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<div>Hi Tania,<br>
<br>
I'd agree with Stephan on the larger z stacks. I usually take >300 um, but it kinda depends on the field of view of the camera, as at some point you're taking images in an area that will no longer overlap.<br>
<br>
400 interest points would be sufficient if they all overlapped, but more are required in general. I believe Stephan's articles suggest 1000-2000 to get the best results. All depends on overlap percentage.<br>
<br>
When defining the beads I aim to use a sigma and threshold that picks out the peak of the bead if possible. Stephan, can I ask if it's detrimental when the threshold is too low and I'm identifying the same bead on many z planes? Also, I assume the sigma value
determines the steepness of the point, but it still seems to pick up on larger aggregated beads etc, so I'm not too sure about that part.<br>
<br>
Neil<br>
<br>
<br>
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<div style="border:none; padding:3.0pt 0cm 0cm 0cm"><span style="font-size:11.0pt; font-family:'Calibri','sans-serif'"><b>From:</b> Stephan.Preibisch@mdc-berlin.de<br>
<b>Sent:</b> Aug 25, 2016 3:41 AM<br>
<b>To:</b> Tania Mendonca<br>
<b>Cc:</b> Anthony, Neil; openspim@openspim.org<br>
<b>Subject:</b> Re: [OpenSPIM] Multiview registration - no inliers<br>
</span></div>
</div>
<br type="attribution">
<div>Hi Tania,
<div class=""><br class="">
</div>
<div class="">from looking at the data I agree with Anthony, there are some problems which make it hard. But trying different calibrations and not getting a result makes me believe that there is maybe no overlap in between the stacks. You have quite a few of
these big blobs apparently, so is it possible that these are different blobs?</div>
<div class=""><br class="">
</div>
<div class="">If you run it again, make the stacks way bigger in z to make sure that there is overlap, increase the laser power, and maybe put some simple auto fluorescent sample in there so it is possible to judge if the calibration is right and if there is
overlap.</div>
<div class=""><br class="">
</div>
<div class="">Good luck,</div>
<div class="">Stephan<br class="">
<div class="">
<div class="" style="color:rgb(0,0,0); letter-spacing:normal; orphans:auto; text-align:start; text-indent:0px; text-transform:none; white-space:normal; widows:auto; word-spacing:0px; word-wrap:break-word">
<div class="" style="color:rgb(0,0,0); letter-spacing:normal; orphans:auto; text-align:start; text-indent:0px; text-transform:none; white-space:normal; widows:auto; word-spacing:0px; word-wrap:break-word">
<div class="" style="orphans:2; widows:2">---</div>
<div class="" style="orphans:2; widows:2"><br class="">
</div>
<div class="" style="orphans:2; widows:2">Dr. Stephan Preibisch</div>
<div class="" style="orphans:2; widows:2">Group Leader</div>
<div class="" style="orphans:2; widows:2"><br class="">
</div>
<div class="" style="orphans:2; widows:2">Berlin Institute of Medical Systems Biology of the MDC</div>
<div class="" style="orphans:2; widows:2">Building 89, 1.08b</div>
<div class="" style="orphans:2; widows:2"><br class="">
</div>
<div class="" style="orphans:2; widows:2">email:<span class="Apple-converted-space"> </span><a href="mailto:stephan.preibisch@mdc-berlin.de" class="">stephan.preibisch@mdc-berlin.de</a></div>
<div class="" style="orphans:2; widows:2">web: <a href="http://fly.mpi-cbg.de/preibisch" class="">http://www.preibisch.net/</a></div>
</div>
</div>
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<br class="">
<div>
<blockquote type="cite" class="">
<div class="">On 24 Aug 2016, at 19:49, Tania Mendonca <<a href="mailto:tvmendonca1@sheffield.ac.uk" class="">tvmendonca1@sheffield.ac.uk</a>> wrote:</div>
<br class="Apple-interchange-newline">
<div class="">
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Hi Neil
<div class=""><br class="">
</div>
<div class="">Thanks for your email! The stacks should have the voxel size correctly set to 0.235 x 0.235 x 0.750. I'm assuming you've looked at the data on µManager as I'm unfamiliar with the ViewSetup tag that you mention. My system is not quite an OpenSPIM
- I use LabView to control and don't have the Picard 4D stage (I use a Piezo for scanning instead). </div>
<div class=""><br class="">
</div>
<div class="">Your manual transform looks about right, the stacks are about 120º apart with the big bead somewhere near the middle. I have tried a few different µbeads and the ones in these stacks are 1µm green beads but as you say they are nowhere near as
bright as the big beads. Perhaps I do need to dump the large beads and find something else to try and reconstruct. I've been finding about 400-ish interest points in my stacks- is there a magic number that works for you? I've been imaging to about 100µm depth,
how much would you recommend? Don't the stacks start getting too large to handle? </div>
<div class=""><br class="">
</div>
