<html><head></head><body><div style="color:#000; background-color:#fff; font-family:HelveticaNeue, Helvetica Neue, Helvetica, Arial, Lucida Grande, Sans-Serif;font-size:14px"><div id="yui_3_16_0_ym19_1_1473234471964_16918"><span id="yui_3_16_0_ym19_1_1473234471964_17163">Hi </span></div><div id="yui_3_16_0_ym19_1_1473234471964_16918"><span><br></span></div><div id="yui_3_16_0_ym19_1_1473234471964_16918"><span id="yui_3_16_0_ym19_1_1473234471964_17111">Do you have only one cryo cycle a week?! How many cryo sessions you have in a week? </span></div><div id="yui_3_16_0_ym19_1_1473234471964_16918"><span><br></span></div><div id="yui_3_16_0_ym19_1_1473234471964_16918"><span id="yui_3_16_0_ym19_1_1473234471964_17162">Best Regards</span></div><div id="yui_3_16_0_ym19_1_1473234471964_16918"><span><br></span></div><div id="yui_3_16_0_ym19_1_1473234471964_16918"><span>Andy </span></div><div></div><div id="yui_3_16_0_ym19_1_1473234471964_16835"> </div><div class="signature" id="yui_3_16_0_ym19_1_1473234471964_16806"><div id="yui_3_16_0_ym19_1_1473234471964_16805"><font class="Apple-style-span" id="yui_3_16_0_ym19_1_1473234471964_16804"><span class="Apple-style-span" style="line-height: 19px;" id="yui_3_16_0_ym19_1_1473234471964_16803"><b id="yui_3_16_0_ym19_1_1473234471964_16802"><blockquote type="cite" style="line-height: normal;" id="yui_3_16_0_ym19_1_1473234471964_16801"><div id="yui_3_16_0_ym19_1_1473234471964_16800"><div id="yui_3_16_0_ym19_1_1473234471964_16799"><div id="yui_3_16_0_ym19_1_1473234471964_16798"><span class="Apple-style-span" style="border-collapse: separate; font-style: normal; font-variant: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px;" id="yui_3_16_0_ym19_1_1473234471964_16797"><span class="Apple-style-span" style="border-collapse: separate; font-style: normal; font-variant: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px;" id="yui_3_16_0_ym19_1_1473234471964_16796"><div style="word-wrap: break-word;" id="yui_3_16_0_ym19_1_1473234471964_16795"><div id="yui_3_16_0_ym19_1_1473234471964_16794"><div id="yui_3_16_0_ym19_1_1473234471964_16834" style="color: rgb(0, 0, 0); font-family: Helvetica; font-size: 16px; font-weight: normal;"><font class="Apple-style-span" color="#341dbb" face="'Times New Roman'" size="4" id="yui_3_16_0_ym19_1_1473234471964_16833"><b id="yui_3_16_0_ym19_1_1473234471964_16832">Dr. Anindito Sen (Ph.D)</b></font></div><div id="yui_3_16_0_ym19_1_1473234471964_16831"><br></div></div></div></span></span></div></div></div></blockquote><div style="color: rgb(0, 0, 255); font-family: garamond, 'new york', times, serif; font-size: 28px;" id="yui_3_16_0_ym19_1_1473234471964_16915"><div id="yui_3_16_0_ym19_1_1473234471964_16914"><div id="yui_3_16_0_ym19_1_1473234471964_16913"><div id="yui_3_16_0_ym19_1_1473234471964_16912"><span class="Apple-style-span" style="border-collapse:separate;color:rgb(0, 0, 0);font-family:Helvetica;font-style:normal;font-variant:normal;font-weight:normal;letter-spacing:normal;line-height:normal;orphans:2;text-indent:0px;text-transform:none;white-space:normal;widows:2;word-spacing:0px;font-size:16px;" id="yui_3_16_0_ym19_1_1473234471964_16911"><span class="Apple-style-span" style="border-collapse:separate;color:rgb(0, 0, 0);font-family:Helvetica;font-style:normal;font-variant:normal;font-weight:normal;letter-spacing:normal;line-height:normal;orphans:2;text-indent:0px;text-transform:none;white-space:normal;widows:2;word-spacing:0px;font-size:16px;" id="yui_3_16_0_ym19_1_1473234471964_16910"><div style="word-wrap:break-word;" id="yui_3_16_0_ym19_1_1473234471964_16909"><div id="yui_3_16_0_ym19_1_1473234471964_16908"><div id="yui_3_16_0_ym19_1_1473234471964_16907"><font class="Apple-style-span" color="#341dbb" face="'Times New Roman'" size="4"><b><br></b></font></div></div></div></span></span></div></div></div></div></b></span></font></div></div><div class="qtdSeparateBR" id="yui_3_16_0_ym19_1_1473234471964_16906"><br><br></div><div class="yahoo_quoted" id="yui_3_16_0_ym19_1_1473234471964_17116" style="display: block;"> <div style="font-family: HelveticaNeue, Helvetica