Newbie question - Depth of Field considerations...

Just bought that first macro lens? Post here to get helpful feedback and answers to any questions you might have.

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rick
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Newbie question - Depth of Field considerations...

Post by rick »

Hi Everyone,

I have a few questions and concerns about Depth of Field (DoF) calculations for my soon to be real setup. I have a Nikon Nikkor Micro 200mm f/4 ED-IF and will be using it as my tube lens. Recently I purchased an adapter ring to mount Mitutoyo objectives to the front. While I wait for the adapter ring to arrive I've been trying to find a resource that would help me understand and calculate DoF for various combinations I'm envisioning. I'm hopeful that someone can help me figure out how much overlap I'll need between images to increase stacking success. Soon I'll be acquiring a Mitutoyo M Plan APO 10x objective. Over time I'll more than likely add similar objectives with 2x, 5x and 20x magnifications.

Camera/Lens Setup:
Nikon D800e, full frame sensor
Nikon Nikkor Micro 200mm f/4 ED-IF, used as tube lens
RafCamera M26 to 62mm adapter ring
Mitutoyo objectives

How does having a microscope objective combined with the 200mm tube lens play in the formulas that calculate DoF?

Beyond Depth of Field, I'm also wondering about a few other things. What aperture should the tube lens be set at, does it matter? Can I shift magnification on the tube lens by rotating the focus ring from one extreme to another? Will moving the focus ring impact the quality of the images being captured?

Thanks in advance for your guidance and expertise!

--Rick

Adalbert
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Re: Newbie question - Depth of Field considerations...

Post by Adalbert »

Hi Rick,
see glossary in https://www.mitutoyo.com/wp-content/upl ... 010611.pdf
BTW, I use the simple rule DOF = 0,00055 / (NA*NA)
Best,
ADi

rick
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Joined: Tue Feb 02, 2021 8:49 am
Location: Los Angeles, California

Re: Newbie question - Depth of Field considerations...

Post by rick »

Thanks ADi for the helpful Mitutoyo glossary referral link and the simplified formula! I have found these two seemingly helpful sites yet they don't address all my questions & concerns. Plus at this point I'm not 100% sure how to utilize the calculators when the objectives are combined with my 200mm tube lens. Maybe the tube lens is not part of the equation?

MACRO DEPTH OF FIELD (DOF) CALCULATOR
https://www.photopills.com/calculators/dof-macro

Focus Stacking
http://extreme-macro.co.uk/focus-stacking/#calculator

Yesterday I ordered a MicroMate from WeMacro to help with image collection. My macro platform uses an old microscope base that provides 100µm of travel with a full turn of the fine focus knob (with 0 backlash). I'll be able to make steps as short as 0.25µm when needed.

--Rick

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Re: Newbie question - Depth of Field considerations...

Post by rjlittlefield »

You have my sympathy about the difficulty of good references. I've been studying DOF for 50 years, and writing about it for more than 10, and I have never figured out how to explain it thoroughly yet concisely. But let me try to answer your questions.

First, as background summary...

In general, DOF is determined by two very different factors:
(1) how quickly the image becomes blurred as it goes out of focus, and
(2) how much blur can be tolerated by the viewer of the image.

Classic formulas like DOF = 2*C*N*(m+1)/m^2 incorporate these two factors by
(1) pretending that blur is entirely determined by ray tracing, ignoring the wave properties of light, and
(2) inserting a specific value for "circle of confusion", that reflects typical situations.
These formulas work well at low magnifications, say from landscape to close-up photography.

But at higher magnifications, a wave property of light called "diffraction" becomes important. By the time you're using a microscope objective, diffraction is the main controlling factor. Diffraction determines how quickly the image becomes blurred as it goes out of focus, and it does that so strongly that all other factors can be ignored. In this regime, it works well to use formulas based on the NA = "numerical aperture" of the objective. The formula quoted by ADi is a good one.

Now, about your specific questions...
Maybe the tube lens is not part of the equation?
The tube lens has no effect on NA, so it also has no effect on DOF. You can even change magnification by changing focal length of the tube lens, and still NA and DOF are not changed.
What aperture should the tube lens be set at, does it matter?
Wide open is a good starting point. Stopping down may give a small increase in image quality, mostly due to elimination of stray reflections. But if you stop down too far, you will definitely get vignetting. The amount of vignetting depends on the sensor size, some internal properties of the tube lens, and your own tolerance for corner darkening. You'll have to experiment to see what works best with your setup.
Can I shift magnification on the tube lens by rotating the focus ring from one extreme to another?
Rotating the focus ring will shift focus, but it typically has only a small effect on magnification. One way to shoot a stack is by rotating the focus ring by small amounts.

If you want to change magnification, the trick is to use a tube lens with a different focal length. At first glance, a zoom telephoto would seem to be ideal. Unfortunately zoom telephotos typically give a lot of vignetting if you zoom them very far from their longest setting. This is especially troublesome with full frame sensors.
Will moving the focus ring impact the quality of the images being captured?
Yes, but mostly at high magnifications and for large turns of the focus ring. All of these aspects are discussed in the thread titled "AF motor focusing with a microscope objective", at viewtopic.php?t=14569 . But you won't be needing any of that because you'll be stepping focus with the microscope stage.

I hope this helps!

--Rik

rick
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Location: Los Angeles, California

Re: Newbie question - Depth of Field considerations...

Post by rick »

After a little shipping delay my new MicroMate arrived from China today, yay! I'll be investigating and learning how to use it in the coming days, hopefully it won't take me weeks. I have time as the Mitutoyo adapter plate I ordered is still in Russia, dancing with customs clearance for almost two weeks. The plate was shipped March 10th, hoping it will arrive soon.

