Question about DOF when using a shorter tube lens.

[Kieran Jones]

I'm using a tube lens with a focal length that's shorter than that specified for my microscope objective.
I'm using a 105mm instead of a 200mm tube lens with a 10x /NA 0.28 infinity corrected objective.

I'd like to know if doing this changes the DOF of my setup.

Thanks.

Kieran

[rjlittlefield]

The objective is still running at NA 0.28, so the diffraction limited 1/4-lambda DOF will be unchanged.

However, the resulting optical image may be so sharp that the usable resolution becomes limited by pixel size, either in the sensor or in the final display medium. In this case the DOF will increase at lower magnification. See HERE for some discussion and illustration that NA 0.25 at 5X outresolves a 15 megapixel APS-C sensor.

So unfortunately the answer is "it depends". For image quality, you can't go wrong by assuming that the DOF remains unchanged, but that may mean shooting more images than you would really need for your application. If that matters, there's no substitute for testing.

--Rik

[Kieran Jones]

Hello Rik:

Thanks for the quick and informative reply.

I'm currently using the same focus-stacking step-size that I use with the longer tube lens (5 microns),
but I'd like to use a larger increment to reduce the stack depth if possible.

As you point out, there's no substitute for testing.

Regards,

Kieran

[Kieran Jones]

As a test of using a larger stack step size, I've posted two images of the same fly here.

Both were taken with the shorter 105mm tube lens, but one used a stack step size of 5 microns and the other a step size of 10 microns.

[pontop]

In Zerene Stacker there is a setting to stack every n'th frame. That way you can use the same stack to test how big steps are OK.

[Kieran Jones]

Hello pontop:

Thanks for that very useful tip - it will save me much time in the future (I should have thought of that myself!).

Kieran