Neue, Helvetica, Arial, Lucida Grande, Sans-Serif; font-size: 14px;" id="yui_3_16_0_ym19_1_1473234471964_17115"> <div style="font-family: HelveticaNeue, Helvetica Neue, Helvetica, Arial, Lucida Grande, Sans-Serif; font-size: 16px;" id="yui_3_16_0_ym19_1_1473234471964_17114"> <div dir="ltr" id="yui_3_16_0_ym19_1_1473234471964_17113"> <font size="2" face="Arial" id="yui_3_16_0_ym19_1_1473234471964_17112"> <hr size="1" id="yui_3_16_0_ym19_1_1473234471964_17161"> <b><span style="font-weight:bold;">From:</span></b> "Grassucci, Robert" <rg2502@cumc.columbia.edu><br> <b><span style="font-weight: bold;">To:</span></b> Sayan Bhakta <19sayanbhakta89@gmail.com> <br><b><span style="font-weight: bold;">Cc:</span></b> "3dem@ncmir.ucsd.edu" <3dem@ncmir.ucsd.edu><br> <b><span style="font-weight: bold;">Sent:</span></b> Wednesday, 7 September 2016, 2:42<br> <b><span style="font-weight: bold;">Subject:</span></b> Re: [3dem] Regarding Image Quality from FEI Tecnai G2 Polara<br> </font> </div> <div class="y_msg_container" id="yui_3_16_0_ym19_1_1473234471964_17117"><br><div id="yiv6259617052">
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<div class="yiv6259617052MsoNormal" id="yui_3_16_0_ym19_1_1473234471964_17178"><span style="font-size:11.0pt;color:#1F497D;">Hi Sayan</span></div>
<div class="yiv6259617052MsoNormal" id="yui_3_16_0_ym19_1_1473234471964_17180"><span style="font-size:11.0pt;color:#1F497D;" id="yui_3_16_0_ym19_1_1473234471964_17179">What is the vacuum in your projection chamber? Generally contamination comes from the projection chamber assuming there is no leak previously. Did the falcon
camera just get installed? That could be the source of your contamination. Do you use film? If it is not desiccated then that could be the source but if you have a falcon you probably do not use film. Since the cryo box on the Polara has a relatively large
hole in the bottom use of an objective aperture can help restrict the area that is contaminated on your grid. Do you keep Valve B open and the MSC attached? That could also be your vacuum leak. Good luck.</span></div>
<div class="yiv6259617052MsoNormal" id="yui_3_16_0_ym19_1_1473234471964_17181"><span style="font-size:11.0pt;color:#1F497D;">Best,</span></div>
<div class="yiv6259617052MsoNormal" id="yui_3_16_0_ym19_1_1473234471964_17190"><span style="font-size:11.0pt;color:#1F497D;">Bob</span></div>
<div class="yiv6259617052MsoNormal" id="yui_3_16_0_ym19_1_1473234471964_17182"><span style="font-size:11.0pt;color:#1F497D;"> </span></div>
<div class="yiv6259617052MsoNormal" id="yui_3_16_0_ym19_1_1473234471964_17123"><span style="font-size:11.0pt;color:#1F497D;"> </span></div>
<div class="yiv6259617052MsoNormal" id="yui_3_16_0_ym19_1_1473234471964_17120"><b><span style="font-size:10.0pt;">From:</span></b><span style="font-size:10.0pt;" id="yui_3_16_0_ym19_1_1473234471964_17183"> Sayan Bhakta [mailto:19sayanbhakta89@gmail.com]
<br>
<b>Sent:</b> Tuesday, September 06, 2016 1:35 PM<br>
<b>To:</b> Grassucci, Robert<br>
<b>Cc:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> Re: [3dem] Regarding Image Quality from FEI Tecnai G2 Polara</span></div>
<div class="yiv6259617052MsoNormal" id="yui_3_16_0_ym19_1_1473234471964_17126"> </div>
<div id="yui_3_16_0_ym19_1_1473234471964_17212">
<div class="yiv6259617052MsoNormal">Hi Bob,</div>
<div>
<div class="yiv6259617052MsoNormal">Thanks for your suggestion. I would surely convey the message to the FEI engineer.</div>
</div>
<div id="yui_3_16_0_ym19_1_1473234471964_17211">
<div class="yiv6259617052MsoNormal" id="yui_3_16_0_ym19_1_1473234471964_17210">Apart from this, we also have another issue and that is constant ice formation inside the column. The engineer who has come, kept a grid inside the column in the weekend and on Monday there was ice contamination formed onto the grid. After
that when we inspect, we see continuous decrease in quality of images taken subsequently on the first day itself. This might be because of the fact that the ice starts growing from the day 1 itself. We don't see it on the screen because of the beam may be???
