<html><head><meta http-equiv="Content-Type" content="text/html; charset=UTF-8"></head><body dir="auto">Hi Miriam,<div dir="auto">Regarding your mail to the 3DEM list,</div><div dir="auto">I tend to avoid the use of molecular sieve in solvents for freeze substitution. (Fun fact, in the old book 'Crytechniques in biological electron microscopy' Hans Moor warns against the use of molecular sieve with freeze substitution) </div><div dir="auto">The main problem (besides the chance of small particles in your sample) is that any trace of the molecular sieve will directly react with any osmium used during the freeze substitution turning it black within a few hours. </div><div dir="auto">I've successfully used glass distilled solvents (EMS), VWR anhydrous solvents and Merck seccosolv solvents (though the latter have been discontinued last year). I keep them dry by flushing the bottle with dry nitrogen gas and storing the bottle in a container with silica pearls under dry nitrogen (And all lids taped off with parafilm).</div><div dir="auto"><br></div><div dir="auto">Alternatively if you do not use water in the freeze-substitution mix you can also use acidified 2,2-dimethoxypropane (1-2 drops of 37% hcl in 100ml dimethoxypropane) and add 2 mg of this to 98ml 96% ethanol. Any water present will be converted to acetone and methanol. In contrast to other methods of making dry ethanol this does'nt react with osmium.</div><div dir="auto"><br></div><div dir="auto">If you're not using omium then the only issue will be the dust particles. You could opt for using the 'porous containers' EMS sells for critical point drying (catalog nr. 70187-20) instead of dialysis tubing.</div><div dir="auto"><br></div><div dir="auto">Best wishes!</div><div dir="auto">Rob</div><div dir="auto"><br></div><div dir="auto">---------</div><div dir="auto">Dr. Rob Mesman</div><div dir="auto">Post-Doc</div><div dir="auto">Department of Microbiology (postbox. 72)<br class="">
Institute for Water & Wetland Research<br class="">
Faculty of Science<br class="">
Radboud University<br class="">
Heyendaalseweg 135<br class="">
6525 AJ Nijmegen<br class="">
The Netherlands</div><div><br></div><div style="font-size:100%;color:#000000" dir="auto"><!-- originalMessage --><div>-------- Oorspronkelijk bericht --------</div><div>Van: 3dem-request@ncmir.ucsd.edu </div><div>Datum: 15-01-20 20:28 (GMT+01:00) </div><div>Aan: 3dem@ncmir.ucsd.edu </div><div>Onderwerp: 3dem Digest, Vol 149, Issue 14</div></div><br dir="auto">----------------------------------------------------------------------<br dir="auto"><br dir="auto">Message: 1<br dir="auto">Date: Wed, 15 Jan 2020 13:56:47 +0000<br dir="auto">From: Lucas Miriam Susanna <miriam.lucas@scopem.ethz.ch><br dir="auto">To: Reinhard Rachel <Reinhard.Rachel@biologie.uni-regensburg.de><br dir="auto">Cc: "3dem@ncmir.ucsd.edu" <3dem@ncmir.ucsd.edu><br dir="auto">Subject: Re: [3dem] Precipitation observed after making freeze<br dir="auto"> substitution cocktail containing 5% water<br dir="auto">Message-ID: <2781b74b66cb44f8901ffcdb6a0b7382@scopem.ethz.ch><br dir="auto">Content-Type: text/plain; charset="iso-8859-1"<br dir="auto"><br dir="auto">Dear Reinhard, <br dir="auto">dear All,<br dir="auto"><br dir="auto">Thanks a lot for your comments! <br dir="auto">Following up on this topic, I'd like to ask, if you are preparing the dry ethanol and/or acetone yourself, and if so - how do you do it?