[3DEM] Vitrobot and a Question?

Tue Feb 15 02:40:22 PST 2005

Hi all,

I've also had similar experiences to Bob. I use liposomes which start in size
from ~100 nm upwards in my cryoEM experiments, and it is normal to find them
huddled together at the edge of a hole  where the ice is thicker than in the
centre of the hole. I use manual blotting and a gravity plunger. 

When freezing I also aim to have an ice thickness gradient across the grid, as
I'd rather have a smaller area that I can use than none at all.

regards

Sarah

Quoting Bob Grassucci <bob.grassucci at wadsworth.org>:

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> 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
> >
> >
> >**** Messages to this list are automatically archived ***
> >**** Please limit quoting of previous postings to the bare minimum ****
> >
> >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
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