As suggested by many there must be some leakage in the column. But the vacuum of the system is constant at 6. May be that minor leakage is somewhere undetectable. If you share your view on this, It would be highly appreciated.</div>
</div>
<div>
<div class="yiv6259617052MsoNormal">Sayan</div>
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<div id="yui_3_16_0_ym19_1_1473234471964_17224">
<div class="yiv6259617052MsoNormal"> </div>
<div id="yui_3_16_0_ym19_1_1473234471964_17223">
<div class="yiv6259617052MsoNormal">On Tue, Sep 6, 2016 at 8:25 PM, Grassucci, Robert <<a rel="nofollow" ymailto="mailto:rg2502@cumc.columbia.edu" target="_blank" href="mailto:rg2502@cumc.columbia.edu">rg2502@cumc.columbia.edu</a>> wrote:</div>
<div id="yui_3_16_0_ym19_1_1473234471964_17222">
<div id="yui_3_16_0_ym19_1_1473234471964_17221">
<div class="yiv6259617052MsoNormal" style=""><span style="font-size:11.0pt;color:#1F497D;">Hi Sayan,</span></div>
<div class="yiv6259617052MsoNormal" style=""><span style="font-size:11.0pt;color:#1F497D;">I believe what you are seeing is terracing or ring collapse. This happens when the feg tip shape
goes from a sharp 9high intensity) tip to a more blunt (dim) tip. Sometimes you will even see it split and look like 2 sources. This happens at our facility about once a month so we monitor the intensity very carefully. The good news is that it generally
recovers quickly and then remains stable for a long while. FEI service engineers know about this phenomenon and have some things to do to make it less frequent. One is to decrease the tip temperature and the other is to increase the extraction voltage.
You can also reshape the tip by bringing down the Ht and extraction and then bringing it back up but this does not always work well. If your tip is damaged then it just may need to be changed. Speak to your engineer so he can correct this. I hope that
helps. Good luck.</span></div>
<div class="yiv6259617052MsoNormal" style=""><span style="font-size:11.0pt;color:#1F497D;">Bob</span></div>
<div class="yiv6259617052MsoNormal" style=""><span style="font-size:11.0pt;color:#1F497D;"> </span></div>
<div class="yiv6259617052MsoNormal" style=""><span style="font-size:11.0pt;color:#1F497D;"> </span></div>
<div class="yiv6259617052MsoNormal" style=""><b><span style="font-size:10.0pt;">From:</span></b><span style="font-size:10.0pt;"> 3dem [mailto:<a rel="nofollow" ymailto="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank" href="mailto:3dem-bounces@ncmir.ucsd.edu">3dem-bounces@ncmir.ucsd.edu</a>]
<b>On Behalf Of </b>Sayan Bhakta<br>
<b>Sent:</b> Saturday, September 03, 2016 6:39 AM<br>
<b>To:</b> <a rel="nofollow" ymailto="mailto:3dem@ncmir.ucsd.edu" target="_blank" href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br>
<b>Subject:</b> [3dem] Regarding Image Quality from FEI Tecnai G2 Polara</span></div>
<div id="yui_3_16_0_ym19_1_1473234471964_17220">
<div id="yui_3_16_0_ym19_1_1473234471964_17219">
<div class="yiv6259617052MsoNormal" style=""> </div>
<div id="yui_3_16_0_ym19_1_1473234471964_17218">
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<div>
<div class="yiv6259617052MsoNormal" style="">Hi,</div>
</div>
<div class="yiv6259617052MsoNormal" style="margin-bottom:12.0pt;">We are facing many problems with our Cryo EM facility. I am giving some details of the machine and one of the main problems we are facing. It would be very helpful if people comment on
this and help us to find the actual problem.</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="">Machine:</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="">FEI Tecnai G2 Polara</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="">300Kv</div>
</div>
<div id="yui_3_16_0_ym19_1_1473234471964_17217">
<div class="yiv6259617052MsoNormal" style="" id="yui_3_16_0_ym19_1_1473234471964_17216">Have been running for last two years.<br>
<br>
Camera:</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="">FEI Eagle 4k*4K</div>
</div>
<div>
<div>
<div>