<div class="">Thanks for the tip on the camera noise! I think I'll try again tomorrow with brighter beads, no big beads and larger volumes - I'll let you know how it goes! </div>
<div class=""><br class="">
</div>
<div class="">Best wishes</div>
<div class="">Tania</div>
</div>
<div class="gmail_extra" style="font-family:Helvetica; font-size:12px; font-style:normal; font-weight:normal; letter-spacing:normal; orphans:auto; text-align:start; text-indent:0px; text-transform:none; white-space:normal; widows:auto; word-spacing:0px">
<br class="">
<div class="gmail_quote">On 24 August 2016 at 17:55, Anthony, Neil<span class="Apple-converted-space"> </span><span dir="ltr" class=""><<a href="mailto:nantho2@emory.edu" target="_blank" class="">nantho2@emory.edu</a>></span><span class="Apple-converted-space"> </span>wrote:<br class="">
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<div lang="EN-US" class="">
<div class="">
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif">Hi Tania,<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><br class="">
Can I please ask what the z voxel size is? I see in the test.xml that you have a voxel size of 0.235 in xy and z for id 0,1,2 in the ViewSetup tag. When I create a xml from scratch here, setting xy to 0.235 and z to 1.5 (the smallest on the standard Picard
4D stage) I see each id in the xml has a size of “0.235 0.235 1.5”.<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif">Stephan, I assume these are the voxel sizes used for the BDV. Would I be correct in assuming some more and saying that if only one value is seen it’s used for xy and
z?<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif">… interlude … etc. etc. lalalalala … coffee etc.<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif">I now see what the problem is. Well, what some of the problems might be.<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif">I did a manual transform around the y axis and manually overlapped the big bead. See attached. Is this how is should look? If I have it laid out correctly I think
you have a combination of not enough beads, combined with not enough overlap of physical volumes given the low number of beads. On top of that I feel the small bead signal is so low that it’s approaching the camera noise*.<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif">It is difficult as the big beads are so bright, and you might want to find a better sample to test with. I’ve done some testing on spheroids that are really bright,
and I had to swap my 100nm tetra spec beads for some really bright 1um green beads to be able to get good signal on the beads and not saturate the sample of interest.<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif">I started with just little beads to figure out the basics, and I take just sub-res bead data to get my PSF output from the decon too.<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif">Neil<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif">* inspect the data stacks with orthogonal view and you’ll see the vertical lines running down the stack. For this kind of noise you can run a few line scans across your
beads and noise to get an idea of the intensity difference and then use remove outliers<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><b class=""><span class="" style="font-size:11pt; font-family:Calibri,sans-serif">From:</span></b><span class="" style="font-size:11pt; font-family:Calibri,sans-serif"><span class="Apple-converted-space"> </span>OpenSPIM [mailto:<a href="mailto:openspim-bounces@openspim.org" target="_blank" class="">openspim-bounces@<wbr class="">openspim.org</a>]<span class="Apple-converted-space"> </span><b class="">On
Behalf Of<span class="Apple-converted-space"> </span></b>Tania Mendonca<br class="">
<b class="">Sent:</b><span class="Apple-converted-space"> </span>Friday, August 19, 2016 3:31 PM<br class="">
<b class="">To:</b><span class="Apple-converted-space"> </span><a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank" class="">Stephan.Preibisch@mdc-berlin.<wbr class="">de</a><br class="">
<b class="">Cc:</b><span class="Apple-converted-space"> </span><a href="mailto:openspim@openspim.org" target="_blank" class="">openspim@openspim.org</a><span class=""><br class="">
<b class="">Subject:</b><span class="Apple-converted-space"> </span>Re: [OpenSPIM] Multiview registration - no inliers<u class=""></u><u class=""></u></span></span></p>
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
<div class="">
<p class="MsoNormal">Hi<u class=""></u><u class=""></u></p>
<div class="">
<div class="h5">
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">Just tried it but it still doesn't work. <u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">Thanks<u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">Tania<u class=""></u><u class=""></u></p>
</div>
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<div class="">
<div class="h5">
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
<div class="">
