<html xmlns:v="urn:schemas-microsoft-com:vml" xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns:m="http://schemas.microsoft.com/office/2004/12/omml" xmlns="http://www.w3.org/TR/REC-html40">
<head>
<meta http-equiv="Content-Type" content="text/html; charset=utf-8">
<meta name="Generator" content="Microsoft Word 15 (filtered medium)">
<style><!--
/* Font Definitions */
@font-face
{font-family:Helvetica;
panose-1:2 11 6 4 2 2 2 2 2 4;}
@font-face
{font-family:"Cambria Math";
panose-1:2 4 5 3 5 4 6 3 2 4;}
@font-face
{font-family:DengXian;
panose-1:2 1 6 0 3 1 1 1 1 1;}
@font-face
{font-family:Calibri;
panose-1:2 15 5 2 2 2 4 3 2 4;}
@font-face
{font-family:Tahoma;
panose-1:2 11 6 4 3 5 4 4 2 4;}
@font-face
{font-family:"\@DengXian";
panose-1:2 1 6 0 3 1 1 1 1 1;}
/* Style Definitions */
p.MsoNormal, li.MsoNormal, div.MsoNormal
{margin:0in;
margin-bottom:.0001pt;
font-size:11.0pt;
font-family:"Calibri",sans-serif;}
a:link, span.MsoHyperlink
{mso-style-priority:99;
color:blue;
text-decoration:underline;}
a:visited, span.MsoHyperlinkFollowed
{mso-style-priority:99;
color:purple;
text-decoration:underline;}
p.MsoListParagraph, li.MsoListParagraph, div.MsoListParagraph
{mso-style-priority:34;
margin-top:0in;
margin-right:0in;
margin-bottom:0in;
margin-left:.5in;
margin-bottom:.0001pt;
font-size:11.0pt;
font-family:"Calibri",sans-serif;}
p.msonormal0, li.msonormal0, div.msonormal0
{mso-style-name:msonormal;
mso-margin-top-alt:auto;
margin-right:0in;
mso-margin-bottom-alt:auto;
margin-left:0in;
font-size:11.0pt;
font-family:"Calibri",sans-serif;}
span.apple-converted-space
{mso-style-name:apple-converted-space;}
span.EmailStyle20
{mso-style-type:personal-reply;
font-family:"Calibri",sans-serif;
color:windowtext;}
.MsoChpDefault
{mso-style-type:export-only;
font-size:10.0pt;}
@page WordSection1
{size:8.5in 11.0in;
margin:1.0in 1.0in 1.0in 1.0in;}
div.WordSection1
{page:WordSection1;}
/* List Definitions */
@list l0
{mso-list-id:1187908754;
mso-list-type:hybrid;
mso-list-template-ids:-516915642 67698705 67698713 67698715 67698703 67698713 67698715 67698703 67698713 67698715;}
@list l0:level1
{mso-level-text:"%1\)";
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;}
@list l0:level2
{mso-level-number-format:alpha-lower;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;}
@list l0:level3
{mso-level-number-format:roman-lower;
mso-level-tab-stop:none;
mso-level-number-position:right;
text-indent:-9.0pt;}
@list l0:level4
{mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;}
@list l0:level5
{mso-level-number-format:alpha-lower;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;}
@list l0:level6
{mso-level-number-format:roman-lower;
mso-level-tab-stop:none;
mso-level-number-position:right;
text-indent:-9.0pt;}
@list l0:level7
{mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;}
@list l0:level8
{mso-level-number-format:alpha-lower;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;}
@list l0:level9
{mso-level-number-format:roman-lower;
mso-level-tab-stop:none;
mso-level-number-position:right;
text-indent:-9.0pt;}
ol
{margin-bottom:0in;}
ul
{margin-bottom:0in;}
--></style><!--[if gte mso 9]><xml>
<o:shapedefaults v:ext="edit" spidmax="1026" />
</xml><![endif]--><!--[if gte mso 9]><xml>
<o:shapelayout v:ext="edit">
<o:idmap v:ext="edit" data="1" />
</o:shapelayout></xml><![endif]-->
</head>
<body lang="EN-US" link="blue" vlink="purple">
<div class="WordSection1">
<p class="MsoNormal">The modified procedure from the traditional one for our L120C is
<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<ol style="margin-top:0in" start="1" type="1">
<li class="MsoListParagraph" style="margin-left:0in;mso-list:l0 level1 lfo1">Start the turbo pump and wait it reaching 100% speed and set ‘airlock pump time’ to 30 – 60 seconds<o:p></o:p></li><li class="MsoListParagraph" style="margin-left:0in;mso-list:l0 level1 lfo1">Set the alpha at -55 to -60 degree and click on ‘set alpha’ to tilt the compustage
<o:p></o:p></li><li class="MsoListParagraph" style="margin-left:0in;mso-list:l0 level1 lfo1">Insert the 626 holder to the engaging position and select the cryo-ST holder from the screen<o:p></o:p></li><li class="MsoListParagraph" style="margin-left:0in;mso-list:l0 level1 lfo1">Hold the cryo-holder and click on ‘set alpha’ to rotate the compustage back to 0 degree at least 10 seconds before the airlock pump time expired<o:p></o:p></li><li class="MsoListParagraph" style="margin-left:0in;mso-list:l0 level1 lfo1">Wait until the red light on the compustage off, rotate the cryo-holder to the insertion position and insert the holder in smoothly.<o:p></o:p></li><li class="MsoListParagraph" style="margin-left:0in;mso-list:l0 level1 lfo1">Feel free to breath<o:p></o:p></li></ol>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Best,<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Dan<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<div>
