[OpenSPIM] Rotation Axis

[Anthony, Neil]

Hi Jan, thanks for the detailed response.

I’ll look forward to taking my first data set soon and I’m sure they’ll be more posts from me to keep people updated :)

And thanks for the Bassi reference.  I like their little xy translation mount.

Neil


From: Jan Huisken [mailto:huisken at mpi-cbg.de]
Sent: Tuesday, February 23, 2016 3:11 AM
To: Anthony, Neil
Cc: openspim at openspim.org
Subject: Re: [OpenSPIM] Rotation Axis

Hi Neil

here are a few thoughts:

- The rotational axis can be determined very precisely by mounting a cylinder of fluorescent beads, switching on the laser and spinning the sample. You will see a “snow storm” of beads. You keep on moving your sample in x and z until you see a line of beads that do not move. This is your rotational axis. Above this line you will see beads coming towards the lens, below away (or the other way around). You position this line of beads in your FOV’s center and call this stage position (0,y,0).

- Once you have determined the rotational axis you can find and center your sample in the FOV. (The sample will not necessarily be on the rational axis.) When your sample is in the FOV the stage position tells you how far away from the rational axis you are. Rotating now by an angle theta should go along with a translation along x and z that you can easily calculate with Sin(theta) and Cos(Theta) and the distance from the axis. The sample will now rotate around the FOV’s center. (I do not know how the openSPIM software handles this.)

- You can also build a little translational stage that allows you to reposition the sample with respect to the rotational axis (very important for OPT, useful for SPIM, see Bassi et al, Development 2015). This way you can always position the sample precisely on the rotational axis.

- The rotational axis is never perfectly vertical (parallel to y). Generally this is not a big deal. If you want to do MV imaging you anyway need to register the data sets.

- Ideally you should have stages that are precise and reliable, i.e. they find the same position even after a few hours. Then you can image a bunch of beads from different directions at the beginning of your experiment, register the beads (Preibisch et al.) and you know once and forever how to register data from these directions. No need to embed ugly beads with your sample. Again, this assumes that you have a nice set of motors.

- It is important to understand that the registration of data is independent of the actual sample’s position. It does not matter if the FEP tube is bent, if the sample is at an angle or off-axis. The transformations necessary to register the data are solely determined by how (well) you have assembled the (hopefully precise) motors.

Hope this helps.

Jan


—
Jan Huisken
Head of Max Planck Research Group

Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)
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www.mpi-cbg.de/huisken<http://www.mpi-cbg.de/huisken>
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+49 351 2102487





On Feb 23, 2016, at 3:39 AM, Anthony, Neil <nantho2 at emory.edu<mailto:nantho2 at emory.edu>> wrote:

Hi all, I hope the scopes are treating you well.

I have a general question for all.  I haven't yet taken any data (almost everything is in place and nearly ready to roll), but I'm curious to know about the axis of rotation when rotating the sample to different views.

Maybe I'm over thinking this, or the effects of being off axis are smaller than I imagine, but I'm really curious to know how people are experiencing and counteracting this part of the acquisition.  In a perfect world my theta would rotate around an axis parallel to the y axis and the sample would be placed on the rotation axis.  I could then translate the axis into the center of my camera and z-stack and rotate from there.  In reality, I'm guessing here as you know, you'd have an axis of rotation that's tilted to some degree, and the sample would likely be off center.  This would lead to samples moving in an arc when rotating, and xyz translation required to bring the region of interest back into view on each rotation.

Any experiences or thoughts in this direction are much appreciated.

Thanks in advance for your time.

Neil



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