[3dem] Re: 3dem Digest, Vol 49, Issue 20
DocDave50 at aol.com
DocDave50 at aol.com
Thu Sep 29 09:48:51 PDT 2011
Hi Liz,
I would agree with Rachael's question re: vesicle blebbing from the cell
or, if there is a serum component in your media, there could be excess lipid
desorbing from the serum. Given the density of the blobs and the relative
amount of them over the image space, they appear very much like detergent
or amphiphile globules that one would see in water that isn't cleaned via a
"clean" reverse phase column in the water purification system used to
prepare the buffers. A second source of these little 'buggers'...since I am
moving to London, I have to start using some appropriate phraseology I'd
heard while there in July...is the glassware cleaning process. If glassware
used to prepare buffers is washed in a dishwasher and the wash cycles are not
appropriately set OR the glassware is not rinsed frequently enough...12
times to remove regular dishwashing soap...a lot huh?...then 'soap' is left
to dry on the glass surface and then you prepare your buffers, media,
sterilize it and what to your wondering eyes should appear in every prep and
every time you try to make a change...blobs!
I'd suggest this one option. First, don't solvent wash your grids. If
they need solvent washing, get fresh grids. If you are going to solvent wash
them, wash with Chloroform and then pure hexane. After washing, you must
leave your grids under vacuum for ~3 min to ensure that the solvent is no
longer on/in the hydrophobic carbon surface. If you are concerned about
contaminants coming from the grids, you could try very short carbon
evaporation on your holey carbon grids...both sides...prior to sample addition. My
suggestion is that you get some of your purified water...often times if you
have a glass bottle reservoir you can see it there. Take a surface angle
glance...since fat floats...and see if you can see any sheen on the surface
of the water. Remember that double glass distillation does not remove
organics just inorganics. If there is a sheen, there is your problem. When it
gets really bad, there is a slight soap ring on your reservoir. I would
then take your water being used to prepare buffers and just use this to
prepare a grid with the very same freeze plunge process you are currently
using. Are the blobs there in this image? Yet another potential source of
these blobs...and I've seen them myself...are you possibly maintaining your
grids under vacuum using either house vacuum or an oil vacuum pump to
establish vacuum. Both of these systems provide very low vacuum AND, most notably,
TONS of oil back-streaming which, over time, contaminate your grids. I
found this out the hard way when I was starting the process of making our own
periodic holey carbon with the PDMS stamping procedure. If I stored the
grids long enough under a vacuum pump driven by oil or house vacuum, you
could actually see 'puddles' of oil on the grid surface when you looked at
them from the side. You now have a very prominent source of 'blobbish'
looking things all over the grid and in the ice.
I would be interested to know the storage conditions you are using for
your grids...remember, that in many Biochemistry labs, there are folks using
house vacuum or oil vacuum pumps such that there is oil contaminants in the
lab air...been there, done that...and whether a quick look at your base
water supply yields the same 'blobs'.
Hope this helps...and, yes, I have seen them and these were the causes in
my case.
David W. Chester, Ph.D.
Department of Biochemistry
Center for Structural Biology
Imperial College of London
In a message dated 9/28/2011 3:00:07 P.M. Eastern Daylight Time,
3dem-request at ncmir.ucsd.edu writes:
http://dl.dropbox.com/u/23095990/Caulobacter_2sept2011.tif
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