<div class="yiv6259617052MsoNormal" style=""> </div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="">Problem:</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="margin-bottom:12.0pt;">On the first day when we are putting our sample into the Cryo EM, we are getting good images. From the second day the quality of the images deteriorates.</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="">1. If we look at the images taken on the first day and second day, the intensity values of the images decreased on the second day.</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="">2. From the FFT, we see nice thorn rings from the images taken on the first day. But for the images taken in the second day, the FFT is very bad. Most of them are having drifts.</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="">3. Irrespective of the days, there are some images for which the 1D spectrum is having a cut in between. What I mean by that is there is spectrum upto certain distance in the 1D
spectrum, then there is no waves, then again there are some waves.</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="">4. Irrespective of the days, images are sometimes with blakish shadow on one side and with whitish shadow on other side. We need to do gain correction more frequently than usual-
a number of times in a day.</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style=""> </div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="margin-bottom:12.0pt;">For your information:</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="">1. The vacuum is stable on the second day.</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="margin-bottom:12.0pt;">2. We are giving Cryo-cycle each weekend.</div>
</div>
<div>
<div class="yiv6259617052MsoNormal" style="">If anyone has ever faced the same problem or has any advice on this regard, it would be very helpful. We have been struggling very hard to standardise many issues with our microscope.
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<div style="margin:0in;margin-bottom:.0001pt;"><span style="font-size:9.5pt;color:black;">Sayan Bhakta</span></div>
<div style="margin:0in;margin-bottom:.0001pt;">
<span style="font-size:9.5pt;color:black;">Research Scholar</span></div>
<div style="margin:0in;margin-bottom:.0001pt;">
<span style="font-size:9.5pt;color:black;">Group of Dr. Jayati Sengupta</span></div>
<div style="margin:0in;margin-bottom:.0001pt;">
<span style="font-size:9.5pt;color:black;">CSIR - Indian Institute of Chemical Biology</span></div>
<div style="margin:0in;margin-bottom:.0001pt;">
<span style="font-size:9.5pt;color:black;">Kolkata, West Bengal</span></div>
<div style="margin:0in;margin-bottom:.0001pt;">
<span style="font-size:9.5pt;color:black;">India</span></div>
</div>
</div>
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<div class="yiv6259617052MsoNormal" id="yui_3_16_0_ym19_1_1473234471964_17228"> </div>
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<div>
<div style="margin:0in;margin-bottom:.0001pt;"><span style="font-size:9.5pt;color:black;">Sayan Bhakta</span><span style="font-size:9.5pt;"></span></div>
<div style="margin:0in;margin-bottom:.0001pt;">
<span style="font-size:9.5pt;color:black;">Research Scholar</span><span style="font-size:9.5pt;"></span></div>
<div style="margin:0in;margin-bottom:.0001pt;">
<span style="font-size:9.5pt;color:black;">Group of Dr. Jayati Sengupta</span><span style="font-size:9.5pt;"></span></div>
<div style="margin:0in;margin-bottom:.0001pt;">
<span style="font-size:9.5pt;color:black;">CSIR - Indian Institute of Chemical Biology</span><span style="font-size:9.5pt;"></span></div>
<div style="margin:0in;margin-bottom:.0001pt;">
<span style="font-size:9.5pt;color:black;">Kolkata, West Bengal</span><span style="font-size:9.5pt;"></span></div>
<div style="margin:0in;margin-bottom:.0001pt;">
<span style="font-size:9.5pt;color:black;">India</span><span style="font-size:9.5pt;"></span></div>
</div>
</div>
</div>
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</div>
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