Sensible starting point for step-size when stacking @ 6.75x?

[stevekale]

I know a lot of trial and error may be required but what would be a sensible starting point? Nikon plan 10x infinity objective on 135m "tube lens" (Nikon Nikkor 2.8 )

[rjlittlefield]

10 microns should work well. Depending on your subject and criteria, you may be happy with a bigger step.

In general, the calculator at http://www.microscopyu.com/tutorials/java/depthoffield/index.html seems to give pretty good numbers. Its big limitations are that 1) you can only specify an objective used at rated magnification, and 2) the usual concept of circle of confusion is (confusingly) represented as a parameter "e" described as "the smallest distance ... that can be resolved by a detector that is placed in the image plane of the microscope objective." An effective way to use the calculator is to just choose the objective, ignore the actual magnification, push the e-slider full left to its minimum value, and use the depth of field number that pops out. For a 10X NA 0.25 objective, this produces the value 10.4 microns.

To check, I went back and experimented with one of my 5X NA 0.25 stacks (10X NA 0.25 on a 100 mm tube lens). Flashed to compare at actual pixels, the result is obviously softer at 20 microns than at 10, but if you didn't have the two to compare, the 20 micron would look fine. In fact I used 20 microns step (stacked every 2nd frame) to make the stereo at http://www.photomacrography.net/forum/viewtopic.php?p=104190#104190. Really even that was overkill for the displayed resolution.

--Rik

[stevekale]

Thanks!

[ChrisR]

Yes, but, ... um, I'll start a new thread.