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<div>Great! I will be there on Wednesday giving a course on Multiview reconstruction...hope to see you around!</div>
<div id="AppleMailSignature"><br>
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<div id="AppleMailSignature">All the best,</div>
<div id="AppleMailSignature">Stephan <br>
<br>
Sent from my iPhone</div>
<div><br>
On Aug 29, 2016, at 16:29, Tania Mendonca <<a href="mailto:tvmendonca1@sheffield.ac.uk">tvmendonca1@sheffield.ac.uk</a>> wrote:<br>
<br>
</div>
<blockquote type="cite">
<div>
<div dir="ltr">Hi Neil and Stephan
<div><br>
</div>
<div>Thanks for the tips and all the help! I tried again with a higher concentration of the 1µm beads, got rid of the bright big beads and increased the z acquisition distance to increase the region of overlap and was finally successful!</div>
<div><br>
</div>
<div>I hope to meet many of you OpenSPIMmers in Sheffield this week! </div>
<div><br>
</div>
<div>Best wishes</div>
<div>Tania</div>
</div>
<div class="gmail_extra"><br>
<div class="gmail_quote">On 25 August 2016 at 15:08, Anthony, Neil <span dir="ltr">
<<a href="mailto:nantho2@emory.edu" target="_blank">nantho2@emory.edu</a>></span> wrote:<br>
<blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">
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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>
</div>
<div><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>
<a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank">Stephan.Preibisch@mdc-berlin.<wbr>de</a><br>
<b>Sent:</b> Aug 25, 2016 3:41 AM<br>
<b>To:</b> Tania Mendonca<br>
<b>Cc:</b> Anthony, Neil; <a href="mailto:openspim@openspim.org" target="_blank">
openspim@openspim.org</a>
<div>
<div class="h5"><br>
<b>Subject:</b> Re: [OpenSPIM] Multiview registration - no inliers<br>
</div>
</div>
</span></div>
</div>
<div>
<div class="h5"><br type="attribution">
<div>Hi Tania,
<div><br>
</div>
<div>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><br>
</div>
<div>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><br>
</div>
<div>Good luck,</div>
<div>Stephan<br>
<div>
<div style="color:rgb(0,0,0);letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;word-wrap:break-word">
<div style="color:rgb(0,0,0);letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;word-wrap:break-word">
<div>---</div>
<div><br>
</div>
<div>Dr. Stephan Preibisch</div>
<div>Group Leader</div>
<div><br>
</div>
<div>Berlin Institute of Medical Systems Biology of the MDC</div>
<div>Building 89, 1.08b</div>
<div><br>
</div>
<div>email:<span> </span><a href="mailto:stephan.preibisch@mdc-berlin.de" target="_blank">stephan.preibisch@mdc-<wbr>berlin.de</a></div>
<div>web: <a href="http://fly.mpi-cbg.de/preibisch" target="_blank">http://www.preibisch.net/</a></div>
</div>
</div>
</div>
<br>
<div>
<blockquote type="cite">
<div>On 24 Aug 2016, at 19:49, Tania Mendonca <<a href="mailto:tvmendonca1@sheffield.ac.uk" target="_blank">tvmendonca1@sheffield.ac.uk</a>> wrote:</div>
<br>
<div>
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Hi Neil
<div><br>
</div>
<div>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><br>
</div>
<div>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><br>
</div>
<div>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><br>
</div>
<div>Best wishes</div>
<div>Tania</div>
</div>
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<br>
<div class="gmail_quote">On 24 August 2016 at 17:55, Anthony, Neil<span> </span><span dir="ltr"><<a href="mailto:nantho2@emory.edu" target="_blank">nantho2@emory.edu</a>></span><span> </span>wrote<wbr>:<br>
<blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left-width:1px;border-left-color:rgb(204,204,204);border-left-style:solid;padding-left:1ex">
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<div>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif">Hi Tania,<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;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”.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span 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></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif">… interlude … etc. etc. lalalalala … coffee etc.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span 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></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span 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></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span 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></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span 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></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif">Neil<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span 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></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><b><span style="font-size:11pt;font-family:Calibri,sans-serif">From:</span></b><span style="font-size:11pt;font-family:Calibri,sans-serif"><span> </span>OpenSPIM [mailto:<a href="mailto:openspim-bounces@openspim.org" target="_blank">openspim-bounces@opens<wbr>pim.org</a>]<span> </span><b>On
Behalf Of<span> </span></b>Tania Mendonca<br>
<b>Sent:</b><span> </span>Friday, August 19, 2016 3:31 PM<br>
<b>To:</b><span> </span><a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank">Stephan.Preibisch@mdc-<wbr>berlin.de</a><br>
<b>Cc:</b><span> </span><a href="mailto:openspim@openspim.org" target="_blank">openspim@openspim.org</a><span><br>
<b>Subject:</b><span> </span>Re: [OpenSPIM] Multiview registration - no inliers<u></u><u></u></span></span></p>
<p class="MsoNormal"><u></u> <u></u></p>
<div>
<p class="MsoNormal">Hi<u></u><u></u></p>
<div>
<div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<p class="MsoNormal">Just tried it but it still doesn't work. <u></u><u></u></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<p class="MsoNormal">Thanks<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">Tania<u></u><u></u></p>
</div>
</div>
</div>
</div>
<div>
<div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
<div>
