<html><head><meta http-equiv="Content-Type" content="text/html charset=us-ascii"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><div class="">Hi Tommi,</div><div class=""><br class=""></div><div class="">It can depend on the settings you are using. I always advice users to keep focus and exposure settings exactly the same, but I have seen many people who always use a higher magnification for focus and then things become alignment dependent: in the alignment procedures there is a magnification dependent defocus step which often is neglected.</div><div class=""><br class=""></div><div class="">What are your settings for focus and exposure in this case?</div><div class=""><br class=""></div><div class="">Cheers,</div><div class=""><br class=""></div><div class="">Wim Hagen</div><div class="">EMBL Heidelberg</div><div class=""><br class=""></div><div><blockquote type="cite" class=""><div class="">On Apr 27, 2015, at 11:52 PM, Tommi White <<a href="mailto:whitetommi@gmail.com" class="">whitetommi@gmail.com</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><div dir="ltr" class=""><div class=""><div class=""><div class=""><div class=""><div class=""><div class=""><div class=""><div class=""><div class="">Dear 3DEM.<br class=""><br class="">Perhaps the community can assist with this issue we are struggling with. In Low Dose on our Tecnai F30, the Low Dose focus image has a different defocus value (using FFT) versus Low Dose Exposure. <br class=""><br class="">I have been checking this on a relatively flat cross-grating (not cryo) specimen. I set LD exposure to be zero defocus (with focus knob), but when I switch over to LD focus (~ 3 micron away on tilt axis) the image is defocused (~ -2 micron). <br class=""><br class="">I believe there is some 200 kV procedure alignment that must not be set properly - but am not quite sure which one...any suggestions on what to tweak to get this exposure/focus area reflecting the same defocus?<br class=""><br class=""></div>Thanks for any suggestions...<br class=""></div>Tommi<br class=""><br class=""></div>Tommi A. White, Ph.D.<br class=""></div>Associate Director, Electron Microscopy Core Facility<br class=""></div>Assistant Professor of Biochemistry<br class=""></div>University of Missouri<br class=""></div>W125 Veterinary Medicine<br class=""></div>Columbia, MO 65211<br class=""></div>573-882-8304<br class=""><a href="http://emc.missouri.edu/" class="">http://emc.missouri.edu</a><br class=""><br class=""></div>
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