[3dem] C-flats

[Jean Watermeyer]

Dear Thorsten

I posted a similar query a (long!) while ago - but I don't think I ever got
to posting a summary of responses.

Below are all the responses I received - I hope they are useful to you. A
belated thank you to everyone for your advice.

In the end, I succeeded in keeping the carbon intact by
- blotting by hand at an angle to the grid,
- making sure the plunger didn't go too deep into the ethane
- plunging when the ethane had only just begun to ice at the edges (not
actually icing over - I'm still not sure that this was not too warm
though).
However, I have since switched to quantifoil grids with Ottensmeyer thin
carbon as an alternative strategy to keep my sample flat.

Regards
Jean Watermeyer

Collection of C-flat advice:

Sergej Masich to me

show details 10/12/2008

Dear Jean,

I usually coat some extra carbon to stabilize the grids.

If you use freshly discharged grids, they are far too hydrophilic.
Therefore, it is extremely difficult to blot quantitatively. I prefer
glow-discharging the grids approximately 1 hour in advance. The grids remain
yet reasonably hydrophilic.

Hope it will help. Best regards,

Sergej


Stephen Mick to me

show details 10/12/2008

Jean,

This is Stephen from Protochips and we make C-flat. Thank you for posting
your question on the 3DEM listserver. It will be interesting to hear from
the community the specimen prep protocols that people have found useful with
C-flat.  When we hear from customers, we typically see people use much
higher humidity and shorter blot times -- closer to 90% - 100% humidity and
<5sec blotting.  However, it seems that every sample requires its own
specific prep conditions.

One thing to note, we make C-flat with a normal carbon thickness of ~20nm,
but we also make a version with thicker carbon of ~40nm. The thicker carbon
is more robust to handling and still preserves the flat film and clean
surface. If you would like to try the thicker carbon film, then please let
me know what mesh type and hole pattern you use.  I would be happy to send
you some samples.

Best Regards,
Stephen

Williams, Dewight R to me

show details 10/12/2008

Hi Jean,

I have used C-flat grids routinely for about two years now and have had good
results with ice thickness, particle preservation, and retention of carbon
support. I manually blot my grids on a gravity driven plunge arm into a 2 cm
deep cup of liquid ethane in a small dewar of liquid nitrogen. I blot from 5
to 10 seconds depending on buffer, protein sample concentration, humidity (I
don't use a humidity controlled chamber), and type of blotting paper. I
always freshly coat my c-flats before working with them. I essentially just
add a dusting of fresh carbon onto both sides of the grid, enough to barely
see on a piece of filter paper beneath the grids. This I have found to be
essential for consistent blotting results.

I also sometimes lose more carbon support than I would like, but typically
this is because of my clumsiness or zealous blotting. Our vitrobot loves to
remove the carbon from C-flats and I don't tend to use it much anymore. I
always apply my liquid to the side of the grid that is away from the carbon
layer and blot from the side with the carbon layer. I try not to drag the
filter paper along the surface.

Finally, I have found certain detergents or buffers tend to make the carbon
more fragile. I don't know why this would be the case, unless protochips has
something in the water they float the carbon supports or something on the
grids before they are coated. I now try to avoid any detergents, in
particular Triton-X series, but these sorts of things are voodoo that arise
from one bad experience and need to be empirically tested by adjusting ones
buffer to see if that is a possible source. Try your plunging with just
mili-Q quality water to see if the c-flats behave similarly.

I hope these helps,

Dewight

Dewight R. Williams Ph.D.
Department Molecular Physiology and Biophysics
Vanderbilt University
708 Light Hall, 2215 Garland Ave
Nashville, TN. 37232
Lab: (615) 322-7898

Terje Dokland to me, 3dem

show details 10/12/2008

We have used C-flat grids and manual blotting for some time, until we
recently got our Vitrobot. Like you, we find that if we glow discharge the
grids, we cannot get thin ice. The problem seems to be that the water goes
through the holes, and since we blot only from one side we get thick ice
forming on the other side. In the Vitrobot, we don't have this problem,
since the grids are blotted from both sides. Perhaps it would work better if
you blot from manually from both sides. We have never tried that (too
complicated).

If we don't glow discharge and blot for about 2-3 sec we get some thin ice,
but the thickness is very variable from one area to the other and you tend
to get huge gradients of ice thickness ("lumps" of ice of which only the
edge is usable) as you have also observed, but that's how we have been
operating, until we got the Vitrobot.

On the other issue that you mention, we find that the film on the 200 mesh
grids is too fragile and is almost always completely broken by the time we
get it in the microscope. The 400 mesh grids are more robost and retain most
of the film, however.

Terje

Joel Quispe to me, stephen

show details 10/12/2008

Hello Jean,
In response to your email about Cflats.
The carbon is thin and the open area on a 200 mesh grid is large and the
carbon will break a lot more than the 400mesh grids. We mainly use 400mesh,
but if we use 200mesh we add an extra layer of carbon to keep it from
breaking during blotting and plunging.
We have a Vitrobot and found that the parameters sometimes need to be
changed if the sample migrates to both sides of the grid. We blot 1-2
seconds longer if this happens. This might be happening to you. I have never
blotted on both sides manually, but other labs have done it by attaching
filter paper to large forceps and using that to blot.
I don't have that much experience with a glow discharger, I use a plasma
cleaner. Other people here do use a glow discharger but I don't know their
settings.

Best,
Joel


Joel D. Quispe
(858)784-9012
CB113

Puey Ounjai to me, 3dem

show details 10/12/2008

Hi Jean,
There are several things you can try! Some of them work for me!

