[OpenSPIM] 3D Reconstruction

Tue Nov 10 10:57:21 CST 2015

Hi Pete,

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.

Kind regards,

Dylan

From: Peter Gabriel Pitrone [mailto:pitrone at mpi-cbg.de]
Sent: 10 November 2015 17:41
To: Windell, Dylan <dw388 at exeter.ac.uk>
Cc: Pavel Tomancak <tomancak at mpi-cbg.de>; openspim at openspim.org
Subject: Re: [OpenSPIM] 3D Reconstruction

Hello Dylan,

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…

Best Regards,
Pete

Peter Gabriel Pitrone - DipRMS TechRMS FRMS
Light Sheet Fluorescence Microscopist and Imaging Specialist
for Dr. Pavel Tomancak's research group at the
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstraße 108
01307 Dresden, Saxony
Germany

http://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html &
http://www.openspim.org

"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

On Nov 10, 2015, at 5:20 PM, Windell, Dylan <dw388 at exeter.ac.uk<mailto:dw388 at exeter.ac.uk>> wrote:

Dear Pavel,

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:

http://1drv.ms/1M426qo

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.

Let me know if you need any more information.

Thanks again,

Dylan

From: Pavel Tomancak [mailto:tomancak at mpi-cbg.de]
Sent: 10 November 2015 13:33
To: Windell, Dylan
Cc: openspim at openspim.org<mailto:openspim at openspim.org>
Subject: Re: [OpenSPIM] 3D Reconstruction

Dear Dylan,

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.

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.

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.

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?

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.

I will help more after you send the logs or even deposit the data somewhere.

All the best

PAvel

If anyone has had difficulty in this or have done work on Zebrafish with SPIM I would really appreciate any help.

Kind regards,

Dylan

Dylan Windell
Postgraduate Researcher
College<http://business-school.exeter.ac.uk/research/areas/centres/isr/> of Life and Environmental Sciences
University of Exeter, Geoffrey Pope
Stocker Road, Exeter EX4 4QD, UK
phone: +44 (0) 7596725056
email: dw388 at exeter.ac.uk<mailto:dw388 at exeter.ac.uk>

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