<html><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; ">Dear Dylan,<div><br></div><div><br class="Apple-interchange-newline"><blockquote type="cite"><div lang="EN-GB" link="blue" vlink="purple"><div class="WordSection1" style="page: WordSection1; "><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 11pt; ">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></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p></o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; 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.</div></div></div></blockquote><div><br></div>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><blockquote type="cite"><div lang="EN-GB" link="blue" vlink="purple"><div class="WordSection1" style="page: WordSection1; "><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p></o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; 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?</div></div></div></blockquote><div><br></div><div>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.</div><div><br></div><div>I will help more after you send the logs or even deposit the data somewhere.</div><div><br></div><div>All the best</div><div><br></div><div>PAvel</div><div><br></div><br><blockquote type="cite"><div lang="EN-GB" link="blue" vlink="purple"><div class="WordSection1" style="page: WordSection1; "><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p></o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; 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></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; ">Kind regards,<o:p></o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; ">Dylan<o:p></o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10pt; color: rgb(31, 73, 125); ">Dylan Windell<o:p></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10pt; color: rgb(31, 73, 125); ">Postgraduate Researcher<o:p></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10pt; color: rgb(31, 73, 125); "><a href="http://business-school.exeter.ac.uk/research/areas/centres/isr/" style="color: purple; "><span style="color: rgb(31, 73, 125); ">College</span></a> of Life and Environmental Sciences<o:p></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10pt; color: rgb(31, 73, 125); ">University of Exeter, Geoffrey Pope<o:p></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10pt; color: rgb(31, 73, 125); ">Stocker Road, Exeter EX4 4QD, UK<o:p></o:p></span></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><i><span style="font-size: 10pt; color: rgb(31, 73, 125); ">phone: +44 (0) 7596725056<o:p></o:p></span></i></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><i><span style="font-size: 10pt; color: rgb(31, 73, 125); ">email: <u><a href="mailto:dw388@exeter.ac.uk" style="color: purple; "><span style="color: blue; ">dw388@exeter.ac.uk</span></a></u><o:p></o:p></span></i></div><div style="margin: 0cm 0cm 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></div></div>_______________________________________________<br>OpenSPIM mailing list<br><a href="mailto:OpenSPIM@openspim.org" style="color: purple; ">OpenSPIM@openspim.org</a><br><a href="http://openspim.org/mailman/listinfo/openspim" style="color: purple; ">http://openspim.org/mailman/listinfo/openspim</a></div></blockquote></div></body></html>