<html><head><meta http-equiv="Content-Type" content="text/html charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">Hello Dylan,<div class=""><br class=""></div><div class="">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… </div><div class=""><br class=""></div><div class=""><div apple-content-edited="true" class=""><div style="color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px;" class=""><div class="">Best Regards,</div><div class="">Pete</div><div class=""><br class=""></div><div class="">Peter Gabriel Pitrone - DipRMS TechRMS FRMS</div><div class="">Light Sheet Fluorescence Microscopist and Imaging Specialist</div><div class="">for Dr. Pavel Tomancak's research group at the</div><div class="">Max Planck Institute of Molecular Cell Biology and Genetics</div><div class="">Pfotenhauerstraße 108</div><div class="">01307 Dresden, Saxony</div><div class="">Germany</div><div class=""><br class=""></div><div class=""><br class=""></div><div class=""><a href="http://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html" class="">http://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html</a> &</div><div class=""><a href="http://www.openspim.org" class="">http://www.openspim.org</a></div><div class=""><br class=""></div><div class="">"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</div></div><div style="color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px;" class=""><br class=""></div><br class="Apple-interchange-newline">
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<br class=""><div><blockquote type="cite" class=""><div class="">On Nov 10, 2015, at 5:20 PM, Windell, Dylan <<a href="mailto:dw388@exeter.ac.uk" class="">dw388@exeter.ac.uk</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><div class="WordSection1" style="page: WordSection1; font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px;"><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Dear Pavel,<o:p class=""></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">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 class=""></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""><a href="http://1drv.ms/1M426qo" style="color: purple; text-decoration: underline;" class="">http://1drv.ms/1M426qo</a><o:p class=""></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">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 class=""></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Let me know if you need any more information.<o:p class=""></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Thanks again,<o:p class=""></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""><br class="">Dylan<o:p class=""></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div class=""><div style="border-style: solid none none; border-top-color: rgb(181, 196, 223); border-top-width: 1pt; padding: 3pt 0cm 0cm;" class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><b class=""><span lang="EN-US" style="font-size: 10pt; font-family: Tahoma, sans-serif;" class="">From:</span></b><span lang="EN-US" style="font-size: 10pt; font-family: Tahoma, sans-serif;" class=""><span class="Apple-converted-space"> </span>Pavel Tomancak [<a href="mailto:tomancak@mpi-cbg.de" style="color: purple; text-decoration: underline;" class="">mailto:tomancak@mpi-cbg.de</a>]<span class="Apple-converted-space"> </span><br class=""><b class="">Sent:</b><span class="Apple-converted-space"> </span>10 November 2015 13:33<br class=""><b class="">To:</b><span class="Apple-converted-space"> </span>Windell, Dylan<br class=""><b class="">Cc:</b><span class="Apple-converted-space"> </span><a href="mailto:openspim@openspim.org" style="color: purple; text-decoration: underline;" class="">openspim@openspim.org</a><br class=""><b class="">Subject:</b><span class="Apple-converted-space"> </span>Re: [OpenSPIM] 3D Reconstruction<o:p class=""></o:p></span></div></div></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">Dear Dylan,<o:p class=""></o:p></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><br class=""><br class=""><o:p class=""></o:p></div><div class=""><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class="">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 class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""> <o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class="">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 class=""></o:p></span></div></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">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 class=""><br class=""><o:p class=""></o:p></div><div class=""><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""> <o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class="">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 class=""></o:p></span></div></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">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 class=""></o:p></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">I will help more after you send the logs or even deposit the data somewhere.<o:p class=""></o:p></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">All the best<o:p class=""></o:p></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">PAvel<o:p class=""></o:p></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><br class=""><br class=""><o:p class=""></o:p></div><div class=""><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""> <o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class="">If anyone has had difficulty in this or have done work on Zebrafish with SPIM I would really appreciate any help.<o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""> <o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class="">Kind regards,<o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""> <o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class="">Dylan<o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""> <o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 10pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Dylan Windell</span><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 10pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Postgraduate Researcher</span><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 10pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""><a href="http://business-school.exeter.ac.uk/research/areas/centres/isr/" style="color: purple; text-decoration: underline;" class=""><span style="color: rgb(31, 73, 125);" class="">College</span></a> of Life and Environmental Sciences</span><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 10pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">University of Exeter, Geoffrey Pope</span><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 10pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Stocker Road, Exeter EX4 4QD, UK</span><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><i class=""><span style="font-size: 10pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">phone: +44 (0) 7596725056</span></i><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><i class=""><span style="font-size: 10pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">email: <u class=""><a href="mailto:dw388@exeter.ac.uk" style="color: purple; text-decoration: underline;" class="">dw388@exeter.ac.uk</a></u></span></i><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif;" class=""> <o:p class=""></o:p></span></div></div><div style="margin: 0cm 0cm 0.0001pt; 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