<div dir="ltr">Hi Nadav,<div><br></div><div>Is it possible that your 4L dewars are not dry enough so that your LN2 in dewars is contaminated? What is the humidity in your room where you dry the dewars?</div><div><br></div><div>Best regards,</div><div>Dong-Hua Chen</div><div>Department of Structural Biology</div><div>Stanford University</div></div><div class="gmail_extra"><br><div class="gmail_quote">On Thu, Sep 1, 2016 at 7:14 AM, Wim Hagen <span dir="ltr"><<a href="mailto:hagen@embl.de" target="_blank">hagen@embl.de</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div style="word-wrap:break-word"><div>Hi Nadav,</div><div><br></div><div>If the holder is too warm due bad clip-ring clamping, things are too warm, but I would then expect a time dependence, first you get “leopard pattern” which then migrates into cubic spots. Are the spots cubic? This process always happens but it is faster when too warm, here’s an example of a badly clipped Polara cartridge after one night in the column:</div><div><br></div><div></div></div><br><div style="word-wrap:break-word"><div> </div></div><br><div style="word-wrap:break-word"><div></div><div><br></div><div>Could also be your humidifier is "raining too course”, maybe try a wet sponge in the Leica chamber instead of the humidifier.</div><div><br></div><div>Is your cryo-box cold and stable? Take images every minute in LM mode with the box visible, cool down the cold trap, you should see movement only over the first 30 minutes, then it needs to remain stable. If it keeps going back and forth between certain positions, it starts touching something, so it warms up, stops touching, cools down again etc.</div><div><br></div><div>An example of 30 min from our properly working Tecnai 12 cryo-box:</div><div><br></div><div></div></div><br><div style="word-wrap:break-word"><div></div><div><br></div><div>Best</div><div><br></div><div>Wim</div><br><div><blockquote type="cite"><div>On Sep 1, 2016, at 11:00 AM, Nadav Elad <<a href="mailto:nadav.elad@weizmann.ac.il" target="_blank">nadav.elad@weizmann.ac.il</a>> wrote:</div><br><div><div style="font-family:Helvetica;font-size:12px;font-style:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px"><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">Dear all<u></u><u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">We are having a serious contamination problem in our cryo preparations and would be grateful for any suggestion. Although this issue has been discussed in the past in this forum, the solutions raised do not seem to work. The attached image shows the contamination on a bad day. I am referring mainly to the lighter, smaller particles (10-20 nm) which are the most persistent. They seem to be above or below the ice layer and are the first to evaporate under the electron beam. The concentration of contaminants is variable.<span> </span><u></u><u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">We are using a Leica EM GP plunger for freezing, and Tecnai T12 and F20 microscopes with Gatan side entry holders. We have tried to replace the ethane cylinder and are in the process of ordering a third one, cleaning the glow discharger machines, different cryo holders, different cryo-transferring units and grids of varying sources. There seems to be no leaks in the compustages of the microscopes.<u></u><u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">Thank you for your help<u></u><u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">Nadav<u></u><u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">--<u></u><u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">Nadav Elad, Ph.D<span dir="RTL"></span><span dir="RTL"></span><span lang="HE" dir="RTL" style="font-family:Arial,sans-serif"><span dir="RTL"></span><span dir="RTL"></span>.<br></span>Electron Microscopy Unit<span lang="HE" dir="RTL" style="font-family:Arial,sans-serif"><br></span>Weizmann Institute of Science<u></u><u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">Rehovot, Israel<span lang="HE" dir="RTL" style="font-family:Arial,sans-serif"><br></span>Tel: <a href="tel:%2B972%20%280%29%208%20934%202115" value="+97289342115" target="_blank">+972 (0) 8 934 2115</a><u></u><u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">Mobile: +972 (0) 52 3909 420<u></u><u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">Email: <span> </span><a href="mailto:nadav.elad@weizmann.ac.il" style="color:purple;text-decoration:underline" target="_blank">nadav.elad@weizmann.<wbr>ac.il</a><u></u><u></u></div><div style="margin:0in 0in 0.0001pt;font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></div></div><span><contamination.jpg></span><span style="font-family:Helvetica;font-size:12px;font-style:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;float:none;display:inline!important">___________<wbr>______________________________<wbr>______</span><br style="font-family:Helvetica;font-size:12px;font-style:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px"><span style="font-family:Helvetica;font-size:12px;font-style:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;float:none;display:inline!important">3dem mailing list</span><br style="font-family:Helvetica;font-size:12px;font-style:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px"><a href="mailto:3dem@ncmir.ucsd.edu" style="color:purple;text-decoration:underline;font-family:Helvetica;font-size:12px;font-style:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px" target="_blank">3dem@ncmir.ucsd.edu</a><br style="font-family:Helvetica;font-size:12px;font-style:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px"><a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem" style="color:purple;text-decoration:underline;font-family:Helvetica;font-size:12px;font-style:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px" target="_blank">https://mail.ncmir.ucsd.edu/<wbr>mailman/listinfo/3dem</a></div></blockquote></div><br></div><br>______________________________<wbr>_________________<br>
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