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<p class="MsoNormal"><span style="mso-fareast-language:EN-US">It could also come at the moment you insert the grid in the microscope, and even inside the microscope, through a tiny leak. If without cooling down the anticontaminator you cannot reach an IGP value
of 6 (on F20) when a holder is inserted but you can reach it when no holder is in, it speaks for a leak. Even if you reach IGP value within specification (e.g. 14), this might be creating this ice contamination. You heat your holder to -90 until the contamination
is gone (it should go away if it is water). Then let it cool down again and wait. If you see it reappear then you have a tiny leak. We had this problem and an intervention on the compustage solved it.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US">Further possible issues that we also experienced : The anticontaminator had been taken out of position… When we noticed it and brought it back in position (unfortunately after several weeks of head
scratching !) the leopard skin disappeared !<o:p></o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US">Benoit<o:p></o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"><o:p> </o:p></span></p>
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<p class="MsoNormal"><b><span style="color:black">De : </span></b><span style="color:black">3dem <3dem-bounces@ncmir.ucsd.edu> au nom de "Shi, Dan" <shid@janelia.hhmi.org><br>
<b>Date : </b>jeudi, 1 septembre 2016 21:52<br>
<b>À : </b>Nadav Elad <nadav.elad@weizmann.ac.il>, "3dem@ncmir.ucsd.edu" <3dem@ncmir.ucsd.edu><br>
<b>Objet : </b>Re: [3dem] contamination in cryo samples</span><span style="font-size:12.0pt;color:black"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-family:"Times New Roman""><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.5pt;color:black">Hi Nadav,<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.5pt;color:black">The contaminations could be either ice or impurity of ethane, if it’s from ethane, you may heat the holder tip to –150 C to see if the particles evaporate, if not, they are ice.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.5pt;color:black">Such nanometer granule shape of ice contamination seems to be formed in cold air first and deposited onto the cold grid, warmer holder would tend to crystallize original amorphous ice forming the
‘leopard –cubic pattern’ of contamination as Win described. Minor vacuum leaking into microscope should generate evenly distributed ice coverage and not nucleated ice particles, because the column is at RT.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.5pt;color:black"> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.5pt;color:black">There are two places could generate such contamination, most likely, the contamination is from the Leica EM plunger’s grid transfer chamber, the positive pressure from LN2 evaporation is not enough
to keep humid air off the chamber; the second place may be Gatan loading station, if you open the lid to mount the clip ring, wet air can sneak in to contaminate your grids. You may compare with Vitrobot and/or mount a grid with the plastic lid on all the
time.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.5pt;color:black">Good luck,<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.5pt;color:black">Dan <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.5pt;color:black"><o:p> </o:p></span></p>
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<p class="MsoNormal"><b><span style="color:black">From: </span></b><span style="color:black">3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu">3dem-bounces@ncmir.ucsd.edu</a>> on behalf of Nadav Elad <<a href="mailto:nadav.elad@weizmann.ac.il">nadav.elad@weizmann.ac.il</a>><br>
<b>Date: </b>Thursday, September 1, 2016 at 5:00 AM<br>
<b>To: </b>"<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a>" <<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a>><br>
<b>Subject: </b>[3dem] contamination in cryo samples<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.5pt;color:black"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="color:black">Dear all<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black"> <o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">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. <o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black"> <o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">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.<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black"> <o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">Thank you for your help<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black"> <o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">Nadav<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black"> <o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">--<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">Nadav Elad, Ph.D</span><span dir="RTL"></span><span lang="HE" dir="RTL" style="font-family:Arial;color:black"><span dir="RTL"></span>.<br>
</span><span lang="EN-US" style="color:black">Electron Microscopy Unit</span><span lang="HE" dir="RTL" style="font-family:Arial;color:black"><br>
</span><span lang="EN-US" style="color:black">Weizmann Institute of Science<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">Rehovot, Israel</span><span lang="HE" dir="RTL" style="font-family:Arial;color:black"><br>
</span><span lang="EN-US" style="color:black">Tel: +972 (0) 8 934 2115<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">Mobile: +972 (0) 52 3909 420<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">Email: <a href="mailto:nadav.elad@weizmann.ac.il">
nadav.elad@weizmann.ac.il</a><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black"> <o:p></o:p></span></p>
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