ADi, I did a few calculations based on your simplified formula and objective NA values from the link you provided. Do the below resultant values look accurate? I've included all the objectives I may acquire, yet I'll be starting with 10x.

Rough Objective Dof calculations using 0.00055/NA² (NOTE: UPDATED to reflect proper micron values, also updated copy in below paragraph)
• M Plan APO 1x = 0.00055/(0.025)² = 0.880 mm or 880.0 microns
• M Plan APO 2x = 0.00055/(0.055)² = 0.1818 mm or 181.8 microns
• M Plan APO 5x = 0.00055/(0.14)² = 0.0281 mm or 28.1 microns
• M Plan APO 10x = 0.00055/(0.28)² = 0.007 mm or 7.0 microns
• M Plan APO 20x = 0.00055/(0.42)² = 0.00312 mm or 3.12 microns
• M Plan APO 50x = 0.00055/(0.55)² = 0.001818 mm or 1.818 microns
• M Plan APO 100x = 0.00055/(0.7)² = 0.00102 mm or 1.02 microns

If these are accurate, do I now need to make steps with 30% overlap? If yes, the values above would be lessened by 1/3. As previously mentioned my setup, now with the MicroMate controller, can be stepped as short as 0.25 microns. This means I'll be able to use a Mitutoyo 100x objective and still have enough to overlap between steps. Once I get the adapter plate and objectives I'll capture reference coverage areas and images from my full frame sensor to share. I have a feeling that the focus collar on my Nikon Micro 200mm f4 tube lens will allow some magnification. Perhaps not, based on one of Rik's comments. With a lower power Close-Up lense I have seen focus collar magnification shifts. I use this shift to gauge which objective/close-up lens combination to use relative to the size of the specimen I'm imaging (not to focus stack with the collar). I typically want to fill the sensor as much as possible to minimize cropping.

Rik, thank you for your comments. I really appreciate your wealth of experience, insight and more than anything else your response to my seemingly simple questions.

--Rick
Last edited by rick on Tue Mar 23, 2021 5:21 pm, edited 2 times in total.

rjlittlefield
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Re: Newbie question - Depth of Field considerations...

Post by rjlittlefield »

Rick, thanks for the kind words.

About your calculations...

Your mm numbers are OK, but your micron numbers [were] too small by a factor of 10. 1 mm = 1000 microns, not 100 microns.

0.25 micron = 0.00025 mm, so you'll have no trouble using any objective.

--Rik

rick
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Location: Los Angeles, California

Re: Newbie question - Depth of Field considerations...

Post by rick »

Rik, thank you for that clarification. I'm happily surprised in the event I step up to 100x in the future. Not sure if this is correct protocol yet I updated my previous post to reflect the micron changes. I also updated some of the copy to reflect this new awareness. I did this so my comments don't confuse anyone in the future. Thanks again!

--Rick

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Re: Newbie question - Depth of Field considerations...

Post by rjlittlefield »

Edit-in-place with explanation is a good approach to this sort of glitch. No worries about protocol.

--Rik

Pau
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Re: Newbie question - Depth of Field considerations...

Post by Pau »

Yesterday I ordered a MicroMate from WeMacro to help with image collection. My macro platform uses an old microscope base that provides 100µm of travel with a full turn of the fine focus knob (with 0 backlash). I'll be able to make steps as short as 0.25µm when needed.
Be aware that the Wemacro controller software is not well suited for their MicroMate*, the "µm" shown at the screen only correspond to actual micron with their focus rail with 200step/turn motor.

The MicroMate has a 400 step/turn and you have it coupled to 100µm/turn focus block. It takes "2000µm" in software for a complete turn, so with a 100µm/turn block you have

100/2000 = 0.05 actual µm each "µm" in software and so 20"µm" in software for 1 actual µm

Yo can do much smaller steps if the precision of the mechanics allows it.

*I've communicated about this with William, the Wemacro maker, but modifications have not been made after two years.
Pau

Adalbert
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Re: Newbie question - Depth of Field considerations...

Post by Adalbert »

Hi Rick,
do I have to take steps with 30% overlap?
I usually take 3 photos per DOF, and more than 3 at high magnification.
It is my experience; some photographers take many more pictures per DOF.
If you wanted to mount the rail yourself, please take a look at my rail:
https://www.photomacrography.net/forum/ ... 41#p186841
Best,
ADi

Pau
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Re: Newbie question - Depth of Field considerations...

Post by Pau »

Adalbert wrote:
Wed Mar 24, 2021 3:38 am
do I have to take steps with 30% overlap?
I usually take 3 photos per DOF, and more than 3 at high magnification.
Aren't they about the same? (well, 33.33 overlap)
Pau

rjlittlefield
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Re: Newbie question - Depth of Field considerations...

Post by rjlittlefield »

Pau wrote:
Wed Mar 24, 2021 4:31 am
Adalbert wrote:
Wed Mar 24, 2021 3:38 am
do I have to take steps with 30% overlap?
I usually take 3 photos per DOF, and more than 3 at high magnification.
Aren't they about the same? (well, 33.33 overlap)
I interpret "3 photos per DOF" as meaning 66.66% overlap, that is, 2/3 of the DOF of each frame is also covered by the next frame.

--Rik

Adalbert
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Re: Newbie question - Depth of Field considerations...

Post by Adalbert »

Hello Pau,

Actually, I just wanted to stress that this is how I proceed.

The higher the NA, the smaller the steps.
The smaller the steps, the greater the deviation from the step size.
The greater the deviation, the greater the number of photos within the DOF.

A look at the evaluation of the test of the behavior of a rail explains everything 😊.
https://www.photomacrography.net/forum/ ... 7&start=15

Best,
ADi

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