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<div>Hi Pete, thanks for the info on the alignment.</div>
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<div>Can I ask how the alignment of the cylindrical lens might affect things when you place it back in the path? You slide it to create the best focus on the conjugate mirror(?), but how sensitive are things to it being off axis, not quite perpendicular, or
slightly rotated? Also, can you view these things via the camera using the scattered light too? </div>
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<div>I apologise if these are silly questions; I've not aligned the system yet, just started bolting it down.</div>
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<div>Thanks for your time</div>
<div>Neil</div>
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<span style="font-size:11.0pt; font-family:'Calibri','sans-serif'"><b>From:</b> Peter Gabriel Pitrone <pitrone@mpi-cbg.de><br>
<b>Sent:</b> Nov 11, 2015 10:45 AM<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)<br>
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<div>Howdy Dylan,
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<div class="">Go ahead and use an ND filter, it shouldn’t effect the process much at all… Sometimes I use them as well when the signal is too intense, but one can’t see any beads when it is in place.<br class="">
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<div class="">Best Regards,</div>
<div class="">Pete</div>
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<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>
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<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>
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<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>
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<div class="">On Nov 11, 2015, at 4:32 PM, Windell, Dylan <<a href="mailto:dw388@exeter.ac.uk" class="">dw388@exeter.ac.uk</a>> wrote:</div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">Hey Pete,</span></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">Thanks for the instructions, I will try them out ASAP. I am guessing you meant replacing the cylindrical lens after the alignment?</span></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">My laser cannot be attenuated that low but if I were to use an ND filter, would that affect the alignment process?</span></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">Kind regards,</span></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">Dylan</span></div>
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<b class=""><span lang="EN-US" class="" style="font-size:10pt; font-family:Tahoma,sans-serif">From:</span></b><span lang="EN-US" class="" style="font-size:10pt; font-family:Tahoma,sans-serif"><span class="Apple-converted-space"> </span>Peter Gabriel Pitrone
[<a href="mailto:pitrone@mpi-cbg.de" class="">mailto:pitrone@mpi-cbg.de</a>]<span class="Apple-converted-space"> </span><br class="">
<b class="">Sent:</b><span class="Apple-converted-space"> </span>11 November 2015 15:22<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" class="">openspim@openspim.org</a><br class="">
<b class="">Subject:</b><span class="Apple-converted-space"> </span>Re: [OpenSPIM] Light Sheet Alignment Procedure (Was: 3D Reconstruction)</span></div>
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Of course you need to replace the Cylindrical lens to SPIMage again… Sent that last one too fast.</p>
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On Nov 11, 2015, at 4:14 PM, Peter Gabriel Pitrone <<a href="mailto:pitrone@mpi-cbg.de" class="" style="color:purple; text-decoration:underline">pitrone@mpi-cbg.de</a>> wrote:</div>
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Hey Dylan,</div>
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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:</div>
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- Either take out the sample, or move it well beyond the focal plane (far away from the detection objective)</div>
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- Take out all the filters blocking the laser from the camera (make sure that the laser is modulated way down to around ±1 mW)</div>
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- Take out the cylindrical lens(es)</div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">- Go “Live” at a short exposure time (10-25 ms)</span></div>
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<span class="" style="font-size:9pt; 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.</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">Best Regards,</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">Pete</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">Peter Gabriel Pitrone - DipRMS TechRMS FRMS</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">Light Sheet Fluorescence Microscopist and Imaging Specialist</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">for Dr. Pavel Tomancak's research group at the</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">Max Planck Institute of Molecular Cell Biology and Genetics</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">Pfotenhauerstraße 108</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">01307 Dresden, Saxony</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">Germany</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif"><a href="http://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html" class="" style="color:purple; text-decoration:underline">http://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html</a><span class="Apple-converted-space"> </span>&</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif"><a href="http://www.openspim.org/" class="" style="color:purple; text-decoration:underline">http://www.openspim.org</a></span></div>
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<span class="" style="font-size:9pt; 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></div>
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On Nov 10, 2015, at 5:57 PM, Windell, Dylan <<a href="mailto:dw388@exeter.ac.uk" class="" style="color:purple; text-decoration:underline">dw388@exeter.ac.uk</a>> wrote:</div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">Hi Pete,</span></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">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></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">Kind regards,</span></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">Dylan</span></div>
