DoF/3 = step size?

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dhmiller
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DoF/3 = step size?

Post by dhmiller »

This is probably very old news to most, but I just saw a post on FB recommending that the step size for any given stack should be at least the DoF/3. Is that the "industry" standard? (Poster also recommends DoF/6 if printing large prints.)

Adalbert
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Re: DoF/3 = step size?

Post by Adalbert »

Hello Dennis,

I always use the step size DOF/3, only for very high NA I use a larger divisor like 4 or 5.

My rail is precise and can handle very small steps, but the deviations from the desired size are inevitable.
If you are unlucky and the sum of the deviations of the two following steps is larger than the DOF, then you lose a layer.

Since I have a camera with a fully electronic shutter, the number of photos taken
does not matter in the consumption of the camera.

Best, ADi

dhmiller
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Re: DoF/3 = step size?

Post by dhmiller »

Thanks very much. (Probably the reason I am having trouble with 20x and 50x.) Never too late to learn!

Macrero
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Re: DoF/3 = step size?

Post by Macrero »

At less than 20X I use 70% DoF steps. Stacks comes out just as they should. I have made comparisons using shorter steps and I am unable to see any difference. Well, the difference is that shorter steps imply more work, time (shooting and processing) and probably some more noise accumulated in Zerene (PMax). I see no reason to use steps that short.

At high magnifications I do use shorter steps (about 1/3 DoF) to compensate for mechanical (rail, motor) flaws.

But try and see for yourself. Make a stack with different step size, say 70%, 50% and 30% DoF and compare the results.

Best,

- Macrero

Edit:
dhmiller wrote:
Sun Aug 22, 2021 9:11 am
Poster also recommends DoF/6 if printing large prints.
What? :| Did the "poster" post some proof? Let me guess, he did't...
https://500px.com/macrero - Amateurs worry about equipment, Pros worry about money, Masters worry about Light

dhmiller
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Re: DoF/3 = step size?

Post by dhmiller »

"Did the "poster" post some proof"
There was noting polemical about the comment. It was just a suggestion. I'm trying it currently to determine if it will help my high magnification stacks.

Macrero
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Re: DoF/3 = step size?

Post by Macrero »

dhmiller wrote:
Tue Aug 24, 2021 7:17 am
"Did the "poster" post some proof"
There was noting polemical about the comment. It was just a suggestion. I'm trying it currently to determine if it will help my high magnification stacks.
I understand that, but I find that suggestion unfounded, to say the least.

Yep, the best way to clear up any doubts is to try and see for yourself.

There are several ways to improve your stacks: getting better gear, improving your technique...

Using hilariously short steps is not one of them, at least not in my book and experience.

Edit: Oh, sorry, I'm multitasking... Just noticed the "high magnification stacks" part. You didn't mention that in your OP.

At very high magnifications it is a good idea to use shorter than necessary steps to compensate for possible mechanical errors. Though if you use a good rail, 1/6 DoF still seems overdoing it.

dhmiller
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Re: DoF/3 = step size?

Post by dhmiller »

Thanks, Macrero. I am always trying to improve my technique - a never ending challenge ;-) And this was the first I had heard of using anything smaller than the DoF. I live by Rik's table (2-C) in the Zerene manual, which says : "Determine DOF (step size) [sic] from Numerical Aperture (NA)," so I had always just used the values in those tables for step size (5x, 10x, 20, ...100x). But yes, I understand using a fraction of DOF is more important at higher magnifications and is not so essential at lower.

Thanks again for your help. Now back to lighting and cleaning struggles ;-)

Macrero
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Re: DoF/3 = step size?

Post by Macrero »

dhmiller wrote:
Tue Aug 24, 2021 9:18 am
Thanks, Macrero. I am always trying to improve my technique - a never ending challenge ;-) And this was the first I had heard of using anything smaller than the DoF. I live by Rik's table (2-C) in the Zerene manual, which says : "Determine DOF (step size) [sic] from Numerical Aperture (NA)," so I had always just used the values in those tables for step size (5x, 10x, 20, ...100x). But yes, I understand using a fraction of DOF is more important at higher magnifications and is not so essential at lower.

Thanks again for your help. Now back to lighting and cleaning struggles ;-)
My pleasure!

There is always something to be done and improved. Good luck, looking forward to seeing your high-mag stacks.

Best,

- Macrero
https://500px.com/macrero - Amateurs worry about equipment, Pros worry about money, Masters worry about Light

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Re: DoF/3 = step size?

Post by rjlittlefield »

There is another reason why small steps are sometimes helpful at high magnification.

This is the "squirming around laterally" effect that may look something like this illustration from viewtopic.php?p=149187#p149187 :

Image

The physical reason for the effect is difficult to explain. It has to do with light bouncing off the subject asymmetrically, so that much more light enters one side of the objective than the other. This results in the objective essentially "looking sideways" at features of the subject. But the result is simple: as focus changes, those features appear to move laterally. Depending on the nature of the subject and the illumination, subject features can move laterally much faster than they soften and sharpen due to defocus.

Unfortunately, this problem is often the most troublesome with subjects and illumination schemes that are visually the most interesting. It gets worse with directional illumination, shiny subjects, and wide entrance cones. High magnification makes those last two aspects much worse because at high mag most surfaces are shiny, and at high mag you need wide entrance cones to get good resolution.

