How to calculate the step-size for the stacking

Adalbert · Post by **Adalbert** » Thu Feb 25, 2016 3:51 am

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 · Post by **zzffnn** » Thu Feb 25, 2016 5:49 am

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

JH · Post by JH » Thu Feb 25, 2016 11:10 am

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/publicatio ... 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

Adalbert · Post by **Adalbert** » Fri Feb 26, 2016 5:29 am

Hello Jörgen,

“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

rjlittlefield · Post by **rjlittlefield** » Fri Feb 26, 2016 12:46 pm

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 · Post by **pulsar123** » Fri Feb 26, 2016 12:58 pm

For this I simply use online macro calculators:

http://coinimaging.com/calculator.html

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

http://www.cambridgeincolour.com/tutori ... lenses.htm

Adalbert · Post by **Adalbert** » Fri Feb 26, 2016 1:12 pm

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

BR, Adi

Adalbert · Post by **Adalbert** » Fri Feb 26, 2016 1:22 pm

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 · Post by **rjlittlefield** » Fri Feb 26, 2016 1:30 pm

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 · Post by **René** » Sun Feb 28, 2016 4:51 am

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

Adalbert · Post by **Adalbert** » Mon Feb 29, 2016 8:13 am

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 · Post by **Pau** » Mon Feb 29, 2016 9:00 am

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/v ... hp?t=15607 (second picture)

Adalbert · Post by **Adalbert** » Mon Feb 29, 2016 9:30 am

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