[OpenSPIM] 3D Reconstruction

[Pavel Tomancak]

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 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
>  
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