<p class="MsoNormal">On 19 August 2016 at 20:13,<span class="Apple-converted-space"> </span><a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank" class="">Stephan.Preibisch@mdc-berlin.<wbr class="">de</a><span class="Apple-converted-space"> </span><<a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank" class="">Stephan.Preibisch@mdc-berlin.<wbr class="">de</a>>
wrote:<u class=""></u><u class=""></u></p>
<blockquote class="" style="border-style:none none none solid; border-left-color:rgb(204,204,204); border-left-width:1pt; padding:0in 0in 0in 6pt; margin:5pt 0in 5pt 4.8pt">
<div class="">
<div class="">
<p class="MsoNormal">Hi, I just think that in the beginning when you define your dataset the pixel sizes were wrong which potentially corrupts the downstream workflow. Try to start from the very beginning, and define a new dataset and manually put in the correct
calibration. This might just work ...<u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">All the best,<u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">Stephan <br class="">
<br class="">
Sent from my iPhone<u class=""></u><u class=""></u></p>
</div>
<div class="">
<div class="">
<div class="">
<p class="MsoNormal" style="margin-bottom:12pt"><br class="">
On Aug 19, 2016, at 21:00, Tania Mendonca <<a href="mailto:tvmendonca1@sheffield.ac.uk" target="_blank" class="">tvmendonca1@sheffield.ac.uk</a>> wrote:<u class=""></u><u class=""></u></p>
</div>
<blockquote class="" style="margin-top:5pt; margin-bottom:5pt">
<div class="">
<div class="">
<p class="MsoNormal">Hi<span class="Apple-converted-space"> </span><u class=""></u><u class=""></u></p>
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">I've just checked to make sure but yes I have corrected the voxel size on the image stacks and there shouldn't be a mistake in that. However, your reply gave me an idea and I did a quick test. I duplicated one of the images and ran the
multiview reconstruction plugin for the two duplicates. It worked, so it makes me think that there's nothing wrong with the files themselves and you might be right about the issue being with my voxel depth. I've referred to SVI's Nyquist calculator to workout
my step size, is there a recommended step size that people use?<u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">Best wishes<u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">Tania<u class=""></u><u class=""></u></p>
</div>
</div>
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
<div class="">
<p class="MsoNormal">On 19 August 2016 at 16:58,<span class="Apple-converted-space"> </span><a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank" class="">Stephan.Preibisch@mdc-berlin.<wbr class="">de</a><span class="Apple-converted-space"> </span><<a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank" class="">Stephan.Preibisch@mdc-berlin.<wbr class="">de</a>>
wrote:<u class=""></u><u class=""></u></p>
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<div class="">
<div class="">
<p class="MsoNormal">Hi, my first guess would be that your calibration (pixel size is x,y and z) is wrong ... Can you make sure this is correct when you define your dataset?<u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">All the best,<u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">Stephan <u class=""></u><u class=""></u></p>
</div>
<div class="">
<div class="">
<div class="">
<p class="MsoNormal" style="margin-bottom:12pt"><br class="">
On Aug 19, 2016, at 17:17, Tania Mendonca <<a href="mailto:tvmendonca1@sheffield.ac.uk" target="_blank" class="">tvmendonca1@sheffield.ac.uk</a>> wrote:<u class=""></u><u class=""></u></p>
</div>
<blockquote class="" style="margin-top:5pt; margin-bottom:5pt">
<div class="">
<div class="">
<p class="MsoNormal">Hi all<span class="Apple-converted-space"> </span><u class=""></u><u class=""></u></p>
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">Hope your microscopes are aligned and treating you well! I'm sorry if I've missed this conversation before but I was wondering if any of you have had similar problems as me with trying to get the Fiji multiview reconstruction plugin to
like my data. <u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">I've tried a range of microspheres - different colours, different sizes and different concentrations but it never seems to find enough correspondences between views. <span class="" style="font-size:9.5pt">I've been primarily experimenting
with interactively detecting interest points. I've tried the presets as well but with no luck. I've tried to systematically work through various combinations of sigma and threshold but always end up with 0 inliers. Registrations always terminates with the
following error: "There are no connected tiles, cannot do an optimization. Quitting."</span><u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
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<div class="">