<div style="border:none;border-top:solid #E1E1E1 1.0pt;padding:3.0pt 0in 0in 0in">
<p class="MsoNormal"><b>From:</b> Denis Chrétien <denis.chretien@univ-rennes1.fr>
<br>
<b>Sent:</b> Monday, February 24, 2020 4:54 PM<br>
<b>To:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> Re: [3dem] Insertion of Holder 626<o:p></o:p></p>
</div>
</div>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Sorry, I mentioned Thermo (for the O-rings), but for the 626 it would be Gatan (still cost about one arm and a leg ...)<o:p></o:p></p>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">I forgot to mention also the -60° insertion procedure that Wim also quoted. It really improved our ability to insert cryo-holders without leaks (and risks of warming-up the specimen by spilling all the LN2 outside the cryo-holder):<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">1) Rotate the cryo-stage to -60°<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">2) Pre-pump the airlock (wait until red light goes off)<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">3) insert the cryo-holder and wait (again) until red light goes off (time fixed by yourself, between 30 sec and one min of pumping).<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">4) stop breathing*<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">5) Hold the cryo-holder and reset the stage to 0°. Again, wait until red light goes off!<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">6) Rotate the cryo-holder and insert it gently (don’t let the vacuum aspire it abruptly inside the stage!)<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">7) Check that the vacuum didn’t went to high during insertion<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">8) Breath*<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">* optional<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
<div>
<p class="MsoNormal">Denis<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><br>
<br>
<o:p></o:p></p>
<blockquote style="margin-top:5.0pt;margin-bottom:5.0pt">
<div>
<p class="MsoNormal">Le 24 févr. 2020 à 22:01, Denis Chrétien <<a href="mailto:denis.chretien@univ-rennes1.fr">denis.chretien@univ-rennes1.fr</a>> a écrit :<o:p></o:p></p>
</div>
<p class="MsoNormal"><o:p> </o:p></p>
<div>
<div>
<p class="MsoNormal">If we order O-rings from Thermo, it cost one arm… Does anyone have references for « standard » O-rings that we can buy elsewhere for a much cheaper price?<o:p></o:p></p>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">To come back to the original questions, we still use 626 and other side-entry holders. Sometime we face vacuum problems. In our experience these are mainly due to (as already said):<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">- Dusts on the O-ring (or inside the airlock system). Inspect the O-ring and also try to blow dry-air inside the airlock. Maybe the airlock tube is damage, then ask Thermo for repair … :-(<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">- Dry O-rings: regularly put some (not a lot !) vacuum grease on it. Be careful not to scratch the metal when removing it or damaging the O-ring. Change it if dry or obviously damaged.<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">- One frequent issue with cryo-holders: do not fill the cryo-holder station with to much LN2, you may freeze the O-ring and it will inevitably lead to a vacuum leak (very reproducible!). To limit this issue, we slightly lift the cryo-holder
station with a rectangular grid box to avoid LN2 going into contact with the O-ring. We (not every one of us) cool the cryo-stage inside the microscope. Then the work-station is cooled just before inserting the specimen grid on the holder. This also limits
the risk of cooling the O-ring.<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">- We « pre-pump » the airlock, and pump for 1 min before inserting (on a Tecnai G2 Sphera). 3 min of pumping seams a lot!<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">- Change of the zeolithe, grease on the valve O-ring of the cryo-holder, periodic zeolithe cycle (we do it every session): it improves stability on our side.<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">Best<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">Denis<o:p></o:p></p>
