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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Hey Pete,<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 for the instructions, I will try them out ASAP. I am guessing you meant replacing the cylindrical lens after the alignment?<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">My laser cannot be attenuated that low but if I were to use an ND filter, would that affect the alignment process?<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">Kind regards,<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">Dylan<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>
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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""> Peter Gabriel Pitrone [mailto:pitrone@mpi-cbg.de]
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<b>Sent:</b> 11 November 2015 15:22<br>
<b>To:</b> Windell, Dylan<br>
<b>Cc:</b> openspim@openspim.org<br>
<b>Subject:</b> Re: [OpenSPIM] Light Sheet Alignment Procedure (Was: 3D Reconstruction)<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal" style="margin-bottom:12.0pt">Of course you need to replace the Cylindrical lens to SPIMage again… Sent that last one too fast.<o:p></o:p></p>
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<p class="MsoNormal">On Nov 11, 2015, at 4:14 PM, Peter Gabriel Pitrone <<a href="mailto:pitrone@mpi-cbg.de">pitrone@mpi-cbg.de</a>> wrote:<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Hey 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">I’ve started using a different technique for alignment that doesn’t require any thing at all (except for the media) in the sample chamber. I’ll describe it in short below:<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">- Either take out the sample, or move it well beyond the focal plane (far away from the detection objective)<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">- Take out all the filters blocking the laser from the camera (make sure that the laser is modulated way down to around ±1 mW)<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">- Take out the cylindrical lens(es)<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"">- Go “Live” at a short exposure time (10-25 ms)<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">You should see an in-focus beam in the middle of the field of view that cuts across the image, and it should slightly converge and then diverge with the narrowest point
in the middle of the field of view. Now you can adjust the conjugate plane (corner) mirror horizontally to correct any misalignment in terms of focus, and vertically to place it in the center across the field. The two lens telescope between the mirror and
the illumination lens can be adjusted along the rail so the narrowest part of the beam is centered in the exact middle.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif""><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">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"">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""><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">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"">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"">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"">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"">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"">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"">Germany<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif""><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif""><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""><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""><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">"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"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif""><o:p> </o:p></span></p>
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<p class="MsoNormal">On Nov 10, 2015, at 5:57 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"><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">Hi Pete,</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 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.</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">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";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">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:11.0pt;font-family:"Calibri","sans-serif"">From:</span></b><span class="apple-converted-space"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif"">Peter
Gabriel Pitrone [<a href="mailto:pitrone@mpi-cbg.de">mailto:pitrone@mpi-cbg.de</a>]<span class="apple-converted-space"> </span><br>
<b>Sent:</b><span class="apple-converted-space"> </span>10 November 2015 17:41<br>
<b>To:</b><span class="apple-converted-space"> </span>Windell, Dylan <<a href="mailto:dw388@exeter.ac.uk">dw388@exeter.ac.uk</a>><br>
<b>Cc:</b><span class="apple-converted-space"> </span>Pavel Tomancak <<a href="mailto:tomancak@mpi-cbg.de">tomancak@mpi-cbg.de</a>>;
<a href="mailto:openspim@openspim.org">openspim@openspim.org</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">Hello 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">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"">Best Regards,</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">Pete</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">Peter Gabriel Pitrone - DipRMS TechRMS FRMS</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">Light Sheet Fluorescence Microscopist and Imaging Specialist</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">for Dr. Pavel Tomancak's research group at the</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">Max Planck Institute of Molecular Cell Biology and Genetics</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">Pfotenhauerstraße 108</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">01307 Dresden, Saxony</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">Germany</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif""><a href="http://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html"><span style="color:purple">http://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html</span></a><span class="apple-converted-space"> </span>&</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif""><a href="http://www.openspim.org/"><span style="color:purple">http://www.openspim.org</span></a></span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica","sans-serif"">"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</span><o:p></o:p></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"><span style="color:purple">dw388@exeter.ac.uk</span></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">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"><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"> <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"> <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"> <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>
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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>
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</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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<p class="MsoNormal">_______________________________________________<br>
OpenSPIM mailing list<br>
<a href="mailto:OpenSPIM@openspim.org">OpenSPIM@openspim.org</a><br>
<a href="http://openspim.org/mailman/listinfo/openspim">http://openspim.org/mailman/listinfo/openspim</a><o:p></o:p></p>
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