Zerene: How to stop the rotation of the stable subject

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Adalbert
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Zerene: How to stop the rotation of the stable subject

Post by Adalbert »

Hello everybody,

Does anyone know how to stop the rotation of the subject on the stack?
Originally I tried stacking in one pass, but there were too many artifacts.

So then I tried the sub stacks.
First I aligned the images and then created the sub-stacks.
- Number of single shots =501
- Size of the sub-stack=15, Overlap=5 ; Number of sub-stacks=50, PMax

This is what the single shots look like:
single_shots_1024.jpg
And the sub-stacks look like that:
sub-stacks_1024.jpg
BTW, the subject is a slide with a cover glass; magnification 100x.
The photo subject is not rotated on the single shots,
but the dots on the cover glass move.

Best, ADi

rjlittlefield
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Re: Zerene: How to stop the rotation of the stable subject

Post by rjlittlefield »

Go to Options > Preferences > Alignment and remove the checkmark on Rotation.

For this level of magnification you should also remove the checkmark on Scale.

If your illumination is stable, remove the checkmark on Brightness.

If your rig is stable enough, then remove the checkmarks on Shift X and Shift Y also.

In general, it is better to remove checkmarks on all aspects that are physically stabilized by the apparatus. This prevents the alignment algorithm from being misled by how the size and shape of things appear to change as they go in and out of focus.

--Rik

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Adalbert
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Re: Zerene: How to stop the rotation of the stable subject

Post by Adalbert »

Hello Rik,

Thanks for the hint!
The next question is: to rotate or not to rotate.

With the alignment/rotation enabled, the result looks like this:
alingment_on.jpg

And with the alignment/rotation disabled, the result looks like that:
alignment_off.jpg

I have tried to create a usable stack, but it has been very difficult:
Hypostom_100x_300px.jpg
Best,
ADi

rjlittlefield
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Re: Zerene: How to stop the rotation of the stable subject

Post by rjlittlefield »

You mentioned that "the dots on the cover glass move". I can see in the top two images of your last post that for sure all that extra moving content would wreak havoc with keeping the subject aligned.

But I am puzzled by what is physically going on in this case. Exactly what are those dots, and why do they move?

--Rik

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Adalbert
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Re: Zerene: How to stop the rotation of the stable subject

Post by Adalbert »

Hello Rik
the dots on the cover glass move
That's just my guess, I don't have any hard evidence.
But I couldn't explain it any other way .

It's quite funny if you scroll through the pictures quickly,
you can see the stationary hypostome and the passing dots.

Best,
ADi

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Re: Zerene: How to stop the rotation of the stable subject

Post by rjlittlefield »

I have seen effects like this with bubbles in fluid. Were you looking through any fluid, or was everything solid?

--Rik

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Adalbert
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Re: Zerene: How to stop the rotation of the stable subject

Post by Adalbert »

Hi Rik,
Preparation in Euparal, but no immersion oil; Nikon E Plan 100x/1,25
ADi

rjlittlefield
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Re: Zerene: How to stop the rotation of the stable subject

Post by rjlittlefield »

And the Euparal had hardened?

If so then this is a huge mystery!

Is the problem repeatable? Can you see it through the eyepieces (and distinguish it from floaters in your own eyes)?

--Rik

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Adalbert
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Re: Zerene: How to stop the rotation of the stable subject

Post by Adalbert »

Hi Rik,
And the Euparal had hardened?
no, and that is also the explanation :-)

This is a fresh and very thick preparation of a whole tick.
To stack the whole hypostome, the lens has pressed on the cover glass
so that the air bubbles in the euparal have shifted.

Best,
ADi

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Re: Zerene: How to stop the rotation of the stable subject

Post by rjlittlefield »

Excellent -- the mystery is solved!

--Rik

MarkSturtevant
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Re: Zerene: How to stop the rotation of the stable subject

Post by MarkSturtevant »

I wonder if there is a way to de-gas the mountant to remove air bubbles before it hardens. OR de-gas the mountant before its applied to the specimen?
I know putting the slides on a slide warmer accelerates the hardening, and this also helps to shrink bubbles but maybe not eliminate them.
Mark Sturtevant
Dept. of Still Waters

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