<p class="MsoNormal">On 19 August 2016 at 20:13,<span> </span><a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank">Stephan.Preibisch@mdc-<wbr>berlin.de</a><span> </span><<a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank">Stephan.Preibisch@<wbr>mdc-berlin.de</a>>
wrote:<u></u><u></u></p>
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<div>
<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></u><u></u></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<p class="MsoNormal">All the best,<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">Stephan <br>
<br>
Sent from my iPhone<u></u><u></u></p>
</div>
<div>
<div>
<div>
<p class="MsoNormal" style="margin-bottom:12pt"><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:<u></u><u></u></p>
</div>
<blockquote style="margin-top:5pt;margin-bottom:5pt">
<div>
<div>
<p class="MsoNormal">Hi<span> </span><u></u><u></u></p>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<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?<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<p class="MsoNormal">Best wishes<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">Tania<u></u><u></u></p>
</div>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
<div>
<p class="MsoNormal">On 19 August 2016 at 16:58,<span> </span><a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank">Stephan.Preibisch@mdc-<wbr>berlin.de</a><span> </span><<a href="mailto:Stephan.Preibisch@mdc-berlin.de" target="_blank">Stephan.Preibisch@<wbr>mdc-berlin.de</a>>
wrote:<u></u><u></u></p>
<blockquote 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>
<div>
<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></u><u></u></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<p class="MsoNormal">All the best,<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">Stephan <u></u><u></u></p>
</div>
<div>
<div>
<div>
<p class="MsoNormal" style="margin-bottom:12pt"><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:<u></u><u></u></p>
</div>
<blockquote style="margin-top:5pt;margin-bottom:5pt">
<div>
<div>
<p class="MsoNormal">Hi all<span> </span><u></u><u></u></p>
<div>
<p class="MsoNormal"><u></u> <u></u></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. <u></u><u></u></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></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><u></u><u></u></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<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/<wbr>file/d/0B-he-_wcys_-aUtRZ0pidF<wbr>hyUlk/view?usp=sharing</a><u></u><u></u></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><u></u><u></u></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<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><u></u><u></u></p>
</div>
<div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><u></u> <u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">I hope I'm just missing a trick somewhere. Any help would be greatly appreciated!<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><u></u> <u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Best wishes<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Tania<u></u><u></u></span></p>
</div>
<p class="MsoNormal">--<span> </span><u></u><u></u></p>
<div>
<div>
<p class="MsoNormal">-------------------------<span> </span><u></u><u></u></p>
<div>
<p class="MsoNormal">Tania Mendonca<span> </span><u></u><u></u></p>
<div>
<p class="MsoNormal">Doctoral Research Student<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">University of Sheffield<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">S10 2TN<u></u><u></u></p>
</div>
</div>
<div>
<p class="MsoNormal">-------------------------<u></u><u></u></p>
</div>
</div>
</div>
</div>
</div>
</div>
</blockquote>
</div>
</div>
<blockquote style="margin-top:5pt;margin-bottom:5pt">
<div>
<p class="MsoNormal">______________________________<wbr>_________________<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/li<wbr>stinfo/openspim</a><u></u><u></u></p>
</div>
</blockquote>
</div>
</blockquote>
</div>
<p class="MsoNormal"><br>
<br clear="all">
<u></u><u></u></p>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<p class="MsoNormal">--<span> </span><u></u><u></u></p>
<div>
<div>
<p class="MsoNormal">-------------------------<span> </span><u></u><u></u></p>
<div>
<p class="MsoNormal">Tania Mendonca<span> </span><u></u><u></u></p>
<div>
<p class="MsoNormal">Doctoral Research Student<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">University of Sheffield<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">S10 2TN<u></u><u></u></p>
</div>
</div>
<div>
<p class="MsoNormal">-------------------------<u></u><u></u></p>
</div>
</div>
</div>
</div>
</div>
</blockquote>
</div>
</div>
</div>
</blockquote>
</div>
<p class="MsoNormal"><br>
<br clear="all">
<u></u><u></u></p>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<p class="MsoNormal">--<span> </span><u></u><u></u></p>
<div>
<div>
<p class="MsoNormal">-------------------------<u></u><u></u></p>
<div>
<p class="MsoNormal">Tania Mendonca<u></u><u></u></p>
<div>
<p class="MsoNormal">Doctoral Research Student<u></u><u></u></p>
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<div>
<p class="MsoNormal">University of Sheffield<u></u><u></u></p>
</div>
<div>
<p class="MsoNormal">S10 2TN<u></u><u></u></p>
</div>
</div>
<div>
<p class="MsoNormal">-------------------------<u></u><u></u></p>
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<br>
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--<span> </span><br>
<div>
<div dir="ltr">-------------------------
<div>Tania Mendonca
<div>Doctoral Research Student<br>
</div>
<div>University of Sheffield<br>
</div>
<div>S10 2TN<br>
</div>
</div>
<div>-------------------------</div>
</div>
</div>
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-- <br>
<div class="gmail_signature" data-smartmail="gmail_signature">
<div dir="ltr">-------------------------
<div>Tania Mendonca
<div>Doctoral Research Student<br>
</div>
<div>University of Sheffield<br>
</div>
<div>S10 2TN<br>
</div>
</div>
<div>-------------------------<br>
</div>
</div>
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