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<b class=""><span lang="EN-US" class="" style="font-size:11pt; font-family:Calibri,sans-serif">From:</span></b><span class="apple-converted-space"><span lang="EN-US" class="" style="font-size:11pt; font-family:Calibri,sans-serif"> </span></span><span lang="EN-US" class="" style="font-size:11pt; font-family:Calibri,sans-serif">Peter
Gabriel Pitrone [<a href="mailto:pitrone@mpi-cbg.de" class="" style="color:purple; text-decoration:underline">mailto:pitrone@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 17:41<br class="">
<b class="">To:</b><span class="apple-converted-space"> </span>Windell, Dylan <<a href="mailto:dw388@exeter.ac.uk" class="" style="color:purple; text-decoration:underline">dw388@exeter.ac.uk</a>><br class="">
<b class="">Cc:</b><span class="apple-converted-space"> </span>Pavel Tomancak <<a href="mailto:tomancak@mpi-cbg.de" class="" style="color:purple; text-decoration:underline">tomancak@mpi-cbg.de</a>>;<span class="Apple-converted-space"> </span><a href="mailto:openspim@openspim.org" class="" style="color:purple; text-decoration:underline">openspim@openspim.org</a><br class="">
<b class="">Subject:</b><span class="apple-converted-space"> </span>Re: [OpenSPIM] 3D Reconstruction</span></div>
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Hello Dylan,</div>
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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>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">Best Regards,</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif">Pete</span></div>
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<span class="" style="font-size:9pt; font-family:Helvetica,sans-serif"><a href="http://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html" class="" style="color:purple; text-decoration:underline"><span class="" style="color:purple">http://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html</span></a><span class="apple-converted-space"> </span>&</span></div>
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<span class="" style="font-size:9pt; 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></div>
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On Nov 10, 2015, at 5:20 PM, Windell, Dylan <<a href="mailto:dw388@exeter.ac.uk" class="" style="color:purple; text-decoration:underline"><span class="" style="color:purple">dw388@exeter.ac.uk</span></a>> wrote:</div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">Dear Pavel,</span></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">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></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)"><a href="http://1drv.ms/1M426qo" class="" style="color:purple; text-decoration:underline"><span class="" style="color:purple">http://1drv.ms/1M426qo</span></a></span></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">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></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">Let me know if you need any more information.</span></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">Thanks again,</span></div>
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Dylan</span></div>
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<b class=""><span lang="EN-US" class="" style="font-size:10pt; font-family:Tahoma,sans-serif">From:</span></b><span class="apple-converted-space"><span lang="EN-US" class="" style="font-size:10pt; font-family:Tahoma,sans-serif"> </span></span><span lang="EN-US" class="" style="font-size:10pt; font-family:Tahoma,sans-serif">Pavel
Tomancak [<a href="mailto:tomancak@mpi-cbg.de" class="" style="color:purple; text-decoration:underline"><span class="" style="color:purple">mailto:tomancak@mpi-cbg.de</span></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" class="" style="color:purple; text-decoration:underline"><span class="" style="color:purple">openspim@openspim.org</span></a><br class="">
<b class="">Subject:</b><span class="apple-converted-space"> </span>Re: [OpenSPIM] 3D Reconstruction</span></div>
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Dear Dylan,</div>
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<span class="" style="font-size:11pt; 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></div>
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<span class="" style="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.</span></div>
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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="">
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<span class="" style="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?</span></div>
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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>
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I will help more after you send the logs or even deposit the data somewhere.</div>
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All the best</div>
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PAvel</div>
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<span class="" style="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.</span></div>
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<span class="" style="font-size:11pt; font-family:Calibri,sans-serif">Kind regards,</span></div>
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<span class="" style="font-size:10pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)"><a href="http://business-school.exeter.ac.uk/research/areas/centres/isr/" class="" style="color:purple; text-decoration:underline"><span class="" style="color:rgb(31,73,125)">College</span></a> of
Life and Environmental Sciences</span></div>
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<span class="" style="font-size:10pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">Stocker Road, Exeter EX4 4QD, UK</span></div>
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<i class=""><span class="" style="font-size:10pt; font-family:Calibri,sans-serif; color:rgb(31,73,125)">email: <u class=""><a href="mailto:dw388@exeter.ac.uk" class="" style="color:purple; text-decoration:underline"><span class="" style="color:purple">dw388@exeter.ac.uk</span></a></u></span></i></div>
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