<br dir="auto"><br dir="auto">We are using molecular sieve, which we wash (and store) in alcohol to remove abrasion/dust, then fill it into dialysis tubes (with clips to close both ends) and add this into the bottles in which the alcohol is supplied. We refill a bottle once. However, the handling of these dialysis-tubes is a pain and they tend to burst when introduced into the bottles. <br dir="auto">Several suppliers sell extra-dry ethanol or acetone with molecular sieve floating freely in the bottles. <br dir="auto"><br dir="auto">We had a discussion in the lab, weather abrasion dust from molecular sieve is transferred onto the samples, and weather this happens in an extent that it really influences the imaging, e.g. for critically-point dried samples, as well as for resin-embedded specimen. <br dir="auto"><br dir="auto">What's your opinion?<br dir="auto"><br dir="auto"><br dir="auto">Kind regards,<br dir="auto">Miriam<br dir="auto"><br dir="auto"><br dir="auto">--- <br dir="auto">Dr. Miriam Lucas<br dir="auto">ETH Z?rich | ScopeM - Scientific Center for Optical and Electron Microscopy<br dir="auto">HPM C57.2<br dir="auto">Otto-Stern-Weg 3<br dir="auto">CH-8093 Zurich<br dir="auto">Switzerland<br dir="auto"><br dir="auto">Phone:?? +41 44 633 4424<br dir="auto">Fax:???????? +41 44 633 1336<br dir="auto"><br dir="auto">miriam.lucas@scopem.ethz.ch<br dir="auto">www.scopem.ethz.ch<br dir="auto"><br dir="auto"><br dir="auto"><br dir="auto">-----Urspr?ngliche Nachricht-----<br dir="auto">Von: 3dem <3dem-bounces@ncmir.ucsd.edu> Im Auftrag von Reinhard Rachel<br dir="auto">Gesendet: Freitag, 20. Dezember 2019 11:47<br dir="auto">An: Christopher.Peddie@crick.ac.uk; aparvate@lji.org<br dir="auto">Cc: 3dem@ncmir.ucsd.edu<br dir="auto">Betreff: Re: [3dem] Precipitation observed after making freeze substitution cocktail containing 5% water<br dir="auto"><br dir="auto">>>> 19.12.2019 at 20:41:<br dir="auto">> I would agree with the 7-8% stock concentration for UA. Even at that <br dir="auto">> concentration, it would take a while to dissolve in methanol. <br dir="auto">> Additionally, I would filter it with a 0.22 micron filter, make <br dir="auto">> aliquots and then freeze in LN2. Dont know if LN2 was overkill but i <br dir="auto">> have never seen cloudy precipitation in my FS mixes and i was using 3% <br dir="auto">> OS and 0.5% UA along with some TA and glutaraldehyde.<br dir="auto"><br dir="auto">with some delay, here is a brief comment from my side.<br dir="auto">some people do not mention the solvent, in their comments (or in the M&M in the papers). <br dir="auto">in theory, you can push the stock solution in pure water to 7-8% , but ...<br dir="auto">this depends whether you have CO2 free water (UO2 carbonate might precipitate, first!). The CO2 / carbonate anion is one of our enimies, in these solutions. Just keep this in mind. <br dir="auto">Thus, you may boil Water (= get rid of CO2) before solving the UAc. Or use freshly distilled (!!) water. Yes, distilled, not the Millipore stuff: no, never. -- And if you like, you may add minute amounts = a mini drop of glacial acetic acid (which tries to keep the pH such that the CO2 is favored against the bicarbonate anion and the carbonate!)