<div dir="ltr">One possibility is that the clip ring is not holding the cryo grid well and tightly in the Gatan holder tip. I would suggest trying a new clip ring first.<div><br></div><div>Best regards,</div><div>Dong-Hua</div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Wed, Jan 5, 2022 at 6:47 AM Grassucci, Robert <<a href="mailto:rg2502@cumc.columbia.edu">rg2502@cumc.columbia.edu</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left-width:1px;border-left-style:solid;border-left-color:rgb(204,204,204);padding-left:1ex">
<div lang="EN-US">
<div class="gmail-m_661158921493411425WordSection1">
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">Regarding pump down times for side entry holders. We have used up to 120 s for pump down but you can play with it to find the sweet spot. If it is too long
you could get devitrified ice but that is unlikely at 120 S.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">Regarding checking the grid clamping simply used fine tweezers to try and move the clamped grid. It should be tightly secured and shred if moved. If it moves
relatively freely then either your clamp ring needs to be changed (worn out) or the spring that holds the ring in the holder needs to be replaced.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">As Wim said the cooling of the cryo shield can be checked by going to low mag and watching it move as it cools. This was what the video he attached shows. Unfortunately
they do not have a read out of the shield temperature as they had in the early cryo shields.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">Good luck.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">Bob<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"><u></u> <u></u></span></p>
<div>
<div style="border-style:solid none none;border-top-width:1pt;border-top-color:rgb(225,225,225);padding:3pt 0in 0in">
<p class="MsoNormal"><b><span style="font-size:11pt;font-family:Calibri,sans-serif">From:</span></b><span style="font-size:11pt;font-family:Calibri,sans-serif"> 3dem [mailto:<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank">3dem-bounces@ncmir.ucsd.edu</a>]
<b>On Behalf Of </b>jb<br>
<b>Sent:</b> Wednesday, January 5, 2022 9:17 AM<br>
<b>To:</b> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br>
<b>Subject:</b> [EXTERNAL] Re: [3dem] contamination...leopard?<u></u><u></u></span></p>
</div>
</div>
<p class="MsoNormal"><u></u> <u></u></p>
<div>
<p class="MsoNormal">Hello together,<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">what would be the best or maximum time to pump the airlock at the Tecnai Spirit Scope??<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">I changed allready from 30 seconds to 45 seconds.<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">Vacuum values look good.<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">We suffer a lot from contamination and different ice formations since quite some time... every grid looks different.<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">How can we check if the clip ring at the Gatan holder 626 is perfectly adjusted?<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">I mean this is the place where the grid has to have the perfect contact a heat bridge to the holder.<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">How can we check cryo-box or cryo-shield?<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">Kind regards <u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">Jörg<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<pre>****************************<u></u><u></u></pre>
<pre>Jörg Bürger<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>Charité - Universitätsmedizin Berlin<u></u><u></u></pre>
<pre>Institut für Medizinische Physik und Biophysik<u></u><u></u></pre>
<pre>Charitéplatz 1, CCO - Virchowweg 6<u></u><u></u></pre>
<pre>10117 Berlin<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>Max Planck Institut für molekulare Genetik<u></u><u></u></pre>
<pre>Microscopy and Cryo-EM microscopy group<u></u><u></u></pre>
<pre>Ihnestr. 63-73<u></u><u></u></pre>
<pre>14195 Berlin<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>tel. 030-8413-1644/1278<u></u><u></u></pre>
<pre>fax. 030-8413-1385<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>email. <a href="mailto:buerger@molgen.mpg.de" target="_blank">buerger@molgen.mpg.de</a><u></u><u></u></pre>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<p class="MsoNormal">On 05.01.2022 14:51, Wim Hagen wrote:<u></u><u></u></p>
</div>
<blockquote style="margin-top:5pt;margin-bottom:5pt">
<pre>In the very early days it was even suggested to use ethane to "coat the sample” as a measure of preventing transfer contamination:<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<p class="MsoNormal"><br>
<br>
<u></u><u></u></p>
<pre><u></u> <u></u></pre>
<pre><u></u> <u></u></pre>
<pre>Best,<u></u><u></u></pre>
<pre>Wim<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<blockquote style="margin-top:5pt;margin-bottom:5pt">