The tables at https://zerenesystems.com/cms/stacker/d ... romicrodof only deal with the softening effect of diffraction. They give step sizes that you need to reach the 1/4 lambda level of wavefront error, which is the classically accepted arbitrary criterion for being "diffraction limited". But the tables do not take into account at all the "squirming around laterally" effect.

Anyway, when the "squirming around laterally" effect is in play, using a step size that is much smaller than calculated from 1/4-lambda DOF gives the stacking software the best chance to see the subject at near perfect focus, which also coincides with the feature's being seen as its true physical position. When subject features tend to be small and linear, like small bristles or the striations on large bristles or the small groves on scales, using something like 1/3 DOF or even smaller can do a lot to avoid problems like "stairsteps" along those features.

--Rik

quenoteam
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Re: DoF/3 = step size?

Post by quenoteam »

Very interesting Rik, I think this problem is one that I have not solved well yet

dhmiller
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Re: DoF/3 = step size?

Post by dhmiller »

Thanks, Rik. Interesting assessment. Is there a known cutoff for magnification factor above which you think fractional DoF per step becomes essential?

dhmiller
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Re: DoF/3 = step size?

Post by dhmiller »

On a related note, I have a MIty 100 for sale and someone asked me for the suggested step size. This objective has an NA of .70 and the formula Zerene/Rik suggests is .00055/(NA*NA), which calculates out to .00055(.70*.70), or .00055/.49, which is .00112, so I guess 1 um would be about right. The fellow asked me if that would be the same for a full frame and APS-C sensor - I don't know the answer. Is sensor size a factor in this equation?

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Re: DoF/3 = step size?

Post by rjlittlefield »

dhmiller wrote:
Thu Aug 26, 2021 7:41 am
Is there a known cutoff for magnification factor above which you think fractional DoF per step becomes essential?
I don't know one.

I do most of my work at the nominal calculated values, rounded down to some nearby convenient number. For 10X NA 0.28, the calculation gives 0.006875 mm (per Zerene Stacker's DOFcalculator), which I typically round down to 0.005. For 20X NA 0.42, the calculation gives 0.0029737, which is so close to 0.003 that I just use that value. NA 0.42 step 0.003 mm is what I used a few days ago for the fly proboscis at http://www.photomacrography.net/forum/v ... 58#p276158 .

But note... I am usually interested in the structure of the subject, not so much in getting best possible esthetics in the final image. So my illumination tends to be flat and my subjects are not chosen to be "flashy". Plus my focusing mechanics (a microscope focus block) are so precise that I don't have to allow for variation in the step sizes. Somebody who is interested in esthetics and is working with different illumination, different subjects, and different hardware may well find that fractional DOF is valuable or even mandatory at any magnification.

I think the bottom line has to be "try it and see".

One good way to do that is to shoot a test stack using step size that is a small fraction of a DOF, then process it multiple times with Options > Preferences > Preprocessing > "Stack every N'th frame" set to 1, 2, 3, etc. Compare the outputs, determine what number gives the best tradeoffs for you, then use the correspondingly larger step size for future shoots. For example at 10X NA 0.28, you might shoot the test stack with 0.001 mm step size, then find that "Stack every 6'th frame" is the best choice, and shoot subsequent stacks at 0.006 mm. (Just remember to set "Stack every N'th frame" back to 1 after running your tests!)
dhmiller wrote:
Thu Aug 26, 2021 8:31 am
On a related note, I have a MIty 100 for sale and someone asked me for the suggested step size. This objective has an NA of .70 and the formula Zerene/Rik suggests is .00055/(NA*NA), which calculates out to .00055(.70*.70), or .00055/.49, which is .00112, so I guess 1 um would be about right. The fellow asked me if that would be the same for a full frame and APS-C sensor - I don't know the answer. Is sensor size a factor in this equation?
No, sensor size is not a factor in determining step size here. At 100X NA 0.70, the optics will be working at effective f/71. That means the optical image will be deep into diffraction territory on any size of modern sensor. The full frame camera will capture more total detail because it will capture a larger total field. But the resolution on subject, and the DOF, will be identical for full frame and APS-C.

--Rik

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Re: DoF/3 = step size?

Post by Chris S. »

dhmiller wrote:
Thu Aug 26, 2021 8:31 am
. . . I have a Mity 100 for sale and someone asked me for the suggested step size. This objective has an NA of .70 . . . .
With a Mitty 100/0.70 on my rig, I most often use 0.5 micron (0.0005 mm) steps. But as has been said, there is considerable "season to taste" range in choosing step size, depending on the subject, lighting, macro rig, available time, etc. I would not disagree with someone who preferred 1 micron steps, with this objective, in their work.

--Chris S.

dhmiller
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Re: DoF/3 = step size?

Post by dhmiller »

Rik - great idea to use Nth frame - had not been aware of that feature. I assume, however, that the fraction of DoF for step size used will need to be be different with different subjects and level of detail (not to mention lighting). But that just makes the task more of a challenge and the rewards ever greater. ;-)

Chris - thanks for the comment. The 100x is sitting in its box waiting for a buyer, but I will pass along your tip if/when an interested party shows up. ;-)

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