[OpenSPIM] Light Sheet Alignment Procedure (Was: 3D Reconstruction)

Tue Nov 17 06:28:51 CST 2015

Hi Pete, thanks for the info on the alignment.

Can I ask how the alignment of the cylindrical lens might affect things when you place it back in the path? You slide it to create the best focus on the conjugate mirror(?), but how sensitive are things to it being off axis, not quite perpendicular, or slightly rotated? Also, can you view these things via the camera using the scattered light too?

I apologise if these are silly questions; I've not aligned the system yet, just started bolting it down.

Thanks for your time
Neil

From: Peter Gabriel Pitrone <pitrone at mpi-cbg.de>
Sent: Nov 11, 2015 10:45 AM
To: Windell, Dylan
Cc: openspim at openspim.org
Subject: Re: [OpenSPIM] Light Sheet Alignment Procedure (Was: 3D Reconstruction)

Howdy Dylan,

Go ahead and use an ND filter, it shouldn’t effect the process much at all… Sometimes I use them as well when the signal is too intense, but one can’t see any beads when it is in place.

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 11, 2015, at 4:32 PM, Windell, Dylan <dw388 at exeter.ac.uk<mailto:dw388 at exeter.ac.uk>> wrote:

Hey Pete,

Thanks for the instructions, I will try them out ASAP. I am guessing you meant replacing the cylindrical lens after the alignment?

My laser cannot be attenuated that low but if I were to use an ND filter, would that affect the alignment process?

Kind regards,

Dylan

From: Peter Gabriel Pitrone [mailto:pitrone at mpi-cbg.de]
Sent: 11 November 2015 15:22
To: Windell, Dylan
Cc: openspim at openspim.org<mailto:openspim at openspim.org>
Subject: Re: [OpenSPIM] Light Sheet Alignment Procedure (Was: 3D Reconstruction)

Of course you need to replace the Cylindrical lens to SPIMage again… Sent that last one too fast.
On Nov 11, 2015, at 4:14 PM, Peter Gabriel Pitrone <pitrone at mpi-cbg.de<mailto:pitrone at mpi-cbg.de>> wrote:

Hey Dylan,

I’ve started using a different technique for alignment that doesn’t require any thing at all (except for the media) in the sample chamber. I’ll describe it in short below:

- Either take out the sample, or move it well beyond the focal plane (far away from the detection objective)

- Take out all the filters blocking the laser from the camera (make sure that the laser is modulated way down to around ±1 mW)

- Take out the cylindrical lens(es)

- Go “Live” at a short exposure time (10-25 ms)

You should see an in-focus beam in the middle of the field of view that cuts across the image, and it should slightly converge and then diverge with the narrowest point in the middle of the field of view. Now you can adjust the conjugate plane (corner) mirror horizontally to correct any misalignment in terms of focus, and vertically to place it in the center across the field. The two lens telescope between the mirror and the illumination lens can be adjusted along the rail so the narrowest part of the beam is centered in the exact middle.

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<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 5:57 PM, Windell, Dylan <dw388 at exeter.ac.uk<mailto:dw388 at exeter.ac.uk>> wrote:

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<mailto:dw388 at exeter.ac.uk>>
Cc: Pavel Tomancak <tomancak at mpi-cbg.de<mailto:tomancak at mpi-cbg.de>>; openspim at openspim.org<mailto: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<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>

_______________________________________________
OpenSPIM mailing list
OpenSPIM at openspim.org<mailto:OpenSPIM at openspim.org>
http://openspim.org/mailman/listinfo/openspim
<5um_140exp_3_angles.txt><3um_3_angles.txt>_______________________________________________
OpenSPIM mailing list
OpenSPIM at openspim.org<mailto:OpenSPIM at openspim.org>
http://openspim.org/mailman/listinfo/openspim

_______________________________________________
OpenSPIM mailing list
OpenSPIM at openspim.org<mailto:OpenSPIM at openspim.org>
http://openspim.org/mailman/listinfo/openspim

________________________________

This e-mail message (including any attachments) is for the sole use of
the intended recipient(s) and may contain confidential and privileged
information. If the reader of this message is not the intended
recipient, you are hereby notified that any dissemination, distribution
or copying of this message (including any attachments) is strictly
prohibited.

If you have received this message in error, please contact
the sender by reply e-mail message and destroy all copies of the
original message (including attachments).
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://openspim.org/pipermail/openspim/attachments/20151117/9b132f93/attachment-0002.html>