<div dir="ltr"><div><div><div>Hi William,<br><br></div>If everything is OK on the fluorescent screen, then it seems the problem is occurring post screen/pre camera. This may be unrelated but on our system of FEI T20 and Eagle camera, it is very important to select the post specimen shutter (ie mechanical and part of camera system), rather than pre-specimen shutter (extreme gun tilt), when doing room temp tomography. If the pre-specimen shutter is left on, say after a low dose session, then I sometimes see this effect that looks like astigmatism and it can even randomly appear half way through the day, thus looking confusingly like some charge build up problem. For cryo tomography it is not a problem as both pre and post shutters are employed.<br><br></div>Regards,<br><br></div>Judith Mantell<br></div><div class="gmail_extra"><br clear="all"><div><div class="gmail_signature"><div dir="ltr"><b>Judith Mantell</b><div><br></div><div>Electron Microscopy Specialist<div>Wolfson Bioimaging Facility</div><div>Faculty of Medical and Veterinary Sciences</div><div>Medical Sciences Building</div><div>University of Bristol</div><div>Bristol. BS8 1TL</div><div><br></div><div>0117 3312178</div></div></div></div></div>
<br><div class="gmail_quote">On 3 June 2015 at 20:01, <span dir="ltr"><<a href="mailto:3dem-request@ncmir.ucsd.edu" target="_blank">3dem-request@ncmir.ucsd.edu</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">Send 3dem mailing list submissions to<br>
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Today's Topics:<br>
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1. Image distortion issues (William Nicolas)<br>
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Message: 1<br>
Date: Wed, 3 Jun 2015 19:04:18 +0200<br>
From: William Nicolas <<a href="mailto:wjnicolas@hotmail.com">wjnicolas@hotmail.com</a>><br>
To: <a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br>
Subject: [3dem] Image distortion issues<br>
Message-ID: <BLU436-SMTP165AF703875DBE82F2C1944A7B40@phx.gbl><br>
Content-Type: text/plain; charset="utf-8"<br>
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Hello everyone,<br>
<br>
I am actually having trouble with coating my grids with gold particles to use as fiducial markers prior to tilt series acquisition.<br>
<br>
I usually use outdated secondary antibodies linked to 5nm gold particles but it went out. I thus bought a gold particle solution from BBI solution and can?t get the parameters straight.<br>
I tested different concentrations and different ways to put them on my grid and have found optimal settings.<br>
<br>
The problem is now that I want to use them on 200nm thick HM20 embedded sample slices which are deposited on a slot-grid coated with formvar, I have distorted images on the camera BUT not on the fluorescent screen.<br>
The beads and image seems to be stretched in one direction and it is not astigmatism nor sample drifting. I thought of charges building up on the surface of the sample, distorting the electrons that are transmitted, maybe because of the BBI solutions liquid media?<br>
<br>
Do you have a solution to prevent charges from building up on the surface of my sample ?<br>
<br>
Thanks in advance.<br>
<br>
Regards,<br>
<br>
William Nicolas, PhD student, CNRS UMR 5200<br>
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