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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Dear Pavel,<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">Thank you for your email. I just ran the registrations again on a couple of datasets I had. They were taken at 60 degrees apart at 10 or 20x magnification.
The logs correspond with the raw data I have uploaded on my onedrive if you want to have a look at those too:<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"><a href="http://1drv.ms/1M426qo">http://1drv.ms/1M426qo</a><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">I have purchased some wider borosilicate capillaries that might help in both having more volume for beads and ensuring the tube is dead straight. Zhung, you
might find that helpful? I will be running tests on them very soon so would let you know. I will also try Vaa3D, thanks for the suggestion.<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">Let me know if you need any more information.<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">Thanks again,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><br>
Dylan<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""> Pavel Tomancak [mailto:tomancak@mpi-cbg.de]
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<b>Sent:</b> 10 November 2015 13:33<br>
<b>To:</b> Windell, Dylan<br>
<b>Cc:</b> openspim@openspim.org<br>
<b>Subject:</b> Re: [OpenSPIM] 3D Reconstruction<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Dear Dylan,<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif"">I have been trying to register Z-stacks of Zebrafish using the SPIM plugin with various concentrations of beads but despite everything I try I cannot get the beads to match
with different angles.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif"">I am not sure if this is because of the light sheet not being aligned properly (I am calculating the PSF very soon and will re-align the system from scratch) or due to sample
prep etc. I have corrected bead drifting (as this was evident in low concentrations of agarose) and I am pretty sure the xy and z resolution numbers are correct. I will attempt the Multiview reconstruction package, FluoRender or even ClearVolume but without
bead registration working I am not sure if that will work either.<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Did you already run the multi-view reconstruction? Can you attach the ImageJ log. I am particularly looking for numbers of beads and numbers of RANSAC inliers. That would be the way to start troubleshooting.<br>
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<o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif"">Would it be possible that if the FEP tube is not straight, the manual positioning of the next angles would misalign bead registration? I have also thought that as the embryo
fills up the tube, I am not getting enough volume of beads on either side of the stack for bead registration?<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">That maybe a problem. There may be not enough beads in the OVERLAP between the views. You can visualise that in the Multiview Reconstruction application.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">I will help more after you send the logs or even deposit the data somewhere.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">All the best<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">PAvel<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif"">If anyone has had difficulty in this or have done work on Zebrafish with SPIM I would really appreciate any help.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif"">Kind regards,<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif"">Dylan<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Dylan Windell</span><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Postgraduate Researcher</span><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><a href="http://business-school.exeter.ac.uk/research/areas/centres/isr/"><span style="color:#1F497D">College</span></a> of Life and Environmental Sciences</span><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Calibri","sans-serif";color:#1F497D">University of Exeter, Geoffrey Pope</span><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Stocker Road, Exeter EX4 4QD, UK</span><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""><o:p></o:p></span></p>
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<p class="MsoNormal"><i><span style="font-size:10.0pt;font-family:"Calibri","sans-serif";color:#1F497D">phone: +44 (0) 7596725056</span></i><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""><o:p></o:p></span></p>
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<p class="MsoNormal"><i><span style="font-size:10.0pt;font-family:"Calibri","sans-serif";color:#1F497D">email: <u><a href="mailto:dw388@exeter.ac.uk">dw388@exeter.ac.uk</a></u></span></i><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif""> <o:p></o:p></span></p>
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<p class="MsoNormal">_______________________________________________<br>
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