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How to calculate the step-size for the stacking

 
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Adalbert



Joined: 30 Nov 2015
Posts: 727

PostPosted: Thu Feb 25, 2016 3:51 am    Post subject: How to calculate the step-size for the stacking Reply with quote

Hello everyone,
Does anybody know any rule for the calculation of the size of the steps for the focus-stacking?
Especially for the microphotography with an ocular and a relay-lens.
BTW, for the macrophotography (without any ocular) I am using the rule based on NA
550nm/(NA*NA)) / 2
Thank you in advance.
BR, Adi
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zzffnn



Joined: 22 May 2014
Posts: 1818
Location: Texas USA

PostPosted: Thu Feb 25, 2016 5:49 am    Post subject: Reply with quote

I would like to know as well, thank you very much!
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JH



Joined: 09 Mar 2013
Posts: 1216
Location: Vallentuna, Stockholm, Sweden

PostPosted: Thu Feb 25, 2016 11:10 am    Post subject: Reply with quote

I use the formula from the article "Depth-of-Focus in Microscopy" written by I.T. Young, R. Zagers, L.J. van Vliet, J. Mullikin, F. Boddeke, H. Netten

https://www.researchgate.net/publication/27352734_Depth-of-Focus_in_Microscopy

To get the two sided depth I just multiply the formulas with the factor 2, hence the "lamda/(NA^2)"

If I get the math right the "simple" lamda/(2*NA^2) formula is totaly OK in air until NA goes up to 0,5.

Above that you have to do some new calculations - or stop doubling the "simple" formula.

I do not change the step size with different relay lenses.

Regads

Jörgen
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Adalbert



Joined: 30 Nov 2015
Posts: 727

PostPosted: Fri Feb 26, 2016 5:29 am    Post subject: Reply with quote

Hello Jörgen,
Quote:
“I do not change the step size with different relay lenses.”

Are you using the same rule for the calculation of the step-size for the photos with an ocular and without any ocular?
BR, Adi
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rjlittlefield
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Joined: 01 Aug 2006
Posts: 20029
Location: Richland, Washington State, USA

PostPosted: Fri Feb 26, 2016 12:46 pm    Post subject: Reply with quote

The optical DOF, such as 1/4-lambda wavefront error, depends only on the NA of the objective.

Changes to other lenses in the system will not alter the optical DOF, unless you introduce some sort of small aperture that further stops down the whole system.

The only other way that DOF will change is if you reduce the magnification or display size far enough that what the viewer sees is limited by sensor or display resolution, instead of optical resolution.

--Rik
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pulsar123



Joined: 12 Jun 2015
Posts: 130

PostPosted: Fri Feb 26, 2016 12:58 pm    Post subject: Reply with quote

For this I simply use online macro calculators:

http://coinimaging.com/calculator.html

http://extreme-macro.co.uk/calculators/

http://www.cambridgeincolour.com/tutorials/macro-lenses.htm
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Adalbert



Joined: 30 Nov 2015
Posts: 727

PostPosted: Fri Feb 26, 2016 1:12 pm    Post subject: Reply with quote

Hello Rik,
Can the DOF be affected by the manipulation of the iris of my relay-lens (Helios 44M) ?

BR, Adi
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Adalbert



Joined: 30 Nov 2015
Posts: 727

PostPosted: Fri Feb 26, 2016 1:22 pm    Post subject: Reply with quote

Hello pulsar123,
I am looking for the rule because I would like to implement it to my self-made macro-rail.
At the moment the NA is entered and the step –size is calculated by the program.
BR, Adi
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rjlittlefield
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Joined: 01 Aug 2006
Posts: 20029
Location: Richland, Washington State, USA

PostPosted: Fri Feb 26, 2016 1:30 pm    Post subject: Reply with quote

Adalbert wrote:
Can the DOF be affected by the manipulation of the iris of my relay-lens (Helios 44M) ?

There is no simple yes/no answer.

Stopping down the relay lens can increase DOF but it can also introduce vignetting.

Whether you can stop down far enough to increase DOF, before you also get too much vignetting, depends on details of the relay lens and how you have the objective mounted.

So, you would have to test to be sure.

In any case, remember that getting more DOF by stopping down also introduces more diffraction. Stopping down anywhere in the system is equivalent to using an objective with smaller NA.

--Rik
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René



Joined: 08 Oct 2006
Posts: 433

PostPosted: Sun Feb 28, 2016 4:51 am    Post subject: Reply with quote

Hi Adi,
By practical comparison: 20x: 1um, 40x: 0.67um, 60x oil: 0.5um.
This is based on the micrometer screw; due to the refractive index of the mountant this is not the same distance as for the sample.

Hth, Rene
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Adalbert



Joined: 30 Nov 2015
Posts: 727

PostPosted: Mon Feb 29, 2016 8:13 am    Post subject: Reply with quote

Hello Rene,
OK, but my calculation is based on the aperture (550nm/(NA*NA)) / 2



At the moment I am taking two photos a level of DOF.
BR, Adi
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Pau
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Joined: 20 Jan 2010
Posts: 4826
Location: Valencia, Spain

PostPosted: Mon Feb 29, 2016 9:00 am    Post subject: Reply with quote

rjlittlefield wrote:
Adalbert wrote:
Can the DOF be affected by the manipulation of the iris of my relay-lens (Helios 44M) ?

There is no simple yes/no answer.

Stopping down the relay lens can increase DOF but it can also introduce vignetting.

Whether you can stop down far enough to increase DOF, before you also get too much vignetting, depends on details of the relay lens and how you have the objective mounted.


With the lenses shown, you'll mostly can get just vignette, the camera lens aperture will show almost focused, it's useful to close it just before vignette to help preventing external light but not to have more DOF/contrast. With the microscope this is done with the condenser aperture.
Take a look: http://www.photomacrography.net/forum/viewtopic.php?t=15607 (second picture)
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Pau
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Adalbert



Joined: 30 Nov 2015
Posts: 727

PostPosted: Mon Feb 29, 2016 9:30 am    Post subject: Reply with quote

Hi Pau,
Thank you for the link!
My solution is similar but I’m using 58mm relay-lens (close to 63mm) for the full-frame.

BR, Adi
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