[3DEM] Vitrobot and a Question?

[Cervantes, Jessica]

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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