<p class="MsoNormal">My home built system doesn't run on µManager and my images are acquired as .cxd files which I convert to .tifs before processing. I'm attaching some of my test data -<a href="https://drive.google.com/file/d/0B-he-_wcys_-aUtRZ0pidFhyUlk/view?usp=sharing" target="_blank" class="">https://drive.google.com/file/<wbr class="">d/0B-he-_wcys_-<wbr class="">aUtRZ0pidFhyUlk/view?usp=<wbr class="">sharing</a><u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">the sample here is 1µm yellow-green beads <span class="" style="font-size:9.5pt">(1:4000 dilution) and 10µm beads (1:50 dilution) with the idea of using the 1µm beads to reconstruct the 10µm beads. I use the same objectives as on the OpenSPIM
system (10X for illumination and 20X for detection), magnification on the system is 27.7X. The illumination here is 473nm and detection at 520nm.</span><u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
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<div class="">
<p class="MsoNormal"><span class="" style="font-size:9.5pt">A log file has been included in the zip file to give you an idea of what I've been trying. </span><u class=""></u><u class=""></u></p>
</div>
<div class="">
<div class="">
<p class="MsoNormal"><span class="" style="font-size:9.5pt"><u class=""></u> <u class=""></u></span></p>
</div>
<div class="">
<p class="MsoNormal"><span class="" style="font-size:9.5pt">I hope I'm just missing a trick somewhere. Any help would be greatly appreciated!<u class=""></u><u class=""></u></span></p>
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<div class="">
<p class="MsoNormal"><span class="" style="font-size:9.5pt"><u class=""></u> <u class=""></u></span></p>
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<p class="MsoNormal"><span class="" style="font-size:9.5pt">Best wishes<u class=""></u><u class=""></u></span></p>
</div>
<div class="">
<p class="MsoNormal"><span class="" style="font-size:9.5pt">Tania<u class=""></u><u class=""></u></span></p>
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<p class="MsoNormal">--<span class="Apple-converted-space"> </span><u class=""></u><u class=""></u></p>
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<div class="">
<p class="MsoNormal">-------------------------<span class="Apple-converted-space"> </span><u class=""></u><u class=""></u></p>
<div class="">
<p class="MsoNormal">Tania Mendonca<span class="Apple-converted-space"> </span><u class=""></u><u class=""></u></p>
<div class="">
<p class="MsoNormal">Doctoral Research Student<u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">University of Sheffield<u class=""></u><u class=""></u></p>
</div>
<div class="">
<p class="MsoNormal">S10 2TN<u class=""></u><u class=""></u></p>
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<p class="MsoNormal">-------------------------<u class=""></u><u class=""></u></p>
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<blockquote class="" style="margin-top:5pt; margin-bottom:5pt">
<div class="">
<p class="MsoNormal">______________________________<wbr class="">_________________<br class="">
OpenSPIM mailing list<br class="">
<a href="mailto:OpenSPIM@openspim.org" target="_blank" class="">OpenSPIM@openspim.org</a><br class="">
<a href="http://openspim.org/mailman/listinfo/openspim" target="_blank" class="">http://openspim.org/mailman/<wbr class="">listinfo/openspim</a><u class=""></u><u class=""></u></p>
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<p class="MsoNormal">--<span class="Apple-converted-space"> </span><u class=""></u><u class=""></u></p>
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<p class="MsoNormal">-------------------------<span class="Apple-converted-space"> </span><u class=""></u><u class=""></u></p>
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<p class="MsoNormal">Tania Mendonca<span class="Apple-converted-space"> </span><u class=""></u><u class=""></u></p>
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<p class="MsoNormal">Doctoral Research Student<u class=""></u><u class=""></u></p>
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<p class="MsoNormal">University of Sheffield<u class=""></u><u class=""></u></p>
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<p class="MsoNormal">S10 2TN<u class=""></u><u class=""></u></p>
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<p class="MsoNormal">Tania Mendonca<u class=""></u><u class=""></u></p>
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<p class="MsoNormal">Doctoral Research Student<u class=""></u><u class=""></u></p>
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<p class="MsoNormal">University of Sheffield<u class=""></u><u class=""></u></p>
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<div class="">Tania Mendonca
<div class="">Doctoral Research Student<br class="">
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<div class="">University of Sheffield<br class="">
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<div class="">S10 2TN<br class="">
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