[3dem] Subject: Precipitation observed after making freeze substitution cocktail containing 5% water

[r.mesman]

Hi Qiaohui Yang,There are two solutions to your problem,One is to make the freeze-substition mixture without water (so for 5ml end volume dissolve the osmium and uranyl acetate in 4,75 ml acetone), cool it down to -90°C and then pipette in the water (this needs to be done quickly otherwise the water freezes in the pipette tip). Then vortex the solution until all visible ice crystals have dissolved and cool down the mixture again. The othet solution is to use a stock solution of 20% uranyl acetate dissolved in dry methanol.(This was also done in tyhe walther & ziegler paper on the addition of water in freeze substitution).In my hands with using the first method 1% of water is enough to enhance membrane contrast, going to 3% enhances the visibility of glycogen and not contrast is gained from going to 5%. The small ammount of methanol added in method 2 allows for more water to be dissolved in the substitution mix at lower temperatures, so more water needs to be added to get the same contrast enhancement effect.With best regards,Rob MesmanDr. Rob MesmanPost-DocDepartment of MicrobiologyFaculty of ScienceRadboud University NijmegenHeyendaalseweg 135NL-6525 AJ NijmegenThe NetherlandsMessage: 2Date: Thu, 19 Dec 2019 08:39:29 +0000 (UTC)From: Qiaohui Yang <qiaohui.yang at ymail.com>To: 3dem at ncmir.ucsd.eduSubject: [3dem] Precipitation observed after making freeze	substitution cocktail containing 5% waterMessage-ID: <1061929162.800832.1576744769397 at mail.yahoo.com>Content-Type: text/plain; charset="utf-8"Hi everyone!We recently tried to make freeze substitution cocktailconsisting of 1% osmium tetroxide, 0.1% uranyl acetate and 5% water but observedthat the mixture turned slightly cloudy within 1-2 minutes. We tried to see if the precipitation would occur in the absence of osmium tetroxide, and we observed some cloudiness too after mixing uranyl acetate solution in water with pure acetone.Just wondering if anyone has encountered similar problem andif so, would appreciate some tips on how to overcome this problem.Thank you!?Best regards,Qiaohui-------------- next part --------------An HTML attachment was scrubbed...URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20191219/aa9c3a85/attachment.html>------------------------------Subject: Digest Footer_______________________________________________3dem mailing list3dem at ncmir.ucsd.eduhttps://mail.ncmir.ucsd.edu/mailman/listinfo/3dem------------------------------End of 3dem Digest, Vol 148, Issue 22*************************************
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