<br dir="auto"><br dir="auto">and solubility depends on the temperature (dissociation constants depend on the temperature! physical chemistry). <br dir="auto">Thus, I do not recommend to put such stock solutions into the cold room, below 10 C, or fridge. <br dir="auto">Nor does it make sense to freeze them ... why? make small amounts and use them up. Freezing: you enforce the stuff to precipitate ... and, for the solution at room temp.: If you know of a bug that starts growing in a stock solution of UAc 5% or higher, please let me know; I would be interested (I am also microbiologist). <br dir="auto"><br dir="auto">we and others have tested: UAc dissolves nicely in MeOH, up to 20%. This does not last that long; I would make small amounts, only, which are needed within a week. - but this is easy to do. <br dir="auto"><br dir="auto">Have fun, and to all of you: <br dir="auto">merry Christmas - a happy New Year, and all the best! <br dir="auto">Reinhard<br dir="auto"><br dir="auto"><br dir="auto">--<br dir="auto">Prof. Dr. Reinhard Rachel<br dir="auto">University of Regensburg<br dir="auto">Centre for EM / Anatomy<br dir="auto">Faculty of Biology & Preclin. Med.<br dir="auto">Universitaetsstrasse 31<br dir="auto">D-93053 Regensburg - Germany<br dir="auto">tel +49 941 943 -2837, -1720<br dir="auto">mail reinhard.rachel@biologie.uni-regensburg.de<br dir="auto">office: VKL 3.1.29<br dir="auto">member of the IFSM board<br dir="auto"><br dir="auto">Next microscopy conferences:<br dir="auto">- EMC2020 in Kopenhagen, 23.-28.8. 2020 (European conference)<br dir="auto">- MC2021 in Vienna (D-A-CH conference)<br dir="auto">- IMC20 in Busan, South Korea: Sept 25-30, 2022<br dir="auto">- next Microbiol. conferences: VAAM 8.-11.03. 2020 Leipzig<br dir="auto"><br dir="auto"><br dir="auto">_______________________________________________<br dir="auto">3dem mailing list<br dir="auto">3dem@ncmir.ucsd.edu<br dir="auto">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem<br dir="auto"><br dir="auto"><br dir="auto">------------------------------<br dir="auto"><br dir="auto">Message: 2<br dir="auto">Date: Wed, 15 Jan 2020 19:27:59 +0000<br dir="auto">From: "Gibson, Kimberley" <kimberley.gibson@yale.edu><br dir="auto">To: Lucas Miriam Susanna <miriam.lucas@scopem.ethz.ch>, Reinhard<br dir="auto"> Rachel <Reinhard.Rachel@biologie.uni-regensburg.de><br dir="auto">Cc: "3dem@ncmir.ucsd.edu" <3dem@ncmir.ucsd.edu><br dir="auto">Subject: Re: [3dem] Precipitation observed after making freeze<br dir="auto"> substitution cocktail containing 5% water<br dir="auto">Message-ID:<br dir="auto"> <BN7PR08MB48848B3B8C3B2E465F316B0DEB370@BN7PR08MB4884.namprd08.prod.outlook.com><br dir="auto"> <br dir="auto">Content-Type: text/plain; charset="iso-8859-1"<br dir="auto"><br dir="auto">Hi Miriam,<br dir="auto"><br dir="auto">When using molecular sieves, we would mix them directly in a bottle with Ethanol (i.e. free floating). Once capped, invert/shake the bottle a few times to mix the sieves/Ethanol and let the Ethanol solution sit for at least a day or more before using it. The sieves and any particulate matter should settle to the bottom of the bottle. Afterwards, when pipetting or pouring the pure Ethanol, make sure to not invert or shake the bottle.<br dir="auto">I've never had any issues with contamination of molecular sieve dust in resin-embedded samples - which would have been very noticeable when sectioning with diamond knives.<br dir="auto"><br dir="auto">All the best,<br dir="auto"><br dir="auto">Kim<br dir="auto">________________________________<br dir="auto">From: 3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Lucas Miriam Susanna <miriam.lucas@scopem.ethz.ch><br dir="auto">Sent: Wednesday, January 15, 2020 08:56<br dir="auto">To: Reinhard Rachel <Reinhard.Rachel@biologie.uni-regensburg.de><br dir="auto">Cc: 3dem@ncmir.ucsd.edu <3dem@ncmir.ucsd.edu><br dir="auto">Subject: Re: [3dem] Precipitation observed after making freeze substitution cocktail containing 5% water<br dir="auto"><br dir="auto">Dear Reinhard,<br dir="auto">dear All,<br dir="auto"><br dir="auto">Thanks a lot for your comments!