have seen 
instances where our ribosomes (25nm) have been segregated to the edge of 
the hole in the carbon we use, while there is nothing in the center.  My 
guess is that the particle goes to where it can fit and if it no longer 
fits it comes into contact with the blotting paper and is then wicked 
away.  Have you ever seen this type of behavior with your grids?  You might 
just need thicker ice or if you want a gentler blot with the Vitrobot use a 
negative displacement.  Good luck.
         Bob

At 02:31 PM 2/11/2005 -0800, Cervantes, Jessica wrote:
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>Hello All:
>
>I've been reading the Gatan cryoplunge thread with interest as we have an 
>FEI Vitrobot.  We are very happy with its ease of use and the quality of 
>samples produced.
>
>Here's a question that has come up recently: we work with nanoparticle 
>samples that have a wide size-distribution, anywhere from 10s to 100s of 
>nanometers.  We find that when we prepare these samples for cryo TEM, the 
>10s of nm sized particles are retained on the grid, but there is little or 
>no evidence of the + 100 nm particles.  The conjecture is that the 
>particles have been "blotted off" by the Vitrobot.  We are currently 
>performing experiments to confirm this hypothesis (for instance, comparing 
>the results of hand-blotting, a gentler(?) method, to machine-blotted 
>grids), but I wonder if any of you have come across this problem?  Is the 
>answer as simple as the thickness of the ice layer on the grid?
>
>Thanks very much,
>Jessica Cervantes
>
>Bend Research, Inc
>64550 Research Rd
>Bend, OR  97701
>(541) 382-4100 page
>(541) 382-0212 x240
>(541) 382-6177 fax
>
>
>-----Original Message-----
>From: owner-3dem at ncmir.ucsd.edu [mailto:owner-3dem at ncmir.ucsd.edu]
>Sent: Wednesday, February 09, 2005 2:04 PM
>To: 3dem at ucsd.edu
>Subject: [3DEM] Gatan cryoplunge
>
>
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>Dear All,
>
>Seems to be a lot of interest in this topic.
>
>I have used both the Gatan and the Vitrobot.
>
>The Gatan model was a very early version, several years ago so it might
>have been improved since then. I had problems with contaminated grids and I
>put it down to the design at the time having the grid box mounted on mesh
>above the liquid nitrogen so it sat in nitrogen vapor. The best advice
>would be to try and test a working set up yourself.
>
>We recently acquired a Vitrobot and are happy with it, especially as others
>mention for novices or people plunging for the first time. The humidity and
>temperature controlled chamber is excellent for sensitive samples. It does
>seem to be very consistent with a variety of samples once you have got your
>parameters right
>People with a lot of hand blotting experience still like to do it as it can
>be a bit quicker to set up and with experience you might get a greater ice
>coverage. Its also hard to change old habits.
>
>According to FEI, the ice gradient is intentional so that at least
>somewhere on the grid you will have a strip section with reasonable ice
>thickness. This is why the dual blotting pads are inclined at an angle. It
>would be nice though if you had the possibility to change this angle.
>
>One way to test the blotting efficiency using different parameters is to
>blot droplets of dye (any light microscopy stain will do) so you can see
>the distribution on the filter paper. We did this and found with some
>settings that only one side of the grid gets blotted. This may vary from
>machine to machine as the alignment of the plunger might vary slightly and
>I have not found a way to adjust it.
>Also make sure both blotting pads rotate after each blot as they are
>supposed to do. Sometimes one side of ours gets stuck so you end up
>blotting on saturated filter paper if you are not watching for this. As
>others mention always stick with the same filter paper. We use Whatman 1
>qualitative. The key is to be consistent whatever you use.
>
>Regarding Quantifoil grids, my experience from hand blotting is the foils
>break if the grids are over blotted. The filter paper then adsorbs more
>strongly to the Quantifoil surface and pulls it away when the blotting
>paper retracts. You can get away with this somewhat by blotting less or
>from the opposite side when hand blotting.This obviously does not work with
>blotting pads from both sides.
>We also get more broken film with the Vitrobot than with hand blotting and
>I think this is as Paul suggests from the relatively strong blotting
>pressure of the Vitrobot pads.
>
>Happy Plunging.
>
>Ken
>
>
>
>
>
>
>**********************************************************************
>Ken Goldie
>European Molecular Biology Laboratories
>Meyerhofstrasse 1
>69117 Heidelberg
>GERMANY
>
>Tel:  0049 6221 387 8362 (Office),   Fax 0049 6221 387 8306
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********************************
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