<pre>On 5. Jan 2022, at 14:45, Reinhard Rachel <a href="mailto:Reinhard.Rachel@biologie.uni-regensburg.de" target="_blank"><Reinhard.Rachel@biologie.uni-regensburg.de></a> wrote:<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>I see your point, Wim. <u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<blockquote style="margin-top:5pt;margin-bottom:5pt">
<pre>On systems with side-entry cryo-holders, the pump-time for holder insertion<u></u><u></u></pre>
</blockquote>
<pre><u></u> <u></u></pre>
<blockquote style="margin-top:5pt;margin-bottom:5pt">
<pre>is limited and pressure is not measured by the vacuum system. <u></u><u></u></pre>
</blockquote>
<pre><u></u> <u></u></pre>
<pre>it is measured on our 2100F. <u></u><u></u></pre>
<pre>I doubt that 20 sec is a good time for cryo-transfer. Vacuum always needs<u></u><u></u></pre>
<pre>time!<u></u><u></u></pre>
<pre>the sample temperature should be kept low enough for quite some time (120 /<u></u><u></u></pre>
<pre>180 sec, at least). <u></u><u></u></pre>
<pre>but the vacuum improves quite considerable when going from short pump times<u></u><u></u></pre>
<pre>(20sec) up to 180 sec. or even longer. <u></u><u></u></pre>
<pre>this is at least the case for the air-lock/gonio of our 2100F (holder with two<u></u><u></u></pre>
<pre>O-rings ...). <u></u><u></u></pre>
<pre>thus: <u></u><u></u></pre>
<pre>- tilting to -30 or -35 degrees is fully sufficient<u></u><u></u></pre>
<pre>- pumping up to 120/180 sec (or even longer?) is fine, and the sample looks<u></u><u></u></pre>
<pre>fine, too. <u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<blockquote style="margin-top:5pt;margin-bottom:5pt">
<pre>If a grid <u></u><u></u></pre>
<pre>contains a lot of frozen ethane (from a fast transfer from ethane into<u></u><u></u></pre>
</blockquote>
<pre>liquid <u></u><u></u></pre>
<blockquote style="margin-top:5pt;margin-bottom:5pt">
<pre>nitrogen on the plunge freezer), this ethane will sublimate in vacuum: too <u></u><u></u></pre>
<pre>much ethane and/or short pump time -> column vacuum crashes as the ion<u></u><u></u></pre>
</blockquote>
<pre>getter <u></u><u></u></pre>
<blockquote style="margin-top:5pt;margin-bottom:5pt">
<pre>pumps see a lot of ethane coming in when the holder is inserted. <u></u><u></u></pre>
</blockquote>
<pre><u></u> <u></u></pre>
<pre>this is the point - thank you, Wim. <u></u><u></u></pre>
<pre>thus, I think Tobias & Co may try longer times for pre-pumping in the<u></u><u></u></pre>
<pre>air-lock? <u></u><u></u></pre>
<pre>I do not know this type of air-lock in the Halo - but it might work. <u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>kind regards,<u></u><u></u></pre>
<pre>Reinhard<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>-- <u></u><u></u></pre>
<pre>Prof. Dr. Reinhard Rachel<u></u><u></u></pre>
<pre>University of Regensburg<u></u><u></u></pre>
<pre>Centre for EM / Anatomy<u></u><u></u></pre>
<pre>Faculty of Biology & Preclin. Med.<u></u><u></u></pre>
<pre>Universitaetsstrasse 31<u></u><u></u></pre>
<pre>D-93053 Regensburg - Germany<u></u><u></u></pre>
<pre>tel +49 941 943 -2837, -1666<u></u><u></u></pre>
<pre>mail <a href="mailto:reinhard.rachel@biologie.uni-regensburg.de" target="_blank">reinhard.rachel@biologie.uni-regensburg.de</a><u></u><u></u></pre>
<pre>office: VKL 3.1.29 <u></u><u></u></pre>
<pre>member of the IFSM board<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>Next microscopy conferences:<u></u><u></u></pre>
<pre>- MC2023, DGE: 26.2.-2.3. 2023<u></u><u></u></pre>
<pre>- next European EM conf.: EMC2024 in Kopenhagen, August 2024<u></u><u></u></pre>
<pre>- IMC20 in Busan, South Korea: Sept, 2023<u></u><u></u></pre>
<pre>- next Microbiol. conferences: VAAM 20-23 Febr 2022 Düsseldorf<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
</blockquote>
<pre><u></u> <u></u></pre>
<p class="MsoNormal"><br>
<br>
<u></u><u></u></p>
<pre>_______________________________________________<u></u><u></u></pre>
<pre>3dem mailing list<u></u><u></u></pre>
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</blockquote>
<p><u></u> <u></u></p>
<pre>-- <u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>****************************<u></u><u></u></pre>
<pre>Jörg Bürger<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>Charité - Universitätsmedizin Berlin<u></u><u></u></pre>
<pre>Institut für Medizinische Physik und Biophysik<u></u><u></u></pre>
<pre>Charitéplatz 1, CCO - Virchowweg 6<u></u><u></u></pre>
<pre>10117 Berlin<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>Max Planck Institut für molekulare Genetik<u></u><u></u></pre>
<pre>Microscopy and Cryo-EM microscopy group<u></u><u></u></pre>
<pre>Ihnestr. 63-73<u></u><u></u></pre>
<pre>14195 Berlin<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>tel. 030-8413-1644/1278<u></u><u></u></pre>
<pre>fax. 030-8413-1385<u></u><u></u></pre>
<pre><u></u> <u></u></pre>
<pre>email. <a href="mailto:buerger@molgen.mpg.de" target="_blank">buerger@molgen.mpg.de</a><u></u><u></u></pre>
</div>
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