<br dir="auto">Following up on this topic, I'd like to ask, if you are preparing the dry ethanol and/or acetone yourself, and if so - how do you do it?<br dir="auto"><br dir="auto">We are using molecular sieve, which we wash (and store) in alcohol to remove abrasion/dust, then fill it into dialysis tubes (with clips to close both ends) and add this into the bottles in which the alcohol is supplied. We refill a bottle once. However, the handling of these dialysis-tubes is a pain and they tend to burst when introduced into the bottles.<br dir="auto">Several suppliers sell extra-dry ethanol or acetone with molecular sieve floating freely in the bottles.<br dir="auto"><br dir="auto">We had a discussion in the lab, weather abrasion dust from molecular sieve is transferred onto the samples, and weather this happens in an extent that it really influences the imaging, e.g. for critically-point dried samples, as well as for resin-embedded specimen.<br dir="auto"><br dir="auto">What's your opinion?<br dir="auto"><br dir="auto"><br dir="auto">Kind regards,<br dir="auto">Miriam<br dir="auto"><br dir="auto"><br dir="auto">---<br dir="auto">Dr. Miriam Lucas<br dir="auto">ETH Z?rich | ScopeM - Scientific Center for Optical and Electron Microscopy<br dir="auto">HPM C57.2<br dir="auto">Otto-Stern-Weg 3<br dir="auto">CH-8093 Zurich<br dir="auto">Switzerland<br dir="auto"><br dir="auto">Phone: +41 44 633 4424<br dir="auto">Fax: +41 44 633 1336<br dir="auto"><br dir="auto">miriam.lucas@scopem.ethz.ch<br dir="auto">https://nam05.safelinks.protection.outlook.com/?url=www.scopem.ethz.ch&data=02%7C01%7Ckimberley.gibson%40yale.edu%7Ca11d5d25595946e6f12708d799c2cb1f%7Cdd8cbebb21394df8b4114e3e87abeb5c%7C0%7C0%7C637146934251328073&sdata=CoIE%2BaE6JAoQixsthcSry7fIZEUVQnXp0sP9dvlO7lY%3D&reserved=0<br dir="auto"><br dir="auto"><br dir="auto"><br dir="auto">-----Urspr?ngliche Nachricht-----<br dir="auto">Von: 3dem <3dem-bounces@ncmir.ucsd.edu> Im Auftrag von Reinhard Rachel<br dir="auto">Gesendet: Freitag, 20. Dezember 2019 11:47<br dir="auto">An: Christopher.Peddie@crick.ac.uk; aparvate@lji.org<br dir="auto">Cc: 3dem@ncmir.ucsd.edu<br dir="auto">Betreff: Re: [3dem] Precipitation observed after making freeze substitution cocktail containing 5% water<br dir="auto"><br dir="auto">>>> 19.12.2019 at 20:41:<br dir="auto">> I would agree with the 7-8% stock concentration for UA. Even at that<br dir="auto">> concentration, it would take a while to dissolve in methanol.<br dir="auto">> Additionally, I would filter it with a 0.22 micron filter, make<br dir="auto">> aliquots and then freeze in LN2. Dont know if LN2 was overkill but i<br dir="auto">> have never seen cloudy precipitation in my FS mixes and i was using 3%<br dir="auto">> OS and 0.5% UA along with some TA and glutaraldehyde.<br dir="auto"><br dir="auto">with some delay, here is a brief comment from my side.<br dir="auto">some people do not mention the solvent, in their comments (or in the M&M in the papers).<br dir="auto">in theory, you can push the stock solution in pure water to 7-8% , but ...<br dir="auto">this depends whether you have CO2 free water (UO2 carbonate might precipitate, first!). The CO2 / carbonate anion is one of our enimies, in these solutions. Just keep this in mind.<br dir="auto">Thus, you may boil Water (= get rid of CO2) before solving the UAc. Or use freshly distilled (!!) water. Yes, distilled, not the Millipore stuff: no, never. -- And if you like, you may add minute amounts = a mini drop of glacial acetic acid (which tries to keep the pH such that the CO2 is favored against the bicarbonate anion and the carbonate!)