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<p class="MsoNormal"><a name="_MailEndCompose"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">HI Tommi,<o:p></o:p></span></a></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">If the defocus series is unticked then your issue comes from the alignment file. I am guessing you have a different magnification for Focus and Acquisition.
If that is not the case , then I don’t know. If that is the case then check that the Alignment parameter “Image Shift and Focus” at the magnifications (I am guessing either MH and SA or just SA) of interest are correct and redo them if they are not.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Regards<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Eric<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
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<p class="MsoNormal"><b><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:navy">-----------------<o:p></o:p></span></b></p>
<p class="MsoNormal"><b><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:navy">Dr Eric Hanssen<o:p></o:p></span></b></p>
<p class="MsoNormal"><b><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:navy"><br>
</span></b><b><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:#1F497D">Senior Research Fellow<o:p></o:p></span></b></p>
<p class="MsoNormal"><b><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:#1F497D">Head - Melbourne Advanced Microscopy Facility
</span></b><b><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:#1F497D">–</span></b><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:#1F497D">
<b>Bio21 Institute node<o:p></o:p></b></span></p>
<p class="MsoNormal"><b><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:#1F497D">Adjunct Senior Fellow – Department of Biochemistry and Molecular Biology<o:p></o:p></span></b></p>
<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:#1F497D"><br>
Bio21 Molecular Science and Biotechnology Institute<br>
</span><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:gray">30 Flemington Road - The University of Melbourne - Victoria 3010 - Australia<br>
email: </span><a href="mailto:ehanssen@unimelb.edu.au"><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:gray">ehanssen@unimelb.edu.au</span></a><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:gray"> | Office: +61 3 83442449 |
Microscope: +61 3 83442509 | Fax: +61 3 9347 8265<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:gray">Web:
</span><a href="http://www.microscopy.unimelb.edu.au/"><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:blue">www.microscopy.unimelb.edu.au</span></a><span style="font-size:10.0pt;font-family:"Comic Sans MS";color:gray"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><img border="0" width="627" height="174" id="Picture_x0020_1" src="cid:image001.jpg@01D0819B.405439C0" alt="BANNER_foremail"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
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<p class="MsoNormal"><b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif"">From:</span></b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif""> 3dem [mailto:3dem-bounces@ncmir.ucsd.edu]
<b>On Behalf Of </b>Grassucci, Robert<br>
<b>Sent:</b> Tuesday, 28 April 2015 8:45 AM<br>
<b>To:</b> Tommi White; 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> Re: [3dem] Low Dose Focus/Exposure<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Hi Tommi<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">There is an option in low dose to take a defocus series. Make sure that that is not activated. It is designed to take an exposure at a certain
defocus from where you set it in focus mode. That would me my best guess. Good luck.<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Bob<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif"">From:</span></b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif""> 3dem [<a href="mailto:3dem-bounces@ncmir.ucsd.edu">mailto:3dem-bounces@ncmir.ucsd.edu</a>]
<b>On Behalf Of </b>Tommi White<br>
<b>Sent:</b> Monday, April 27, 2015 5:53 PM<br>
<b>To:</b> <a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br>
<b>Subject:</b> [3dem] Low Dose Focus/Exposure<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><span lang="EN-US">Dear 3DEM.<br>
<br>
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>
<br>
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>
<br>
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?<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US">Thanks for any suggestions...<o:p></o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><span lang="EN-US">Tommi<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US">Tommi A. White, Ph.D.<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US">Associate Director, Electron Microscopy Core Facility<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US">Assistant Professor of Biochemistry<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US">University of Missouri<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US">W125 Veterinary Medicine<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US">Columbia, MO 65211<o:p></o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><span lang="EN-US">573-882-8304<br>
<a href="http://emc.missouri.edu">http://emc.missouri.edu</a><o:p></o:p></span></p>
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