1) float a thin layer of C on the C-flat and blot normally
2) vary the drain time (waiting time between blot and plunge)
3) blot from both side
4) use 2-3 layers of dry blotting paper instead of one
Hope that helps,
Puey

Matt Swulius to Puey, me, 3dem

show details 10/12/2008

Terje's response pretty much sums up my experience with c-flat. The 200 mesh
grids simply fell apart no matter how I blotted them. I tried evaporating
additional carbon on to them (as suggested by the manufacturer), but it has
little effect if any. They were very generous though and had a new batch
sent to me free of charge and they gave me some 400 mesh grids to try as
well. As others have noted the carbon support is more durable on the 400
mesh grids, but I was still surprised to see 50% or more of the carbon gone.
I've since stuck with quantifoil and have had no problems getting thin ice.

Matthias Wolf to 3dem, me

show details 11/12/2008

Hi all,

I use C-flat copper 400 mesh, 1 um hole size, 2 um distance.
Glow discharge 20 sec at +20 mV (a longer time pulls my particles towards
the carbon and 10 sec is usually the minimum to render the surface
hydrophilic). I blot 4 ul sample manually in the cold room at 4C from the
carbon side using Whatman Nr.40 (slow, calcium free) for 25-30 sec. This
results in reproducible ice thickness between 400-800 A. It is possible to
get thinner ice by increasing the blot time, but the amount of dry grid
squares will increase dramatically. The cold room is essential to maintain
humidity near saturation throughout the seasons.

I have successfully used both C-flats and Quantifoils. However, I have never
experienced charge-induced drift on C-flats, but often on Quantifoils
(soaking in ethyl acetate to remove plastic residue helps, but not
significantly). In my hands, C-flats result in thinner and more homogeneous
ice with no charging and less cryo-crinkling than Quantifoils. I have
reached my highest resolutions only when using C-flats.

The carbon film on the C-flats I have used so far is indeed thin, but just
right. Yes, it is fragile and hence requires handling with care. In my
opinion, the vitrobot is far too harsh on grids and allows no control  over
blotting force or closing speed and its standard filter paper wicks too fast
for adequate control by blotting time alone. I hope that this discussion
won't prompt the manufacturer of C-flat grids to modify their excellent
product by thickening the carbon film.

  Matthias

  Christopher Kennaway to me

show details 11/12/2008

Hi Jean,

I have been using C-flat grids (200 mesh, 1um holes), with an automated
blotting apparatus (blotting both sides 2 seconds), and also found that
almost all the carbon has been stripped off of most grids. I have used glow
discharge both with and without amylamine. It might be that 400 mesh are
more sturdy, but my guess is that my batch (and yours?) has too thin a
carbon film. On many C-flat grids I have found that the focus spot blasts a
hole through the carbon within ~30 seconds, and you can often see small
holes through it at 150000x.
Chris Kennaway

Hello All,

As a note to the community, for those who need a more rigid film we also
offer a double-thick carbon C-flat.  Many have found that the thicker film
adds flexibility to the sample prep process, particularly for 200 mesh
grids. Hope this helps.


Best Regards,
Stephen

Jean,
regarding the answer by Terje. If you want to try double-blotting by hand,
is
not complicated. There is a step by step description of how to make the
device
for double-blotting (with a figure) in one of our articles:
T. Ruiz and M. Radermacher. (2006) Three-dimensional analysis of single
particles by electron microscopy: sample preparation and data adquisition.
Methods in Molecular Biology 319, 403-425.
It is a Humana Press book, with a very strict copyright, I cannot just send
you
the pdf. Tell me if you cannot find it in your library and I can either fax
you
the page or rewrite that part for you.
Regards

Teresa

**************************************************************************
Teresa Ruiz, Ph.D.
Associate Professor and Chair of the Graduate Education Committee
Dept. of Molecular Physiology and Biophysics
College of Medicine
University of Vermont
Burlington, VT 05405
(802) 656-4835

On 25 May 2011 11:08, Thorsten Mielke <mielke at molgen.mpg.de> wrote:

> Dear all,
>
> we are trying to vitrify some small proteins on c-flat grids (type
> CF-2/4-4C) using a Vitrobot from FEI. However, after plunge-freezing, the
> carbon film is heavily destroyed. Additionally, we find only a few good
> holes on the remaining intact meshes. The carbon film also seems to be
> extremely fragile and easily disrupts upon focusing (300 kV, 155kx Polara).
> The grids seem to be OK (with or without glow-discharching) until
> plunge-freezing. We varied the blotting offset at the Vitrobot to play with
> the blotting force, no success.  Did you observe similar problems with
> C-flats? Any idea how to prevent the carbon film from breaking?
>
> Thanks
>
> Thorsten
>
>
>
>
> --
>
> *******************************************
> Dr. Thorsten Mielke
> - UltraStrukturNetzwerk -
>
> Max Planck Institute for Molecular Genetics
> Ihnestr. 63-73
> D-14195 Berlin
>
> Tel.: +49-(0)30 8413-1644/1141
> Fax: +49-(0)30 8413-1385
> E-mail: mielke at molgen.mpg.de
>
> _______________________________________________
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> 3dem at ncmir.ucsd.edu
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>



-- 
-----------------------------

Dr. Jean Watermeyer
Electron Microscope Unit
R.W. James Building
University of Cape Town
Rondebosch
7700
South Africa

Tel. +2721 6502820
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