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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">Hi Tania,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><br>
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”.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;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?<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">… interlude … etc. etc. lalalalala … coffee etc.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">I now see what the problem is. Well, what some of the problems might be.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;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*.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;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.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;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.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">Neil<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;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
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><b><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">From:</span></b><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"> OpenSPIM [mailto:openspim-bounces@openspim.org]
<b>On Behalf Of </b>Tania Mendonca<br>
<b>Sent:</b> Friday, August 19, 2016 3:31 PM<br>
<b>To:</b> Stephan.Preibisch@mdc-berlin.de<br>
<b>Cc:</b> openspim@openspim.org<br>
<b>Subject:</b> Re: [OpenSPIM] Multiview registration - no inliers<o:p></o:p></span></p>
<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Hi<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Just tried it but it still doesn't work. <o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Thanks<o:p></o:p></p>
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<p class="MsoNormal">Tania<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">On 19 August 2016 at 20:13, <a href="mailto:Stephan.Preibisch@mdc-berlin.de">
Stephan.Preibisch@mdc-berlin.de</a> <<a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank">Stephan.Preibisch@mdc-berlin.de</a>> wrote:<o:p></o:p></p>
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<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 ...<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">Stephan <br>
<br>
Sent from my iPhone<o:p></o:p></p>
</div>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><br>
On Aug 19, 2016, at 21:00, Tania Mendonca <<a href="mailto:tvmendonca1@sheffield.ac.uk" target="_blank">tvmendonca1@sheffield.ac.uk</a>> wrote:<o:p></o:p></p>
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<p class="MsoNormal">Hi <o:p></o:p></p>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
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<div>
<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?<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Best wishes<o:p></o:p></p>
</div>
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<p class="MsoNormal">Tania<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">On 19 August 2016 at 16:58, <a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank">
Stephan.Preibisch@mdc-berlin.de</a> <<a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank">Stephan.Preibisch@mdc-berlin.de</a>> wrote:<o:p></o:p></p>
<blockquote style="border:none;border-left:solid #CCCCCC 1.0pt;padding:0in 0in 0in 6.0pt;margin-left:4.8pt;margin-top:5.0pt;margin-right:0in;margin-bottom:5.0pt">
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<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?<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>
</div>
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<p class="MsoNormal">Stephan <o:p></o:p></p>
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<div>
<div>
<p class="MsoNormal" style="margin-bottom:12.0pt"><br>
On Aug 19, 2016, at 17:17, Tania Mendonca <<a href="mailto:tvmendonca1@sheffield.ac.uk" target="_blank">tvmendonca1@sheffield.ac.uk</a>> wrote:<o:p></o:p></p>
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<p class="MsoNormal">Hi all <o:p></o:p></p>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<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. <o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<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 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><o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<div>
<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">
https://drive.google.com/file/d/0B-he-_wcys_-aUtRZ0pidFhyUlk/view?usp=sharing</a><o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">the sample here is 1µm yellow-green beads <span 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><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:9.5pt">A log file has been included in the zip file to give you an idea of what I've been trying. </span><o:p></o:p></p>
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<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
</div>
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<p class="MsoNormal"><span style="font-size:9.5pt">I hope I'm just missing a trick somewhere. Any help would be greatly appreciated!<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
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<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Best wishes<o:p></o:p></span></p>
</div>
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<p class="MsoNormal"><span style="font-size:9.5pt">Tania<o:p></o:p></span></p>
</div>
<p class="MsoNormal">-- <o:p></o:p></p>
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<div>
<p class="MsoNormal">------------------------- <o:p></o:p></p>
<div>
<p class="MsoNormal">Tania Mendonca <o:p></o:p></p>
<div>
<p class="MsoNormal">Doctoral Research Student<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">University of Sheffield<o:p></o:p></p>
</div>
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<p class="MsoNormal">S10 2TN<o:p></o:p></p>
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<p class="MsoNormal">-------------------------<o:p></o:p></p>
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<p class="MsoNormal">_______________________________________________<br>
OpenSPIM mailing list<br>
<a href="mailto:OpenSPIM@openspim.org" target="_blank">OpenSPIM@openspim.org</a><br>
<a href="http://openspim.org/mailman/listinfo/openspim" target="_blank">http://openspim.org/mailman/listinfo/openspim</a><o:p></o:p></p>
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<p class="MsoNormal"><br>
<br clear="all">
<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">-- <o:p></o:p></p>
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<div>
<p class="MsoNormal">------------------------- <o:p></o:p></p>
<div>
<p class="MsoNormal">Tania Mendonca <o:p></o:p></p>
<div>
<p class="MsoNormal">Doctoral Research Student<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">University of Sheffield<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">S10 2TN<o:p></o:p></p>
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</div>
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<p class="MsoNormal">-------------------------<o:p></o:p></p>
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<p class="MsoNormal"><br>
<br clear="all">
<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">-- <o:p></o:p></p>
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<p class="MsoNormal">-------------------------<o:p></o:p></p>
<div>
<p class="MsoNormal">Tania Mendonca<o:p></o:p></p>
<div>
<p class="MsoNormal">Doctoral Research Student<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">University of Sheffield<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">S10 2TN<o:p></o:p></p>
</div>
</div>
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<p class="MsoNormal">-------------------------<o:p></o:p></p>
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