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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US">Hi Pete,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US">I have a cut micrometre slide stuck in a syringe which I use to check the laser sheet straightness as per OpenSPIM instructions followed
by checking the embryos for image sharpness. I probably should be doing it with beads but I am more comfortable with an embryo as I am not 100% what an optimal bead scan would look like.<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;mso-fareast-language:EN-US">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;color:#1F497D;mso-fareast-language:EN-US">Dylan<o:p></o:p></span></p>
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<p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif">From:</span></b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif"> Peter Gabriel Pitrone [mailto:pitrone@mpi-cbg.de]
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<b>Sent:</b> 10 November 2015 17:41<br>
<b>To:</b> Windell, Dylan <dw388@exeter.ac.uk><br>
<b>Cc:</b> Pavel Tomancak <tomancak@mpi-cbg.de>; openspim@openspim.org<br>
<b>Subject:</b> Re: [OpenSPIM] 3D Reconstruction<o:p></o:p></span></p>
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<p class="MsoNormal">Hello Dylan,<o:p></o:p></p>
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<p class="MsoNormal">Can you tell me your light sheet alignment procedure that you do, hopefully before each experiment, maybe it could help exclude that possibility from the equation… <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:9.0pt;font-family:"Helvetica",sans-serif;color:black">Best Regards,<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:black">Pete<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:black">Peter Gabriel Pitrone - DipRMS TechRMS FRMS<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:black">Light Sheet Fluorescence Microscopist and Imaging Specialist<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:black">for Dr. Pavel Tomancak's research group at the<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:black">Max Planck Institute of Molecular Cell Biology and Genetics<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:black">Pfotenhauerstraße 108<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:black">01307 Dresden, Saxony<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:black"><a href="http://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html">http://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html</a> &<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:black"><a href="http://www.openspim.org">http://www.openspim.org</a><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:black">"I KEEP six honest serving-men (they taught me all I knew); their names are What and Why and When and How and Where and Who." Rudyard Kipling - The Elephant's
Child<o:p></o:p></span></p>
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<p class="MsoNormal">On Nov 10, 2015, at 5:20 PM, Windell, Dylan <<a href="mailto:dw388@exeter.ac.uk">dw388@exeter.ac.uk</a>> wrote:<o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D">Dear Pavel,</span><o:p></o:p></p>
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<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:</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D"><a href="http://1drv.ms/1M426qo"><span style="color:purple">http://1drv.ms/1M426qo</span></a></span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D"> </span><o:p></o:p></p>
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<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.</span><o:p></o:p></p>
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<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.</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D">Thanks again,</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D"><br>
Dylan</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D"> </span><o:p></o:p></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 class="apple-converted-space"><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma",sans-serif"> </span></span><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma",sans-serif">Pavel
Tomancak [<a href="mailto:tomancak@mpi-cbg.de"><span style="color:purple">mailto:tomancak@mpi-cbg.de</span></a>]<span class="apple-converted-space"> </span><br>
<b>Sent:</b><span class="apple-converted-space"> </span>10 November 2015 13:33<br>
<b>To:</b><span class="apple-converted-space"> </span>Windell, Dylan<br>
<b>Cc:</b><span class="apple-converted-space"> </span><a href="mailto:openspim@openspim.org"><span style="color:purple">openspim@openspim.org</span></a><br>
<b>Subject:</b><span class="apple-converted-space"> </span>Re: [OpenSPIM] 3D Reconstruction</span><o:p></o:p></p>
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<p class="MsoNormal"> <o:p></o:p></p>
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<p class="MsoNormal">Dear Dylan,<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.</span><o:p></o:p></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.</span><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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<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?</span><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">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">PAvel<o:p></o:p></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.</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">Kind regards,</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">Dylan</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Calibri",sans-serif;color:#1F497D">Dylan Windell</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Calibri",sans-serif;color:#1F497D">Postgraduate Researcher</span><o:p></o:p></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><o:p></o:p></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><o:p></o:p></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><o:p></o:p></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><o:p></o:p></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"><span style="color:purple">dw388@exeter.ac.uk</span></a></u></span></i><o:p></o:p></p>
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<p class="MsoNormal">_______________________________________________<br>
OpenSPIM mailing list<br>
<a href="mailto:OpenSPIM@openspim.org"><span style="color:purple">OpenSPIM@openspim.org</span></a><br>
<a href="http://openspim.org/mailman/listinfo/openspim"><span style="color:purple">http://openspim.org/mailman/listinfo/openspim</span></a><o:p></o:p></p>
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<p class="MsoNormal"><5um_140exp_3_angles.txt><3um_3_angles.txt><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">_______________________________________________<br>
OpenSPIM mailing list<br>
</span><a href="mailto:OpenSPIM@openspim.org"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:purple">OpenSPIM@openspim.org</span></a><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif"><br>
</span><a href="http://openspim.org/mailman/listinfo/openspim"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif;color:purple">http://openspim.org/mailman/listinfo/openspim</span></a><o:p></o:p></p>
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