</div>
<div>
<div>
<p class="MsoNormal"><br>
<br>
<o:p></o:p></p>
<blockquote style="margin-top:5.0pt;margin-bottom:5.0pt">
<div>
<p class="MsoNormal">Le 24 févr. 2020 à 21:26, David Stokes <<a href="mailto:stokes@nyu.edu">stokes@nyu.edu</a>> a écrit :<o:p></o:p></p>
</div>
<p class="MsoNormal"><o:p> </o:p></p>
<div>
<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">I used to do the following prior to every session:<br>
Remove o-ring from holder.<br>
Clean o-ring off with kimwipes and fingers (I didn't bother to use any solvent).<br>
Clean o-ring groove with kimwipes.<br>
Blow away fibers with compressed air.<br>
Lightly grease o-ring.<br>
Inspect o-ring and o-ring groove under dissecting scope and carefully remove any fibers prior to re-installation onto the holder.<br>
Don't scratch the o-ring groove. In fact, don't touch it with forceps. Use compressed air.<br>
<br>
During the inspection process, you will see whether there are any nicks or flat spots in the o-ring. If so, then consider a replacement. If there is a scratch in the o-ring groove, then you might need a new holder (or maybe not).<br>
Also make sure you really have the right sized o-ring. At some point in ancient history, they changed the size slightly, such that CM200 o-ring was a bit fatter than the one for the CM12 stage.<br>
<br>
David<br>
<br>
<br>
On 2/24/2020 3:10 PM, Sharon G Wolf wrote:<br style="caret-color: rgb(0, 0, 0);font-variant-caps: normal;text-align:start;-webkit-text-stroke-width: 0px;word-spacing:0px">
<br>
</span><o:p></o:p></p>
<blockquote style="margin-top:5.0pt;margin-bottom:5.0pt">
<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">Terje<br>
We also have suffered for years from vacuum breaks. We tried EVERYTHING. The most effective thing was to (believe it or not) change the o-ring every single session.<br>
Recently we understood this was due to the fibers and other "dirt" that we observed on the o-rings every time we looked with the binocular after inserting and removing the holder from the microscopes, transfer stations or pumping station. We embarked on a
“cleaning program” where we gently swab all the above mentioned devises. It’s now slowly getting better. We are still changing the o rings but we hope it will soon no longer be necessary, once all the "stuff" that accumulated over the years is cleaned out
from those places.<br>
Sharon<br>
<br>
<br>
Sent from my iPhone<br>
<br>
<br>
<o:p></o:p></span></p>
<blockquote style="margin-top:5.0pt;margin-bottom:5.0pt">
<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">On 24 Feb 2020, at 19:28, Dokland, Terje <<a href="mailto:dokland@uab.edu">dokland@uab.edu</a>> wrote:<br>
<br>
</span><span style="font-size:9.0pt;font-family:"Tahoma",sans-serif"></span><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">We have also had persistent vacuum break problems with our 626 holder in our Tecnai F20 over the past two years. About
50% of the time inserting the holder breaks the vacuum. I though it was unique to this holder, but with the Elsa holder that we recently acquired it is even worse: vacuum break about 90% if the time. We’ve changed O rings, had the airlock disassembled and
cleaned, but the problem persists.<br>
<br>
That being said, it never affected ice quality, and the temperature usually doesn’t go above -165 C. When the vacuum recovers it’s usually at 25-30 log or so and back to <10 within a half hour. It’s really annoying though.<br>
<br>
In my experience as long as the temperature doesn’t rise above -150C you should be ok.<br>
<br>
It helps to make sure the tip is very cold by submerging it completely in lN2 before inserting.<br>
<br>
Terje<br>
<br>
Sent from my iPhone<br>
<br>
<br>
<o:p></o:p></span></p>
<blockquote style="margin-top:5.0pt;margin-bottom:5.0pt">
<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">On Feb 24, 2020, at 7:13 AM, TALAVERA PEREZ Ariel <<a href="mailto:Ariel.Talavera@ulb.be">Ariel.Talavera@ulb.be</a>> wrote:<br>
<br>
Dear all,<br>
<br>
I am using a Gatan 626 holder on a side entry TALOS microscope. I am<br>
getting problems with very frequent crystalline ice formation on the<br>
grids. Some times I get a mix of vitreous and crystalline water, but<br>
must of the time I only get crystalline water. I have been extra careful<br>
during sample preparation, and transferring the grind on to the holder,<br>
