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(Forgot to CC the list, sorry!)<br>
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<th align="RIGHT" nowrap="nowrap" valign="BASELINE">Subject:
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<td>Re: [OpenSPIM] Off Center Rotation & Calibration
Tools in Open SPIM Software</td>
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<th align="RIGHT" nowrap="nowrap" valign="BASELINE">Date: </th>
<td>Wed, 12 Mar 2014 02:06:59 -0500</td>
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<th align="RIGHT" nowrap="nowrap" valign="BASELINE">From: </th>
<td>Luke Stuyvenberg <a class="moz-txt-link-rfc2396E" href="mailto:stuyvenberg@wisc.edu"><stuyvenberg@wisc.edu></a></td>
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<th align="RIGHT" nowrap="nowrap" valign="BASELINE">To: </th>
<td>LIM Soon Yew John (IMB)
<a class="moz-txt-link-rfc2396E" href="mailto:john.lim@imb.a-star.edu.sg"><john.lim@imb.a-star.edu.sg></a></td>
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<pre>Hi John,
Solving this problem is precisely the goal of the automatic rotational
axis calibration tool (opened by clicking 'Calibrate' on the stage
control tab).
I'm afraid it's poorly documented at the moment -- a failure on my part.
I believe we have a ticket out to provide more documentation (or if we
don't, we soon will).
Here's a rough guide that should get you started:
- Set the rotation stage to a low value -- -90 degrees or so.
- Find a nice, bright bead in the live view, then open the calibrator.
- Use FIJI's rectangle ROI tool to select a region around that bead. The
region should be as large as possible without including other/brighter
beads.
- Click 'Obtain Next' to begin the axis detection. (This button will
change to 'Interrupt' while the software is processing.) The system will
scan through Z to localize the bead, then rotate the stage by one step
and repeat, building a list of bead positions.
- After about 90 degrees (though, the more locations it has, the
better!), you can stop it with 'Interrupt', then click 'Recalculate' to
determine the rotation axis location and direction.
This is an optimistic guide, however; in my experience, the calibrator
sometimes has trouble tracking a bead -- or a bead that looked bright
quickly becomes dim and difficult to distinguish. The 'Scan Tweaks'
button opens an advanced panel for controlling the tracking process:
first/second delta control the Z scan size, X/Y determination is for
localizing in the image plane, and so on. In particular, changing X/Y
determination to 'Max Intens.' often produces better results than
'Gaussian (Beads)'.
Once the axis is detected, the calibration window can be closed or
minimized. At this point, rotations with the mouse (i.e. holding Alt and
dragging the mouse back and forth) will attempt to rotate around the
detected axis. (I should note that the rotation slider does *not*
implement this calculation at the moment! Only the mouse controls are
properly affected.)
I'm sorry I can't be more helpful presently; I'll check on that
documentation ticket/create it if necessary, to make sure this feature
is documented more thoroughly.
Luke
On 3/10/2014 11:08 PM, LIM Soon Yew John (IMB) wrote:
> Dear All,
>
> I had a question regarding how to deal with off center rotation of the sample? And can this issue be deal with using the calibration tools in the Open SPIM software and is there instructions available on how to use the calibration tools in the software?
>
> Any help will be greatly appreciated,
> John
> ________________________________________
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