[3dem] Re: Ethane - propane mix
sindelar at alum.mit.edu
Wed May 2 10:40:04 PDT 2012
Dear Bill, I am glad you raised this issue. We also regularly use the ethane/propane mixture here at Yale, and while others do not report vacuum issues similar to what you have experienced, I myself have seen them. It appears to be tweezer - specific; others use our Vitrobot, while I have been using a home-built cryo-plunger with a different set of tweezers.
What I noticed was as follows. If a freshly frozen grid is inserted immediately into our F20, the vacuum is initially good ***until the specimen holder shutter is opened***. Then, the IGP pressure readout will slowly rise up to a maximum of 20-25, before slowly subsiding again to the expected value of ~6-10. I observe this consistently with my own specimens, while others (using the Vitrobot) do not report the effect. I believe that in earlier experiments I sometimes saw a similar thing with liquid ethane, but not as consistently.
We actually "fixed" the problem by accident when we damaged our tweezers by dropping them; I repaired them by using sandpaper to file down the bent ends, making the ends considerably more blunt. Unexpectedly the vacuum issue went away; when we replaced the tweezers, the effect returned. I subsequently examined the grids closely after freezing, and saw that the ethane/propane mixture is indeed being retained after freezing in the area where the tweezers grasp the grid.
We decided we were able to live with this issue, because for us it only seemed to delay the vacuum recovery slightly; however, it does raise the obvious concern that the IGP may be aged prematurely by the extra stress. I think the suggestions by others to dry the grid with filter paper after freezing (or to store the grids for a day, which also fixes the problem) make a lot of sense and I look forward to using them myself!
Date: Tue, 01 May 2012 16:02:40 -0400
From: William Rice <rice at nysbc.org>
Subject: [3dem] Ethane - propane mix
To: 3dem at ncmir.ucsd.edu
Message-ID: <4FA04160.4030705 at nysbc.org>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed
Have many of you had experience using a mix of 37% ethane / 63% propane
as a cryogen? It has the advantage that it stays liquid at liquid
nitrogen temperature. We had thought it would be easier to handle, but
we found that it seems to leave a thin layer of liquid cryogen on the
surface of the grid. Putting a freshly frozen grid into the microscope
seemed to cause problems with the IGP as this liquid seemed to evaporate
only in the high vacuum of the microscope. Solid ethane frozen on the
grid usually cracks and breaks off or sublimes in the airlock during
pumping before insertion. Has anyone else seen this effect?
William J. Rice, Ph.D.
New York Structural Biology Center
89 Convent Avenue, NY, NY 10027
-------------- next part --------------
An HTML attachment was scrubbed...
More information about the 3dem