and I have done 48 hours holder bake out before insertion, but I still<br>
get ice on the grid.<br>
<br>
I monitored the temperature of the holder during the insertion process,<br>
including 3 minutes for the Airlock pump. During those 3 minutes the<br>
temperature rises up to -152.2. Can this be the reason of the unwanted<br>
ice formation? How high can be the temperature of the holder to ensure a<br>
nice vitreous water?<br>
<br>
I am also getting vacuum problems while inserting the holder. This<br>
process has resulted to be extremely, extremely sensitive. After the 3<br>
minutes Airlock pumping (I cannot give longer time because of the<br>
aforementioned temperature issue) as soon as I open the column port the<br>
vacuum either rises to 99 Log or completely crashes the column vacuum.<br>
This has happened with two different holders either cooled with liquid<br>
nitrogen or at room temperature. So, my question: Is this insertion<br>
process always that picky or it can be that there is something wrong<br>
with the holders/Airlock pump?<br>
<br>
Thanks a lot in advanced.<br>
<br>
Best regards.<br>
<br>
Ariel<br>
<br>
--<span class="apple-converted-space"> </span><br>
Ariel TALAVERA PEREZ, PhD<br>
Center for Microscopy and Molecular Imaging (CMMI)<br>
Laboratory of Microscopy<br>
Université Libre de Bruxelles<br>
Rue Adrienne Bolland, 8<br>
B-6041 Gosselies, Belgium<br>
Phone +32 (0)26509866<br>
<br>
_______________________________________________<br>
3dem mailing list<br>
<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br>
<a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwIGaQ&c=slrrB7dE8n7gBJbeO0g-IQ&r=hgbDG_ZdQaorZsucl01zHg&m=Yn2a7P10qylNPjdZ499rPbNzetpn8RyAV870SJbr0hg&s=wwZt0ruHRcbRcKBOUphvqWKC9QI8lDUPt_L8YiYwln4&e=">https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwIGaQ&c=slrrB7dE8n7gBJbeO0g-IQ&r=hgbDG_ZdQaorZsucl01zHg&m=Yn2a7P10qylNPjdZ499rPbNzetpn8RyAV870SJbr0hg&s=wwZt0ruHRcbRcKBOUphvqWKC9QI8lDUPt_L8YiYwln4&e=</a><o:p></o:p></span></p>
</blockquote>
<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">_______________________________________________<br>
3dem mailing list<br>
<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br>
<a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwIGaQ&c=slrrB7dE8n7gBJbeO0g-IQ&r=hgbDG_ZdQaorZsucl01zHg&m=Yn2a7P10qylNPjdZ499rPbNzetpn8RyAV870SJbr0hg&s=wwZt0ruHRcbRcKBOUphvqWKC9QI8lDUPt_L8YiYwln4&e=">https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwIGaQ&c=slrrB7dE8n7gBJbeO0g-IQ&r=hgbDG_ZdQaorZsucl01zHg&m=Yn2a7P10qylNPjdZ499rPbNzetpn8RyAV870SJbr0hg&s=wwZt0ruHRcbRcKBOUphvqWKC9QI8lDUPt_L8YiYwln4&e=</a><o:p></o:p></span></p>
</blockquote>
<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">_______________________________________________<br>
3dem mailing list<br>
<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br>
<a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwIGaQ&c=slrrB7dE8n7gBJbeO0g-IQ&r=hgbDG_ZdQaorZsucl01zHg&m=Yn2a7P10qylNPjdZ499rPbNzetpn8RyAV870SJbr0hg&s=wwZt0ruHRcbRcKBOUphvqWKC9QI8lDUPt_L8YiYwln4&e=">https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwIGaQ&c=slrrB7dE8n7gBJbeO0g-IQ&r=hgbDG_ZdQaorZsucl01zHg&m=Yn2a7P10qylNPjdZ499rPbNzetpn8RyAV870SJbr0hg&s=wwZt0ruHRcbRcKBOUphvqWKC9QI8lDUPt_L8YiYwln4&e=</a><o:p></o:p></span></p>
</blockquote>
<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif"><br>
--<span class="apple-converted-space"> </span><br>
David L. Stokes<br>
Skirball Institute, Dept. of Cell Biology<br>
NYU School of Medicine<br>
</span><a href="http://stokeslab.med.nyu.edu/"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">http://stokeslab.med.nyu.edu/</span></a><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif"><br>
tel: 212-263-1580<br>
fax: 212-263-2150<br>
<br>
_______________________________________________<br>
3dem mailing list<br>
</span><a href="mailto:3dem@ncmir.ucsd.edu"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">3dem@ncmir.ucsd.edu</span></a><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif"><br>
</span><a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</span></a><o:p></o:p></p>
</div>
</blockquote>
</div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
</div>
<p class="MsoNormal">_______________________________________________<br>
3dem mailing list<br>
<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br>
<a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a><o:p></o:p></p>
</div>
</blockquote>
</div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
</div>
</body>
</html>