[3DEM] Vitrobot and a Question?

[Bill Tivol]

On Feb 11, 2005, at 2:31 PM, Cervantes, Jessica wrote:

> 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?
>
Dear Jessica,
	We have frozen lots of particles in your size range, and there is no 
indication that they are "blotted off", or that ice thickness is a 
problem.  However, when the blot paper comes in contact with the 
specimen, both the fluid and the particles will partition between the 
paper and the water remaining on the grid, so, if your particles bind 
strongly to the paper, it is possible that they will partition onto the 
paper in preference to staying on the grid.  I don't know whether using 
a different paper or other blotting substance would be practical, but 
what you want to do is to have the particles remain in the water and 
not bind to the blotting substance.  It could be that blotting from the 
edge after manual application and setting your parameters to zero blots 
could work.  I have also run across specimens--membranes--that had very 
poor coverage on cryogrids, but much better coverage when negatively 
stained.  I had to fiddle with my blotting technique a lot to get fair 
coverage; I was using a gravity plunger, not a Vitrobot, at that time.
Yours,
Bill Tivol, PhD
EM Scientist and Manager
Cryo-Electron Microscopy Facility
Broad Center, Mail Code 114-96
California Institute of Technology
Pasadena CA 91125
(626) 395-8833
tivol at caltech.edu