40X and ApsC Sony a6100: how calculate step size?

anto74 · Post by **anto74** » Thu Mar 12, 2020 9:32 am

Hello,
i would buy a good 40x ( Nikon M Plan Elwd i think...) but i have one question: i have Wemacro rail and i can set step size to 0,001mm. With 40x how can i calculate right step size?
Thanks
Antonio

rjlittlefield · Post by **rjlittlefield** » Thu Mar 12, 2020 10:01 am

With microscope objectives, a good estimate of DOF can be calculated as DOF = 0.00055/(NA*NA) .

So for example, a 40X NA 0.5 objective would have calculated DOF = 0.00055/(0.5*0.5) = 0.0022 mm.

You definitely will not be able to use a step size that is much larger than this, or you will get focus banding.

Depending on the subject and illumination, you may get cleaner results with a smaller step size.

I suggest trying both 0.001 and 0.002 mm and see which you are happier with.

--Rik

anto74 · Post by **anto74** » Fri Mar 13, 2020 12:42 am

Thanks Rik!
Antonio

rjlittlefield wrote:With microscope objectives, a good estimate of DOF can be calculated as DOF = 0.00055/(NA*NA) .

So for example, a 40X NA 0.5 objective would have calculated DOF = 0.00055/(0.5*0.5) = 0.0022 mm.

You definitely will not be able to use a step size that is much larger than this, or you will get focus banding.

Depending on the subject and illumination, you may get cleaner results with a smaller step size.

I suggest trying both 0.001 and 0.002 mm and see which you are happier with.

--Rik

Macro_Cosmos · Post by **Macro_Cosmos** » Fri Mar 13, 2020 3:33 am

I suggest 1um/0.001mm.

Miljenko · Post by **Miljenko** » Mon Mar 16, 2020 6:41 am

Antonio,
I'm using Nikon MPlan 40x 0.50 ELWD and setting my DIY rail at 1.25 um for this objective since that is default full step for my rail. It seems just about right.
However, Kurt from Switzerland used 0.5 um step with his wonderful Odysseus butterfly wing scales stack using the very same objective.
Maybe he can comment on reasons for using such small step.
Best,
Miljenko