Step Size

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jin
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Step Size

Post by jin »

Apologize if this question had been asked before but i don't seem to find it and the question is how to use the table in Zerene Stacker (cut & paste as below) to determine the step size (so that i'll not over stack):

Microscope Objectives
NA DOF
0.1 0.055 mm
0.14 0.028 mm
0.2 0.014 mm
0.25 0.0088 mm
0.3 0.0061 mm
0.4 0.0034 mm
0.5 0.0022 mm
0.55 0.0018 mm

For example i 'should' be setting the step size to 0.0088mm (or less) if the objective i'm using for stacking has got a NA of 0.25.

Pls. advise if otherwise or offer alternate solution for step size determination.

cheers!

rjlittlefield
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Re: Step Size

Post by rjlittlefield »

jin wrote:For example i 'should' be setting the step size to 0.0088mm (or less) if the objective i'm using for stacking has got a NA of 0.25.
Correct.

But a few words of caution...

It's always best to consider computed numbers for DOF as only a starting point, then either just go smaller to be conservative or experiment if it's critical to get some optimum value.

In the case of this table (Table 2-C at https://zerenesystems.com/cms/stacker/d ... romicrodof), the numbers are computed using the formula DOF = 0.00055/(NA*NA). That formula is based totally on constraining defocus blur to be no worse than 1/4-lambda wavefront error for green light of lambda = 550 nm. That gives a maximum contrast loss of about 26% at the worst level of fine detail, compared to a perfectly focused image that is suffering only from diffraction blur.

The formula does not take into account other troublesome effects that often occur at wide aperture, most notably the "squirming" effects that often occur because of directional reflections off shiny 3D surfaces, for example as shown at https://www.photomacrography.net/forum/ ... 187#149187 .

If you have one of those difficult cases, then you may need a substantially smaller step size in order to get the cleanest possible result.

--Rik

Lou Jost
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Post by Lou Jost »

Rik, do you have some worst-case guidelines to suggest for an adjustment factor (which probably varies with NA)?

metebalci
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Re: Step Size

Post by metebalci »

rjlittlefield wrote: If you have one of those difficult cases, then you may need a substantially smaller step size in order to get the cleanest possible result.
Probably asking a well answered question before, sorry for that. Assuming there is no constraint of time, computational power and space etc., does the output of focus stacking (algorithms) get better with decreasing step size ? Or a concrete example, if I have NA 0.14, would a step size=2um hurt anything compared to a step size of 10um or 20um ?

Mete

metebalci
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Post by metebalci »

I just saw the following thread, so I guess the answer is that a bit more noise may happen with smaller step size but generally no decrease (and potentially an increase) in the output quality.

https://www.photomacrography.net/forum/ ... hp?t=41075

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