<br dir="auto"><br dir="auto">and solubility depends on the temperature (dissociation constants depend on the temperature! physical chemistry).<br dir="auto">Thus, I do not recommend to put such stock solutions into the cold room, below 10 C, or fridge.<br dir="auto">Nor does it make sense to freeze them ... why? make small amounts and use them up. Freezing: you enforce the stuff to precipitate ... and, for the solution at room temp.: If you know of a bug that starts growing in a stock solution of UAc 5% or higher, please let me know; I would be interested (I am also microbiologist).<br dir="auto"><br dir="auto">we and others have tested: UAc dissolves nicely in MeOH, up to 20%. This does not last that long; I would make small amounts, only, which are needed within a week. - but this is easy to do.<br dir="auto"><br dir="auto">Have fun, and to all of you:<br dir="auto">merry Christmas - a happy New Year, and all the best!<br dir="auto">Reinhard<br dir="auto"><br dir="auto"><br dir="auto">--<br dir="auto">Prof. Dr. Reinhard Rachel<br dir="auto">University of Regensburg<br dir="auto">Centre for EM / Anatomy<br dir="auto">Faculty of Biology & Preclin. Med.<br dir="auto">Universitaetsstrasse 31<br dir="auto">D-93053 Regensburg - Germany<br dir="auto">tel +49 941 943 -2837, -1720<br dir="auto">mail reinhard.rachel@biologie.uni-regensburg.de<br dir="auto">office: VKL 3.1.29<br dir="auto">member of the IFSM board<br dir="auto"><br dir="auto">Next microscopy conferences:<br dir="auto">- EMC2020 in Kopenhagen, 23.-28.8. 2020 (European conference)<br dir="auto">- MC2021 in Vienna (D-A-CH conference)<br dir="auto">- IMC20 in Busan, South Korea: Sept 25-30, 2022<br dir="auto">- next Microbiol. conferences: VAAM 8.-11.03. 2020 Leipzig<br dir="auto"><br dir="auto"><br dir="auto">_______________________________________________<br dir="auto">3dem mailing list<br dir="auto">3dem@ncmir.ucsd.edu<br dir="auto">https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fmail.ncmir.ucsd.edu%2Fmailman%2Flistinfo%2F3dem&data=02%7C01%7Ckimberley.gibson%40yale.edu%7Ca11d5d25595946e6f12708d799c2cb1f%7Cdd8cbebb21394df8b4114e3e87abeb5c%7C0%7C0%7C637146934251328073&sdata=bOylnOfiC9sJCCqs47fQjveeWZJVVfQgS8jOuBn0DH0%3D&reserved=0<br dir="auto">_______________________________________________<br dir="auto">3dem mailing list<br dir="auto">3dem@ncmir.ucsd.edu<br dir="auto">https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fmail.ncmir.ucsd.edu%2Fmailman%2Flistinfo%2F3dem&data=02%7C01%7Ckimberley.gibson%40yale.edu%7Ca11d5d25595946e6f12708d799c2cb1f%7Cdd8cbebb21394df8b4114e3e87abeb5c%7C0%7C0%7C637146934251338067&sdata=r5Gqt%2Fm4YJEHTyWa7vafNWzROftgmcx2toqGTXr7Bo0%3D&reserved=0<br dir="auto">-------------- next part --------------<br dir="auto">An HTML attachment was scrubbed...<br dir="auto">URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20200115/b1da9e20/attachment.html><br dir="auto"><br dir="auto">------------------------------<br dir="auto"><br dir="auto">Subject: Digest Footer<br dir="auto"><br dir="auto">_______________________________________________<br dir="auto">3dem mailing list<br dir="auto">3dem@ncmir.ucsd.edu<br dir="auto">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem<br dir="auto"><br dir="auto"><br dir="auto">------------------------------<br dir="auto"><br dir="auto">End of 3dem Digest, Vol 149, Issue 14<br dir="auto">*************************************<br dir="auto"></body></html>