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<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">Dear Nancy,<o:p></o:p></span></p>
<span style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB"><o:p></o:p></span>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">What Sjors is suggesting is a
simplified
version of what we suggested in:<o:p></o:p></span></p>
<span style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB"><o:p></o:p></span>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">"A posteriori correction of camera
characteristics
from large image data sets", P. Afanasyev et al, Nature,
Scientific
Reports.<o:p></o:p></span><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB"><o:p> </o:p></span>
</p>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">The camera correction should
preferably include the
subtraction of the average background and the normalization of
the standard deviations
of the pixel vectors, especially if you are operating your DE
camera in integrating
mode. The movie alignments can
improve significantly after the a posteriori camera
correction.<span style="mso-spacerun:yes"> </span>The other
aspect is the FRC validation of the
camera correction. The FRC is then not used as a ‘gold
standard’ 2D resolution
criterion but rather as an indicator of the independence of
different images
collected with the same sensor. The FRC should not cross the
3-sigma threshold
indicating the expected random noise fluctuations around
FRC=0. For details see the
paper.<o:p></o:p></span></p>
<span style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB"><o:p></o:p></span>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">If you want to test the a
posteriori camera
correction programs, contact <a
href="mailto:michael@imagescience.de"><a class="moz-txt-link-abbreviated" href="mailto:michael@imagescience.de">michael@imagescience.de</a></a>!<o:p></o:p></span></p>
<span style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB"><o:p></o:p></span>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">Hope this helps!<o:p></o:p></span></p>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">Marin<o:p></o:p></span></p>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
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style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB"><o:p> </o:p></span></p>
=====================================<br>
<br>
On 28/01/2016 00:48, Nancy Meyer wrote:<br>
</div>
<blockquote
cite="mid:7B6DD4F49731AB4691DC21234C3BAE985641108A@EXMB06.ohsu.edu"
type="cite">
<pre wrap="">Again, thank you for the reassuring response! Maybe I've lucked out with similar image intensities in previous datasets - I should probably look at a few min/max, mean, and stdev values to convince myself.
I was pretty careful to remove particles on that horiz artefact and if restoring their inclusion is the biggest gain from redoing gain correction, I can maybe let that slide for now. I did just see "A posteriori correction of camera
characteristics from large image data sets", P. Afanasyev et al, Nature, Scientific Reports, after looking up your recommendation - looks like they've implemented it in Imagic - wonder if you could do the same correction in just ImageJ...
Thank you for taking the time to look at the images... slowly getting the hang of Relion, but the input is so invaluable!
Best,
Nancy
______________________________________
From: Sjors Scheres [<a class="moz-txt-link-abbreviated" href="mailto:scheres@mrc-lmb.cam.ac.uk">scheres@mrc-lmb.cam.ac.uk</a>]
Sent: Wednesday, January 27, 2016 1:10 PM
To: Nancy Meyer
Cc: <a class="moz-txt-link-abbreviated" href="mailto:ccpem@jiscmail.ac.uk">ccpem@jiscmail.ac.uk</a>
Subject: Re: [ccpem] Adequate particle normalization prior to 2D classification?
Hi Nancy,
The particles look perfectly fine to me. The overall differences in
greyscale you see is because each of the particles is scaled from white to
black with their own min and max values, which are susceptible to
outliers. The mean will probably be perfectly fine. You can get rid of
this with removing white and black dust, but it will not be necessary to
do so.
The classes also look quite OK. Just select all the nice ones and proceed
with another 2D classification or go into 3D already. Some details are
already coming up, so the 3D structure will look nice.
The micrographs show a sign of a bad gain correction. If you have many of
them (> several hundreds) you could correct the gain a-posteriori by
calculating the average of all micrographs and then divide each micrograph
by that average. This should get rid of the horizontal artefact (as long
as it is present in all micrographs). That may save some particles that
lie on that line.
HTH,
Sjors
</pre>
<br>
</blockquote>
<br>
<pre class="moz-signature" cols="72">--
================================================================
Prof Dr Ir Marin van Heel
Professor of Cryo-EM Data Processing
Leiden University
NeCEN Building Room 05.27
Einsteinweg 55
2333 CC Leiden
The Netherlands
Tel. NL: +31(0)715271424 // Mobile NL: +31(0)652736618
Skype: Marin.van.Heel
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and: mvh.office(A_T)gmail.com
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