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Joined: 30 Nov 2015
Posts: 727

 Posted: Thu Feb 25, 2016 3:51 am    Post subject: How to calculate the step-size for the stacking 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
zzffnn

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

 Posted: Thu Feb 25, 2016 5:49 am    Post subject: I would like to know as well, thank you very much!_________________Selling my Canon FD 200mm F/2.8 lens
JH

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

 Posted: Thu Feb 25, 2016 11:10 am    Post subject: 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

Joined: 30 Nov 2015
Posts: 727

Posted: Fri Feb 26, 2016 5:29 am    Post subject:

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?
rjlittlefield

Joined: 01 Aug 2006
Posts: 20029
Location: Richland, Washington State, USA

 Posted: Fri Feb 26, 2016 12:46 pm    Post subject: 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
pulsar123

Joined: 12 Jun 2015
Posts: 130

 Posted: Fri Feb 26, 2016 12:58 pm    Post subject: 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

Joined: 30 Nov 2015
Posts: 727

 Posted: Fri Feb 26, 2016 1:12 pm    Post subject: Hello Rik, Can the DOF be affected by the manipulation of the iris of my relay-lens (Helios 44M) ? BR, Adi

Joined: 30 Nov 2015
Posts: 727

 Posted: Fri Feb 26, 2016 1:22 pm    Post subject: 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
rjlittlefield

Joined: 01 Aug 2006
Posts: 20029
Location: Richland, Washington State, USA

Posted: Fri Feb 26, 2016 1:30 pm    Post subject:

 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
René

Joined: 08 Oct 2006
Posts: 433

 Posted: Sun Feb 28, 2016 4:51 am    Post subject: 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

Joined: 30 Nov 2015
Posts: 727

 Posted: Mon Feb 29, 2016 8:13 am    Post subject: 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
Pau

Joined: 20 Jan 2010
Posts: 4826
Location: Valencia, Spain

Posted: Mon Feb 29, 2016 9:00 am    Post subject:

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)
_________________
Pau

Joined: 30 Nov 2015
Posts: 727

 Posted: Mon Feb 29, 2016 9:30 am    Post subject: 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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